. . . . .tuua-nLth-u..- -.
.. .. U .. #- .Ll.....-...4-.hn-l-.-........E-.hn\.14-fi
. .. 1.... --.r.-..u-m-HMM\.nr.-fl

Mn”...

 

  

 

“Ht! .
“1:5
3: 3
.f
.33
~ 1
1|:

3

3

3? -.:
y!

a) u

            

         

v

it:
3...:
..E
E.
fl.

 

I '

yummy...”
:m. 3““

.9-

:35;
3:
w!
. '-

  

       

            

 

I

 

.12.?-
’19-'13

. . I
.....u......-...,...m..-. u"... .
1...... :

  

1.. I!

3-3

 

1.3%.»...- 33%.... H

  

 

 

3... - 1.- .
. A l,-
i “.1151...

ubrmqmuwhrmvuumé mud. . - .
. . - ,
.-

 

 

1 I

Huang-3?:

h. “1...?

3—,. 1‘ i. , .

$.50. 1145!: .....
- .1

.- . .
. . .
v-AAV “uh-3.3.4. .1. $.- .
.\ 2.5.5.-..1. .f. .
5.0.9 .9...

I. .lutui.
33:115-. I..\\
. vhjxviz.

                          

 

      

    

    

. 4-.er-1huhv.
.2- V Fey-(.I.\.'ll\|l.t -

            

      

           

  

          
     

     

  

d5. .1
- .- .1 awn-4......-
..... fun 3 I...
. . .. Nu... _ ..vnaflw 21:1!
‘ 11 - . Iii] .
‘0 5‘. fit? .-
-..---...- 35......
11.5.5.1---

Jfika..\-.-.

 

  

            
 

   

 
        

        

  

                 

- - i ,
. ..- -. ...-........-wvi wW-Nwta
. . . ... n. a flan-ax... .33....-
- . .. \- u. .37.... a... .
{luv-U-rldvflvghm55H 14... man... Mmmhmvwn 1.. 3..."...
- 1 kn.- -.rdflql. , $58-11- . .. $5.? 0..-
.I--. J1-.. . aria-.5559...
'5? 3. NIL-ruinfii ‘IUIWJ; .
-\« Ila...- ..
. - . ..-.\--.-2....... . .
. dn-‘ihvtuqu‘vfl‘éflfli II -
.3 3-. 3..- -fivsgfluurun
«A l - 0.5“! 0.... ..r‘lfl .
51.51.154.15 u...- 5 o ..
2.-...1. -..-. 51.1 .8... I...
.- 3355... .
.2...- .

    

E 1.1!...011.‘
2.. .fl.n\£\:1\¢.!ul..¥\
I) A...- . :vaOL... Wil-

t: .1. n. (

 

I ..-

  

.. . .m..Z..-. ..7
. .vrfi.wbniti.-.-h-.K.U-tii.ufixdnwa
.4...“ ‘ADQIVHQ‘ - .- 1|
.. 5.254581? «Jam. 11%....
.99...“ . . .- Ir
5.... 2 . uni-1.3.91.9...“ .
.fi-flu...mv..-.nhb.r-§-...-.u..- ..
I‘ullv“!~\ vlinwt‘d‘lluu .4.
.

ton-\l. .
1 .. 5.... x

     

 

              

           

 

.v-C.-Ill-.
\a'.‘ .-
‘1A93.-| .
. O) ’Xl‘Il. .

 

.

.- -.
. 91.?
Hi...-
- . 1-..-
.h-u-fli- .3

.4“an -.v..- Q .
. a. I’ci. O .
In“... I...- ..U>..Wdfl".fiwaan.:vh. It: 1

 

1...".- -r-H.......n.. - .-
. unto-n ..r- lob-5.1... .31.
. --...I-..-.-l-1...-.\ . .v
. I.9I‘ Al I
-.- 1-. rM-.--1.-...-!..-..-
. ..- .-.......-n.. .- 2.. .-
31... ...-...r....-.-:. . - . .
- ..
2.-..
....-\a--. --
2... .51....1 .- 5.1-.
-.....- 1. awn-flu.

i...
.- .-irI-axlt

 

‘IIY.||.
: 1.1-
....!-.JD‘..J . vL
- .. v... .3 c
- - I.-\~..v.l k.- )v..'. ..
‘1‘..th 13‘ A‘II‘ non.
.. ..v .aruix... .
1...!-

   

 

...nlns.r . .
In . I ..HVDIH .

. . l....¢......-..- gig-mus z.-

.nfivnriu! KN... Zia-(i.-

3. - . .5.....-..u.....-.r

 

           

 

 

         

    

              

 

                   

      

                

     

                   

            

           

        

   

 

v.- 29....“
. . h.-.n-..-.-.I-...3. .. .
. mug-Snunulrw‘ 141-..-»m- 3. . .
.1... ..l. “-51....- ‘U. I"..-:...---.... 5.5””.
C‘- 5.1.-V,-K- a. 1.1.25.1: 1.7.. v .- ku‘l 1".
. 2..- .. .41..-.- . -O-.- 5‘0“»--. «12-13-31...-
.. . .\-|- I... --. ($5.354... -.o-yvoylV-i. T35... .-
t£4b~ltfivn . .‘DAI‘IOI at‘t- ‘- U.“ \u'. 1‘sfitl It‘cnfllhhiu‘filsv, WP!"
..A. urn-95...... ..... rub-.0..- abut-v.5... 13....-.“ .
7. ..--!..- . 4......- -. 2......5... 1-. . ..Sl-t...
:. I. - . . - - .-- x.-- .
.87.... .-. . 5...}..- - :45.- ..-.1... 3-.....
.. .1. .0 .1 . .-1\.v-..\-.-... -..- . :.
.l .-t. .IJ .1. Alv. nit-.11-}! |.)...l-|.,.l.vinvu.-
c I\.V. ‘1 ‘1‘}.\ II a. IXI”‘:I\\-I'PI.4 ’3 V.) .
. .5 3 f .-.5:.\\-.. ..n. - l- 1.. -3...» .
3. .- .k...|.. 1-...5‘. .-...-«.. . .I..1..-J......... Han-.01.“...- .
-- . , . ;1§.u.t....xnkc.vb.tc.b-.xloxV-U¥-.. ..- ..{......-.-r..al’.
. .. . . ..u-xll...1-I. .-lt-.-.v.-..-On...iwn- l--...-....-.v-...
15151-31... ova-II.- .S...v1..- .3252.-- I...
. 5.5-... 17.5%. ..v...!.. ‘13-... 5.1-...-
. ll- $..ln...v... V1... 5-... 5-12:5...
1-. .- 93... .18....- -....-...-."...u.-...D.\
2... 1...-..-
In... 1...... .
.sn. ..1- -
. - inf-a
Mun-11w”. . u.......-.-...- v...-
. - i- 9 1- - .-K.
'tw... ’1 V .‘-‘v.w - .
r‘....... air-.1. l.- -
1.. ..5-.. .. ‘1.
- \.I Ill-I-l-Ildnu.Ԥlll\

       

 

   

.‘:
- AL-. 05.1.3- -
- .i v0bl.vl.. ..II. :50: 7-
.Y. I... o .5... 1'33. h:
5 IOIYII C:l0u«~l| I‘L‘Ifiil uh
. - ks \‘I: ..‘l
‘1... ml... . ..(u-nc‘...
.v.

 

.L I. J .
\s-Nxvhu... .
5.222....

  

.1...- ..-. .1331...” .. - i. .1 .
. - 4.3!. .r' , l)-.\ le-
qfill‘chtl. [Qt-l. I0
.- .- .-- .5.--.... .
.2..-
:-

 

- .- -- .1.-
..t. -- ..n.-$.fl.n.-..v-.-H-. -1..-

    

 

.3...
.I.|. .
:3...- -.----. .
.1- 2-. .ltitnall-w Ply-5....
-. 3-. iii-huh-.."

-

 

.
.8--. -
..-£le.-.-.1......-4. .
.. .- :2...-
‘IVY’I‘UiLIII-ull h-‘J- 1
- L... -J..-.~.-...-.L-.

to... -.
.- 5-. . ..d.
\i‘ 3.4“. \JnV)‘ ..A.
55...... .11....- .-

11..- .5.-. . a. -

.. ..-.--..-....- -. ,

   

 

V.|

       

   

 

 

   

   

  

. 1...)...- -.r 35.5...- -
. . Fifi-3.... 5.1...- v... ..J. fit-umn-HHVh...‘ at.--
.. .. .1- I\-v....’unl|t . Ex. .xvlv‘uVl" titti‘ I‘Db '9
. ..- :fi.m.-... fl -.--.-.I-..-..--:...M.l.?.-.-..-.1-)-.z3...... 90.5... $31.9...“-...c-.$.L..-.u.---:........-u.l... ..... v
.15.- cr 1L.- ..:.-.4 11...-.. or. .llu.-..r-n..A-....-._..h.9..‘.\.v ..n- v.5...- .xb..Lvn.-....|.i-.|-O..Ib 1!...
u-Q...Xl-ku.II-r »0.-\11 ll ulvoJ..I.OOu.. .- .lv.x|v.t II...II.§|I'I?I.I .nv.I.. iv v
1...... . 1-...- -. ..L. - i"...- - - ..-.-\..--..-.-w-..-..-.-.- - ......:.. .-
rsv-ibfiL-bu... 1-. -- .lwbiJl .I .-.h.-. 513. 1 I31: .5151.-.- ... . .3... c... :-
. .-. l.- . . 4!...- .-..... -..-. 31.... $1..--l.11..-.!.-.... a... 1.. 33...»..- .9
.- - ----.!-....-..-.-..-..-....-.-...-.--......-.3...5...-..-...t..-|.-.-..--..--.-.-.--..-......-l-.-.....--l... ht... 3......- ....l . .7.-
!.-!- 3.....-- -...... -... -...-z..--...--.A....-.:h-.--.:-.v .
. |\ ~ 0 ‘- ‘qvlii ..l ot|| v.- ‘1‘3-lvq
h... .4... ,Il...H-.-..-.....--:..-l..-.-....-..:. . -1 . .
. . ill-5093.5..- .-
.. 1-. .....l. .-
u‘v. |‘.. -. {Qt-I
-.-...-.....-.... .

. Pup-.23....r-ii-
5.33:1...-
. -7? .N--.-\-.4...\.. .(2

 

 

    
 

, ..IP-I ‘ -.|
99%.... .. (ah-.11.. .- - ,
- . . .- . .u. 5-.- .l..- . .
l . u. - $.54... . - - - .
. 4 ‘2- \‘iu‘ \IO- val-1.13! I-..\ 1“ .
th... . .1. -.-I $0.91.... 31-33-}!
..- 1 u--. ..I. .1--...-.F-t..-.-.-.-I. v.3“...- .
. . . 33.31..- u...», 1-...n....-.--..-u.-. r...
I. .I‘.‘\ ‘1 ‘l\.10 I I
1 5-.- . .- .-
-5...

 

 

. - -. -
. . . :1 IV. .-
...-.-....3 .-.-...-....-.--L:r-...-..-.-.-..-..-.-.-.3..h .....-u-......-..-.- 3.:

.- l t .1 I .- - -
. - . ..F...p.H-u...-.-..€ LEA-".- .- 1.312.... ..uv N“%«53.HHNmN-hmfihfifl93 .
.3...- .-.i....-.\.-.-.....-.. i..--.w.u.-.~.v...K-..~E ‘0...- -? .94....u......-.-?.1 huh-3...! . ..-
.h..-. 5.35.1.5}... 351:... - 1... l...I.-.-......-.X-. 501-3...-
_ ..s. .- 2..- -.-...,...- ..u-unu-U... .--...-b...r......u.........-..-..- .LILEWIJH... . --I..-..-.- .15...- -
- 1..- v---..- -. . 51.....4ufl4 Ntlr . .
Wu...»- ..-.v.-.t--..-..3L 1......“ ..vw-utnnr-m- "1...... .- 5.” nun-w flag-3.5.4.
3..- ..» .-d-Un-lnfimn-fiun-Hu. «Huang-vb... . .- awn-5491.4.-
»fl... Qua-“W...- - .

 

    

 

      

   

v”.
u.... 5|- 3... ..-
v?)‘\¥\.D-9| . ;
I kit! .6!- 1..
(I.

      

     

        

     

            

 

  
  

   

      

 
  

 

 

. 3.
... .55.... .- .-
.mM-hgmufl-GF 1....
L..- - .- . 4-.- .-
.- -.1..-!..¢.-a...-.x..-.a.s --n...-u-.-.2.-l....1! J-..--.-
.1 00.0.! ..ldV-th-Jucx 1.5-3.1.! c .- ‘9‘
-9...- -2 £4. . - .nn-Q....s....:-.-...(I\.-... ..l. V.
. .. .-- 30..---.- ... 2-.....Uup. -2...- Jr...‘ oat-v. - . .
.u. .41....- I1‘. .2 -59-...- .12..- .I - ..1 .- .-. r-.. .
.1..- £5... - ...--...1..-....- £511.... -.....- ... -|.v ..I-n...“ -.hllnwhffiflnu. -3 .21.:-
, 0. 5.51.14.41-34...» .l u: u...-.....r-.---¥..-I-.b:-1s-fAI..-
.r..-.\.-.-.. . -.l-‘It... ..n-u.-. 1...... 3:516... i....!.---.-
{03-31(30- s...~7r.V.-2|-\.-. -. . -..- -- .I-v .I.....-..-.i5.v\.$5.63...--
. la} -.~3.-b1lr..-.o.l- D... .l .I-.-.I.. .. (IO-..o-l ...|‘.-...-.-||I. .Iv-.--.-.
1.575.»- Ik..--.J\1-...I...-.n..v...hln .XL-u ..l... . .. .5...- “I...“ 5.4. -..-....v..z..-.....-...--..v.-.S-.... .
I I ‘I. ‘. a II ‘1’ o'l-Vll- 0' I - \‘I ' -'\.§lt- I".1II: ‘ ’l.’..:- ‘l|‘V¢ .l’l“. - .. Iv.
«fin-5.1.5.: lag-«.- .- ... . ....->.--.-..-... .31.. ..d.......--.-.\-...- bk.-. .1: 3.?le tun-h”- .
kiwi. .lunvn-‘I. o»...- .-..! 2...}.-. .-..... 3.--}-..-L...-..-...-...-..--Vl.v--...-.--- 1 - . -!
a. . - .. . . 1.01.1}.-.- .IJ-fl'. -.- - .1... -.........-.-.-..-.Ix!-...-. .l.-..-lb.-.,-:-.-. ..d..--t...-...-....--.
.- . 1-; -.. .5..- L.- ...3..-....--.-..--..!..53..-... - 11., t.)- -...--- . .. .
-. 1-...F......u-..-! .-..-P....-.muh..-....r-.-. . .5.-,-1..-h..- ............. .1...-.u.-.....n.- .-..-..-.... . . . . .
x - JON-fir-.. .093. fits-mmuxtn... ail-.14....ua-t1wu-n. . ... - - I .- .
. . .- \. 5...... .......3. . L. 25-. 19%;... .53....5053- -. -- ..v-I-H .n-W MN... ..
- .-‘l‘.’ PJ‘s-4.5 , ..ra.v0\-J~...I1b.-§vvo|v. .. f... . kill- l... «5“. “v?-
I‘ .- llnfg - . («E-.1 \v-N-I- 1-5- Ill... 5.
..5lu.V-§ . - . - .h-.‘.-........1uu-.I. - . . . t. ...-l-.|-- . .Hlilllt
’- c-Itholtlv.).\-H..v. . \.-lV‘ll-§_. ..nJ-h . \V .-
. . i. . . .6...- -...-..-.-..-.......--..---R..-- L.» -....-n.-.-v-..-I;. (-3-1- .
.. , - u .- .-- - ......... - . .-l-.1. -L-.u.-....I-..h.-.Hh\...l-...LIIT-l 1.5.5....I. duh-I155...
- , .. . ..- K-rl in... . A - I...- w-- I 11.8."..5‘0-15f- 1.5.151.-- ,
- . . l .2514.- ..l. n? .- . . . ....... ..5 51‘io.b).umu.5z.flu
.. ..£-.La|.u-Lu-._ . . , . . . 1W-..
5-.. .- -b - ..1 -
- - . 9...“... I. . .1
| . .-..\I-.III- - -

 

AN STATE UNIVERSITY Ll A

IIWIHIIIH m m Ill {Itimlflififll

3 1293 01570 6165

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the
dissertation entitled

Inhibition of Avian Leukosis Virus Replication
by Antisense RNA

presented by

Kyoung—Eun Kim

has been accepted towards fulfillment
of the requirements for

Ph.D- degree in My

 

    
 

D

\A .
O Bap! professor

Date \I/vfl/fl

MS U i: an Affirmative Action/Equal Opportunity Institution

 

LIBRARY

Mlchlgan State
Unlverslty

 

 

 

0-12771

 

PLACE ll RETURN BOX to remove We checkout from your record.
TO AVOID FINES return on or baton dd. duo.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU I. An Afflrmatlvo Action/Equal Opportunlty Inotltulon
Wan-9.1

 

INHIBITION OF AVIAN LEUKOSIS VIRUS REPLICATION BY
ANTISENSE RNA

BY

Kyoung—Eun Kim

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology

1996

ABSTRACT

INHIBITION OF AVIAN LEUKOSIS VIRUS REPLICATION BY
ANTISENSE RNA

BY

Kyoung-Eun Kim

Avian Leukosis Virus (ALV) is a class of retrovirus
which causes lymphoid leukosis in chickens. Its presence
in commercial chicken flocks affects productivity through
disease and death. With the generation of ALV—resistant
chickens as an eventual goal, the possibility of
conferring such resistance using antisense RNA has been
tested in vitro. The 5' end of the ALV genome was employed
as a target for antisense inhibition, and four fragments
of increasing size in this region were amplified by PCR.
Antisense RNA generated from each PCR product was tested
for its effect on replication of recombinant ALV vectors
in stably transfected RPBO cell lines. One antisense
transfectant showed a significant reduction of viral
replication in repeated experiments. Other transfectants,
however, did not show significant inhibition of viral
replication even though a substantial amount of antisense

RNA was detected in some of these cell lines.

Subsequently, an antisense oligodeoxynucleotide (ODN)
approach was employed in hopes of locating the most
effective target sequence in this region. Of the several
ODN tested, one which was complementary to the retroviral
primer binding site showed the most inhibition. However,
when antisense RNA directly complementary to the primer
binding site region was generated within transfected RP30
cells, this RNA again failed to substantially inhibit ALV
replication. Finally, antisense RNA was generated
complementary to the mRNA for the cellular receptor for
subgroup A ALV in hopes of generating ALV resistance by
blocking receptor expression. Again, although a
substantial amount of antisense RNA was generated in
several distinct cell lines, no significant reduction of
viral infectivity was observed. It is concluded that, at
least in our experiments, antisense strategies were not
reliably effective in inhibiting the spread of ALV in
culture, and that therefore this is not a particularly
attractive strategy to generate ALV-resistant transgenic

chickens at this time.

To my husband, my parents, and Eui-Suk’s family for the

love and support

iv

ACKNOWLDGEMENTS

I'd like to thank my mentors, Dr. Jerry Dodgson and
Dr. Donald Salter for all the supports, advice and
wonderful patience. As a real model, they showed me the
ways to think as a scientist and it was a great privilege
for me to learn from them. I'd also like to thank my
committee members, Dr. Michele Fluck, Dr. Richard
Schwartz, and Dr. Steve Triezenberg for their valuable
advice and time which helped broadening the scope of
knowledge as well as pursuing this project. Many thanks to
Dr. Sue Conrad and Dr. Ron Patterson for guide and advice.
I won't forget my lab mates, Bill Payne, Huei—Min Lin,
Steve Suchyta, Christoph Knorr, Yi Li, Natalie Moore,
Wynne Lewis, Chongpo Kim, Kyle Enger, and Ron Okimoto, for
the advice, help and all the good times that we had
together. If it were not for the friendship of Susan Kutas
as well as my fellows, my life here might have not been
the same. It was a great experience to learn and study in
this nice and warm environment with the excellent support

from all the staffs.

TABLE OF CONTENTS

List of Figures ................................... viii
List of Tables ...................................... x
Introduction ....................................... 1

Chapter 1. Literature review

Avian Leukosis Virus ..................... 3
Retroviral Vectors ....................... 17
Antisense RNA ............................ 20
Mechanism of antisense RNA

or ODN action ............................ 26

Chapter 2. Antisense RNA.generation as a strategy for the
induction of cellular resistance to ALV

Abstract ................................. 35
Introduction ............................. 36
Materials and methods ..................... 40
Results .................................. 48
Discussion ............................... 74

Chapter 3. Antisense Oligodeoxynucleotides as Inhibitors
of ALV Growth

Abstract .................................. 89
Introduction .............................. 90
Materials and methods ..................... 93
Results ................................... 94
Discussion ............................... 107

Chapter 4. Antisense RNA.complementary to subgroup.AHALV
recaptor mRNA.erpressed in a quail cell line:
test for effects on virus susceptibility

Introduction ............................ 118

Materials and methods ................... 121
Results ................................. 128
Discussion .............................. 141
Summary and Conclusions ............................ 143
Bibliography ....................................... 145

 

List of Figures

Chapter 1
Figure 1-1. The retrovirus virion ................. 4
Figure 1-2. Diagram of ALV proviral DNA,

genomic RNA and proteins ................ 6
Figure 1-3. The genome organization of

RCOSBP and RCASBP ....................... 19
Figure 1-4. Phosphorothioate and

phosphodiester linkages ................. 25

Chapter 2
Figure 2-1. The regions targeted by

antisense RNA ........................... 39

Figure 2-2A.Diagram illustraiting the procedure

Figure

Figure

Figure
Figure
Figure

Figure

Figure
Figure

Figure

employed to screen constitutive
antisense-expressing colonies ........... 50

2-ZB.Diagram illustrating the procedure

employed to screen antisense-expressing
colonies using the tet-regulatable

expression system ..................... 51
2-3. Southern blot analysis of representive

clones ................................. 52
2-4. RT-PCR analysis of clone BB3 ............ 56
2-5. The effect of cell lines harboring the AE

antisense sequence in the tet-repressible
system ................................ 62

2-6. The effect of cell lines harboring the AD

2-7.

antisense sequence in the tet—repressible
system ................................ 64
The effect of cell lines harboring the AC
antisense sequence in the tet-repressible
system ................................ 67

2-8. Northern blot analysis of the

AD-tet clones ......................... 69

2-9. Northern blot analysis of the

AE—tet clones ......................... 70

2-10. The effect of cell lines harboring the

an

CF antisense sequence in the

tet-repressible system ................ 72
Figure 2-11. Northern blot analysis of the CF-clones

in the tet-inducible

expression system ..................... 76
Figure 2-12. The effect of cell lines harboring the

CF antisense sequence in the

tet-inducible system .................. 77
Figure 2-13. The effect of cell lines harboring the CF

antisense sequence (without poly A.signal)

in the tet-inducible system ........... 82
Figure 2-14. Effects of pooled.383 transfectants

with AE-sense in the tet-repressible

system ................................ 85

Chapter 3

Figure 3-1..A diagram illustrating the procedure
used to test antisense ODN inhibition

of viral replication ..................... 96
Figure 3-2. The regions targeted by
antisense ODN ........................... 97
Figure 3—3A.Effects of antisense ODN #4 compared
to the controls ........................ 105
B.Effects of antisense ODN #4 compared
to the controls ........................ 106

Figure 3-4. Sequence of the region near the PBS
of the RCOS and RCAS viral RNA
targeted by antisense

ODN #4, #4A and #4B .................. 108
Figure 3-5A.Effects of ODN #4, #4A and #4B ........ 109
Figure 3-5B.Effects of ODN #4, #4A and #4B ........ 110
Chapter 4
Figure 4-1. The region targeted by

antisense RNA .......................... 125
Figure 4-2. Northern blot analysis of antisense

QR RNA ................................ 134

Figure 4-3A. Detection of proviral DNA
B. The genome organization of
RCASBPAP(A)provirus ................ 137
Figure 4-4. Northern blot analysis of AP mRNA
expression ............................. 139

ix

Chapter 2

Table 2-1.

