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LIBRARY

Mm§mm

 

This is to certify that the
thesis entitled

ANALYSIS OF RIBONUCLEIC ACID FROM
FELINE LEUKEMIA VIRUS

presented by
DAVID ALVIN BRIAN
has been accepted towards fulfillment

of the requirements for

Ph.D. dpgrep- 1n Microbiology and
Public Health

MW

Major professor

 

 

0-7 639

 

 

 

 

ABSTRACT

ANALYSIS OF RIBONUCLEIC ACID FROM
FELINE LEUKEMIA VIRUS

BY

David A. Brian

The Rickard strain of feline leukemia virus (FeLV-R) is con-
tinuously produced by the permanently infected feline thymus tumor
cell line F-422. Attempts to synchronize F-422 cells using the methods
of thymidine excess and serum-free medium were unsuccessful.

FeLV—R (density of 1.14 g/cc) concentrated by salt precipita—
tion, polyethylene glycol precipitation, or pelleting and purified by
isopycnic centrifugation appears to contain particulate (possibly
vesicular) material as a contaminant derived from the host cell. This
is suggested by electronmicroscopy of purified virus, velocity
sedimentation studies and by RNA analysis of particles resolved by
velocity sedimentation. FeLV-R can be purified from the contaminant
by rate zonal centrifugation or by rapid pelleting through a 20% w/w
sucrose barrier. Purified FeLV—R can infect the Crandell-CCC feline
kidney cell line and produce progeny virus with a density of 1.14 g/cc.

By labeling F-422 cells with a continuous source of 3Huridine
and quantitating the radioactivity in purified virus, the minimum

interval between the synthesis of viral RNA and its release into virus

 

 

 

David A. Brian

was measured to be 30 min. or less. By labeling F—422 cells with a
15 min. pulse, the average interval between the synthesis of viral RNA
and its release into virus was measured to be between 4 and S h.

RNA from FeLV-R was resolved into three size classes, 50 to 608
(comprising 50 to 73% of the total), 88 (comprising 3 to 7% of the
total), and 4 to 58 (comprising 7 to 21% of the total) when analyzed by
electrophoresis on 2.0% polyacrylamide -0.5% agarose gels. The 50 to
605 RNA from virus harvested after 4 h. of labeling electrophoretically

migrated faster and sedimented more slowly than the same RNA harvested

 

after 20 h. of labeling. This argues for an intravirion modification
of the high molecular weight RNA. The high molecular weight subunits
from dissociated 50-608 RNA cosedimented with 28S ribosomal RNA when
analyzed by velocity sedimentation through 99% dimethylsulfoxide
(DMSO) giving them an estimated molecular weight of 1.8 x 106 daltons.

Aggregates intermediate between 50 to 608 (s = 7.09) and 28S

25,DMSO
(525 DMSO = 4.22) suggest that conditions other than 99% DMSO alone are

needed for complete dissociation of the RNA. The 88 RNA in FeLV has

not previously been reported.

 

ANALYSIS OF RIBONUCLEIC ACID FROM

FELINE LEUKEMIA VIRUS
By

. \*‘ .
Dav1d A. Brian

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Microbiology and Public Health

1974

 

 

DEDICATION

To Donna, Matt, and Molly

 

 

ii

ACKNOWLEDGMENTS

I wish to express my appreciation to Dr. Leland F. Velicer for
his guidance and support during my dissertation research. I wish
also to express sincere appreciation to Dr. Fritz M. Rottman and

Mr. Arlan R. Thomason for many hours of discussion, and for instructing

 

me during my entrance into the field of ribonucleic acid chemistry.
I am grateful for National Institutes of Health Traineeship

support which extended through the full course of my Ph.D. training.

 

iii

TABLE OF CONTENTS

LIST OF TABLES .

LIST OF FIGURES.

LIST OF ABBREVIATIONS.

INTRODUCTION.

LITERATURE REVIEW .

1.

Chemical Composition of RNA Tumor Viruses

Lipids.
Proteins .
Nucleic Acids

RNA of RNA Tumor Viruses.

Native 48 RNA .

Seventy 8- associated 4S RNA.

Native SS RNA . .