Table 2-2.

Table 2-3.

Table 2-4.

Chapter 3

Table 3—1.

Table 3-2.

Table 3-3.

Table 3-4.

Chapter 4

Table 4-1.

Table 4-2A.

Table 4-28.

Table 4-3.

List of Tables

Primers used in PCR amplification
of antisense target regions

of ALV .................................. 42
Effects of the transfectants on ALV
replication ............................. 53
The effect of clone 383 on

replication of ALV ....................... 54
The level of transactivation of the CF-tet
inducible clones ......................... 75
Sequence of antisense

oligodeoxynucelotides ................... 98
Dose response of inhibition of ALV
replication by antisense ODN ............ 99
Random sequence control

oligodeoxynucleotides ................... 102
Effects of antisense ODNs compared to

their randomers ........................ 103
The primers used in

PCR amplification ....................... 124
The effect of QT6 clones harboring

the QR2 antisense sequence on viral
infectivity ............................ 130
The effect of QT6 clones harboring the QR3
antisense sequence on viral

infectivity ............................ 131
Assay of AP activity ................... 135

INTRODUCTION

Avian Leukosis Virus (ALV) belongs to the Avian
Leukosis-Sarcoma group of retroviruses. Some of the
ALVs are pathogenic and cause lymphoid leukosis (LL) in
chickens by transforming B cells in the bursa of
Fabricius. These transformed B cells develop into bursal
tumors which can metastasize to other organs resulting
in eventual death. Although there has been significant
progress in eradicating ALV from the breeder stock of
commercial chicken flocks, no commercial vaccines are
currently available. Pathogenic ALVs still exist in
chickens and their effects are enhanced by nonpathogenic
ALVs and other pathogenic avian viruses.

Antisense RNA has been implicated in the regulation
of gene expression in various systems and has been
employed to inhibit virus replication. It is thought to
block gene expression by hybridizing to target RNA and
rendering it functionally inactive, generally by an
unknown mechanism(s). This thesis describes the effects
of antisense RNA on ALV replication in tissue culture

system.

Chapter 1.

Literature Review

Avian Leukosis Virus

Avian Leukosis Virus (ALV) is a class of retrovirus
belonging to the avian leukosis-sarcoma group. ALVs lack
cellular oncogenes but can still cause long latency
neoplasms. DNA copies of the ALV genomes (provirus)
integrate into the host cell genome and can activate
adjacent host proto-oncogenes. Most chickens also contain
one to several proviral remnants within their genomes.
These DNA copies are called endogenous viral (ev) genes
that in some cases can be expressed as complete endogenous
viruses (EV) (reviewed in Crittenden, 1991).

.A typical retrovirus contains two identical copies of
genomic RNA that are packaged in a protein core together
with reverse transcriptase (Figure 1-1). This core
particle is encapsulated by a glycoprotein and cell
envelope to make up the complete virion. The ALV genome is
a dimer of two identical RNAs, each 7.5 kilobases (kb) in
size, held together through the dimer linkage site
Retrovirus genomes are arranged so that almost all
noncoding sequences that contain important recognition
signals are located in terminal regions (long terminal
repeat; LTR), with internal regions given over virtually
entirely to protein coding functions. ALV is the simplest

type of retrovirus, containing three genes (gag, pol and

 

v 1'
g8

RT& IN
NC

 

RNA.gename

envelqpe

 

 

 

Figure 1-1. The retrovirus virion.

env), each of which, however, encodes more than one viral
protein product or activity. The LTR is the primary cis-
acting regulatory region, and it contains an enhancer,
promoter and poly (A) signal. The LTR can be divided into
three parts; U3, R, and U5 (Figure 1-2).

The U3 of ALV is 150-250 nucleotides (nt) long,
depending on whether the virus is endogenous or exogenous.
This region of exogenous ALVs, such as Rous Sarcoma Virus
(RSV), contains a strong enhancer sequence that is
required for high-level expression from viral promoters in
different cell types (Crittenden, 1991; Fadly, 1986). The
U3 region of avian endogenous viruses (ev) are distinct
from those of exogenous viruses in that they lack a
detectable LTR-associated enhancer. The absence of a
strong enhancer in the ev LTR has been correlated with its
low oncogenic potential relative to exogenous viruses
(Fadly, 1986).

The R sequence is terminally redundant and present at
the 5’ and 3' ends of the viral genome. The R sequence is
involved in the transfer of nascent DNA from one end of
the genome to the other during the reverse transcription
process.

Mutational analyses have shown that U5 has multiple

roles in the viral life cycle. Some U5 sequences are

U3 R U5 gag pol env U3 R U5

gag .pol env

env

AAAA

 

 

 

 

 

 

Figure 1-2.Euagram.of ALV proviral DNA, genomic RNA and
proteins. A: proviral DNA, B: Genomic and subgenomic forms
of RNA, C: Virion proteins. The unprocessed proteins are
indicated in the boxes. ALV sites shown include: SD,
splice donor; SA, splice acceptor; MA, matrix protein; CA,
capsid protein; NC, nucleocapsid protein; PR, protease;
RT, reverse transcriptase; IN, integrase; SU, surface

protein and TM, transmembrane protein (Crittenden, 1991;
Coffin, 1991).

essential for reverse transcription (Cobrinik et al.,
1988). The 3’end of U5 contains the att sites (12—15 bp)
necessary for integration , and there is evidence
implicating this region in the packaging of viral RNA
(Murphy and Goff, 1988). The primer binding site (PBS)
binds the tRNA primer which is needed for the initiation
of first strand DNA synthesis during reverse
transcription. Different retrovirus groups use different

trpas its

tRNAs to prime. For example, ALV carries tRNA
primer whereas HIV uses tRNA”S(Coffin, 1991; Crittenden,
1993).

The leader sequence, approximately 250 nt in length,
follows the U5 at the 5'end of the viral genome just prior
to the coding region. The important functions of this
region are to specify incorporation of genome length RNA
into virions (packaging sequence), RNA dimerization (Bieth
et al., 1990), and initiation of reverse transcription
(Aiyar et al., 1994; Cobrinik et al., 1991). Knight et
al. reported that a secondary structure of the packaging
sequence in this region is required for efficient
encapsidation of genomic RNA (Knight et al., 1993).

The gag gene encodes a polyprotein precursor (Pr76)

which is cleaved at the step of viral maturation by the

gag-encoded protease (PR, p15). This cleavage gives rise

to the capsid protein (CA, p27), matrix protein (MA, p19)
and nucleoprotein (NC, p12). The gag gene was reported to
contain a regulatory sequence which confers stability to
the RNA and also an enhancer sequence for viral gene
expression (Federspiel and Hughes, 1994). CA forms the
major internal structural feature of the virion. CA is
also the major ALV-specific antigen that is detected in
assays for the presence of ALV in chickens and cell
cultures. There is a splice donor site 13 nt downstream of
the ATG translation initiation signal in the gag gene and
a splice acceptor site at the 5’end of env. The splicing
step is indispensable in the virus life cycle because it
allows expression of the env gene. The MA protein is in
closest association with the viral membrane. Consistent
with its membrane association, the N-terminus of MA is
modified by the addition of a myristic acid group (Weiss
et al, 1982). However, the MA of ALV has only an acetate
group added at its N-terminus (Coffin, 1991). It has been
suggested that the RSV MA plays a role in membrane binding
during assembly of the virus (Parent et al., 1996). The NC
protein is a small basic protein found in the virion in
association with genomic RNA.

The pol gene codes for the reverse transcriptase (p63

alpha, RT) that is used in transcription of the viral DNA

from the viral RNA genome. Retroviral reverse
transcriptase has an RNase H activity which degrades the
RNA strand of the RNA-DNA duplex during reverse
transcription. It also encodes the integrase (p32,IN) that
is specifically involved in the integration of proviral
DNA into the host chromosome. Enzymatic assays for RT are
often used for detection of ALV particles in chickens and
cell cultures. Translational frameshifting fuses the gag
and pol reading frames producing a 180 kd precursor
protein which is processed into the mature gag proteins
and RT and IN. These proteins are packaged together into
the virion with viral genomic RNA.

env is expressed from spliced subgenomic RNA. Its
protein products (envelope glycoproteins, gp85 and gp37)
are important for attachment of the virus to the receptor
on the cell membrane and penetration into cells. These
proteins are used for classification of the virus into 5
major subgroups (Ishizaki and Vogt, 1966; Vogt and
Ishizaki, 1965; Vogt and Ishizaki, 1966). gp85 and gp37
remain linked to each other by disulfide bonds on the
virion (Leamson and Halpern, 1976). These glycoproteins
confer three major subgroup-specific functions: induction
of neutralizing antibodies; production of cell receptor

interference against members of the same subgroup; and

10

control of host-range among avian and mammalian cell
types. ALVs are divided primarily into the subgroups
based on the host specificity conferred by the envelope
glycoprotein (A through E). Endogenous ALVs belong to
subgroup E. The remaining subgroups make up the exogenous
ALVs. Subgroup F and G show host specificity to pheasants
(Fujita et al., 1974). Recently, subgroup J has also been
isolated (E. Smith, personal communication).

The susceptibility of chickens to infection by ALV is
controlled by three genetic loci: tva, tvb, and tvc
(Crittenden, 1991). The tva and tvc susceptibility alleles
are thought to encode receptors or susceptibility factors
for subgroup A and subgroup C viruses, whereas different
alleles of tvb may encode receptors for subgroup B, D, and
E.

The general features of replication include the
following:

A. Binding the envelope glycoprotein to the receptor on
the cell membrane. The initiation of a replication cycle
begins with the specific binding and interaction of the
receptor molecule on the cell with SU protein on the
virion envelope. Bates et al. (1993) and Young et al.
(1993) used gene transfer to clone the receptor specific

for subgroup A in a quail and a chicken cell line,

11

respectively. This receptor was shown to have some
homology to the light density lipoprotein receptor (Bates
et al., 1993; Young et al., 1993). Neither its mRNA nor
protein product in cells, however, was detected,
indicating that the receptor gene is poorly expressed
and/or expressed early in development followed by rapid
mRNA decay. A recent study by Gilbert et al. (1995)
showed conformational changes of subgroup A.Env protein
induced upon binding the receptor that is relevant to the
activation of its fusion function.

B. Release of capsid into the cytoplasm. Following
attachment, the virus envelope and the cell membrane fuse
to release the virion core into the cytoplasm. This step
is beginning to be understood and the TM protein of the
virion seems to play a role. The internalization process
seems to occur by receptor-mediated endocytosis followed
by fusion of the viral envelope and the endosomal
membrane, possibly provoked by the lower pH of the
endosomal contents. However, neither HIV nor ALV requires
an acidic pH for uncoating (Stein et al., 1987).

C. Reverse transcription of a single-stranded RNA into a
double-stranded DNA. This process takes place inside the
virion core. Reverse transcriptase is carried within the

virus in close contact with genomic RNA. tRNA, which is

12

attached at the PBS of the RNA genome, is used as the
primer for initiation in this step. During reverse
transcription, RNase H degrades the RNA strand of the RNA—
DNA hybrid. This is the step during which a lot of
mutations can be introduced into the genome due to the
error-prone nature of RT. RT has higher error rates
(ranging from 3 x 10"3 to 3 x 1045/ replication) than that
of the eukaryotic RNA polymerase II (less than 10’5 per
animal generation; Gopinathan et al.,l979; Mizutani et
al., 1976). Template switches by RT upon confronting the
strong-stop sequence during this step can generate
recombinant forms of the virus (Coffin, 1979; Zhang and
Temin, 1994).

D. Integration of viral DNA into the host chromosomal DNA
to make the provirus. Linear and several circular DNA
forms of the virus genome are found in the nucleus during
integration. The results of in vitro experiments with
murine leukemia virus favor the linear form as the
structure that is integrated to form the provirus
(Fujiwara et al.,1988). A small sequence at the site of
integration in the host DNA, six base pairs long for ALV,
is repeated at each end of the provirus. While it is
clear that the integration machinery shows little sequence

preference in the choice of integration, it has also been

13

observed that integration sites tend to map in or near
transcriptionally active regions and nuclease-sensitive
regions of host chromatin (Mooselehner and Harbers, 1990).
RSV DNA has been observed to integrate at an unusually
high frequency into certain preferred regions of the
chicken genome, and, within those regions, insertions tend
to occur into the exact same site (Shih and Coffin, 1988).
Experiments done in vitro by Pryciak et al. (1992) were in
accord with a model in which integration machinery has
preferential access to the exposed face of the nucleosomal
DNA helix.

E. Transcription of proviral DNA to viral genomic RNA and
mRNA. Transcription of the provirus is initiated at the
junction of U3-R, and it proceeds through the 3' LTR into
flanking cellular DNA, with the final 3’ end located at
the end of R by cleavage and poly (A) addition. All
retrovirus genomes are transcribed by host RNA polymerase
II and host transcription factors. ALV contains
CCAAT/enhancer motifs that cover most of the LTR enhancer
regions (Ryden and Beemon, 1989). Interestingly, the
CCAAT/enhancer motifs are absent in EV LTRs, which
correlates with the very low transcriptional activity of
those loci (Habel et al.,1993). The avian C/EBP-related

factors designated al/EBP and a3/EBP were shown to bind

14

these enhancer elements (Bowers and Ruddell, 1992; Smith
et al., 1994). cDNAs of both a1 and a3 encode leucine
zipper transcription factors. It was recently found that
an NF-kB/Rel-related protein is a component of the LTR
CCAAT/enhancer binding complex through its interaction
with al/EBP (Bowers et al., 1996).

The ALV genome has a splice donor site 13 nt downstream
of the ATG in gag and a splice acceptor site near the 5’
end of env. Splicing of the full—length mRNA is carried
out by the host machinery and generates a mRNA of 2.0 kb
which is essential for expression of env genes.
F. Translation of viral mRNA into proteins. This process
is dependent on the cellular translation machinery, and
it’s identical to the translation of cellular mRNA (Lewin,
1994). The full-length RNA can have two different fates:
some molecules become new genomes and others serve as
message for the viral proteins. How these are selected to
enter either the mRNA or the genome pool is not well
understood. In most of the retroviruses, the gag reading
frame ends in a translational terminator and is also in a
different reading frame from that of pol. A.shift of
reading frame occurs at the junction between the gag and
pol for translational readthrough. The first demonstration

of this “frameshift suppression” was obtained from ALV

15

(Jacks et al., 1988; Jacks and Varmus, 1985). In ALV, the
probability of this frameshifting is about 5%. By
regulating the frequency of this event, the virus balances
the ratio of these two protein products.

G. Assembly of gag-pol protein-genomic RNA complex and
budding. Assembly and budding of retroviruses depend on
the product of a single viral gene, gag. For ALV, this
process appears to occur at the plasma membrane (Weiss et
al., 1982). A conserved cis-acting packaging sequence (90
in the 5' leader of the avian sarcoma virus genome
identifies the viral RNA and allows it to be incorporated
into the virion (Linial and Miller, 1990). Upon budding,
the virus particle undergoes maturation, in which the gag
polyprotein precursor is cleaved into the several
structural proteins (MA, CA, and NC, and in ALV, PR) by
its own protease.

The disease induced by exogenous ALV is called
lymphoid-leukosis (LL). When an exogenous ALV DNA copy is
integrated by chance upstream in the host genome of a
proto-oncogene, such as c—myc, its 3'LTR drives a high
level of transcription of this gene and thereby causes the
transformation of infected B-cells. The transformed B cell
originates from the bursa of Fabricious and transformed

cells metastasize to the liver, spleen and other visceral

l6

organs, with eventual death (Crittenden, 1993; Fadly,
1992; Fadly, 1986). This underlying mechanism inducing
cellular transformation is different from that of rapidly
transforming retroviruses such as RSV. Most strains of RSV
carry the src oncogene between the env gene and the 3'
LTR. Acute transforming viruses such as RSV carry the
oncogene sequences in their genomes and in most other
cases, they are replication defective due to the deletion
of essential virus gene(s)(Weiss et al., 1982). These
viruses arise from the transduction of a cellular
oncogene, c-src or others. The readthrough transcripts,
containing both viral and cellular sequences, are
copackaged with viral genomic RNA. This is followed by
illegitimate recombination events during reverse
transcription to generate a rapidly transforming
retrovirus (Hajjar et al., 1993; Swain et al., 1992).

The continued presence of ALV in commercial chicken
flocks affects productivity through LL and death. Although
there has been significant progress in eradicating ALV
from the breeder stock of commercial flocks, no commercial
vaccines are currently available. ALV still exists in
commercial chicken flocks, and its effects are enhanced by
endogenous viruses and interactions with other avian

viruses (Crittenden et al., 1984; Smith and Fadly, 1988).

l7

Retroviral vectors

Hughes et al. have developed a series of replication
competent retroviral vectors derived from a cloned copy of
the genome of the Schmidt-Ruppin strain RSV (Hughes and
Kosik, 1984; Sorge and Hughes, 1982; Sorge et al., 1983).
They are called RCASBP (Replication Competent, SR-A LTR,
Splice Acceptor, Bryan high titer polymerase) and RCOSBP
(Replication competent, RAV-O LTR, Splice Acceptor, Bryan
high titer polymerase). RCASBP was made by removing the
src gene and introducing a unique ClaI site that can be
used to insert foreign DNA of up to 2 kb. RCOSBP was
constructed by replacing the LTRs of RCAS with those of
RAV-O (Rous associated virus), which are lacking the
enhancer elements. The RCOS and RCAS vectors express
inserted sequences from the viral LTR. Therefore,
insertion of a reporter gene and assay of its activity
makes it easier to study tissue-specific or development—
specific LTR regulation (Fekete and Cepko, 1993). In
contrast, the RCON and RCAN vectors, which lack the splice
acceptor present in RCOS and RCAS, can be used to express
DNA inserts from internal promoters (Boerkoel et al.,
1993). This makes it possible to separate the expression
of the insert from viral gene expression. As an example,

Petropoulos et al. (1992) somatically infected chickens

18

with an RCAN retroviral vector that contains the CAT gene
linked to the chicken skeletal muscle dflractin promoter

and found high CAT activity only in striated muscle,
whereas the chickens infected with the vector carrying the
B-actin promoter/CAT cassette displayed low levels of the
CAT activity in a wide range of tissues (Petropoulos et
al., 1992). This study suggested that gene expression can
be targeted to a variety of other avian cell types by
constructing similar vectors containing other tissue-
specific promoters. The structures of these vectors are
described in Figure 1-3.

These vectors are replication—competent and helper-
independent. Therefore, rearrangements due to
recombination with helper sequences are eliminated, and
both vectors can be produced to a substantially higher
titer. These vectors are widely used as vehicles of gene
transfer into chickens and recently into transgenic mice
carrying the subgroup A receptor (Hughes et al., 1987;
Federspiel et al., 1995).

The host specificity of these retroviral vectors as
well as ALV mainly lies within the gp85-coding domain of
env (Bova et al., 1986; Bova et al., 1993). Replacement of

the 1.1-kb region in a hypervariable region (hr 1) in this

l9

SRfiA LTR SRtA LTR

 

 

 

 

 

 

gag 'pol env
ClaI
RCASBP (A)
RAY-O LTR RAV-O LTR
gag‘ .pol env
[ I I I J
ClaI
RCOSBP(A)

Figure 1-3. The genome organization of RCOSBP and
RCASBP. SR-A LTR, Schmidt-Ruppin strain A long terminal
repeat; RAV—O LTR, Rous-associated virus type 0 long
terminal repeat.

20

domain with that of another subgroup generates a virus of

that subgroup.

Antisense RNA

1. Natural Antisense

Antisense RNA has a sequence complementary to its
target messenger RNA. It has been thought to directly
repress gene expression by hybridizing to target mRNA and
rendering it functionally inactive. Although the
mechanism of antisense RNA inhibition is not clear,
antisense RNA has been receiving great attention and many
trials are ongoing to test its specific inhibitory effect
on target gene expression in several different systems.

The initial discoveries of natural antisense RNAs were
in prokaryotes. For example, the initiation of ColEl
plasmid DNA replication in E.coli is negatively controlled
at the level of primer formation by a small untranslated
antisense RNA (Tomizawa, 1986). In addition, the
regulation of the life cycles of bacteriophages P1 and P7,
as well as plasmid incompatibility and copy number control
has been shown to involve antisense RNA (Brantl and
Wagner, 1994; Biere et al., 1992; Siemering et al., 1994).

There are also many cases where the participation of

21

antisense transcripts in eukaryotic gene regulation is
thought to occur (Simmons, 1993).
2..Artificial Antisense

Since natural antisense is effective and very
specific, many artificial antisense RNAs have been
designed to inhibit the expression of endogenous genes and
the replication of pathogens (Biasolo et al., 1996;
Ronemus et al., 1996; Scherczinger and Knecht, 1993). The
antisense approach has been particularly effective in
plants (Blockland et al., 1993). The well—known transgenic
tomato, “Flavr Savr", has a longer shelf life by using
antisense RNA against the message of the polygalacturonase
gene, resulting in delaying of the softening. It is also
interesting to note that, in a lot of studies with plants,
antisense RNA against the entire coding sequence of the
target gene was an effective inhibitor (Beffa et al.,
1996; Ronemus et al., 1996).

There have been numerous studies on antisense RNAs
which are stably expressed in either cell culture or in
transgenic animals that inhibit the replication of
(retro)viruses (Biasolo et al., 1996; Han et al., 1991).
The aim of antisense techniques for the inhibition of
viral infection is either to suppress the expression of

the integrated provirus in chronically infected cells or

22

to prevent the virus from establishing itself in
uninfected cells. To accomplish this goal, the obvious
route is to disrupt the viral replication cycle. Antisense
techniques can be especially effective in inhibition of
retrovirus replication. Retroviruses have the advantage of
introducing mutations into their genome during
replication, especially by RT, which helps them escape the
host immune system. Therefore, an antisense RNA which is
designed to target a conserved viral sequence is expected
to confer a strong and longer specific inhibitory effect
on retrovirus replication. Additionally, the requirement
of sequence complementarity increases the specific effect
without interfering with any other cellular RNA.
Retroviruses have many important signals at their 5’ ends,
and those signals are relatively well conserved in a given
virus due to the necessity of their interaction with
either viral or cellular proteins. For example, PBS and 9’
are shown to directly interact with cellular tRNA and the
viral capsid proteins, respectively. Therefore, the 5' end
of retroviral RNA seems to be the target that antisense
approaches have to which most often been directed.
Sczakiel and Pawlita (1991) have shown that stable
expression of antisense RNA complementary to a 407-bp

sequence of the 5' leader-gag region of HIV inhibited

23

viral replication in human T cells. An antisense RNA
against TAR has also been shown to inhibit viral
replication by preventing its interaction with TAT as well
as the other signals at the 5' end (Chatterjee et al.,
1992; Vandendriessche et al., 1995). The effect of
antisense RNA was also demonstrated in a study in which

MoMLV-induced leukemia was reduced by expressing an

antisene RNA against W’in a transgenic mouse system (Han

et al., 1991).

Antisense Oligodeoxynucleotides

Antisense oligodeoxynucleotide (ODN), which are
complementary to certain regions of gene messages or viral
sequences, have been getting a lot of attention because of
their ability to modulate gene expression. With its first
success in inhibiting RSV replication in chicken embryo
fibroblast (CEF) cells (Zamecnik and Stephenson 1978;
Stephenson and Zamecnik, 1978), antisense ODNs have
enjoyed considerable success as antiviral agents in
various biological systems. There are even several
antisense ODNs in clinical trials to regulate HIV
replication in patients (Lisziewicz et al., 1994).

Two critical factors to be considered in antisense ODN

approaches are the efficiency of uptake and the stability

24

of the ODN. Due to the negative charge on the phosphate
backbone of the ODN, direct penetration through the cell
membrane is implausible. To increase its cellular uptake
and intracellular stability , various modifications of
the phosphate backbone have been applied. They are
thiolation (Zhao et al., 1993), methylation (i.e.
alkylation) of the phophodiester bonds (Mckay et al.,
1996) as well as conjugation of ODN with peptides
(Bongartz et al., 1994). Figure 1-4 shows the structures
of phosphodiester and phosphorothioate linkages. The
suggested mechanism for cellular uptake of phosphodiester
and phophorothioate ODNs is an endocytic process (Loke et
al., 1989) whereas methlyphosphonates enter cells by
passive diffusion (Miller et al., 1981). Liposome-mediated
delivery of ODNs has been developed to increase cellular
uptake (Bennet et al., 1992; Thierry and Dritschilo,
1992). Microinjection of oligonucleotides directly into
the cell has shown to be an effective, though cumbersome,
method for delivery (Graessmann et al., 1991; Raviprakash
et al., 1995).
Mbchanism.of antisense RNA.or ODN action

There have been many studies to elucidate the mechanism
of antisense inhibition. However, relatively little is

currently understood. Considering the process of gene

25

0
||

'O'P -O-
'-

O
phosphodiester

0
ll

-0- P -O-
I

S
phosphorothioate

Figure 1-4. Phosphorothioate and phosphodiester linkages.

26

expression, the following were suggested as possible
steps where antisense inhibition might take place
(Mirabelli and Crooke, 1993);
A. Transcriptional arrest. ODN may bind to DNA and prevent
initiation of transcription by preventing effective
binding of factors required for transcription, thus,
producing transcriptional arrest. (Nielsen et al., 1991;
Svinarchuk et al., 1996).
B. Inhibition of post-transcriptional processes. Antisense
RNA or ODN that bind to sequences required for splicing
may prevent binding of necessary factors or physically
prevent the required cleavage reactions. This would result
in inhibition of the production of the mature mRNA
(Zamecnik et al., 1986). Another possible mechanism
includes inhibition of 5' capping (Westermann et al.,
1989). Inhibition of 3’ polyadenylation has not been
directly proven. However, antisense ODNs targeting the 3’
untranslated region (UTR) have shown inhibitory effects
(Chiang et al., 1991).
C. Translational arrest. The mechanism for which the
majority of antisense RNA or ODN have been designed is
translational arrest, in which recognition and binding of
target mRNA by ribosome are prevented (Agrawal et al.,

1988; Lemaitre et al., 1987; Sburlati et al., 1991;

27

Sullenger et al., 1990). It was demonstrated in HIV-1 that
sequences essential for packaging the viral RNA are
located around the gag initiation codon and can form a
stable secondary structure. An ODN which is complementary
to this region was found to be an effective inhibitor of
HIV replication. This ODN might block the translation of
gag mRNA and also disrupt the secondary structure of RNA
(Agrawal and Tang, 1992). The positioning of the
initiation codon within the area of complementarity and
the length of antisense RNA or ODNs have varied
considerably.

D. Disruption of RNA structure. RNA adopts a variety of
three-dimensional structures induced by intramolecular
hybridization, the most common of which is the stem—loop
structure. These structures have been shown to play
crucial roles in a variety of functions. As an example,
antisense ODNs designed to target the transactivation
response (TAR) element in HIV were shown to disrupt the
structure of the stem-loop and inhibit TAR—mediated
expression of a reporter gene (Vickers et al.,1991).

E. Activation of RNase H. RNase H is a ubiquitous enzyme
that degrades the RNA strand of an RNA-DNA duplex. It has
been identified in organisms as diverse as E.coli and

human cells (Mirabelli and Crooke, 1993). It was

28

demonstrated that many ODNs may activate RNase H in cell
lysates and purified assays (Gagnor et al., 1987; Walder
and Walder, 1988). ODN with a phophodiester bond seemed to
be a better RNase H activity inducer than phophorothioate
ODN (Boiziau et al., 1995). However, direct proof has not
been found that RNase H activation is the true mechanism
of antisense action in cells. It was also demonstrated
that RNase L activity can be induced in infected cells by
2', 5' oligoadenylate (2-5A). This is formed by 2’, 5'
oligoadenlyate synthetase (2-50AS), activity of which
depends on the presence of viral or cellular double—
stranded RNA (dsRNA). (Maitra et al., 1995). RNase L is an
endonucleolytic enzyme and it degrades both cellular and
viral RNA, resulting in removal of the infected cells.
Schroder et al. (1994) have developed new strategies which
yield a selective avtiviral effect of 2-5A against HIV
infection by application of the LTR-2-50AS hybrid genes.
An antisense RNA molecule which can cleave the target
RNA upon binding would further increase its efficiency. A
ribozyme is a RNA molecule with a certain sequence motif
which can recognize and cleave the target RNA molecule
(Zaug et al., 1986). There have been many applications
incorporating a ribozyme motif into the antisense

sequence, and this improved the antisense effect (Sun et

29

al., 1995). Sullenger and Cech incorporated a tethering
ribozyme to a retroviral packaging signal for
colocalization with the target RNA and showed cleavage of
the target RNA which leads to the protection of uninfected
cells (Sullenger and Cech, 1993).

There are several factors which may affect the
efficiency of antisense inhibition. They are as follows:
A. The place of action. Many investigators have
questioned whether different cellular sites are involved
in antisense control of gene expression. A transgenic
tobacco experiment showed a reduced level of translation
efficiency of target mRNA and suggested a cytoplasmic
interaction between the antisense RNA and the target
message (Cornelissen and Vandewiele, 1989). Meanwhile,
Liu and Carmichael (1994) have suggested from their study
with polyoma virus that antisense RNAs that are retained
in the nucleus bind to target transcripts and appear to
lead to the degradation of their targets. This suggested
that nuclear antisense RNAs were significantly more
effective than were conventional antisense molecules,
which were processed by polyadenylation (Liu and
Carmichael, 1994).

B. The abundance of antisense RNA. Viral RNA may

contribute up to 10 % of the total polyadenylated RNA in

30

infected cells (Coffin, 1991). Therefore, an efficient
antisense system will have to introduce into the target
cell a sufficient amount of antisense RNA in order to
overcome this level of expression and do so without
overwhelming the normal functions of the host cell by
their sheer quantity (Izant and Weintraub, 1985). In
several other cases, antisense RNA was not detected by
typical techniques even though an antisense effect was
obtained (Koschel et al., 1995). It was suggested that
this could be due to the instability of the antisense
transcripts.

RNA Polymerase III (Pol III) is a ubiquitous enzyme
with a transcription efficiency higher than that of RNA
polymerase II (Gabrielsen and Sentenac, 1992). Sullenger
et al. (1990) and Biasolo et al. (1996) showed a high
level expression of antisense transcripts against the gag
and pol genes of MoMLV and the first exon of tat of HIV-1,
respectively, and significant inhibition of viral
replication using Pol III-tRNA promoter systems.

C. The target regions. In most cases, including the
repression of retrovirus replication, the 5’ end of the
target RNA has proven most effective as a site for

antisense expression. Typical retrovirus contains the

PBS, 92 the leader sequence and the ATG translation

31

initiation signal, and, in some cases, the splice
donor/acceptor sites. Secondary structures of the
packaging signal have been implicated in efficient
encapsidation of the virus particles. Also the leader
sequence contains the PBS, onto which antisense RNA might
compete with tRNA for binding. In the case of HIV—1,
although the 5' end of its RNA has been proven to be a
good target in various experiments, antisense RNA against
the first coding exon of tat showed a significant
reduction of viral replication as well (Biasolo et al.,
1996). However, in other experiments in different systems,
the inhibitory effect was achieved with the antisense
transcript targeting the entire coding sequences or
regions at the 3' end (Scherczinger and Knecht, 1993;
Sullenger et al., 1990). Therefore, it is difficult to
predict the optimal design of an antiviral antisense
strategy.

D. The accessibility to the target mRNA sequence. Since
RNAs can fold into various secondary structures, there
have been many investigations of how to improve the
binding efficiency between the antisense RNA or ODN and
its target sequence. Although secondary structures can be
estimated by computer programs based on thermodynamic

stability (Hackett et al., 1991), they may not reflect the

32

structure of the RNA molecule in the cell or when it's

bound by certain proteins.

Research Preposal

Avian lymphoid leukosis (LL) is a neoplastic disease of
chickens caused by ALV. An ALV infection spreads
congenitally from dams to progeny or by chicken—to-chicken
contact in the same flock. ALV infection in commercial
stock is controlled by virus-eradication schemes that
prevent vertical transmission of ALV from one generation
to the next. No efficient vaccines are available and the
pathogenecity of ALV is augmented by the presence of ev
(Smith and Fadly, 1988).

Antisense RNA is complementary to its target RNA. It
inhibits gene expression by hybridizing to the target RNA
and rendering it functionally inactive. It has been widely
tested in a variety of systems including inhibition of
replication of other retroviruses such as HIV and MoMLV.

The long-term goal of this project is to generate
transgenic chickens resistant to ALV infection by using
antisense RNA techniques. In this study, we have tried to
test the efficacy of using antisense RNA in an in vitro
cell culture system. We have focused at the 5' end of ALV

genomic RNA as the target since this region is relatively

33

well conserved among different subgroups of ALV and has
many important regulatory signals. To approach this goal,
we have applied two different methods of expressing
antisense sequences against the viral RNA: the
transcription of antisense RNA in a stable expression
system and the use of antisense oligodeoxynucleotides to
locate the best target region.

As another approach to inhibit replication of ALV, we
have expressed antisense RNA against the message for
subgroup A ALV receptor in a quail cell line. The gene
(tva) for subgroup A virus receptor has been recently
cloned (Bates et al., 1993). However, neither its
transcript nor protein product are detectable, indicating
that the receptor gene is poorly expressed. Therefore, it
seemed likely that the expression of this gene could be
repressed more efficiently by an antisense RNA than the
viral RNA might be. In this study, we have focused at the
5’ end of tva, including its ATG translation initiation

signal.

Chapter 2 .

Antisense RNA generation as a strategy for the induction

of cellular resistance to ALV

34

35

ABSTRACT
Avian leukosis virus (ALV) belongs to the avian leukosis-
sarcoma group of retroviruses. Upon infection, the ALV
provirus can integrate into the 5' end of c—myc cellular
oncogene, leading to over-expression of that oncogene and
resulting in a disease called lymphoid leukosis. No
vaccines are currently available to prevent the spread of
ALV in commercial chicken flocks. In other systems,
antisense RNA has been used to inhibit retroviral
replication in infected cells and to protect uninfected
cells. We have examined the use of antisense RNA to
inhibit ALV replication in an avian cell line, RP30. In an
expression system where an antisense RNA is transcribed
constitutively, one cell line showed a significant
inhibitory effect on replication of test ALV strains. A
low level of the antisense transcript was detected in this
cell line by RT-PCR. However, this inhibitory effect was
not reproducibly observed in other transfected cell lines
or in cells in which the antisense transcript was
generated by a tetracycline-regulatable promoter, even
though a substantial amount of antisense transcript was

detected in such cells.

36

INTRODUCTION

Avian leukosis virus (ALV) is a class of retrovirus
belonging to the avian leukosis-sarcoma group (Crittenden,
1991). The genome structure of ALV is simple compared to
those of some other retrovirus groups, containing three
genes (gag, pol and env) and a regulatory region or long
terminal repeat (LTR). The LTR contains an enhancer,
promoter and poly (A) signal which are recognized by the
cellular transcription machinery.

The general features of ALV replication include the
following: binding of the envelope glycoprotein to the
receptor on the cell membrane, release of the capsid into
the cytoplasm, reverse transcription of a single-stranded
genomic RNA into a double—stranded DNA, integration of the
viral DNA into the host chromosomal DNA to make a
provirus, transcription of viral DNA to viral genomic RNA
and mRNA, some of which is spliced to form a subgenomic
viral message, translation of viral mRNA into proteins and
assembly of the protein—genomic RNA complex at the cell
membrane followed by budding (Coffin, 1991).

ALV can be transmitted either through close contact
or congenitally through the egg. ALV lacks host oncogenes
and is not therefore an acutely transforming virus.

Exogenous ALV can induce a variety of neoplasms, but

37

principally lymphoid leukosis (LL), while endogenous ALV
is rarely oncogenic. This difference is determined
primarily by the enhancer element being present only in
the exogenous viral LTR (Crittenden, 1991). When an
exogenous ALV provirus integrates by chance upstream of
the c-myc gene in the host genome, (usually) its 3' LTR
can enhance the transcription of this gene and cause
transformation of infected B-cells in the Bursa of
Fabricious. Transformed B-cells metastasize to the liver,
spleen and other visceral organs leading eventually to
death (Fadly, 1992). The continued presence of ALV in
commercial chicken flocks affects productivity both
through LL disease and death (Crittenden, 1993).

The use of antisense RNA to inhibit RNA function
within cells and whole organisms has the potential to
provide a versatile molecular tool against viral
infection. There are many reports of the use of antisense
RNA to inhibit retroviral replication. For example,
various regions of the HIV genome have been tested as
targets for antisense RNA, with a resultant reduction in
virus replication (Sczakiel and Pawlita, 1991;
Vandendriessche et al., 1995). Antisense RNA can bind in a
highly specific manner to complementary sequences in mRNA

or viral genomic RNA, potentially blocking processing or

38

translation of the RNA or, possibly, its interaction with
sequence-specific binding proteins.

To test the efficacy of antisense RNA in the
inhibition of ALV replication, avian cell lines containing
antisense RNA sequences against the conserved elements of
the ALV genome were generated and challenged with the ALV—
derived retroviral vectors: RCASBP and RCOSBP (Greenhouse
et al., 1988). The 5’ end of ALV contains a variety of
important regulatory signals and is well conserved among
different subgroups of ALV (Figure 2-1). Important signals
in this region include promoter/enhancer elements in the
LTR, the primer binding site (PBS) for tRNAtrp which is
essential for initiation of reverse transcription, the
packaging sequence (90, the ATG translation initiation
codon for gag-pol and env proteins, and a splice donor
site. Therefore, this region is likely to be an ideal
target against which to direct antisense RNA to inhibit

ALV replication.

39

 

 

 

 

 

 

 

 

01 SA env
A. I LTR I 93? p j LTR
F—> ._gag ........
A_. so *
IR] US I PBS
3
U / I W I dlfl
¢_ ATG
mm B «— c ._ D ._ r.
CF 160 bp
AB 110 bp
AC 260 bp
AD 380 bp
AB 540 bp

 

Figure 2-1. The regions targeted by antisense RNA.
A. The proviral structure of ALV.

B. The 5’ end of the ALV provirus is shown.

The primers are indicated by A, C, D, E, and F.

ALV sites shown include: TATA, promoter;‘¥, packaging
signal; PBS, primer binding site; ATG, translation
initiation signal; SD, splice donor; SA, splice
acceptor and dls, dimer linkage sequence
(Coffin,199l). Various target amplified fragment
regions are shown by lines below the ALV diagram with
their sizes indicated at the right.

40

MEIERIALS AND METHODS

cell culture

RP30 clone5 is a Marek’s disease virus-transformed
turkey lymphoid cell line and is free of endogenous virus.
This cell line and all of its derivatives were maintained
under 5% cm» in Leibovitz L-15/ McCoy’s 5A medium (Life
Technologies, Gaithersburg, MD 20877) containing 10%
chicken serum, 5% fetal bovine serum, 2.5% tryptose

phosphate broth supplemented with gentamycin (10 ug/ml)

and amphotericin B (2.5 ug/ml).
.Electrqporation

Cells were washed twice with phosphate-buffered
saline (PBS) and resuspended in 0.5 ml cold PBS in a 0.4
cm Gene Pulser cuvette (Bio-Rad, Hercules, CA 94547). Ten
pg of each construct was added and the mixture was placed
on ice for 5 min. The electroporation was performed at
room temperature using the Gene Pulser set at 960 uF and

250 V. After electroporation, cells were incubated on ice
for 5 min and then resuspended in 10 ml of growth media.
After 24 h of incubation, cells were spun down,

resuspended in 24 ml of selection media (0.4 mg/ml

Geneticin [Life Technologies] i 1 ug/ml of puromycin

41

[Sigma, St. Louis, MO 63178]) and plated onto a 24-well
plate. Resistant colonies typically developed in 10 d.
Luciferase assay

Colonies to be tested for effectiveness of
tetracycline (tet) regulation were transiently transfected
with 10 pg of pUHCl3-3, containing a luciferase (luc) gene
expressed from the tet-regulatable promoter (Gossen and
Bujard, 1992). After 24 h in growth media, cells were

split into media i tet (4 pg/ml)or doxycycline (dox, 1
pg/ml) and incubated overnight. Cells were counted, washed
once with PBS and lysed in 1x cell culture lysis buffer

(Promega, Madison, WI, 53706). Fifty pl of each cell

lysate was mixed with 100 pl of luciferase substrate,
prepared according to the manufacturer (Promega). The
activity of luciferase was measured using a Turner TD-20e
luminometer (Turner Designs, Inc., Sunnyvale, CA 94086)
and normalized to the cell number.
Polymerase Chain Reaction (PCR) and‘plasmid construction
The sequences of the PCR primers used to amplify the
5' end of the ALV proviral genome are listed in Table 2-1.
Each primer was synthesized by the DNA Core Facility at
Marshall University, Huntington, WV 25704. PCR reactions

were performed with a common 5’ primer (pA or pF) and

42

Table 2-1. Primers used in PCR amplification of antisense

target regions of ALV.

 

 

Primer Sequence
A GTG GAA TTC TAA ACG CCA TTT GAC CAT
B TTG GAA TTC AAT GAA GCC TTC TGC TTC
C TAT GAA TTC GAG CTC CCT CCG ACG
D CTT GAA TTC CTT GAT CCG CAG GCC G
E AAT GAA TTC CGC AGT GAT GGG ATC C

F TGG TGA CCC CGA CGT GAT CG

 

43

various 3' primers (pB, pC, pD or pE; see Figure 2-1)
using a RCOSBPCAT-3(A) plasmid (Greenhouse et al., 1988)
as template as described (Sambrook et al., 1989). Each PCR
product was digested with EcoRI and ligated into
pBluescript II plasmid (Stratagene, La Jolla, CA 92037)
and sequenced by the dideoxy chain termination method
(Sanger et al., 1977). The CF fragment was directly
subcloned into the TA vector (Invitrogen, San Diego, CA
92121) and sequenced. Each PCR fragment was then
transferred into the pRC—CMV eukaryotic expression vector
(Invitrogen) in both orientations by digestion with
HindIII and XbaI followed by ligation. In the tet-
responsive system, PCR products were transferred to
pUHD10-3Neo or pUHDlO-Bpuro using the EcoRI site. Plasmid
DNA purification, restriction and ligation of DNA and
isolation of subclones were as described (Sambrook et al.,
1989). pUHD10-3, pUHD15-1, pUHC13-3 and pUHD172-1Neo were
provided by Herman Bujard (Gossen et al., 1995). pUHDlO-

3Neo was generated by inserting the neomycin-resistance
cassette (with the chicken B-actin promoter and the SV40

poly A signal) from TFANeo (Federspiel et al., 1989) into
pUHD10—3 via HindIII digestion and ligation. pUHD10-3Puro

was generated by inserting the puromycin-

44

resistance cassette from pouro (Li,1996) into pUHD10-3:
pUHD10—3 was digested with HindIII followed by filling in
the ends and ligated with the puromycin—resistance
cassette from pouro by digestion with XhoI and BamHI
followed by filling in the ends as described (Sambrook et
al., 1989)
Reverse-transcription (RT) PCR

First-strand cDNA was synthesized using oligo(dT)18(5
mM) and 1-2 pg of total RNA as described by the
manufacturer of reverse transcriptase (Life Technologies).
A reaction without reverse-transcriptase (RT) served as a
negative control. PCR was performed as described above
with 1 pl of each RT reaction using pA and pE (Figure 2-
1). Each PCR product was run on 1.2 % agarose gel in 1X
Tris-acetate buffer (0.04 M Tris-acetate, 0.001M EDTA).
Virus preparation

RCOSBPCAT(A) virus stock was generated after
electroporation of RP30-5 with the RCOSBPCAT-3(A) plasmid.
When necessary, transfected cells were passaged to allow
virus spread. Culture fluid was collected after
centrifugation at 1600 rpm for 5 min at 4°C. The viral
titer was determined by infecting RP30-5 and chicken

embryo fibroblasts (CEF) from line 1581 and Line 0 (Astrin

45

et al., 1979) using the limiting dilution method, followed
by ELISA assay (Smith et al., 1979) for p27. RAV—49 is a
field isolate of ALV. 1581 CEF infected with RCASBPCAT(A)
was provided by Mr. Bill Payne (Department of
Microbiology, Michigan State University, East Lansing, MI
48824) and RAV—49 was obtained from the USDA Avian Disease
and Oncology Lab, East Lansing, MI, 48824.
Challenge with virus

The general scheme of a challenge experiment is
described in Figure 2—2. Drug resistant cell clones were
counted and 4 x 105cells were seeded onto 60 mm plates in
duplicates. For the tet-responsive expression system, each
transfectant was split into -/+ tet media (4 pg/ml) 2 d
prior to infection. Cells were infected with RCOSBPCAT(A)
and/or RCASBPCAT(A) at various multiplicities of infection
(MOI). Four d post-infection, the culture supernatant was
collected by pelleting cells at 1500 rpm for 4 min at 4°C.
The cell pellet was washed with PBS and lysed in 0.1%
Tween 80/PBS by two cycles of freezing and thawing. Both
were assayed by p27 ELISA (Smith et al., 1979). Colonies
which showed a reduction in p27 were further tested by

varying the M013 and by virus titer assays.

46

Enzyme Linked Immunosorbent.Assay (ELISA)

Samples were frozen and thawed twice in 0.1% Tween80
in PBS prior to the assay. 96—well immulon plates
(Dynatech Laboratories, Inc., Chantilly, VA 22021) were
coated with rabbit anti—p27 antibody (1 pg/ml, SPAFAS,
Inc., Storrs, CT 06268) in coating buffer (0.01 M'Na2C03,
0.03 M NaHCCb, pH 9.5) at 4 °C overnight. The ELISA assay
was performed as described (Smith et al., 1979) using 1
mg/ml of 5-aminosalicylic acid (Sigma) in the phosphate
buffer (0.02 M, pH 6.0) as the substrate. Optical density
was measured at 490nm using a EIA autoreader model EL 310
(Bio-tek Instruments, Inc., Winooski, VT 05404). The OD
was normalized to cell number and/or the total cell
protein, as measured by the BCA Protein Assay Kit (Pierce
Chemical Co., Rockford, IL 61105).
southern hybridization

Genomic DNA was extracted by digestion with
proteinase K and extraction with phenol-chloroform as
described (Sambrook et al., 1989). DNA samples were
digested with HindIII and subjected to 0.7% agarose gel
electrophoresis, followed by transfer to a nylon membrane
(Zeta probe, Bio Rad) and hybridization as described

(Sambrook et al., 1989). The hybridization probe was a

47

32P-labeled AE DNA fragment made by EcoRI digestion of AE-
pBS and labeled by random primer extension (Stratagene).
RNA isolation and northern hybridization

Total RNA was isolated from cells by lysis in 4 M
guanidine thiocyanate, 42 mM sodium citrate, 0.83% N—
lauryl sarcosine and 0.2 mM 2-mercaptoethanol followed by
phenol/chloroform/isoamylalcohol extraction and
isopropanol precipitation (Promega). In other cases, total
RNA was isolated by lysis using Trizol (Sigma) as
described by the manufacturer. 30 pg/lane of RNA was run
on a 1.2% agarose gel containing 1x MOPS
(morphilinepropanesulfonic acid), 0.66 M formaldehyde, and
1 pg of ethidium bromide per m1, and blotted to Magna
charge membrane (Micron Separation Inc., Westborough, MA
01581) in 10x SSC. The blots were hybridized as described

for Southern blot analysis.