Seventy 8— associated SS RNA.

Native 78 (8S) RNA. . . . . . . . .

Seventy 8- associated 78 (8S) RNA . . . . . .

Native 18S and 28S RNA . . .

Seventy 8- associated 18S and 28S RNA.

Native SSS RNA . . . .

Seventy 8- associated SSS RNA

Seventy 8 RNA and Its Subunits.

Poly(A) . . . . . . .

Methylation . .

Summary of Questions on the High Molecular Weight
Subunits . . . . . . . . . . .

Replication .

Kinetics of Labeling .
Synchronization.

iv

Page
viii
ix

xi

10
10
10
11
11
11
11
11
11
16
17

18
19

19
21

 

Page

MATERIALS AND METHODS

 

PART I
Source of Cells and Virus . . . . . . . . . . . . 23
Determining Viable Cell Numbers . . . . . . . 23
Electronmicroscopy of F- 422 Cells and FeLV- R. . . . . . 23
Embedding. . . . . . . . . . . . . . . . . 23
Negative Staining . . . . . . . . . . . . . . 24
Synchronization of F-422 Cells . . . . . . . . . . 24
Thymidine Excess . . . . . . . . . . . . . . 24
Serum—Free Medium . . . . . . . . . . . . . . 2S
BSA Gradients . . . . . . . . . . . . . . 25
Isoleucine— Free Medium . . . . . . . . . . . . 25
Isotopic Labeling of FeLV-R . . . . . . . . . . . 27
Purification of FeLV- R . . . . . . . . . . . . . 27
Radioactivity Assay . . . . . . . . . . . . . 29
Infecting Crandell- CCC Cells . . . . . . . . . . . 29
Cordycepin Treatment of F- 422 Cells. . . . . . . . . 30
PART II
Source of Cells and Viruses . . . . . . . 108
Adaptation of F- 422 Cells for Monolayer Growth . . . . . 108
Isotopic Labeling of FeLV- R RNA . . . . . . . . . . 108
Purification of FeLV-R . . . . . . . . . . . . . 108
RNA Extraction. . . . . . . . . 109
Preparation of Cytoplasmic RNA Markers. . . . . . . . 110
Preparation of NDV RNA . . . . . . . . . . 110
Polyacrylamide- Agarose Gel Electrophoresis . . . . . 111
Velocity Sedimentation of RNA Through Aqueous Sucrose
Gradients. . . . . 112
Velocity Sedimentation of RNA Through Dimethylsulfoxide . . 112
01igo(dT) Cellulose Chromatography . . . . . . . . . 112
Radioactivity Assay . . . . . . . . . . . . 113
RESULTS
PART I
Electronmicrographs of F-422 Cells and FeLV-R . . . . . 31
Synchronization of F-422 Cells . . . . . . . . . . 31
Thymidine Excess . . . . . . . . . . . . . . 31
Serum-Free Medium . . . . . . . . . . . . . . 42

Page

 

Isoleucine-Free Medium . . . . . . . . . . . . . 47
BSA Gradients . . . . . . . . . . . . . . . . 47
Purification of FeLV- R. . . . . . . . . . . . . 48
Infection of Crandell- CCC Cells. . . . . . . . . . . 53
Effect of Cordycepin on Cell Viability . . . . . . . . 60
Effect of Cordycepin on Virus Production. . . . . . . . 60
Kinetics of Incorporation of Uridine into FeLV-R . . . . . 60
Continuous Labeling, Minimum Interval . . . . . . . . 63
Pulse Labeling, Average Interval. . . . . . . . . . 68
PART II
Kinetics of Viral Labeling . . . . . . 114
Gel Electrophoresis and Velocity Sedimentation of. Native
Viral RNA . . . . . . . . . . . 119
Effects of Labeling Time on Viral RNA. . . . . . 125
Velocity Sedimentation of Viral RNA Through 99°o DMSO. . . . 132
Oligo(dT) Cellulose Chromatography. . . . . . . . . . 138
DISCUSSION
PART I
Electronmicrographs of F-422 Cells and FeLV-R . . . . . . 84
Synchronization of F-422 Cells . . . . . . . . . . . 84
Thymidine Excess . . . . . . . . . . . . . . . 84
Serum-Free Medium. . . . . . . . . . . . . . . 85
Isolencine—Free Medium . . . . . . . . . . . . . 85
BSA Gradients . . . . . . . . . . . . . . . . 85
Purification of FeLV-R. . . . . . . . . . . . . . 85
Infection of Crandell-CCC Cells. . . . . . . . . . . 87
Effects of Cordycepin on Virus Production . . . . . . . 89
Kinetics of Incorporation of Uridine Into FeLV-R . . . . . 90
PART II
Kinetics of Uridine Labeling. . . . . . . . . . . 139
Size and Percentage Composition of Native FeLV- R RNAs . . . 139
Effect of Labeling Time on Viral RNA . . . . . . . . . 141
FeLV-R RNA Subunits. . . . . . . . . . . . . . . 142