48

RESULTS

.Effect of constitutive expression of antisense RNA on.ALV'
susceptibility

The target region against which antisense RNA was
generated is shown in Figure 2-1. This region was chosen
because of its high density of conserved, functional
retroviral sequence elements. Various portions of the
sequence of this region were amplified by PCR for
subsequent cloning into appropriate antisense RNA—
expressing vectors. Initially, a PCR reaction was
performed using RCOSBPCAT(A) plasmid as a template and the
primer pair of pA and pE as shown (Figure 2-1). The 540
base pair (bp) product, AF, was inserted into the pRC-CMV
expression vector in both sense and antisense
orientations. This plasmid uses the strong
cytomegalovirus (CMV) promoter to drive transcription of
the inserted DNA fragment, in this case, the AE sequence
Fourteen G418-resistant RP30 colonies transfected with the
AE—pRC—CMV (antisense) construct were obtained. Those
transfectants were screened for any inhibitory effect on
the replication of ALV by infecting them with RCASBPCAT(A)
or RCOSBPCAT(A). After 4 d of infection, p27 viral

protein production was assayed by ELISA in both the

49

culture supernatant and cell lysate (Figure 2-2A).
Initial results of the viral susceptibility assay for the
14 cell lines, along with vector alone controls and
transfected cells with the AE fragment cloned in the sense
direction are shown in Table 2—2. I, II, III, and IV
represent each independent experiment and each
transfectant containing AE-antisense construct is
indicated as 1 to 13.
Further analysis of the reduced ALV susceptibility of 383
Only one out of the 14 transfectants showed a
significant reduction of RCOSBPCAT(A) replication when
compared to that of control cells (Table 2-2), and it
repeatedly demonstrated a reduced susceptibility to
RCOSBPCAT(A) in more than five repeated experiments (Table
2-3). To confirm the presence of the correct AE construct
in the genome of this transfectant, named 3B3, genomic DNA
was isolated and probed with the AE sequence (Figure 2-3A,
lane 3). The 6 kilobase pair (kb) band observed
corresponds to the full-length (linear) plasmid, generated
by digestion with HindIII, which cuts the plasmid at one
site. The pattern observed is consistent with the
transfecting plasmid integrating into the genome in one
site as a tandem multimer (Figure 2—3B). By comparing the

intensity of the full length 6 kb band with that of the

50

AE-CMV antisense construct

l Electroporate and wait for
10 d

Isolate individual G418-resistant colonies

1

Challenge 4x105cells with the
recombinant ALV at various MOI

14d

P27 viral protein produciton in culture
supernatant and cell lysate assayed by ELISA

Figure 2-2An Diagram illustrating the procedure
emplyed to screen constitutive antisense-
expressing colonies.

51

pUHDlO-BNeo PUHDIS-l (tTA)

construct with an
antisense sequence

Cotransfect by
electroporation and
wait for 10 d

iOOOOOOOOOI

Isolate individual G418-resistant colonies

Transient transfection with pUHC13-3
followed by luciferase assay in +/- tet

1

Challenge with the recombinant
ALV in +/- tet

14d

Same as in 2—2A.

Figure 2-23. Diagram illustrating the procedure
employed to screen antisense-expressing colonies
using the tet-regulatable expression system.

A.

52

 

 

kb
23.1 -
9.4 -
6.6 ’ Fl
4.4 - ‘
2.2 -
Hindlll - - - Hindlll
l l
Hind lll Hind lIl Hind Ill

- AE-CMV construct (~6kb)

- probe

genomic DNA

Figure 2-3. Southern blot analysis of represntative
clones.

A. Genomic DNA was extracted and digested with
HindIII, fractionated on an agarose gel, blotted and
hybridized to a 32P-labeled AE-DNA fragment. The
molecular sizes in kb are indicated. Lanes:l, a sense
clone; 2, an antisense clone(3C5); 3, an antisense
clone (383); 4, a vector-alone clone; 5, RP30 cells.
B. Diagram of probable orientation of transfected DNA
in 383.

53

Table 2-2. Effects of the transfectants on ALV

 

 

 

 

 

 

replication.
cell-free p27/mg cell-asso. p27/mg
prot prot

clone average SE average SE

I RP30 20 6 29 5
vector 1 13 7 41 5
AE-sense 22 4 55 10

383 O O 3 O

1 79 1 1 10 3

2 44 12 74 2

3 39 17 75 7

4 29 6 58 0

5 38 17 67 8

6 44 9 34 8

7 14 5 25 3

8 16 2 42 3
9 36 24 71 63
I I vector 1 86 3 87 22

10 120 3 6O 1

11 110 19 60 3

12 120 8 59 4

III RP30 71 7 27 1
13 150 O 34 2

IV RP30 120 14 15 2
vector 2 71 15 49 2

383 6 1 7 2

 

I; samples collected on day 5 post infection

II; samples collected on day 4 post infection

III and IV; samples collected on day 6 post infection.

SE; standard error

vector 1 and vector 2; transfectants with the vector—alone.

54

.maaoo ommm Houucou mo Aqueououm ma uov maaoo mow nod Houfiu
Hmufl> ecu >2 mmm mo Aucflououa 08 nov maaoo SON nod uwuwu Hmua> ecu mcwpw>wp an poucasoamo mm: uouflu msufl> m
.maaoo ommm Houucou mo Aacaououd OE uov maaou sea nod no (mHAm
aNa was an mmm do i.cNmuoud as uoc mHNmo .oH pea 90 «qum de we» oanN>Ne in emanasoamu mm: Nouucoo eNd N

.mcop uoc «oz

 

 

 

 

 

N m L. em N L. NN No.0 3.32

oz s L. Ne N L. 8 N

oz a L. so 3 L. mm To

oz ms L. 3 3 L. am 8.0 levemommmeom

oz m L. NH N L. NH N

dz 2 L. 3 mL. 3 To

..N .H L. 3 L L. 3 8.0 338302
Homecoo moumnaaoo poumfloommmuaaoo Hoe msua>
panes
msufl> w

 

Houucoo emu m

.>A¢ mo coHumoHHdou co mmm mcoao mo

muowuum are .muu wanna

55

3 kb band, which most likely represents the integration
junction fragment containing the probe region, we estimate
that there are 3-5 AE-constructs tandemly integrated in
the genomic DNA of 383. Northern blot analysis was
performed with total RNA from 3B3 to detect the expression
of an antisense RNA, but no such transcript was observed
(results not shown), suggesting that the steady-state
level of antisense AE transcript in this transfectant may
be very low. However, a substantial amount of AE
transcript was detected from the clones harboring the
sense AE—construct. Therefore, RT-PCR was performed to
increase the sensitivity of RNA detection. Figure 2-4,
lane 4, demonstrates a detectable level of RT—PCR product
AE fragment templated by 383 cDNA. As expected, the same
fragment was generated using a small amount of the
transfecting plasmid DNA (lane 5) as template and when
cDNA was used from an AE—sense direction transfectant
known to express detectable levels of RNA (lane 3). The
control which received no RT in the cDNA reaction did not
generate any detectable RT—PCR product (Figure 2-4, lane
1), thereby demonstrating that the fragment did not arise
from genomic DNA that could have contaminated the 383 RNA.
RCASBPCAT(A) has the same antisense target region

sequence as RCOSBPCAT(A) except for 19 nucleotides (nt) in

Figure 2-4.

56

M12345

 

RT—PCR analysis of clone 383.

After PCR amplification of the first-strand
cDNA reverse-transcribed from the total RNA
extracted from a vector-alone clone (lane 2),
sense clone (lane 3) and clone 383 (lane 4)
as described in MATEIRALS AND METHODS in
Chapter 2. Lane 1 is a no—DNA control where
no reverse transcriptase was added in RT
reaction. Lane 5 is a positive control where
the AE-CMV plasmid was used as the template
for PCR reaction. Marker (M) was 100—bp
ladder. A PCR product of correct size, 540—bp
is present in lane 3, 4, and 5.

57

the leader region. Due to its more active LTR, the RCAS
virus gives at least ten fold higher titers than
RCOSBPCAT(A). Clone 383 was challenged with RCASBPCAT(A)
and showed a reduced inhibitory effect on RCASBPCAT(A)
replication at several MOIs tested (Table 2-3). Thus, the
inhibitory effect observed in transfectant 383 is a
partial one, primarily observed when assaying the more
slowly replicating RCOSBPCAT(A). This may relate to the
limited amount of antisense RNA detected in 383 and the
ability of a more active virus to make excess viral RNA or
mRNA.
Reduced.ALV‘susceptibility of 383 as determined.by titer
Clone 383 was further tested for its reduced ability
to grow the RCOSBPCAT(A) target virus by direct titration
of virus grown on these cells (Table 2-3). While 383 still
can grow the RCOS virus, the titer of virus produced from
383 was much lower than that from control RP30 cells. This
indicates that viral spread was significantly impaired in
383 cells. Interestingly, the reduction in virus titer is
more dramatic than that in p27 production. Whatever the
block to replication in 383 cells, it may result in the
production of the non-infectious empty virus particles

which lack RNA but still contain p27 (Han et al., 1991).

58

Reduced susceptibility of 333 to subgroup C ALV

Since only one of 20 antisense transfectants showed a
significant inhibition of ALV replication, we questioned
whether this inhibition was due to antisense RNA
expression or a random clonal variation. A likely
possibility for the latter would be the fortuitous loss or
mutation of the subgroup A-specific receptor on the cell
membrane for ALV (Bates et al., 1993). Rous—associated
virus 49 (RAV-49) belongs to subgroup C and, therefore,
recognizes a different receptor on the cell surface, but
it retains the same antisense target sequence as that of
RCOSBPCAT(A). 383 was challenged with RAV—49 and still
showed a significant reduction in virus replication in
both ELISA.assays and viral titer (Table 2-3). This
suggests that the inhibition of viral growth in 383 cells
occurs after the attachment and entry of the virus into
the cells. At this time we have been unable to design a
test to unambiguously distinguish between antisense RNA
expression and a fortuitous host mutation that alters a
later step in viral growth and spread as the cause of the
383 resistance. Unfortunately, we have been unable to
identify a test virus which lacks the target antisense
sequence but still replicates well in the control RP30

cells. If such a virus were to grow normally on 383, it

59

would provide some (but not conclusive) support for the
possibility that 383 exerts its effect through antisense
expression.
Tetracycline-regulatable antisense expression system

As the analysis of transfectant 383 demonstrates, it
is difficult to definitively distinguish putative
antisense RNA effects from potential unrelated clonal
genetic variance. However, if antisense RNA is expressed
from an experimentally inducible promoter, and viral
resistance is demonstrated to be similarly inducible, each
cell line provides its own genetically identical interns“
control. Therefore, we decided to study the effect of
antisense RNA by tightly regulating its transcription
using the tet-regulatable expression system described by
Bujard et al. (Gossen and Bujard, 1992; Gossen et al.,
1995). This system has several advantages over other
regulated gene expression systems (Gossen et al., 1993).
First, it generally shows a lower baseline expression
level and a higher level of induction than others such as
lactose or heavy metal inducible systems. Second, most
vertebrate cells can tolerate tet to a certain extent. In
this system, gene expression is regulated by a hybrid
transcription factor, tTA, that consists of the

transactivation domain of herpes simplex virus VP16 fused

60

to the carboxy terminus of the tetracycline-repressor
(tetR). The gene of interest is placed downstream of a
minimal promoter linked to seven tandem copies of the
tetR—binding site (tetO). The activation of transcription
from this promoter depends on the binding of tTA to the
tetO site, a process which is tightly regulated by the
presence or absence of the drug. In the presence of tet,
binding of the tTA to tetO is blocked and gene expression
is silent or greatly reduced. Upon removal of tet, tTA
binds to tetO and induces a high level of transcription.

Figure 2—28 illustrates the procedure used to screen
effective antisense colonies in the tet-system. As before,
upon electroporation with the constructs, G418—resistant
RP 30 cells were selected and single colonies were
expanded. Each was then screened for its level of
transactivation activity by transient transfection with
the lac plasmid (pUHC13-3). This plasmid employs the same
tet-operator/minimal CMV promoter as pUHD10—3Neo to drive
expression of luc. Therefore, the level of luciferase
induction after transient transfection provides
confirmation that the regulatable expression system is
operating effectively in any given clonal cell line. Since
the antisense RNA cassette is driven by an identical

promoter to that of luc, it is likely that transcription

61

initiation of the antisense gene will show a similar level
of inducibility. Of course, post-transcriptional effects
likely will cause the relative expression level of
antisense RNA to differ from that of luciferase activity,
but the transactivation test allows one to eliminate cell
clones in which inducible promoter control is non-
functional or poorly functional (presumably due to
integration effects on the pUHD15-1 plasmid).

Fourteen G418—resistant cell transfectants harboring
the AE sequence in the antisense orientation were
screened. Seven of these showed significant
transactivation activity when grown in the media without
tet (Figure 2-5). However, none of those seven showed a
significant inhibition of viral growth upon challenge with
RCOSBPCAT(A) when grown in the media without tet verses
that observed with tet (Figure 2-5).

Two cell lines harboring the AD portion of the target
sequence (Figure 2-1) in the antisense orientation showed
a significant transactivation activity, but, again,
neither of these showed a tet-regulated inhibition of
viral replication (Figure 2—6). Similarly, 15 stably—
transfected cell lines containing the AC (Figure 2—1)
sequence in the antisense orientation showed a high level

of tet-inducible luciferase activity without any

62

Figure 2-5. The effect of cell lines harboring the AE
antisense sequence in the tet-repressible system.

% p27 in -tet was calculated by this formula: [(p27
ELISA.OD/106cells in —tet)+(p27 ELISA 013/106 cells in

+tet)]x100. Fold activation* was based on luciferase

activity and calculated by this formula: (luciferase

activity /106 cells in -tet)+(luciferase activity /106
cells in +tet).

 

 

63

 

 

 

 

 

 

 

 

 

I:] call free
////////// cell-assocnated

 

 

 

 

 

 

 

 

 

   
  

 

 

 

in
_ _ _ fl _
o O o o o o 0
MW n“ mm mm mw :5
or

«on. 5 sun

 

12 14

10

62

47

260 50 64 47 64

to Id
acflvaflon*

Figure 2-5

 

64

Figure 2-6. The effect of cell lines harboring the AD
antisense sequence in the tet-repressible system.

% p27 in -tet was calculated by this formula: [(p27 ELISA
OD/106cells in -tet)+(p27 ELISAOD/lO6 cells in
+tet)]x100. Fold activation* was based on luciferase
activity and calculated by this formula: (luciferase
activity /106 cells in -tet)+(luciferase activity /106
cells in +tet).

 

 

 

 

 

 

 

 

66

statistically significant reduction in viral replication
(Figure 2-7).

Northern blot analysis of two cell lines harboring
the AD sequence (Figure 2—1) demonstrated the substantial
expression of an antisense transcript in a tet-regulated
fashion (Figure 2-8). Similarly, specific transcription
of AE-antisense RNA was detected in two transfectants
only in the absence of tet (Figure 2-9). As described
above, none of these transfectants demonstrated tet-
regulated viral resistance.

CT'antisense RNA expression did not induce.ALV'resistance

The results described in the previous section
demonstrate that several different conserved regions of
the ALV genome have no anti-viral effect, even when they
can be shown to be expressed as antisense RNA at
relatively high levels. Others (Goodchild et al., 1988;
Sczakiel et al., 1992) have shown that the choice of a
target region for antisense inhibition can be critical to
its efficacy. As an experimental method to enhance our
choice of a target, we employed antisense oligonucleotides
in hopes of identifying sequences that are particularly
sensitive to antisense inhibition. These results are
described in Chapter 3 of this thesis. While antisense

oligonucleotides did not show a dramatic anti-viral

67

Figure 2-7. The effect of cell lines harboring the AC
antisense sequence in the tet-repressible system.

% p27 in —tet was calculated by this formula: [(927
ELISA OD/106cells in -tet)+(p27 ELISA 013/106 cells in
+tet)]x100. Fold activation* was based on luciferase

activity and calculated by this formula: (luciferase

activity /106 cells in -tet)+(luciferase activity /106
cells in +tet).

 

 

 

 

68

 

 

 

[:1 cell-free
W cell-associa ted

 

 

7//////////////////////////////////////////////////////
I

 

 

'-////////////////////////////////////////////////////////////

 

 

 

 

 

 

    

7/////////////////////////////////////////////

 

///////////////////////////////////////////////

 

 

 

 

 

 

 

 

 

 

 

 

 

'1
I 7/////////////////////////////////////////l//////17/;
Tblltlé
J
- d — _ — _
o 0 o O O o o
o 5 o 5 o 5
3 2 2 4| 4|

.8- 5 2.. s

 

17 1319 21

16

61011121314

2

clone

150 59 300 33 37

26 330 3 240 2 90

2 150 6

64

flfld

acuvauon'

Figure 2-7

69

clone D (-) 01 D (-) 02
Tet + — + —

 

Figure 2-8. Northern blot analysis of the AD—tet clones.
Each clone (D(—)Ol and D(—)02) was grown in the presence
or absence of tet (4 pg/ml) 2 d prior to total RNA
extraction as described in MATERIALS AND METHODS. Thirty
pg of each RNA was electrophoresed, blotted and hybridized
with 32P-labeled AD-DNA fragment. A band of approximately
400 nt was detected.

70

clone 1 3 4 14
Tht

 

Figure 2-9. Northern blot analysis of the AE-tet clones.
Each clone was grown in the presence or absence of tet (4
pg/ml) for 2 d prior to total RNA extraction as described
in MATERIALS AND METHODS. Thirty pg of RNA per lane was
electrophoresed, blotted and hybridized with a 32-P
labeled AE-DNA fragment.

”Fl.

71

effect, statistically significant inhibition was observed
with oligonucleotides targeted to a Short sequence ranging
from the PBS to the middle of the leader sequence (Table
3-4, Chapter 3). Based on these results, we chose to
target the CF region (Figure 2-1) for regulated expression
of antisense RNA in stably transfected RP30 cells.

The sequence covering the CF region was amplified by
PCR (Figure 2-1) and cloned into both tet—repressible and
-inducible expression system vectors. In the tet-
repressible expression system, each transfectant was
screened for transactivation as described before. Six
transfectants which showed significant transactivation
activity, however, did not show any inhibitory effect on
viral replication as assayed by ELISA (Figure 2-10).
In the tet-inducible expression system, the plasmid
pUHD172-1Neo contains a mutated tetR fused with the
transactivation domain of VP16, so that, in the presence
of dox (a derivative of tet), tTA binds to the tetO in the
pUHD10-3 vector and induces a high level transcription of
the gene located downstream of the promoter (Gossen et
al., 1995). Using this system, RP30 cells were
cotransfected with CF-10-3/puro and pUHD172—1Neo. Eight
transfectants which are both 6418- and puromycin-resistant

were selected. These cell lines were

72

Figure 2-10. The effect of cell lines harboring the CF
antisense sequence in the tet-repressible system.

% p27 in -tet was calculated by this formula: [(p27 ELISA
OD/106cells in -tet)+(p27 ELISA 013/106 cells in

+tet)]x100. Fold activation* was based on luciferase
activity and calculated by this formula: (luciferase
activity /106 cells in -tet)+(luciferase activity /106
cells in +tet).

73

 

 

 

 

[ZZZ] cefl4tee
W cell-associated

 

 

 

 

 

.lw%%%%%%%%%%%%%%%%%%%%

 

  

%%%%%%%%%%%%%%%%

 

 

 

 

 

 

250 -

200 -

_
o
5
1

.8. 5 Re .x.

100 -

50 -

13

11

1

200

168

290

«Md

acflvaflon'

Figure 2-10

74

tested for transactivation activity as previously
described. The results Shown in Table 2-4 identified Six
transfectants with low baseline luciferase expression and
high levels of transactivation. The fold of activation was
in a range of 150 to 1000, which was significantly higher
than that of the tet-repressible expression system
(ranging up to 300 fold). Although detectable antisense
RNA levels were observed in a few transfectants (Figure 2-
11), none of these Showed an inhibitory effect on viral

replication, as shown in Figure 2-12.

DISCUSSION

In this report, the potential inhibition of ALV
retroviral growth and Spread by expression of antisense
RNA was examined. In preliminary studies, one cell line,
383, was detected that was significantly more resistant to
the RCOSBPCAT(A) test strain than the RP30 parental cell
line. 383 expresses an antisense transcript at very low
levels which is complementary to most of the regulatory
Signals at the 5' end of ALV, including the PBS, Th the
leader sequence, the ATG translation initiation signal and
the SD. 383 showed a considerably greater reduction of
RCOS virus titer than of capsid protein production

compared to the controls. This observation suggests that a

75

Table. 2-4. The level of transactivation of the CF-tet
inducible clones.

 

clone fold activationa

 

l 150
2 2

3 940
4 370
5 330
6 300
8 160
10 1

 

3

Fold activation was calculated as described in Figure 2-12.

76

clonel 3 4 5 8
Dox+—+—+—+—+_

   

Figure 2-11. Northern blot analysis of the CF—clones in
the tet-inducible expression system. Each clone was grown
in the presence or absence of dox (1 pg/ml) for 1 d prior
to total RNA extraction as described in MATERIALS AND
METHODS. Thirty pg of RNA per lane was electrophoresed,
blotted and hybridized with a 32-P-labeled CF-DNA
fragment. The predicted size of the transcript is
approximately 200 nt. In clone 1 and 3, the antisense
transcript was detected despite the absence of inducer,
possibly due to cointegration of CF—pUHD10—3puro and
pUHD172-1Neo, resulting in deregulated transcription of
the CF sequence.

77

Figure 2-12. The effect of cell lines harboring the CF
antisense sequence in the tet-inducible system.

% p27 in +dox was calculated by this formula: [(p27
ELISA OD/106cells in +dox)-:-(p27 ELISA OD/106 cells in —
dox)]x100. Fold activation* was based on luciferase
activity and calculated by this formula: (luciferase
activity /106 cells in +dox)+(luciferase activity /106
cells in -dox).

dox=doxycycline

78

 

250

l

200

150 -

% p27 in +dox

100 -

50-

 

 

 

!: cell-free
W cell-associated

 

 

 

 

 

 

 

 

 

 

0

clone

fold
activatlon‘

1 3 4

1 50 940 370

330

Figure 2-11

160

 

79

defect in viral growth on 383 cells might occur in the
packaging step, resulting in the production of non-
infectious particles. Southern blot analysis showed at
least three copies of the antisense plasmid constructs
were integrated tandemly in the 383 genome. It has been
Shown that S’is recognized by a specific motif (Cys-His
box) of the NC protein during viral assembly (Aronoff et
al., 1993;

Dupraz et al., 1990), and this sequence could therefore
act as a potential target for antisense inhibition.
Alternatively, the interaction between antisense RNA and
its target could lead to the induction of double-strand
Specific cellular RNases, resulting in the degradation of
both RNAS and in production of empty virus particles.
Since only one antisense RNA-producing transfectant
demonstrated reduced viral susceptibility, it is certainly
possible that the inhibitory effect in 383 cells may
derive from a clonal variance unrelated to antisense RNA
production. In other words, the 383 line might have
incurred an unrelated mutation which reduces its ability
to grow the target virus. The replication of ALV, like
that of other retroviruses, depends on the host gene
expression machinery. The best characterized genetic

mechanism for host cell resistance to ALV involves changes

80

in the subgroup-specific receptor genes such as tva and
tvb (Crittenden, 1991). In this case our test virus is
subgroup A and if 383 had acquired a mutation in tva, one
would expect no change in its susceptibility to RAV-49, a
subgroup C virus. Our results showed that 383 also had a
reduced ability to grow RAV-49, and, again, the titer of
the virus was reduced more significantly than was
production of the capsid protein. Since it is unlikely
that 383 Spontaneously acquired mutations in both loci
encoding the subgroup A and subgroup C receptors, receptor
variance does not appear to explain viral resistance in
383. Unfortunately, the converse control, using a virus of
subgroup A with an altered antisense target sequence that
grows in RP30 is not presently available. However,
challenge of 383 with the RCAS virus showed much reduced,
if any, viral resistance. This could be due to overcoming
the antisense effect with the larger amount of viral RNA
generated by the stronger promoter in the RCAS virus.
Alternatively, 383 may provide a reduced level (relative
to parental RP30 cells) of a trans-acting host factor
required for replication of RAV-O-based viruses but not by
the RSVeA-based viruses. To further examine the properties
of 383 cells, the line was transfected with a sense RNA-

expressing (AE) construct cloned in the tetracycline-

81

repressible expression system in hopes of overcoming the
viral resistance, if it is due to antisense RNA. Pools of
sense-transfected 383 cells were analyzed in bulk. If
anything, removal of tet to allow expression of AE sense
RNA led to a further reduction of viral growth rather than
relief of the 383 viral resistance (Figure 2—13). While
these results do not conclusively prove that antisense
expression is not the cause of the 383 viral resistance,
they reinforce doubts raised by our other experiments.