vi

LIST OF REFERENCES

Part I . . .
Part II.

APPENDIX .

vii

Page

93
146

153

 

 

 

 

Table

LIST OF TABLES

Native RNA from RNA tumor viruses . . . . . . .

Subunits of 50-753 RNA from RNA tumor viruses .

PART I
Procedure for reconstituting amino acid-free and
bactopeptone-free Leibovitz-McCoys medium (GIBCO
medium number 72154) for isoleucine deficiency .

RNA content in constituents resolved by velocity
sedimentation . . . . . . . . . . . .

Behavior of exogenously added 14C uridine labeled
FeLV in tissue culture .

PART II

ElectrOphoretic migration and velocity sedimentation
of FeLV 50-608 RNA relative to NDV SOS RNA

viii

Page

26

67

. 126

 

LIST OF FIGURES

Figure Page
PART I
1. Electronmicrograph of F-422 cell . . . . . . . . 33
2. Electronmicrograph of F—422 cells and FeLV-R . . . . 35
3. Electronmicrograph of FeLV-R . . . . . . . . . 37
4. Electronmicrograph of purified FeLV-R . . . . . . 39
5. Growth curve for F-422 cells . . . . . . . . . 41
6. Growth curves for F-422 cells during and after excess
thymidine treatment. . . . . . . . . . . . 44
7. Incorporation of 3H thymidine into F-422 cells and
MOPCI-ZIF cells . . . . . . . . . . . . . 46
8. Isopycnic centrifugation of FeLV-R for varying

periods of time . . . . . . . . . . . . . SO
9. Velocity sedimentation of FeLV-R . . . . . . . . 52

10. Isopycnic centrifugation of FeLV-R obtained from
velocity sedimentation gradient. . . . . . . . 55
11. Infection of Crandell CCC cells with FeLV—R . . . . 59
12. Effects of cordycepin treatment on FeLV-R production . 62

13. Incorporation of 3H uridine into purified virions
with continuous labeling . . . . . . . . . . 65

14. Incorporation of 3H uridine into the acid soluble pool
and into RNA in F-422 cells . . . . . . . . . 7O

15. Incorporation of 3H uridine into purified virions
after pulse labeling . . . . . . . . . . . 73

ix

Figure

16.

17.

18.

19.

20.

Incorporation of 3H uridine into ‘purified virions after
pulse labeling . . . . . . . . . . . .

Incorporation of 3H uridine into purified virions after
pulse labeling . . . . . . . . . . .

Incorporation of 3H uridine into purified virions with
continuous labeling . . . . . . . .

Acid soluble pool in F—422 cells after pulse labeling.

Incorporation of 3H uridine into FeLV during pulse-chase
and continuous labeling .

PART II

3 . . . . . . .
Incorporation of H ur1d1ne into pru1f1€d Virions
during continuous labeling

Incorporation of 3H uridine into FeLV during pulse-
chase and continuous labeling . . . . . . . .

Coelectrophoresis of native FeLV RNA, feline thymus
tumor cell rRNA and tRNA, and NDV RNA on 2.0%
polyacrylamide -0.5% agarose gels.

Velocity sedimentation of FeLV RNA and NDV RNA . .

Coelectrophoresis of FeLV RNA and NDV RNA.

Coelectrophoresis of FeLV RNA and NDV RNA. . . . . .