By employing tet-regulatable expression systems, we could
control for possible complications due to clonal variance.
Although Specific, regulated transcription of antisense
RNA was obtained in several transfectants, no
corresponding reduction in virus replication was observed.
It is especially intriguing to note that the antisense RNA
from the CF sequence, which seemed to be the optimal
target region for an antisense ODN (Chapter 3), had no
antiviral effect. One explanation for this discrepancy
might relate to the need to deliver the antisense nucleic
acid to the appropriate subcellular compartment. Although
northern blots demonstrate the presence of substantial
antisense RNA within several cell lines, it is possible
that this RNA is confined to the nucleus and needs to

reach the cytoplasm to exert the described effect on the

82

Figure 2-13. The effect of cell lines harboring the CF
antisense sequence (without poly A signal) in the tet-
inducible system.

% p27 in -tet was calculated by this formula: [(P27
ELISA.OD/106cells in -tet)+(p27 ELISA OD/lO6 cells in
+tet)]x100. Fold activation* was based on luciferase
activity and calculated by this formula: (luciferase
activity /106 cells in ~tet)+(luciferase activity /106
cells in +tet).

83

 

 

 

    

 

m cell-a ssociated

E cell-free

 

 

 

 

 

 

 

 

 

 

 

 

 

120 —

_ - _ -
O o o

100 —
8

6

4

20

.8. e. 2.. .x.

 

17

15

13

11

(None

Figure 2-13

acflvaflon*

flfld

84

virus. However, Since a variety of vector systems were
used to express antisense RNA, all with no increased viral
resistance, this seems an unlikely explanation. Another
factor may be that in vivo-expressed antisense RNA is
considerably longer than the antisense ODN, containing
sequences flanking the presumed optimal target Site,
including the 3' poly A tail. Longer RNA has a greater
potential to form higher-order structures, which could
block interaction with the presumptive antisense target.
We have transfected RP30 cells with a CF-pUHD10-3Neo
construct devoid of the poly A signal. However, the level
of transactivation as measured by assaying the luciferase
acvtivity was significantly reduced. And when these
trasnfectants were assayed for ALV resistance, again, they
showed no inhibitory effect on ALV replication (Figure 2-
13). Peng et al. (1996) have suggested from studies on HIV
that shorter antisense RNA expressed from a stronger
promoter, in their case, a Pol III system, might be more
effective. A third explanation for the difference in ODN
and antisense RNA results would be that ODN—generated
inhibition (Chapter 3) has a very limited effect on viral
growth, and the transfected cell system may be

insufficiently sensitive to detect such an effect, either

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.-
0 0 o o o 0 O
o 8 6 4 2 O
1

86

in individual or pooled colonies. This lack of sensitivity
may relate to the inherent clonal variability of the
transfectants, the limited number of transfectants that
can reasonably be tested or the potential masking effect
of tet or dox on cell growth.

In conclusion, several cell lines have been obtained
which have been Shown to express substantial amounts of
antisense RNA in a regulated fashion but which have no
significant effect on their ability to support growth of
ALV. A variety of conserved viral sequences at the LTR and
the 5' end of the virus have been targeted, including
regions which showed limited, but significant inhibition
when used as targets for antisense ODN (Chapter 3), all
without significant effects on viral growth. In addition,
several different vector systems have been used to
generate antisense RNA, both with and without poly A
addition. While it would certainly be possible to test
many other regions of the ALV genome as antisense targets,
the well~known ability of retroviruses to rapidly mutate
(Coffin, 1991) is likely to confound this approach, at
least for any attempt to create chickens with resistance
to a wide range of field strains of ALV. At best only
limited conclusions can be drawn from predominantly

negative results, like those Shown in this thesis, but the

87

clear suggestion from our experiments is that in vivo
expression of antisense RNA is unlikely to be an effective
way to generate transgenic poultry that are resistant to
field strains of the virus.

In our experiments involving constitutive expression
of antisense RNA, one cell line (383) was obtained that
consistently demonstrated significantly reduced
susceptibility to the target RCOS-based virus and to an
ALV (RAV-49) of a different subgroup. It remains possible
that the effect observed was due to antisense RNA
expression, but this conclusion is placed in doubt by the
following observations: 1. Only very low levels of
antisense RNA were detected in 383, 2. Little or no
resistance was evidenced against a more virulent ALV
(RCAS) with nearly the same antisense target sequence as
RCOS, 3. Numerous attempts to replicate this observation
in other antisense-expressing cell lines failed, and 4.
Attempts to overcome the resistance observed in 383 by
counter-expressing sense RNA failed. Therefore, it seems
likely that the effect observed in 383 derived from a
mutation in this clone of cells that may be unrelated to
antisense expression, perhaps in the expression of some
trans-acting host factor required for RCOS replication but

dispensable for RCAS replication.

Chapter 3.

Antisense Oligodeoxynucleotides as Inhibitors of ALV

Growth

88

89

ABSTRACT

Antisense oligodeoxynucleotides (ODN) are short
stretches of synthetic DNAS made to be complementary to a
target RNA. Numerous cases of ODN inhibition of viral
replication have been reported. In Chapter 2 of this
thesis, we describe the general ineffectiveness of
expressed antisense RNA in attempts to inhibit ALV
replication in different expression systems. In this
chapter, we have employed antisense ODN in hopes of
finding the most effective antisense target in the 5’ end
of ALV genome. Eight different target Sites were selected
based on their potential capacity to block key processes
that occur during viral replication. A short region from
the primer binding site (PBS) to the middle of the
leader/packaging sequence seemed to be the most effective

antisense target in these experiments.

90

INTRODUCTION

Antisense oligodeoxynucleotides (ODN) are short
synthetic DNAS made to be complementary to a target RNA in
hopes of blocking the function of that RNA. Their effects
were first demonstrated when a 13—mer antisense ODN
complementary to the R region of the long terminal repeat
(LTR) sequence was found to inhibit Rous sarcoma virus
(RSV) replication in chicken embryo fibroblasts (CEF)
(Zamecnik and Stephenson, 1978). Biological efficacy of
antisense ODN is generally considered to depend on their
stability against nucleases, their ability to penetrate
the cell membrane and their binding affinity to the
specific target sequence (Peyman et al., 1995). Antisense
ODN have a negatively charged phosphate backbone structure
which is expected to hamper their uptake by cells, and
they may also be susceptible to DNase-mediated
degradation. Modifications of the phosphate backbone are
often employed to increase cellular uptake and stability.
These include thiolation and alkylation of phosphodiester
linkages and also conjugation of the ODN with peptides,
such as poly(L-lysine) and low-density lipoprotein
(Bongartz et al., 1994; Lemaitre et al., 1987; Mishira et
al., 1995). Most modifications (phosphorothioate being the

exception) result in at least a partial reduction in

91

nuclease susceptibility (Wagner, 1995). In addition,
antisense ODN with partial modifications (i.e.,
modification of terminal linkages) have been tested in
various systems and found to be more effective than the
ones with total modification (Chavany et al., 1995). The
direct mechanism of ODN uptake is not yet known.
Experiments with FITC-conjugated ODN Show that, upon
internalization, they appear as speckles inside the
cytoplasm, and most of them are localized in the nucleus
(Geselowitz and Neckers, 1992; Wagner, 1995). These
results suggest that the majority of ODN are encapsulated
in vesicles, and these vesicles may play a role in
transport to the nucleus. Several studies have reported
inhibition of viral gene expression in cultured cells by
phosphorothioate ODN (Lisziewicz et al., 1994; Matsukura
et al., 1987). Clinical trials to evaluate the efficacy of
antisense ODN against human immunodeficiency virus (HIV)
and human cytomegalovirus (HCMV) are in progress (Agrawal
and Tang, 1992; Lisziewicz et al., 1994).

Avian leukosis virus (ALV) is a retrovirus belonging
to the avian leukosis-sarcoma group. This virus infects
chickens and induces lymphoid leukosis. In the previous
chapter of this thesis, several conserved regions of the

ALV genome were examined as potential targets for inducing

92

viral resistance via expression of antisense RNA in stably
transfected cell lines. With one possible exception, we
were unable to detect an antiviral effect when 540
nucleotides (nt) at the 5’ end of ALV genome were used as
a target for antisense RNA expression. To explore this
region in a more detailed manner for the most effective
target sequence for antisense RNA, we chose to examine the

effect of antisense ODN on ALV replication.

93

MATERIALS AND METHODS

ODN‘synthesis

ODN were synthesized using an Applied Biosystems
model 394 DNA synthesizer at the DNA Core Facility of
Marshall University, Huntington, WV 25704. All ODN were
precipitated in ethanol prior to use.

cell culture and ODN treatment

RP 30-5 cells were maintained as described previously
(Chapter 2). Cells were washed once with serum-free media
prewarmed to 37°C and counted. 2x106 cells were seeded
onto a 35mm plate (Sarstedt Inc., Newton, NC 28658) in 0.8
ml of the serum-free media. Twenty pl of Lipofectin
reagent (1 mg/ml, Life Technologies, Gaithersburg, MD

20877) was mixed with 80 pl serum—free media and incubated

for 30 min. Meanwhile, each ODN was also diluted in 100 pl
of the serum—free media at five different concentrations
(0, 2, 5, 10, and 20 pM). The diluted Lipofectin reagent
was mixed with each ODN preparation and incubated for 15
min at room temperature. The mixture was added to each
corresponding plate of cells. After 13 hr at 40°C, cells
were pelleted by centrifugation for 3 min using the IEC

clinical centrifuge. The cell pellet was resuspended in 4

94

ml of complete media, and the cells were incubated for an
additional 6 hr prior to infection with the target ALV
vectors, RCASBPCAT(A) and RCOSBPCAT(A) (Hughes and Kosik,
1984; Hughes et al., 1987).
Virus infection

Cells which were treated with antisense ODN-liposome
mixtures were counted. 4x105 cells were seeded onto each
60 mm plate in triplicate and infected with 4x103
infectious units (iu) of RCOSBPCAT(A) or RCASBPCAT(A). The
antisense ODN solutions were re-added to the cells at
their original concentrations, and additional antisense
ODN were added 48 hr post-infection, again at the stated
concentrations. Cells and culture supernatants were
collected after 4 d and assayed by ELISA as previously

described (Chapter 2).

RESULTS

Dosage-dependent inhibition of’antisense ODN on
replication of ALV

RP30 cells were transfected with antisense ODN and
infected with recombinant ALV test strains followed by

measurement of p27 viral capsid production as illustrated

95

in Figure 3-1. The 5' end of the ALV genome was the
primary focus of these experiments, as it contains
numerous conserved sequence elements critical to viral
replication (Chapter 2, also see Figure 3-2). The sequence
of each antisense ODN is Shown in Table 3-1. Each ODN was
evaluated at five different extracellular concentrations
(0, 2, 5, 10 and 20 pM) in triplicate for its effect in
RP30 cells on replication of RCOSBPCAT(A) or RCASBPCAT(A).
Both RCOS and RCAS are recombinant ALV vectors but differ
in the U3 of their LTR, which in RCAS contains a strong
enhancer element. The two test viruses also differ in
their leader/packaging region sequences by 19 nt.
Therefore, antisense ODN #5 RCOS and #3 RCOS are
specifically complementary to the RCOS viral RNA , whereas
#5 RCAS and #3 RCAS are complementary to RCAS viral RNA.
The results from the initial test are Shown in Table 3-2.
In this experiment, the RCOS-Specific ODNs were tested
with RCOS virus and the RCAS-specific ODNs were tested
with RCAS virus. Both cell-free and cell-associated p27
production were assayed by ELISA as previously described
(Chapter 2) and normalized to total cell count. The effect
of each antisense ODN is compared to controls which were
treated only with the Lipofectin reagent at the

transfection step.

96

Transfection of RP30 cells with ODN

(day -l)

I 13 hrs

Infection with the recombinant ALV and
addition of ODN

(day 0)

Addition of supplementary ODNs

(day 2)

Collection of cells and culture
supernatant

(day 4)

Figure 3-1. A diagram illustrating the procedure used to
test antisense ODN inhibition of viral replication

97

 

8 3 RCAS, 3 RCOS 2
- 7 - - 6
_ l _5 RCAS, 5 RCOS .l _
- 7’/
PBS ATG
LTR leader/packaging gag

sequence

Figure 3-2. The regions targeted by antisense ODN. LTR,
long terminal repeat; PBS, primer binding Site.

98

Table 3-1. Sequence of antisense oligodeoxynucelotides

 

 

ODN sequence
1 TGA CGG CTT CCA TGC TGG AT
2 CTT AAT GAC GGC TTC CAT GC
3 RCOS CGT TAA GCG AGA CGG ATG AG
3 RCAS CGA TAG ACG AGA CGG ATG GA
4 ATC ACG TCG GGG TCA CCA AA
4A GGT CAC CAA ATG AAG CCT TC
48 TTC CCT AAC TAT CAC GTC GG
5 RCOS ATG AGG GCA GGA TCG CCA CG
5 RCAS ATG GAG ACA GGA TCG CCA CG
6 TAA GCA ACC CTT CCT TTT GT
7 CAT GCA GGT GCT CGT AGT CG

8 GGT GAA TGG TAA AAT GGC GT

 

99

.ocoo no: «02
.csonm one moamfimm ounoaadauu wo mommuo>¢..mou3uaso ooumouuca mo mHHoU Goa nod no

deAm on» an zoo oncomfiucm cues ooumouu maaoo 80H nod 00 (qum on» ocaow>ao ma ovumasoamo mm: Houucoo w

 

 

 

 

 

 

No No omo so we om me mm m
cm om mo ooH Nm me me ova N
mN we we mo mo oN am He e
m om ONN oNN e om ONH coo mqomm
mN mN om me SN mm mm so moose
me om we ONH NN me me No v
a co No on N s as ea modem
es as me we so me on He moomm
oz ONH omo coo oz me co co m
we oNH coo ONH mm om omH coo N
ae.0N 21 OH 2: m z: N ee_ON 2: ca 21m 2: N zoo mmcmmwocm
mountaaoo ooumfloommmnaaou
HOHUCOU w HOHHGOU w

 

zoo omcmmfiucm >Q coflumofiadou >q¢ mo cofluflpficcfl mo uncommon omoo .mlm manna

100

First, a dosage-dependent inhibitory effect of each
antisense ODN was observed. In other cases high
concentrations of ODN exhibited toxic effects on cell
growth (Gao et al., 1991; Stein, 1995). In this
experiment, the highest concentration of ODN (20 pM) had
no effect on cell growth, as determined by microscopic
observation and cell counting (data not shown). However,
every ODN had some inhibitory effect on viral replication
at increasing concentrations, suggesting a non-specific
effect. However, the levels of virus replication appeared
to be lower in the presence of some antisense ODN than
others. To control for the non-specific inhibitory effects
of ODN, selected ODN were further investigated by
comparison to those of random sequence control ODN.
Specificity of'the inhibitory effect of’antisense ODN

Phosphorothioate (PS)-oligonucleotides have been
Shown to induce non-specific effects by binding to cell
surface proteins or other intracellular regulatory factors
(Neckers et al., 1995; Stein, 1995). For example, PS-
oligos, in a length—dependent but relatively sequence-
independent manner, are known to bind to soluble CD4 at or
near the HIV-1 binding site (Yakubov et al., 1993), and
they also bind to the v3 loop of the HIV-1 envelope

glycoprotein, gp120 (Stein et al., 1993). It was also

101

suggested that the non—specific effect is dependent on the
number of phosphorothioate linkages in a given length of
ODN (Cheng et al., 1991). Therefore, random sequence
control ODN with the same base composition and
modifications were designed to analyze the specific
effect of a given antisense ODN on virus replication.
Based on our preliminary results, random combinations of
antisense ODN #3, #4, #5 and #6 were synthesized and
tested. Table 3-3 shows the sequence of each control
random sequence ODN. Each antisense ODN was then tested in
parallel with its control ODN for inhibition of virus
replication (Table 3-4). Antisense ODN #4 demonstrated an
inhibitory effect on viral replication when compared to
that of its control at all concentrations tested (p<0.05).
ODN #6 Showed a slight, but statistically insignificant,
decrease in viral replication when compared to that of its
control. Interestingly, ODN #5 showed a significant
reduction of viral replication compared to the control
only at 10 and 20 pM. ODN #3 did not Show any significant
reduction of viral replication compared to the control.
None of these ODN had an inhibitory effect on cell growth
during the time course of the experiment. However, both

antisense and control ODN still showed non-sequence-

102

Table 3-3. Random sequence control oligodeoxynucleotides.

 

ODN ODN sequence

 

#4 random. AGG ATA CGC ATC GTA CAC GC
#5 RCOS random. .AGA CTG CGT GCC GGA GAG AC
#5 RCAS random. .AAG ACG GCG TCA.ATC GAC GG
#6 random TTC GAT TAT TCC CAT CGA TC
#3 RCOS random. .AGC CGT CGA AGT AGT AGA GG

#3 RCAS random. .AAG GGT ATG GAG.ACG ACC AG

 

103

38...-» someone we» NS mooxoa com moovo.

.coauosooud own mo cowosoou o: mucmmoudou sown: .wOOH mm commoudxo ma OOH M oomucoouod .coHumHsonu was» am
.c30cm ma moHdEmm ooumofladso mo oomuo><..muofioocmu on» cue: oouoouu mousuHso mo mHHoo eoH and no (mHAm or»
>3 zoo oncomeucm cue: ooumouu mousuHso mo mHHou 60H Mom no <mHAm ecu mcwoa>wo an oouwH=UHmo mm: Houucoo w

 

 

 

 

 

 

OOH OOH OOH OOH OOH MN OOH OOH mfiumm

OOH OOH OOH OOH OOH Om OOH OOH moomm

mm Ow HO mm HO HO mm mm no

Hv OOH OOH OOH 0v mo OOH OOH m<0mm

Hm mm OOH OOH Om NO OOH OOH moomm

0v 0v mm mm mm Hm mm mm av
zzom 210H 21m Sam Snow ZnoH 21m Sim zoo oncomflucm

ooHMIHHoo ooumfloowmmnHHoo
Houucoo m Homoeoo w

 

.mpofioocmu uHocu on ooquEou mzoo oncomHucm mo muoomum .vlm manna

104

specific inhibitory effects on virus replication,
especially at higher concentrations.
The primer binding site as an ODN target

The results above suggested that antisense ODN #4
exhibited the most consistent reduction of viral
replication. AS a further control, a cocktail of randomers
(rather than a single oligonucleotide whose sequence was
chosen at random) with the same base composition and
modification pattern as antisense ODN #4 was synthesized.
This “super randomer” theoretically contains 4fl3different
sequences. RP30 cells were treated with antisense ODN #4,
randomer #4 and the super randomer simultaneously and
infected with RCOSBPCAT(A) followed by the p27 ELISA

assay. Again, RP30 cells treated with antisense ODN #4
showed inhibition of virus replication at 5 and 10 pM
(Figure 3-3). Little or no effect was observed at 2 and 20

pM. At 2 pM the ODN concentration may have been too low to

generate a significant inhibition, while at 20 pM the
generic inhibition observed for all ODN may have obscured
any sequence-specific effects. Furthermore, the effect of
the super randomer on viral replication was similar to

that of the single #4 randomer. These results suggest that

105

 

 

 

[:1 #4 super randomer

#4 antlsense

#4 randomer

 

 

 

 

  

yfl.

\

  
 

   

 

    

 

 

y//////////////////////////////////

           

%

 

 

 

 

-

1

2.8 a: L Ra 8:.

=e0

 

20

10

Concentration of ODN (pM)

Cell-free p27 ELISA OD was normalized

Figure 3-3.A. Effects of ODN #4 compared to

the controls.
to 106 cells.

106

 

 

 

[:Z] #4 super randomer

#4 antisense

-%\ #4 randomer

  

        

' \

20

  

 

 

 

 

 
 

 

    
 

N\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\L

  

 

 

 

 

1.0

 

H

5.
0

0.0

n=eoe - 5N.- dean-=00

10

Concentration of ODN (pM)

Cell-associated p27 ELISA CD was

Figure 3-3.B. Effects of ODN #4 compared to the
normalized to 106 cells.

controls.

107

the inhibitory effect of antisense ODN #4 is sequence-
specific.

Since antisense ODN #4 is complementary to the
complete sequence of the PBS, we decided to explore its
effect further by using ODN which are partially
overlapping its target region. The relative location of
each ODN is Shown in Figure 3-4. ODN #4A overlaps #4 by 10
nt at the 3' end, and #48, by 10 nt at the 5’ end. Direct
comparison of the effect of each ODN with one another
demonstrated that #4A was not as effective as #4 (Figure
3-5). However, #48 showed a similar reduction in virus

replication as #4 at all concentrations tested.

DISCUSSION

Since its first demonstration (Zamecnik and
Stephenson, 1978), the antisense ODN approach has been
widely employed to inhibit target gene expression in
various systems (Cowsert, 1993). It has been particularly
useful in inhibiting retrovirus replication, including
that of HIV (Goodchild et al., 1988; Lisziewicz et al.,
1994; Matsukura et al., 1987). In fact, some antisense
ODNS are in trials as potential anti-HIV therapies
(Lisziewicz et al., 1994). While there is no imaginable

prospect that antisense ODN would ever be a cost-effective

108

RCOS GATGG ACAGA CCGTT GAGTC CCTAA CGATT GCGAA CACCT GAATG
RCAS ----- C-G-- ----- --T-- ---G- ---c— A---G ------ c——-

AAACC ACTGG GGCTG CACTA.(#4)
RCOS AAGCA GAAGG CTTCA TTlTGC TGACC‘CCGACGTGATICGTTA GGGAA

 

 

 

 

RCAS ----------------- (Tec'rGAcccceAcemA'rlA ---------
“5 ‘ I GGCTG CACTA GCAATCCCTT
c'r'rcc GAAGT AAACC ACTGG (NA) (#48)

GCACC GCTAG GACGG GAGTA (#SRcos)
RCOS TAGTG GTCGG CCACA GACGG CGTGG CGATC c'rccc CTCAT CCGTC
RCAS --------------------------------- T- TC --------

GCACC GCTAG GACAG AGGTA (tsncas)

RCOS TCGCT
RCAS -----

Figure 3-4. Sequence of the,region near the PBS of the RCOS
and the RCAS viral RNA targeted by antisense
ODN #4, #4A and #4B. The PBS is marked by394~7
The sequence of viral RNA is shown in regular
characters and the sequence of each antisense
ODN is shown in bold characters. The common
nucleotides between the RCAS and the RCOS are
indicated as -.

  

109

 

       
   

§\\\\\\\\\\\\\\\\\\\\\\\\\\\.
V//////////////////////fl////fl

  

 

  

.s\\\\\\\\\\\\\\\\\\\\\\\\\\\\\x
7////////////////////////////////./////////////////////%

 

 

  

\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
w///////////////////////////////////////////W///////////2

        

 

  

.s\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\s
gig???

 
  
 

 

 

 

 

10

 

8 6 4 2 O

”=8 corks 8.2.8

10

Concentration of ODN (pM)

#4A and #4B.