Velocity sedimentation of FeLV RNA and murine cytoplasmic
RNA through 99% DMSO . . .

01igo(dT)-cellulose chromatography .

Page

75

77

79

81

83

116

118

121
124

129
131

135

137

BSA
CCC
DMSO
F-422
FCS
FeLV-R
HBSS

HnRNA

MLV

MMTV
mRNA
MSV—SD
NDV
oligo(dT)
PBS

PEG

Poly (A)

LIST OF ABBREVIATIONS

avian myeloblastosis virus

bovine serum albumin

Crandell feline kidney cells
dimethylsulfoxide

feline thymus tumor cells, Rickard's number
fetal calf serum

feline leukemia virus, Rickard's strain
Hank's balanced salt solution

heterogeneous nuclear RNA

methylated albumin kieselguhr

murine leukemia virus

mouse mammary tumor virus

messenger RNA

murine sarcoma virus, Soehner-Dmochowski strain
Newcastle disease virus
oligodeoxythymidilic acid

phOSphate buffered saline

polyethylene glycol

polyadenylic acid

xi

 

 

POPOP

RAV

RD-114

rRNA

SR-RSV

TCA

TNE

tRNA

UDP

UTP

l,4—bis-2-(4-methyl-S-phenyloxazolyl)-benzene
Rous associated virus

feline oncornavirus, originally thought to be of human
origin

ribosomal RNA

sedimentation coefficient

Rous sarcoma virus, Schmidt-Ruppin strain
trichloroacetic acid

tris-sodium chloride—EDTA buffer
transfer RNA

uridine diphosphate

uridine monophosphate

uridine triphosphate

xii

 

INTRODUCTION

There are several reasons why at least one approach to

oncology should be the study of RNA tumor (oncorna) viruses.

1.

Oncornaviruses are known to cause neoplasia in animals:
leukemia (37) and sarcomas (96) in chickens, leukemia (56),
sarcomas (59) and mammary adenocarcinomas (14) in mice, and
leukemias (66) and sarcomas (104) in cats.

Oncornaviruses transform cells in_vi£ro_(75, 98) and there-
fore allow biochemical analysis of the transformation process.

Mutants, both conditional (76) and non-conditional (77), for

transformation defectiveness exist enhancing the ability to
mechanistically understand transformation. While the number
of genes is prObably no more than 50 (54) the problem of
identifying the transformation gene or its product does not
seem insurmountable.

The genetic material in the virion is single-stranded RNA with
probably the same polarity (+) as intracellular messenger RNA
coding for viral structural components (9). A reasonable
approach to the study of controls functioning during virus

replication (and possibly transformation) would be to study

control mechanisms functioning during in_vi££2_translation of
virion RNA (49, 116)-

S. Oncornavirus infection (and therefore disease) can be trans-
mitted horizontally (i.e., associate to associate) (58). To
what extent oncogenesis could be controled as an infection
process should be thoroughly investigated.

6. Oncornavirus genetic material can reside in the genetic material
of the host (94) giving rise to vertical (parent to offspring)
transmission of the disease. Oncogenesis is therefore more
than just an infectious process and requires study of the
entire cellular biochemistry as well as the virus for
understanding the oncogenic process.

7. Oncornaviruses cross species barriers. While the murine
leukemia and sarcoma virsus have been adapted to grow in rat
(51) and human cells (1), feline leukemia and sarcoma viruses
can infect and cause disease in dogs (89), rabbits, marmosets
and monkeys (111) without adaptation. FeLV and FeSV also grow
very well on human cells (64) transforming them in the latter
case (98) without adaptation. The feline leukemia and sarcoma
viruses are therefore important to study both epidemiologically
and biochemically since their role in human neoplasia is an
open question (114).

The feline thymus tumor cell line F-422 (89) used in this study
has the advantages of growing rapidly and producing large quantities
of virus (3-4 mg viral protein/liter/24 h.) (52). It therefore is an

eXcellent system for studying viral replication and for producing

virus for structural studies. A disadvantage is that no uninfected
thymocyte line is available as a control cell.