Cell-free p27 ELISA OD was normalized to 106cells.

Figure 3-5. A. Effects of ODN #4,

110

 

 

        
 
 

  

I §\\\\\\\\\\\N
gig/ii

          

 

     
  

|R\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\s
iii

         

 

IN\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\N
gig

 

 

 

 

 

2 4| 0

2.8 o 25... 38.3

Concentration of ODN (pM)

#4A and #4B.

Cell-associated p27 ELISA was normalized to 103cells.

Figure 3-5. B. Effects of ODN #4,

lll

antiviral therapeutic in domestic poultry, we chose to
examine the effect of antisense ODN on ALV replication in
RP30 cells in hopes that it might guide us in the
construction of antisense RNA-expressing vectors (Chapter
2).

Each antisense ODN in our experiments was modified by
thiolation at two linkages at the 5' end and four linkages
at the 3’ end to improve its resistance to cellular
nucleases. The internal linkages were kept as normal
phosphodiester bonds to promote efficient hybridization
with the target RNA. After an initial, uncontrolled
survey, four of the original 8 target sites were selected
for further study. (This included two sites at which RCAS
and RCOS differ in sequence.) Each of these ODN was
examined in parallel with an appropriate random sequence
control ODN. Each control ODN had the same length, base
composition and modification pattern as its antisense
counterpart. Of the 4 sites tested versus control ODN, 3
continued to Show inhibition of viral replication, but
one, ODN #4, appeared to be most consistently effective.
ODN #4 is complementary to the PBS region of ALV and
therefore has the potential to compete with the host
'tRNAup primer for binding to the viral RNA at the PBS. In

tflne event that this antisense ODN displaces the tRNA

112

primer, this could impair either the generation of
proviral DNA or the subsequent synthesis of functional
viral RNA. Furthermore, the correct secondary structure at
the 5’ end of ALV RNA near the PBS plays a critical role
in the initiation of reverse transcription as shown by
mutational analysis (Aiyar et al., 1994). Therefore, the
presence of ODN #4 instead of the tRNA primer at the PBS
could also potentially block the initiation step of
reverse transcription. However, other studies have shown
that the ODN accumulate preferentially in the nucleus
(Wagner, 1995). Therefore, we cannot exclude the
possibility that antisense ODN #4, for unknown reasons, is
preferentially bound by ALV RNA in the nucleus leading to
its degradation (Cowsert, 1993). Furthermore, the region
including the PBS also contains an element of the
secondary structure which was proven to be critical in
efficient encapsidation of ALV genomic RNA (Knight. et
al., 1994). Therefore, antisense ODN #4 could also disrupt
this secondary structure leading to inefficient packaging
of viral RNA.

Antisense ODN showed no inhibitory effects on cell
growth as determined by microscopic inspection and cell
counting. However, all ODN exhibited a generic inhibitory

effect on virus replication at higher concentrations,

113

suggesting non-sequence specific effects. Non—specific
antiviral effects of PS-ODN have been described in several
reports (Chavany, 1995; Gao. et al., 1991; Krieg and
Stein, 1995), further stressing the importance of
comparing antisense effects to appropriate control ODN.
However, there is some difficulty in choosing an ideal
control. We have generally used a single, randomly chosen
permutation of the antisense ODN sequence. Since there is
an extremely large number of possible choices (sequences
with similar stretches to the antisense ODN or likely
problems in synthesis are eliminated), and since all ODN
have some effect on viral replication, it is impossible to
completely rule out the possibility that a fortuitously
“good” control has been chosen (leading to a false
positive antisense effect) or a fortuitously “bad” control
has been chosen (leading to a false negative). For ODN #4,
we also used a “super randomer” control, a mixture of all
possible ODN sequence. While this is a better control in
some ways, it could be argued that the effective
concentration of each of the 4x’control ODN sequences is
so low that it is an inappropriate comparison to a large
concentration of a single ODN sequence. We have employed
both control ODN approaches with essentially equivalent

results with respect to ODN #4. In addition, by comparing

114

the effect of ODN #4 to flanking sequences in ODN #4A and
#4B, the different ODN studied in effect act as the
controls for each other.

The maximum antiviral effect that was observed with
ODN #4 was approximately 80%. In other cases (Lisziewicz
et al., 1994, Matsukura et al., 1987), particularly of
HIV, the antiviral effect ranged up to 99%. GEM91
(Lisziewicz et al., 1994) was a 25-nt long
phosphorothioate ODN complementary to the ATG initiation
Signal of the gag reading frame of HIV. However, we did
not observe a Significant inhibition of ALV replication
with ODN #6 which was complementary to the ATG signal of
ALV gag. Goodchild et al. (1988) have Shown in studies of
HIV that a 20-mer phosphodiester ODN complementary to the
PBS demonstrated 30—60% inhibition, and the most effective
inhibition was obtained with ODN complementary to the R
region and to certain splice Sites (85% and 80%,
respectively). Furthermore, cellular uptake efficiency in
each experiment is expected to account for at least part
of the variation in effectiveness. Standifer et al. (1995)
have Shown that up to 1.7 % of total labeled ODN could be
found intact and associated with neuronal cells, and this
amount reduced target mRNA levels by 25-30%. However, an

approximately 18-fold of increase in uptake was Shown when

115

cells were incubated with an ODN in the presence of
Lipofectin reagent (Bennet et al., 1992). Therefore, we
employed the lipofection technique to create an
intracellular “reservoir” of antisense ODN and re-added
ODN every 2 days to replace degraded ODN. At this moment,
we do not have information on the cellular uptake
efficiency of ODN in our system. However, since the cells
were treated with an antisense ODN in parallel with its
control randomer, which also has the same modification
pattern, it seems unlikely that there was an extensive
variation in the uptake efficiency between them. Hoke et
al. (1991) suggested from their studies with herpes
simplex virus that the reduced efficacy of partial
compared to fully PS ODN in HeLa cells may result from
increased degradation of the mixed phophodiester/PS-
oligos. Therefore, the effects of different antisense ODN
are likely to depend on several variables, such as the
length, the modification, the target Site, the mode and
activity of viral replication and the host cells used.

In conclusion, we have found a region from the PBS up
to the middle of leader sequence to be an effective target
for antisense ODN. In particular, antisense ODN #4, which
is complementary to the PBS, showed the most consistent

inhibition (maximum of 80%), relative to its controls. In

116

Chapter 2 of this thesis, we have tested the antiviral
effect of this region in a stable antisense RNA expression

system, without significant antiviral effect.

Chapter 4.

Antisense RNA complementary to subgroup A.ALV receptor

mRNA.expressed in a quail cell line: test for effects on

virus susceptibility

117

118

INTRODUCTION

A critical step in the life cycle of an enveloped
virus is the binding of the virus to the host cell. This
process is mediated by specific interactions between the
viral envelope glycoprotein (SU) and the cell—surface
receptor (Coffin, 1990). Avian retroviruses of the avian
leukosis virus (ALV) group have been divided into several
subgroups. The subgroups of ALV are defined by host range
in chicken cells that differ in susceptibility to
infection, patterns of receptor interference, and virus
neutralization (Crittenden, 1991). These properties are
all regulated by differences in the viral envelope surface
glycoprotein, SU (gp85). There are five major subgroups of
ALV (subgroup A to E), which are determined solely by
amino acid differences in the variable regions of SU. The
susceptibility of chickens to ALV subgroups is controlled
by three genetic loci, tva, tvb, and tvc (Crittenden,
1991). tva and tvc alleles are thought to be linked and
encode receptors for subgroup A and subgroup C viruses,
respectively. Different alleles of tvb might encode
receptors for subgroups B, D, and E.

Recently, the gene encoding the receptor for subgroup
A ALV has been cloned in a quail cell line (QT6) and shown

to be the product of the tva locus (Bates et al., 1993, L.

119

Crittenden, personal communication). tva cDNAs of two
different sizes (800 and 950 nt), named pg800 and pg950,
respectively, were identified, which were Shown to be
generated by alternative splicing. The deduced amino acid
sequences predicted that the extracellular domain of the
receptor had some sequence homology to a ligand-binding
domain of the low-density lipoprotein receptor. Although
neither their mRNAs nor protein products are detectable in
chicken cells, these cDNAs can confer susceptibility to
infection by subgroup A.ALV to an otherwise resistant cell
line. The cellular function of this receptor, however,
remains unknown. A truncated form of the putative subgroup
A ALV receptor (without the transmembrane domain) was able
to protect the cells from infection, by binding to the
viruses and blocking attachment (Connoly et al., 1994).

The tva gene can exhibit both susceptible and
resistant alleles, so it was of interest to see if viral
resistance could also be induced by blocking expression of
the gene. Since “knock-out” chicken technology has yet to
be perfected, one attractive method was to try to use
antisense techniques to block tva expression. The
corresponding mRNA is expressed at undetectable levels in
most avian cells, so it might be relatively easy to

overcome its function by expressing small to moderate

120

amounts of stable antisense RNA. Our attempts at
expressing ALV antisense RNA (Chapter 2) were generally
unsuccessful, possibly due to some inherent properties of
viral RNAS. Thus we wished to try the antisense approach
against a host cell target mRNA.

We have expressed antisense RNAS against the message
for subgroup A ALV receptor in hopes to block the viral
infection at its entry step. To date, our focus has been
on the 5' end of this gene. The transcription start site
has not been mapped yet, but there are two putative TATA
boxes 300 and 200 bp upstream of the ATG translation
initiation signal. Two DNA fragments which covered the ATG
signal including the 5' untranslated region were amplified
by the Polymerase Chain Reaction (PCR). The effect of
antisense RNA complementary to this 5’ region was examined

in a tetracycline(tet)-regulatable gene expression system.

121

MATERIALS AND METHODS

cell culture

QT6 is a chemically transformed quail fibroblast cell
line (Moscovici et al., 1977). This cell line and all of
its derivatives were maintained in Leibovitz L-15/ McCoy's
5A.medium (Life Technologies, Gaithersburg, MD 20877)
under 5% COzcontaining 10% chicken serum, 5% fetal bovine
serum, 2.5% tryptose phosphate broth supplemented with
gentamycin (10 pg/ml) and amphotericin B (2.5 pg/ml).
Transfection

Transfection was performed using Lipofectin as
described by the manufacturer (Life Technologies).
Briefly, cells were split one day prior to transfection

into 60 mm plates. Six h before transfection, the media
was replaced with fresh. In one tube, 2.5 pg of each
plasmid construct was mixed with 5 pg of pUHD15-1
(transactivator-encoding plasmid) in 150 pl of serum-free
media. In another tube, 45 pl of Lipofectin (1 mg/ml) was

diluted to 150 pl with serum-free media and incubated for

30 min at room temperature. The Lipofectin solution was
then added to the DNA solution and incubated for 15 min.

Meanwhile, cells were washed once with 4 ml of serum-free

122

media. Serum—free media was added to the DNA/liposome
mixture to 2 ml, and this mixture was then added to cells
and incubated for 16 h at 40°C. At this point cells were
changed into complete media and incubated for an
additional 24 h. Cells were then split in to two 10 cm
plates in selection media containing 0.4 mg/ml of neomycin
(G418—sulfate, Life Technologies) and 4 pg/ml of tet. The
G418-resistant colonies developed in 10 d. Each colony was
expanded and tested for transactivation by transient
transfection with the lac construct, pUHC13-3.

Luciferase assay

Each G418-resistant transfectant was split into 60mm
plates containing media with and without tet one d prior

to transfection with pUHC13—3. Lipofection was performed
as described above using 1.8 pg of pUHC13-3 and 20 pl of
Lipofectin (1 mg/ml) in 300 pl of serum-free media. Cells
were washed twice with phosphate buffered saline (PBS),
lysed in 300 pl of 1x lysis buffer (diluted from 5x with
water; Promega, Madison, WI 53706), and Spun at 14k at 4°C
briefly to remove cell debris. The supernatant was diluted

lOO-fold with 1x lysis buffer and 10 pl was mixed with 100

pl of the substrate at room temperature. The luciferase

123

activity was then measured using a Turner TD-20e
luminometer (Turner Designs, Inc., Sunnyvale, CA 94086).
Plasmid construction

The quail genomic clone (Q5.5-pBSkS(-)) containing
the subgroup A ALV receptor gene was provided by Paul
Bates (Bates et al., 1993). Figure 4—1 illustrates the
region targeted by antisense RNA. Contigs 2 and 6 are
regions previously sequenced by Dr. Bates' lab, who
provided the sequence to us. The “gap" between these
contigs was sequenced using the primer pRec 5 by the
dideoxy chain termination method (Sanger et al., 1977).
The length of this gap was determined to be 125 base pairs
(bp). The PCR primers used are listed in Table 4-1 and
their positions are shown in Figure 4-1. PCR reactions
were performed as described (Sambrook et al., 1989) with
appropriate pairs of primers as indicated in Figure 4-1.
Each PCR fragment (QR2 or QR3) was directly cloned into
the TA vector (Invitrogen, San Diego, CA 92121) and
sequenced. The fragments were then transferred to pUHDlO-
3Neo by EcoRI digestion and ligation. General procedures
used for subcloning were as described (Sambrook et al.,

1989).

124

Table 4-1. The primers used in PCR amplification

 

 

primer sequence

pRec 2 5' TTA CCG GAC CCG TTA CCG 3'
pRec 5 5’ GCG CCA TGT CGG TAC CGC 3'
pRec 6 5' AGT TTC AGC TGG GCA CGT 3’
pRec 7 5' TTG GGC CGC TGT TCG CTC 3’

 

125

Contig 2 Contig 6

pRec 5

 

 

gap
ATG exon 1 exon 2

pRec 2

pRec 7 ._.

pRec 6

— QR 3, 287 bp

Figure 4-1. The region targeted by antisense RNA. Contig 2
(798 bp) and contig 6 (281 bp) were sequenced by Dr.
Bates. Contig 6 contains exon 1 and contig 2 contains two
putative TATA boxes. Gap was sequenced as described in
MATERIALS AND METHODS and determined to be 125 bp in
length. The 5' untranslated region is expected to be
included in the 3’ end of contig 2 and in gap.

126

Virus preparation

RCASBPAP(A) is a subgroup A recombinant ALV vector
carrying the alkaline phosphatase (AP) gene at the 3' of
env (Federspiel et al., 1995). Line 0 chicken embryo
fibroblasts (CEF) infected with RCASBPAP(A) were obtained
from Mr. Bill Payne (Department of Microbiology, Michigan
State University, East Lansing, MI 48824). The culture
supernatant of these CEF was collected and spun at 2000
rpm at 4°C for 5 min to remove any cellular materials. Its
titer was determined by infecting QT6 cells at limited
dilution and assaying for AP activity. The virus stock was
kept at -70° C.
Infection with RCASBPAP (A) and the AP assay

G418-resistant quail cell transfectants with
significant transactivation levels were split and grown in
media with and without tet (4 pg/ml) for 4 d prior to
infection with RCASBPAP(A). AP activity was assayed by
either direct cell staining or by a soluble assay. .AP
staining: 1x105cells of each cell line in +/- tet were
seeded into 6-well plates in triplicate 1 d before
infection. The media was replaced with 1 ml of fresh and
1x103 infectious units (iu) of RCASBPAP(A) was added to

each well. After 3 h of incubation, the cell monolayer

127

was washed twice with PBS to remove the input virus,
overlayed with 3 m1 of media containing 0.6% low-melting
point (LMP) agarose (Life Technologies) and incubated for
3 d. The agarose overlay was then removed, and the cells
were stained for AP activity as described (Rong and Bates,
1995). Soluble AP assay: 2x106 cells of each transfectant
were seeded into 10 cm plates in duplicate one d prior to
infection. The media was removed from the cells and 9 ml
of new media was added. 2x106 iu of RCASBPAP(A) was added
to each plate and incubated for 48 hr. Two d post-
infection, the cell monolayer was washed twice with PBS
and the AP assay was performed as described (Berger et
al., 1987).
Southern hybridization

Genomic DNA was isolated as described in Chapter 2 of
this thesis. Ten pg of each genomic DNA was digested with
ClaI and subjected to 0.8% agarose gel electrophoresis,
followed by transfer to a nylon membrane (MSI, Inc.,
Westborough, MA 01581) and hybridization as described
(Sambrook et al., 1989). The hybridization probe was a 2
kilobase pair (kb) fragment released from the RCASBPAP(A)
plasmid by ClaI digestion or a 32P-labeled QR2 DNA

fragment prepared by digestion of QR2-10-3/Neo with EcoRI

128

32P-labeled by the random.primer extension method
(Sambrook et al., 1989).
Northern hybridization

Total RNA was isolated by lysis using Trizol (Sigma,

St. Louis, MO, 63178) as described by the manufacturer. 30
pg/lane of RNA was run on a 1.2% agarose gel as previously

described (Chapter 2). Blots were hybridized as described

above for Southern blot analysis.

RESULTS

Generation of QT6 clones harboring an antisense sequence
complementary to the.mRNA for the subgroup.A receptor
DNA fragments from the cloned subgroup A receptor
gene were amplified and cloned into the antisense
expression vector pUHD10-3Neo in the appropriate
orientation as described in MATERIALS AND METHODS. Each
construct was then cotransfected into QT6 cells with the
plasmid pUHD15-1 which constitutively expresses tTA, the
transactivating protein. Eighty G418—resistant colonies
were selected in media with tet (to keep the expression of
antisense RNA uninduced state, in case repression of tva

expression might be deleterious to the cells). Each G418-

129

resistant colony was screened for its level of
transactivation by transient transfection with pUHC13-3
(luc-encoding plasmid), followed by an assay of luciferase
activity in the presence and in the absence of tet. A
total of 40 G418-resistant colonies were found to Show
strong transactivation activity, one of them ranging up to
200 fold. Table 4—2 shows 29 colonies which had
transactivation activity and the results from AP staining.

Unfortunately, neither the message nor the protein
expressed by the subgroup A receptor gene has been
detectable by typical northern or immunoblot analysis (P.
Bates, personal communication). Therefore, the level of
.ALV infectivity is most sensitive and, to date, the only
effective assay for receptor expression. G418-resistant
QT6 cell lines with high levels of transactivation were
further tested by two infectivity assays. Since these
experiments were performed in QT6 cells, we employed a
more facile assay for viral infection based on newly
developed AP-expressing ALV vectors (Federspiel et al.,
1995)
AP staining of'the infected cell lines

RCASBPAP(A) is a recombinant ALV vector carrying an
alkaline phosphatase (AP) gene at the 3’ of env. It was

postulated that those QT6 transfectants in which antisense

130

Table 4-2. A. The effect of QT6 clones harboring the QR2
antisense sequence on viral infectivity.

 

Number of Infection Foci

 

 

Clone Fold activation‘
+tet SD -tet SD
QR2 5 2 49 2 16
45 30 8 14
75 30 7 25
1 1 60 41 3 38 1 0
1 2 7 28 9 34 6
1 3 21 36 3 30 14
1 5 190 47 2 73 9
1 7 140 8 2 70 14
1 9 80 1 4 5 1 8 2
28 85 23 4 14 8
29 64 38 5 38 6
32 13 39 1 0 1 0 6
36 37 51 12 43 8
42 16 28 7 33 1 5
52 36 2 3 0.3 0.6
53 1 8 1 4 4 33 12
54 7 1 4
56 22 7 2
58 53 2 1
60 20 27 7 1 5 3
62 20 1 0 4 9 2

 

‘ Fold activation was based on luciferase activity and calculated by this formula: (luciferase
activity in - tet)+(luclferase activity in +tet).

Cells were grown in the presence and absence of tet (4 pglml) for 4 (1 prior to infection.
Average number of infection foci in triplicate wells are shown.

SD; standard deviation

131

Table 4-2. B. The effect of QT6 clones harboring the QR3
antisense sequence on viral infectivity.

 

Number of infection foci

 

 

clone f0"? . +tet SD -tet SD
activation
QR3 3 96 1 1 6 3 2
16 3 56 6 27 5
17 4 21 4 13 4
28 110 41 6 43 1
31 150 28 5 38 4
33 59 41 5 53 6
4o 2 3 1 3 2
42 51 0.3 0.6 2

 

' Fold activation was based on luciferase activity and calculated by this formula: (luciferase
activity in - tet)-:-(|uciferase activity in +tet).

Cells were grown in the presence and absence of tet (4 pig/ml) for 4 d prior to infection.
Average number of infection foci in triplicate wells are shown.

SD; standard deviation

132

RNA inhibits the expression of the subgroup A ALV
receptor should generate a reduced number of infected cell
centers upon infection with RCASBPAP(A) followed by
staining for AP. The G418-resistant quail transfectants
which showed significant levels of transactivation were
split and incubated for 4 d in media with and without
tetracycline. Although the ALV subgroup A receptor is
likely to be rather stable, given its very low levels of
expression, we hypothesized that this time period would be
long enough to observe any decrease in the synthesis of
new receptor molecules that might result from antisense
inhibition. lOscells of each transfectant were infected
with RCASBPAP(A). Three days later, the cells were stained
for AP activity. Each QT6 transfectant was assayed in
triplicate and the results are shown in Table 4-2. The
data demonstrate that the QT6 transfectants remained
susceptible to infection with the RCAS virus, but some of
the transfectants showed a limited decrease in foci in the
absence of tet.

Transcription of antisense RNA in the absence of tet

To confirm the presence of regulated expression of an
antisense transcript, total RNA was extracted from each
transfectant after growth in the presence and absence of

tet for 4 d. Total RNA was analyzed by Northern blot using

133

a QR2-specific probe (Figure 4-2). Detectable levels of
antisense QR2 transcripts were found in the absence of tet
in transfectants #9 and #52. Transfectants #5, #8, and #60
did not have a detectable level of antisense QR2
transcript, and were not further examined.
Soluble.AP assay

A few transfectants appeared to exhibit a decreased
susceptibility to RCASBPAP(A) infection in the absence of
tet as assayed by histochemical AP staining (Table 4—2).
However, the major drawback of this assay is that it is
based on a relatively limited number of foci, leading to
potentially large variances in the results (e.g., QR2-15
and QR2-l7, Table 4-2A). Therefore, another type of AP
assay was performed which measured the total.AP activity
by spectrophotometry. In this assay, a crude cell extract
containing AP was prepared and assayed by measuring the
amount of AP enzyme reaction product (p-nitrophenol)
(Berger et al., 1987). A QT6 transfectant which becomes
less susceptible to RCASBPAP(A) infection due to decreased
expression of tva engendered by antisense RNA should
display decreased AP units/mg cell protein, in the absence
of tet compared to the presence of tet. Each cell line was
infected with RCASBPAP(A) and, on day 2 post-infection,

the cells were harvested and an AP-enriched fraction was

Figure 4-2.

B4

clone QT6 32 52 9
Tet + — + — + —

 

 

Northern blot analysis of antisense QR RNA.
Total RNA was extractedn electrophoresed,
transferred, and hybridized with a DNA
fragment specific to QR2 and QR3 RNA as
described in MATERIALS AND METHODS.

NV; no virus

135

.manmuocuoo no: mmz 42m NmO omscoon bonucE was» >n con>amcm won one: own van on .mu mcoau

 

.oumowaaso ca UoEMOMMoQ uoc

.ocov no: «92

.mm i\+ cumuaaaso mo ommuw>c cm on commoumxo ma :aououm OE\muac= m4

 

 

momH Mora momH oz 1
N02 N02 mvm Q2 + who
mm -:.OOH H -:.vm m ->.mm N -:.Nm 1
ca -:.omH mm -I.O©H m -I.OOH A.->+mm + ma n ma
m .\+ or m -2 mr H -1 mm m -\+ vm 1
ma -:.m> «H -:.mm m -:.Nm b -:.Nv + mm
mH .: oHH pm A: mm m -1 on mom 1
mm -:.oHH av -:.>oH hm -:.mm mmv + mm
0.4+ mm «a -:.ooH ma -:.ooa me 1
ma -:.om. mH-x+nm HH.;+ ooa «mm + m «.mo
v mum m mum N awe H axe uou ocoHo

 

caduoua dado OE \ uuacz m4

 

 

sua>fluom m< no smmmc .miv manna

136

obtained as described in MATERIALS AND METHODS. The
results of the AP assays are shown in Table 4—3. Although
some of these transfectants seemed to have a reduced
infectivity when assayed by histochemical AP staining and
two of them showed relatively high levels of inducible
antisense tva transcript, no significant difference in AP
activity was detected i tet in several tests of these
transfectants.
Detection of proviral DNA

Once a retrovirus enters its host, it copies its RNA
genome into a double-stranded DNA and this DNA copy
integrates into the host genome to establish infection.
It was reported that QT6 cells don't produce ALV at a high
titer, although they can be normally infected (Friis,
1972). Therefore, we decided to examine the potential
influence of receptor interference at an earlier stage of
the viral life cycle, i.e., that portion up to the
integration step. The time required for a virus to enter a
host cell and to complete the generation of a proviral DNA
has been reported to be 4-8 hr (Coffin, 1990).

The G418-resistant QT6 transfectants were carried in
media with and without tet for 4 d prior to infection with
RCASBPAP(A) at 0.5 MOI. Ten hours post infection, genomic

DNA was isolated and digested with ClaI. This released an

137

clone QR2-9 QR2-52 QR3-16 QT6 QT6

T¢t+-+-+-+-NV

 

B .
LTR AP LTR
E gag ”’1 9”" _[:|

 

C Ial . C 101

Figure 4-3. A. Detection of proviral DNA.

Genomic DNA was isolated 10 hr post infection, digested
with ClaI, electrophoresed, transferred, and hybridized
with 2kb—DNA fragment specific to AP gene as described in
MATERIALS AND METHODS.

B. The genome organization of RCASBPAP(A) provirus

138

internal 2-kb fragment from proviral DNA which corresponds
to the AP gene (Figure 4—3). Southern hybridization with a
probe specific to AP demonstrated that, as expected, the
retroviral DNA of RCASBPAP(A) stably integrated into the
host genome (Figure 4-3). Although QT6 transfectants #9
and #52 showed a substantial amount of antisense
transcript in the northern blot assay (Figure 4-2), they
did not show any decrease in the intensity of the proviral
DNA fragment, suggesting that antisense RNA expression had
had no effect on the early portions of the viral life
cycle of the test virus. This includes the attachment
phase which we might have expected would have been
influenced by the expression of antisense to the receptor
mRNA.
Analysis of viral gene expression

Since it was not feasible to examine virus spread by
p27 ELISA.due to inefficient virus production from QT6, we
decided to investigate viral mRNA synthesis using northern
blotting with a virus-specific probe. On d 3 post-
infection of QT6 transfectants with RCASBPAP(A), total RNA
was extracted and hybridized to a probe specific for a
spliced AP message (Figure 4-4). Again, the results did
not demonstrate any reduction in the amount of AP message

generated when cells were carried in the media without

139

Figure 4-4. Northern blot analysis of AP mRNA expression.
Total RNA was extracted 3 d post infection,
electrophoresed, transferred, and hybridized
with a DNA fragment specific to AP gene as
described in MATERIALS AND METHODS. A:
infection with RCASBPAP(A) and B: infection
with RCASBPAP(E).

NV; no virus

 

Virus RCASBPAP(A)

 

clone 9R2-9 QR2-32 QR2-52 QR3-16 QT6

Tet + — + - + - + -
. n '
Virus RCASBPAP(E)

 

done QR2-9 QR2-32 QR2-52 QR3-16 919 gm

Tet+—+—+—

 

141

tet. RCASBPAP(E) recognizes a different receptor and,
therefore, was included as a control. Previous experiments
indicated that subgroup E ALV infects QT6 cells
considerably less efficiently than does subgroup A ALV
(data not shown). This was confirmed in Figure 4—4B
showing a much lower abundance of the AP message from
RCASBPAP(E)-infected cells. As expected, where it could be
detected, there was no difference in AP gene expression i
tet.
DISCUSSION

Here we report our attempts to use an antisense RNA
approach to suppress the expression of the subgroup A.ALV
receptor in the QT6 cell line. We have employed the tet-
regulated gene expression system to generate antisense tva
RNA. It was expected that, in cells which induced high
levels of antisense QR2 or QR3 RNA in the absence of tet,
expression of the receptor would be suppressed, leading to
a decreased susceptibility to ALV. Results of northern
blot analysis (Figure 4-2) indicate that, in a few QT6
transfectants, substantial amounts of antisense RNA were
transcribed specifically in the absence of tet. However,
these cells were still equally susceptible to infection by
subgroup A.ALV, as judged by DNA provirus formation or

production of AP enzyme or mRNA. While it might be argued

142

that the receptor protein could be extremely stable,
delaying the effect of inhibiting its synthesis, cells
were grown through numerous doublings over 4 d, which
should allow for a substantial reduction in receptor level
on cell surfaces. The affinity between the virus and the
receptor may be very high, such that only one or a few
functional receptors would be required for efficient
infection, thereby making the threshold level at which we
would observe a decrease in viral susceptibility very low.
However, since the protein itself has proven to be
virtually undetectable, one assumes it is present at
extremely low amounts in the first place, and if antisense
inhibition were working that an influence on viral
replication would be detected.

In conclusion, we have obtained several QT6 cell
lines which express an antisense transcript complementary
to the tva message in a regulated fashion. However, none
of these cell lines demonstrated a significant decrease in
susceptibility to subgroup A ALV infection. Several of the
many possible explanations for the failure of antisense
inhibition are discussed in Chapter 2 and need not be

repeated here.

Summary and Conclusions

1. Various regions at the 5' end of the ALV genome were

targeted for expression of antisense RNA.

2. One stably transfected cell line (383) showed a
significant inhibition of viral replication in a
constitutive expression system. A low level of antisense
transcript was detected in this transfectant by RT-PCR. A
very significant decrease in the titer of ALV grown on 383
was observed for ALV members of two different subgroups (A
and C). However, attempts to attribute the decrease in
virus susceptibility in 383 to the production of antisense
RNA were unsuccessful, leading to the suggestion that this

cell line may be a random clonal variant.

3. In the tet-regulatable expression systems, a
substantial amount of antisense transcript was detected in
the presence of inducer or in the absence of repressor.
However, a significant inhibition of viral replication was
not observed in these transfectants despite the use of

several expression vectors or antisense target regions.

143

 

144

4. Antisense oligodeoxynucleotides (ODN) were employed in
hopes of finding the most effective antisense target
region near the 5’ end of the ALV genome. A short region
including the PBS was demonstrated to be the most
effective antisense ODN target with the inhibitory effects
of up to 80%. However, an antisense RNA complementary to
this region did not show any inhibitory effect on viral

replication when tested in a stable expression system.

5. The 5' end of the message encoding the cellular
receptor for subgroup A.ALV in a quail cell line has been
employed as a target for antisense RNA expression, in
hopes of specifically reducing susceptibility to subgroup
A ALV. Although specific expression of antisense RNA was
detected in a few transfectants, again, no reduction in

infectivity was observed.

6. Overall, in vivo expression of antisense RNA.appears
unlikely to be an effective way to generate transgenic

poultry that are resistant to field strains.

Bibliography

BIBLIOGRAPHY

Chapter 1.

.Agrawal, S., Goodchild, J., Civeira, Mg P., Thornton,.A, H,m
Sarin, P. S. and Zamecnik, P. C. Oligodeoxynucleoside
phophoramidates and phophorothioates as inhibitors of human
immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85:7079—
7083 (1988)

.Agrawal, S. and Tang, J. Y., GEM91-an antisense
oligonucleotide phophorothioate as a therapeutic agents for
AIDS. Antisense Research and Development 2:261-266 (1992)

Aiyar, A., Cobrinik, D., Ge, 2., Khng, H.-J., and Leis, J.
Interaction between retroviral U5 RNA and the TWTiloop of

the tRNAtrp primer is required for efficient initiation of
reverse transcription. J. Virol 66:2464-2472 (1992)

Aiyar,.A., Ge, 2. and Leis, J. A.specific orientation of
RNA secondary structure is required for initiation of
reverse transcription. J. Virol 68:611-618 (1994)

Bates, P., Young, J. A. and'Varmus, H. E..A receptor for
subgroup A.Rous sarcoma virus is related to the low density
lipoprotein receptor. Cell 74:1043-1051 (1993)

Beffa, R. 8., Refer, R., Thomas, M., and.Meins, F. Decreased

susceptibility to viral disease of B-l,3-glucanase-deficient
plants generated by antisense transformation. The Plant Cell
8:1001-1011 (1996)

Bennet, C. P., Chiang, M; Y., Shoemaker, J. E., and
Mirabelli, C. Cationic lipids enhance cellular uptake and

activity of phophorothioate antisense oligonucleotides.
Molecular Pharmacology 41:1023-1033 (1992)

Biasolo, A” M., Radaelli, A", Pup, L. D., Franchin, E.,
Giuli-morghen, C. and Palu, G. A new antisense tRNA
construct for the genetic treatment of human
immunodefieciency virus type 1 infection. J. Virol 70:2154-
2161 (1996)

Biere, A" L., Citron, Mg, and Schuster, H. Transcriptional
control via translational repression by c4 antisense RNA of
bacteriophage P1 and P7. Genes & Develop. 6:2409-2416 (1992)

M5

146

Bieth, E., Gabus, C., and Darlix, J.-L. A study of the
dimer formation of Rous sarcoma virus RNA and of its effect

on viral protein synthesis in vitro. Nucleic Acids Res.
18:119-127 (1990)

Blockland, R. V., Lange, P., M01, J. N. Mg, and Kooter, J.
M. Modulation of gene expression in plants by antisense
genes. Antisense Reserach and applications. (ed) Crooke, S.
T. and Lebelu, B. CRC Press, Boca Raton, Florida (1993)

Boerkoel, C. P., Federspiel, Ms J., Salter, D. W., Payne, W.
Crittenden, L. B., Knng, H. J. and Hughes, S. H. A.new
defective retroviral vector system based on the Bryan strain
of Rous sarcoma virus. Virology 195:669-679 (1993)

Boiziau, C., Larrouy, B., Sproat, B. S. and Toulme, J. -J.
Antisense 2’-O-alkyl oligoribonucleotides are efficient
inhibitors of reverse transcription. Nucleic Acids Res.
23:64-71 (1995)

Bongartz, J.-P., Aubertin, Au-Mg, Millhaud, P. G., and
Lebleu, B. Improved biological activity of antisense

oligonucleotides conjugated to a fusogenic peptide. Nucleic
Acids Res. 22:4681-4688 (1994)

Bowers, W. J. and Ruddel,.A. al/EBP: a leucine zipper
protein that binds CCAAT/enhancer elements in the avian
leukosis virus long terminal repeat elements. J. Virol.
66:6578-6586 (1992)

Bowers, W. J., Baglia, L. Au and Ruddel, A. Regulation of
avian leukosis virus long terminal repeat-enhanced
transcription by C/EBP-Rel interactions. J. Virol. 70:3051-
3059 (1996)

Bova, C. A", Manfredi, J. P., and Swanstrcm, R. env genes of
avian retroviruses : nucleotide sequences and molecular
recombinants define host range determinants. Virology
152:343-354 (1986)

Bova, C..A., Olsen, J. C., and Swanstrom, R. The avian
retrovirus env gene family : molecular analysis of host
range and antigenic variants. J. Virol 62:75-83 (1993)

Brantl, S. and Wagner, E. G. H. Antisense RNA—mediated
transcriptional attenuation occurs faster than stable
antisense/target RNA pairing: an in vitro study of plasmid
pIP501. EMBO. 13:3599-3607 (1994)

147

Brown, P. 0., Bowerman, B., varmus, H. E., and Bishop, J. M;
Correct integration of retroviral DNA in vitro. Cell 49:347-
356 (1987)

Chatterjee, S., Johnson, P. R. and KK, Wong. Dual-target
inhibition of HIV-1 in vitro by means of an adeno-associated
virus antisense vector. Science 258: 1485-1488 (1992)

Chiang, M. Y., Chen, H., Zounes, M. A., Freier, S. M., Lima,
W. P., and Bennett, C. F. Antisense oligonucleotides inhibit
intercellular adhesion molecule 1 expression by two distinct
mechanisms. J. Biol. Chem. 266:18162-18171 (1991)

Cobrinik, D., Aiyar, A”, Ge, 2., Katzman, M., Huang, H., and
Leis, J. Overlapping retrovirus U5 sequence elements are

required for efficient integration and initiation of reverse
transcription. J. Virol 65:3864-3872 (1991)

Cobrinik, D., Soskey, L., and Leis, J. A.retroviral RNA
secondary structure required for efficient initiation of
reverse trascription. J. Virol 62:3622-3630 (1988)

Coffin, J. M; Structure, replication, and recombination of
retrovirus genomes: some unifying hypotheses. J. of Gen.
Virol. 42:1-26 (1979)

Coffin, J. M. Retroviridae and their replication.
Fundamental Virology, Second Edition, Fields, B. N., Knipe,
D. M. et al.(ed), Raven Press, Ltd., New York (1991)

Cornelissen, M. and'Vandewiele, M. Both RNA level and
translation efficiency are reduced by anti-sense RNA in
transgenic tobacco. Nucleic. Acids. Res. 17: 833-43 (1989)

Crittenden, L. B. New approaches to genetic resistance in
poultry. Arch. Geflugelk. 57:59-68 (1993)

Crittenden, L. B. Retroviral elements in the genome of the
chicken: implications for poultry genetics and breeding.
Crit. Rev. Poultry Biol. 30:73-109 (1991).

Fadly, A" M. Differential diagnosis of lymphoid leukosis.
Avian Leukosis. G. F. Boer (ed), Martinus Nijhoff
Publishing, Boston, Massachusetts (1986)

Fadly,.Am‘M. Leukosis and reticuloendotheliosis. Veterinary
Diagnostic Virology, Mosby Year Book (1992)

148

Federspiel, M. J., Bates, P., Young, J. A", Varmus, H. 8.,
and Hughes, S. H. A system for tissue-specific gene
targeting: transgenic mice susceptible to subgroup A avian
leukosis virus-based retroviral vectors. Proc. Natl. Acad.
Sci. USA 91: 11241-11251 (1994)

Federspiel, M; J. and Hughes, S. H. Effects of gag region on
genome stability: avian retroviral vectors that contain
sequences form the Bryan strain of RSV. Virology 203:211-220
(1994)

Fekete, D. M; and Cepko, C. L. Replication-competent
retroviral vectors encoding alkaline phosphatase reveal
restriction of viral gene expression/transduction in the
chick embryo. Molecular and Cellular Biology 13:2604-2613
(1993)

Fujiwara, T. and Mizuuchi, K. Retroviral DNA integration:
structure and integration intermediate. Cell 54:497-504
(1988)

FUjita, D. J., Chen, Y. C., Friis, R. R., and‘VOgt, P. K.
RNA tumor viruses of pheasants: characterization of avian
leukosis subgroups F and G. virology 60:558-571 (1974)

Gabrielsen, O. S., and Sentenac,.A. RNA polymerase III (C)
and its transcription factors. Trends Biochem. Sci. 18:255-
259 (1992)

Gagnor,C., Bertrand, J. R., Thenet, S., Lemaitre, ML,
Mbrvan, P., Rayner, B.,IMalvy, C., Lebleu, B., Imbach, J.L.
and Paoletti, C., alpha-DNA VI: Comparative study of alpha-
and beta-anomeric oligodeoxyribonucleotides in hybridization
to mRNA and in cell free translation inhibition. Nucleic.
Acids. Res. 15: 10419-10436 (1987)

Geselowitz, D..A. and Neckers, L. M; Analysis of
oligonucleotide binding, internalization, and intracellular
trafficking utilizing a novel radiolabeled crosslinker.
Antisense Research and Development 2:17—25 (1992)

Gilbert, J. M., Hernandez, L. D., Balliet, J.‘W., Bates, P.
and White, J; M; Receptor-mediated conformational changes in

the subgroup A avian leukosis-sarcoma virus envelope
glycoprotein. J. Virol 69:7410-7415 (1995)

Gopinathan, K. P., weymouth, L. A", Knnkel, T..A. and Loeb,
L. A" Mutagenesis in vitro by DNA polymerase from an RNA
tumour virus. 278:857-859 (1979)

149

Graessmann, Mg, Michaels, G., Berg, 8., and Graessmann, A.
Inhibition of SV40 gene expression by microinjected small

antisense RNA and DNA molecules. Nucleic. Acids. Res. 19:

53-58 (1991)

Greenhouse, J. J., Petropoulos, C. J., Crittenden, L. B.,
and Hughes, S. H. Helper-independent retroviral vectors with
Rous-associated virus type 0 long terminal repeats.

J. Virol. 62:4809-4812 (1988)

Habel, D. E., Dohrer, K. L., and Conklin, K. F. Functional
and defective components of avian endogenous virus long

terminal repeat enhancer sequences. J. Virol. 67: 1545-1554
(1993)

Hackett, P. B., Dalton, M. W., Johnson, D. P. and Peterson,
R. B. Phylogenetic and physical analysis of the 5' leader

RNA sequences of avian retroviruses. Nucleic Acids Res.
19:6929-6934 (1991)

Hajjar, A. M. and Linial, M. L. A model system for
nonhomologous recombination between retroviral and cellular
RNA. J. Virol 67:3845-3853 (1993)

Han, L., Yun, J. S. and Wagner, T. E. Inhibition of Moloney
murine leukemia virus-induced leukemia in transgenic mice
expressing antisense RNA complementary to the retroviral
packaging sequence. Proc. Natl. Acad. Sci. USA 88:4313-4317
(1991)

Hughes, S. and Kbsik, E., Mutagenesis of the region between
env and src of the SR-A strain of rous sarcoma virus for the
purpose of constructing helper-independent vectors. Virology
136:89-99 (1984)

Hughes, S.H., Kbsik, E., Fadly,.Au Mg, Salter, D. W. and
Crittenden, L. B. Design of retroviral vectors for the

insertion of foreign deoxyribonucleic acid sequences into
the avian germ line. Poultry Science 65: 1459-1467 (1986)

Ishizaki, R. and‘VOgt, P. K. Immunological relationships

among envelope antigens of avian tumor viruses. Virology
30:375-387 (1966)

Izant, J. G. and Weintraub, H. Constitutive and conditional
suppression of exogenous and endogenous genes by anti-sense
RNA. Science 229: 345-352 (1985)

150

Jacks, T., Madhani, H. D., Masiarz, F. R. and varmus, H. E.
Signals for ribosomal frameshifting in the Rous sarcoma
virus gag-pol region. Cell 55:447-458 (1988)

Jacks, T. and'Varmus, H. E. Expression of the rous sarcoma
virus pol gene by ribosomal frameshifting. Science 230:1237—
1242 (1985)

Khaled, Z., Benimetskaya, L., Zeltser, R., Khan, T., Sharma,
H. W., Narayanan, R. and Stein, C. A” Multiple mechanisms
may contribute to the cellular anti-adhesive effects of
phophorothioate oligodeoxynucleotides. Nucleic Acids Res.
24:737-745 (1996)

Knight, J. 8., Si, 2. H. and Stoltzfus, Mg .A base-paired

structure in the Avian Sarcoma Virus 5' leader is required
for efficient encapsidation of RNA. J. Virol 68:4493-4502
(1993)

Kbschel, K., Brinckmann, U. and Hoyningen-Huene, V3 V3
Measles virus antisense sequences specifically cure cells
persistently infected with measles virus. Virology 207:168-
178 (1995)

Leamson, R. N. and Halpern, M; S. Subunit structure of the
glycoprotein complex of avian tumor virus. J. Virol 18:956-
968 (1976)

Lemaitre, Mg, Bayard, B. and Lebleu, B. Specific antiviral
activity of poly(L-lysine)-conjugated with
oligodeoxyribonucleotide sequence complementary to vesicular
stomatitis virus N protein mRNA initiation site. Proc.
Natl. Acad. Sci. USA 84:648-652 (1987)

Lescure, P., Blumenfeld, M., Thill, G.,‘Vasseur, M; and
Tanner, N. K. Trans cleavage of RNA substrates by an HDV-
derived ribozyme. Prog. Clin. Biol. Res. 382:99-108 (1993)

Lewin, B. Translation: expressing genes as proteins. Genes V
Oxford University Press and Cell Press Inc. New York (1994)

Linial, M. and.Miller,.A~ D. Retroviral RNA packaging:
sequence requirements and implications. Curr. Top.
Microbiol. 157:125-152 (1990)

Lisziewicz, J., Sun, D., weichold, F. P., Theirry,.A. R.,
Lusso, P., Tang, J., Gallo, R. C. and Agrawal, S. Antisense
oligodeoxynuleotide phosphorothioate complementary to Gag
mRNA blocks replication of human immunodeficiency virus type

151

l in human peripheral blood cells. Proc. Natl. Acad. Sci.
USA 91:7942-7946 (1994)

Liu, z. and Carmichael, G. G. Nuclear antisense RNA. An
efficient new method to inhibit gene expression. Mol.
Biotechnol. 2: 107-118 (1994)

Loke, S. L., Stein, C. A", Zhang, X. H., Mbri, K.,
Nakanishi, Mg, Subasinghe, C., Cohen, J. S., and Neckers, L.
M. Characterization of oligonucleotide transport into living
cells. Proc. Natl. Acad. Sci. USA 86: 3474-348 (1989)

'Maitra, R. K., Li, G., Xiao, W. Dong, B. Terrence, P. F. and
Silverman, R. H. Catalytic cleavage of an RNA target by 2-5A
antisense and RNase L. J. Biol. Chem. 270:15071-15705 (1995)

Maran, A., Maitra, R. K., Kumar, A., Dong, B., Xiao, W. Li,
G., Williams, B. R., Terrence, P. P., and Silverman, R.H.
Blockage of NF-kappa B signaling by selective ablation of an
mRNA target by 2-5A antisense chimeras. Science 265:789-792
(1994)

Matsukura, M., Shinozuka, K., Zon, G., Miysuya, H., reitz,
M., Cohen, J. S. and Broder, S. Phophorothioate analogs of
oligodeoxynucleotides: inhibitors of replication and
cytopathic effects of human immunodeficiency virus. Proc.
Natl. Acad. Sci. USA 84:7706-7710 (1987)

McKay, R. A., Cummins, L. L., Graham, M. J., Lesnik, E. A.,
Owens, 8. R., Winniman, M. and Dean, N. M. Enhanced
activity of an antisense oligonucleotide targeting murine
protein kinase C-a by the incorporation of 2'-O-propyl
modifications. Nucleic Acids Res. 24:411-417 (1996)

iMirabelli, C. K. and Crooke, S. T. Antisense
oligonucleotides in the context of modern drug discovery and
development. Antisense research and applications. Crooke, S.
T. and Lebleu, B. (ed). CRC Press, Inc. Boca Raton, Florida
(1993)

Mishira, R. K., Mbreau, C., Ramazeilles, C., Mbreau, S.,
Bonnet, J. and Touleme, J.-J. Improved leishmanicidal
effect of phophrothioate antisense oligonucleotides by LDL-
mediated delivery. Biochimica et biophysica Acta. 1264:229-
237 (1995)

‘Mizutani, S. and Temin, H. M; Incorporation of
noncomplementary deoxyribonucleotides at high frequencies by

152

ribodeoxyvirus DNA polymerase and escherichia coli DNA
polymerase I. Biochemistry 15:1510-1516 (1976)

Mboslehner, K., Karls, U. and Harbers, K. Retroviral
integration sites in transgenic Mov mice frequently map in
the vicinity of transcribed DNA regions. J.‘Virol. 64:3056-
3058 (1990)

‘Murphy, J. E. and Goff, S. P. Construction and analysis of
deletion mutations in the U5 region of Moloney-murine
leukemia virus: effects on RNA packaging and reverse
transcription. J. Virol 63:319—327

Nielsen, P. B., Egholm, M., Berg, R. H., and Burchardt, O.
Sequence-specific recognition of DNA by strand displacement
with a thymidine-substituted polyamide. Science 254:1497-
1500 (1991)

Offensperger, W.-B., Offensperger, 8., Waiter, B., Teubner,
K., Igloi, G., Blum, H. E. and Gerok, W. In vivo inhibition
of duck hepatitis B virus replication and gene expression by
phophorothioate modified antisense oligodeoxynucleotides.
EMBO. 12:1257—1262 (1993)

Parent, L. J., Wilson, C. B., Resh, M. D. and Wills, J. W.
Evidence for a second function of the MA sequence in the
Rous sarcoma virus Gag protein. J. Virol 70:1016-1026 (1996)

Petropoulos, C. J., Payne, W., Salter, W. and Hughes, 8. H.
Using avian retroviral vectors for gene transfer. J. Virol
66:3391-3397 (1992)

Pryciak, P. M5,.Muller, H. P., and‘Varmus, H. E. Simian
virus 40 minichromosomes as targets for retroviral
integration in vivo. Proc. Natl. Acad. Sci. USA 89:9237-9241
(1992)

Raviprakash, K., Liu, Kg, Matteucci, Mg, wagner, R.
Riffenburgh, R., and Carl, M. Inhibition of dengue virus by
novel, modified antisense oligonucleotides. J. Virol. 69:69-
74 (1995)

Ronemus, M. J., Galbiati, Mg, Ticknor, C., Chen, J., and
Dellaporta, S. L. Demethylation-induced developmental
pleiotropy in Arabidopsis. Science 273:654-656 (1996)

Ryden, T. A. and Beemon, K. Avian retroviral long terminal
repeats bind CCAAT/enhancer-binding protein. Mol. Cell.
Biol. 9:1155-1164 (1989)

153

Sburlati, A” R,, Manrew, R. E. and Berger, S. L.

Prothymosin a antisense oligomers inhibit myeloma cell
division. Proc. Nat. Acad. Sci. USA 88:253-257 (1991)

Scherczinger, C. A. and Knecht, D. A. Systematic analysis of
antisense RNA inhibition of myosin II heavy-chain gene
expression in Dictyostelium discoideum. Antisense Res. Dev.
3: 191-205 (1993)

Schroder, H. C., Kelve, M. and Muller, W. E. The 2-5A system
and HIV infection. Prog. Mol. Subcell. Biol. 14:176—197
(1994)

Sczakiel, G. and Pawlita, M; Inhibition of human
immunodeficiency virus type 1 replication in human T cells
stably expressing antisense RNA. J. Virol 65:468-472 (1991)

Shih, C. and Coffin, J. Highly preferred targets for
retrovirus integration. Cell 53:531-537 (1988)

Siemering, K. R., Praszikier, J., and Pittard, A. J.
Mechanism of binding of the antisense and target RNAS
involved in the regulation of IncB plasmid replication. J.
of Bacteriology 176:2677-2688 (1994)

Simmons, R. W. The control of prokaryotic and eukaryotic
gene expression by naturally occuring antisense RNA. Crooke
(ed), Antisense Research and Applications. CRC press, Inc.,
Boca Raton (1993)

Smith, C. D., Baglia, L..A., Curristin, S. M., and Ruddell,
A" The VBP and al/EBP leucine zipper factors bind

overlapping subsets of avian retroviral long terminal repeat
CCAAT/enhancer elements. J. Virol 68:6232-6242 (1994)

Smith, E. J. and Fadly,.Au M. Influence of congenital
transmission of endogenous virus-21 on the immune response
to avian leukosis virus infection and the incidence of
tumors in chickens. Poultry Science 67:1674-1679 (1988)

Serge, J. B. and Hughes, 8., The splicing of intervening
sequences introduced into an infectious retroviral vector.
J. Mol. Appl. Genet. 1:547-599 (1982)

Serge, J., Ricci, B., and Hughes, 8., cis-acting RNA
packaging locus in the 115-nucleotide direct repeat of Rous
sarcoma virus. J. Virol 48:667-675 (1983)

154

Standifer, K. M., Jenab, 8., Su, W., Chien, C. C., Pan, Y.-
X., Inturrisi, C. E. and Pasternak, G. W. Antisense

oligodeoxynucleotides to the cloned 8 receptor DOR-l:
uptake, stability, and regulation of gene expression. J. of
neurochemistry 65:1981-1987 (1995)

Stein, B. S., Gewda, S. D., Lifsen, J. D., Pennhallew, R.
C., Bensch, K. G. and Engelman, E. G. pH-independent HIV

entry into CD4-positive T cells via virus envelope fusion to
the plasma membrane. Cell 49:659-669 (1987)

Stephenson, M; L. and Zamecnik, P. C. Inhibition of Rous
sarcoma Viral RNA translation by a specific
oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA
75:285-288 (1978)

Sullenger, B. A" and Cech, T. R. Tethering ribozymes to a
retroviral packaging signal for destruction of viral RNA.
Science 262:1566-1569 (1993)

Sullenger, B..A., Lee, T. C., Smith, C. At, Ungers, G. E.
and Gilbea, E. Expression of chimeric tRNA-driven antisense
transcripts renders NIH 3T3 cells highly resistant to
Moloney murine leukemia virus replication. Molecular and
Cellular Biology 10:6512-6523 (1990)

Sun, L. Q., Wang, L., Gerlach, W. L. and Symends, G. Target
sequence-specific inhibition of HIV-1 replicaton by
ribozymes directed to tat RNA. Nucleic Acids Res. 23:2909-
2913 (1995)

Svinarchuk, P., Debin, A”, Bertrand, J.-R. and Malvy, C.
Investigation of the intracellular stability and formation
of a triple helix formed with a short purine oligonucleotie
targeted to the murine c-pim-l proto-oncogene promoter.
Nucleic Acids Res. 24:295-302 (1996)

Swain, A” and Coffin, J. Mechanism of transduction by
retroviruses. Science 255:841-845 (1992)

Thierry,.A. and Dritschilo,.A. Intracellular availability
of unmodified, phosphorothioated and liposomally
encapsulated oligodeoxynecleotides for antisense activity.
Nucleic Acids Res. 21:5691-5698 (1992)

Temizawa, J. Control of ColEl plasmid replication: binding
of RNA I to RNA II and inhibition of primer formation. Cell
47: 89-97 (1986)

155

‘Vandendriessche, T. Chuah, M. K., Chiang, L., Chang, H. K.,
Enseli, B., and Morgan, R. A. Inhibition of clinical human
immunodeficiency virus (HIV) type 1 isolates in primary CD4+
T lymphocytes by retroviral vectors expressing anti-HIV
genes. J. Virol. 69:4045-4052 (1995)

‘Vickers, T., Baker, B. P., Cook, P. D., Zeunes, Mg,
Buckheit, R. W. Jr., Germany, J. and Ecker, D. J.

Inhibition of HIV—LTR gene expression by oligonucleotides
targeted to the TAR element. Nucleic Acids Res. 19:3359-3368
(1991)

‘Vegt, P. K. and Ishizaki, R. Reciprocal patterns of genetic
resistance to avian tumor viruses in two lines of chickens.
Virology 26:664-672 (1965)

vegt, P. K. and Ishizaki, R. Patterns of viral interference
in the avian leukosis and sarcoma complex. Virology 30:368-
374 (1966)

Wagner, R. W. The state of the art in antisense research.
Nature Medicine 1:1116-1118 (1995)

Walder, R.Y. and welder, J. A. Role of RNase H in hybrid-
arrested translation by antisense oligonucleotides. Proc.
Natl. Acad. Sci. USA 85:5011—5015 (1988)

Weiss, R., Teich, N., varmus, H., and Coffin, J. RNA
Tumor Viruses, second edition, CSH laboratory, Cold Spring
Harbor, New York (1982)

Westermann, P., Gross, B., and Heinkis, G. Inhibition of
expression of AV40 virus large T-antigen by antisense
oligodeoxyribunucleotides. Biomed. Biochim..Acta. 48:85-93
(1989)

Young, J..A., Bates, P. and varmus, H. E. Isolation of a
chicken gene that confers susceptibility to infection by
subgroup A.avian leukosis and sarcoma virus. J. Virol
67:1811-1817 (1993)

Zamecnik, P. C., Goodchild, J., Taguchi, Y. and Sarin, P. S.
Inhibition of replication and expression of human T-cell
lymphotropic virus type III in cultured cells by exogenous
synthetic oligonucleotides complementary to viral RNA. Proc.
Natl. Acad. Sci. USA 83:4143—4146 (1986)

Zamecnik, P.C. and Stephenson, M. L. Inhibition of Rous
sarcoma virus replication and cell transformation by a

156

specific oligodeoxynucleotide. Proc. Natl. Acad. Sci.USA 75:
280-284 (1978)

Zaug,.A. J., Been, M. D., and Cech, T. R. The Tetrahymena
ribozyme acts like an RNA restriction endonuclease. Nature
324:429-433 (1986)

Zhang, J. and Temin, H. M; Retrovirus recombination depends
on the length of sequence identity and is not error prone.
J. Virol. 68:2409-2414 (1994)

Zhang, J. and Temin, H. Retrovirus recombination depends on
the length of sequence identity and is not error prone. J.
Virol 68:2409-2414 (1994)

Zhang, J. and Temin, H. E. 3' junctions of oncogene-virus
sequences and the mechanism for formation of highly
oncogenic retroviruses. J. Virol 67:1747-1751 (1993)

Zhang, J. and Temin, H. M. Rate and mechanism of
nonhomologous recombination during a single cycle of
retroviral replication. Science 259:234-238 (1993)

Zhae, Q., Matsen, A., Herrera, C. J., Fisher, E., Y. H. and
Krieg, AWZM. Comparison of cellular binding and uptake of
antisense phophodiester, phophorothioate, and mixed
phophorothioate and methylphosphonate oligonucleotides.
Antisense research and development 3:53-66 (1993)

157

Chapter 2.

.Arenef, R., Hajjar, A" M., and Linial,‘M. L. Avian
retroviral RNA encapsidation: Reexamination of functional 5’
RNA sequences and the role of nucleocapsid Cys-His motifs.
J. Virol 67:178-188 (1993)

.Astrin, S. M., Buss, E. G. and Hayward, W. S. Endogenous
viral genes are non-essential in the chicken. Nature(London)
282:339-341 (1979)

Bates, P., Young, J. and'Varmus, H. A receptor for subgroup
A rous sarcoma virus is related to the low density
lipoprotein receptor. Cell 74:1043-1051 (1993)

Coffin, J. M. Retroviridae and their replication.
Fundamental Virology, Second Edition, Fields, B. N., Knipe,
D. M. et al.(ed), Raven Press, Ltd., New York (1991)

Crittenden, L. B. Retroviral elements in the genome of the
chicken: implications for poultry genetics and breeding.
Crit. Rev. Poultry Biol. 3:73-109 (1991)

Crittenden, L. B. New approaches to genetic resistance in
poultry. Arch. Geflugek. 57:59-68 (1993)

Dupraz, P., Oertle, 8., Marie, C., Damay, P. and Spahr, P.
F. Point mutations in the proximal Cys-His box of Rous
sarcoma virus nucleocapsid protein. J. Virol 64:4978-4987
(1990)

Fadly, A" M; Leukosis and reticuloendotheiosis. Veterinary
Diagnostic Viroloogy, Mosby Year Book (1992)

Federspiel, M; J., Crittenden, L. B. and Hughes, S. J.
Expression of avian reticluoendotheliosis virus envelope
confers host resistance. Virology 173:167—177 (1989)

Goodchild, J., Agrawal, S., Civiera, M; P., Sarin, P. 8.,
Sun, D. and Zamecnik, P. C. Inhibition of human
immunodeficiency virus replication by antisense
oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 85:5507-
5511 (1988)

Gessen, M., Benin, A" L., and Bujard, H. Control of gene
activity in higher eukaryotic and prokaryotic cells by
prokaryotic regulatory elements. TIBS. 18:471-475 (1993)

158

Gessen, M. and Bujard, H. Tight control of gene expression
in mammalian cells by tetracycline-responsive promoters.
Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)

Gessen, M., Freundlieb, S., Bender, Gt, Muller, G., Hillen,
W., and Bujard, H. Transcriptional activation by

tetracycline in mammalian cells. Science 268:1766-1769
(1995)

Greenhouse, J. J., Petropoulos, C. J., Crittenden, L. B.,
and Hughes, S. H. Helper-independent retroviral vectors with
Rous-associated virus type 0 long terminal repeats. J. Virol
62:4809-4812 (1988)

Han, L., Yun, J. S. and Wagner, T. E. Inhibition of moloney
murine leukemia virus-induced leukemia in transgenic mice
expressing antisense RNA complementary to the retroviral
packaging sequences. Proc. Natl. Acad. Sci. USA 88:4313-4317
(1991)

Li, Y. Somatic disruption of chicken HMS-17 and HMG-l4
genes. Ph.D. Dissertaion, Michigan State University (1996)

Peng, H., Callisen, D., Li, P. and Burrel, C. Long-term
protection against HIV-1 infection conferred by tat or rev
antisense RNA was affected by the design of the retroviral
vector. Virology 220:377-389 (1996)

Sambreek, J., Fritsch, E. P. and Maniatis, T. Molecular
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989)

Sanger, P., Nieklen, S. and Ceulsen,.A. R. DNA sequencing
with chain terminating inhibitors. Proc. Natl. Acad. Sci.
74:5463-5467 (1977)

Sczakiel, G., Oppenlander, M., Rittner, K. and Pawlita, M.
Tat- and Rev-directed antisense RNA expression inhibits and
abolishes replication of human immunodeficiency virus type
1: a temporal analysis. J. Virol 66:5576—5581 (1992)

Sczakiel, G. and Pawlita, M. Inhibition of human
immunodeficiency virus type 1 replication in human T cells
stably expressing antisense RNA. J. Virol 65:468-472 (1991)

Smith, E. J., Fadly,.A. and Okazaki, W. An enzyme-linked
immunosorbent assay for detecting avian leukosis-sarcoma
viruses. Avian Dis. 23:698-707 (1979)

159

‘Vandendriessche, T., Chuah, M. K., Chiang, L., Chang, H. K,
Enseli, B. and Mergan R..A. Inhibition of clinical human
immunodeficiency virus (HIV) type 1 isolates in primary CD4+

T lymphocytes by retroviral vectors expressing anti-HIV
genes. J. Virol 69:4045-4052 (1995)

160

Chapter 3.

Agrawal, S. and Tang, J. Y. GEM91-an antisense
oligonucleotide phosphorothioate as a therapeutic agents for
AIDS. Antisense Res. Dev. 2:261-266 (1992)

Aiyar, A", Ge, 2. and Leis, J. A specific orientation of RNA
secondary structure is required for initiation of reverse
transcription. J. Virol 68:611-618 (1994)

Bennet, C. P., Chiang, M; Y., Chan, H., Shoemaker, J. E. E.
and Mirabelli, C. K. Cationic lipids enhance cellular uptake
and activity of phophorothioate antisense oligonucleotides.
Molecular pharmacology 41:1023—1033 (1992)

Bengartz, J.—P., Aubertin, An-Mg, Millhaud, P. G., and
Lebelu, B. Improved biological activity of antisense

oligonucleotides conjugated to a fusogenic peptide. Nucleic
Acids res. 22:4681-4688 (1994)

Chavany C., Cennel, Y. and Neckers, L. Contribution of
sequence and phophorothioate content to inhibition of cell
growth and adhesion caused by c-myc antisense oligomers.
Molecular Pharmacology 48:738-746 (1995)

Cewsert, L. M; Antiviral activities of antisense
oligonucleotides. Antisense Research and Applications, CRC
Press, Inc. Boca Raton, Florida (1993)

Geo, W. Y., Han, F. S., Strem, C., Egan, W. and Cheng, Y. C.
Phosphorothioate oligonucleotides are inhibitors of human
DNA polymerase and RNase H: implications for antisense
technology. Molecular Pharmacology 41:223-229 (1991)

Geselewitz, D. A" and Neckers, L. M; Analysis of
oligonucleotide binding, internalization, and intracellular
trafficking utilizing a novel radiolabeled crosslinker.
Antisense Res. Dev. 2:17-25 (1992)

Goodchild, J}, Agrawal, S., Civieira, M; P., Sun, D., and
zamecinik, P. C. Inhibition of human immunodeficiency virus

replication by antisense oligodeoxynicelotides. Proc. Natl.
Acad. Sci. USA 85:5507-5511 (1988)

Hoke, G. D., Draper, K., Freier, S. M., Gonzalez, C.,
Dricer, V} B., Zeunes, M; C., and Ecker, D. J. Effects of
phophorothioate capping on antisense oligonucleotide
stability, hybridization and antiviral efficacy versus

161

herpes simplex virus infection. Nucleic Acids Res. 19:5743-
5748 (1991)

Hughes, S. and Kesik, E., Mutagenesis of the region between

env and src of the SR-A strain of rous sarcoma virus for the
purpose of constructing helper-independent vectors. Virology
136:89-99 (1984)

Hughes, S.H., Kesik, E., Fadly,.An M., Salter, D. W. and
Crittenden, L. B. Design of retroviral vectors for the

insertion of foreign deoxyribonucleic acid sequences into
the avian germ line. Poultry Science 65: 1459-1467 (1986)

Knight, J. B., Si, Z. H. and Stoltzfus, M; A base-paired

structure in the Avian Sarcoma Virus 5' leader is required
for efficient encapsidation of RNA. J. Virol 68:4493-4502
(1993)

Lemaitre, M., Bayard, B. and Lebleu, B. Specific antiviral
activity of poly(L-lysine)-conjugated with
oligodeoxyribonucleotide sequence complementary to vesicular
stomatitis virus N protein mRNA initiation site. Proc.
Natl. Acad. Sci. USA 84:648-652 (1987)

Lisziewicz, J., Sun, D., Wiecheld, F. P., Thierry,.A.R.,
Lusse, P., Tabg, J., Gallo, R. C., and Agrawal, S. Antisense
oligodeoxynucleotide phophorothioate complementary to Gag
mRNA blocks replication of human immunodeficiency virus type
1 in human peripheral blood cells. Proc. Natl. Acad. Sci.
USA 91:7942-7946 (1994)

Matsukura, M., Shinezuka, K., Zen, G., Mitsuya, H., Reitz,
IM., Cohen, J. S., and Breder, S. Phophorothioate analogs of
olgodeoxynucleotides: Inhibitors of replication and
cytopathic effects of human immunodeficiency virus. Proc.
Natl. Acad. Sci. USA 84:7706-7710 (1987)

Mushira, R. K., Mbreau, C., Ramazeilles, C., Mbreau, S.,
Bennet, J. and Teuleme, J.-J. Improved leishmanicidal
effect of phophrothioate antisense oligonucleotides by LDL-
mediated delivery. Biochimica et biophysica Acta. 1264:229-
237 (1995)

Peyman, A”, Ryte,.A., Helsberg, M., Kretzscher, G., and
Uhlmann, E. Enhanced cellular uptake of G-rich

oligonucleotides. Nucleosides & Nucleotides 14:1077-1081
(1995)

162

Satndifer, K. M., Jenab, 8., Su, W., Chien, C. C., Pan, Y.
K., Inturrisi, C. E. and Pasternak, G. W. Antisense

oligodeoxynucleotides to the cloned 6 receptor DOR-l:
Uptake, stability, and regulation of gene expression. J.
Neurochemistry 65:1981-1987 (1995)

Stein, C. A” Does antisense exist? Nature Medicine 1: 1119-
1121 (1995)

Stein, G. A”, Clearyy A" Mg, Yakubev, L. and Lederman, S.
Phophorothioate oligodeoxynucleotides bind to the third
variable loop domain (v3) of human immunodeficiency virus
Type 1 gp120. Antisense Res. Develop. 3:19-31 (1993)

Stephenson, M; L. and Zamecnik, P. C. Inhibition of Rous
sarcoma viral RNA translation by a specific
oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA
75:285-288 (1978)

Wagner, R. W. The state of the art in antisense research.
Nature Medicine 1:1116-1118 (1995)

Yakubev, L. et al., Oligodeoxynucleotides interact with
recombinant CD4 at multiple sites. J. Biol. Chem. 268:
18818-18823 (1993)

Zamecnik, P.C. and Stephenson, M; L. Inhibition of Rous
sarcoma virus replication and cell transformation by a
specific oligodeoxynucleotide. Proc. Natl..Acad. Sci.USA 75:
280—284 (1978)

163

Chapter 4.

.Astrin, S. M., Buss, E. G., and Hayward, W. S. Endogenous
viral genes are non-essential in the chicken. Nature
282:339-341 (1979)

Bates, P., Young, J. A. T., and'Varmus, H. E. A receptor for
subgroup A rous sarcoma virus is related to the low density
lipoprotein receptor. Cell 74:1043-1051 (1993)

Berger, J., Heward,.A. D., Gerber, L., Cullen, B. R., and
Udenfriend, S. Expression of active, membrane-bound human

placental alkaline phosphatase by transfected simian cells.
Proc. Natl. Acad. Sci. USA 84:4885-4889 (1987)

Coffin, J. M. Retroviridae and their replication.
Fundamental Virology: Second Edition, Fields, B. N., Knipe,
D. M. et al.(ed), Raven Press, Ltd., New York (1991)

Cennely, L., Zingler, K. and Young, J..A..A soluble form of
a receptor for subgroup A avian leukosis and sarcoma viruses
(ALSV-A) blocks infection and binds directly to ASLV. J.
Virol 68:2760-2764 (1994)

Crittenden, L. B. Retroviral elements in the genome of the
chicken: implications for poultry genetics and breeding.
Crit. Rev. Poultry Biol. 30:73-109 (1991).

Federspiel, Mu J., Bates, P., Young, J. A. T., varmus, H. E.
and Hughes, S. H. A.system for tissue-specific gene
targeting: transgenic mice susceptible to subgroup A avian
leukosis sarcoma virus-based retroviral vectors. Proc. Natl.
Acad. Sci. USA 91:11241-11245 (1994)

Friis, R. R. Abortive infection of Japanese quail cells with
avian sarcoma viruses. Virology 50:701-712 (1972)

Gilbert, J. Mg, Bates, P., varmus, H. E., and White, J. M.
The receptor for the subgroup A avian leukosis-sarcoma
viruses binds to subgroup A but not subgroup C envelope
glycoprotein. J. Virol 68:5623—5628 (1994)

Gessen, M; and Bujard, H. Tight control of gene expression
in mammalian cells by tetracycline-responsive promoters.
Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)

Mescevici, C., Mescevici, M. G., Jimenez, H., Lei, M. M.,
Hayman, M; J., and vegt, P. K. Continuous tissue culture

164

cell lines derived from chemically induced tumors of
Japanese quail. Cell 11:95—103 (1977)

Reng, L. and Bates, P. Analysis of the subgroup A avian
sarcoma and leukosis virus receptor: the 40-residue,
Cysteine-rich, low-density lipoprotein receptor repeat motif
of Tva is sufficient to mediate viral entry. J. Virol 4847-
4853 (1995)

Sambreek, J., Fritsch, E. F. and Maniatis, T. Molecular
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY (1989)

Sanger, F., Nicklen, S. and Ceulsen, A” R. DNA sequencing
with chain terminating inhibitors. Proc. Natl. Acad. Sci.
74:5463-5467 (1977)

‘Valsesia-Wittmann, S., Drynda, A”, Deleage, G., Aumailley,
2M., Heard, J.-M;, Danes, 0.,‘Verdier, G., and Cesset, F.-L.
Modifications in the binding domain of avian retrovirus
envelope protein to redirect the host range of retroviral
vectors. J. Virol 68:4609-4619 (1994)

Zingler, K., Belanger, C., Peters, R., Agard, D. and Young,
J..A. T. Identification and characterization of the viral
interaction determinant of the subgroup A avian leukosis
virus receptor. J. Virol 69:4261-4266 (1995)

"Iiriiiiiiliiiiii"111i“