The purpose of this study was three—fold: (l) to study the
effects of the cell cycle on virus replication, (2) to measure the
rate of viral RNA synthesis and incorporation into intravirion RNA,
and (3) to characterize the intravirion RNA with respect to molecular
weight and percentage composition of molecules present and to establish
preparative procedures for further analysis of the RNA.

One objective of this laboratory is to study the intracellular
origin and behavior of FeLV RNA as well as the in_vi£rg_translation of
FeLV RNA. In view of this it was imperative that a careful character-
ization of the intravirion RNA be made. At the time these studies

began one paper existed on the characteristics of FeLV RNA (65).

LITERATURE REVIEW

There are many recent reviews on the oncornaviruses (53, 57,
109, 110, 114, 120). The purpose of the following review is to focus
only on that literature which guided my experimentation and to bring

the results which I have obtained into perspective.

1. Chemical Composition of RNA Tumor Viruses

 

AMV is composed of 35% lipids, 63% protein, and around 2% RNA
(16). Based on similarities in structure, all oncornaviruses are

assumed to have this general composition.

Lipids.--Lipids are present in the form of phospholipids and
are evidently all derived from host cell origin since no unique
phospholipids are found in viruses which are not found in the membranes

of uninfected host cells (88).

Proteins.-—The structural proteins of FeLV—R have been
characterized (523- In summary these include two glycoproteins of
200,000 and 80,000 daltons mol wt, and five other proteins of 27,000,
18,000, 15,000, 12,000, and 12,000 daltons mol wt.

These totaled would translate to a single-stranded RNA gene

eQLdvalent to approximately 1.1 x 106 daltons mol wt in size.

The extent to which the carbohydrates found in the glyco-

proteins is coded for by the virus is unknown.

Nucleic acids.-—The nucleic acid in oncornaviruses is RNA.

 

Very minor amounts of DNA reported are very likely of cellular origin
(117). All RNA is single—stranded and is susceptible to digestion by

pancreatic ribonuclease.

2. RNA of RNA Tumor Viruses

 

Table 1 summarizes the analyses of native (undernatured) RNA
species extracted from several oncornaviruses. Methods of analysis
include both velocity sedimentation in neutral sucrose gradients and
gel electrophoresis.

Table 2 summarizes the analyses of denatured high molecular
weight (genomic) RNA from several oncornaviruses. Methods of analysis
include velocity sedimentaion in neutral sucrose gradients and gel
electrophoresis.

The native RNA from all oncornaviruses studied to date is
resolved for the most part into two major size classes: a 50-758 class
and a 45 class. In some cases minor amounts of RNA are seen between
these two extremes. The high molecular weight 50—758 RNA is the
putative genome, and itself is an aggregate of 3-5 smaller 28-388
molecules and some 48 molecules. What follows is a detailed review
of viral RNA. Native RNA refers to undernatured RNA as it is found
in the virion and 7OS-associated in the titles refers to that RNA
which becomes apparent after denaturing 50—708 RNA; 708 in this case
is used as a general term for the high molecular weight genomic RNA

vflrich actually ranges from 50 to 75$ depending upon the report.

.mumv vogmfifinsm Scum accuse up vouaflsoflmum

 

 

Amos «fiswvme mnsmvmm anwwvmms a snfiumwm . . «n«~ovm05 >m= Augean: mama
menu . . unwonvmoan . . . . . . mnwwoumwm .: vm mu>wom man
“vow . . a somvmofl-e . . . . . . anwmoSmom .n v ¢->moa mumfi
nemv . . unwomvmofl-e . . . . . . masoovmwm .; «N m->soa mama
fivmv . . mflsomumofi-v . . . . . . anaoovmom .; v a->qom mama
AmmV . . «fisusvmofl-v . . . . . . «Aamwumwm .g «N om->mz mama
hmmV . . a wmfivmofi-e . . . . . . anwmmvmom .gv am->mz mums
”any aflsomvmv . . . . . . . . unwomvmmo >Sz->wz mamfi
nNNC «Ashavmv . . . . . . «Aseflvmom «msmovmwo .: 4N osuaua >mm Namfi
nNNV unwofivmv . . «nwmsvmmfl unwoflvmmw «fisoflvmon anavamwo cos m osmaua >mm Nama
nNNHV . . nwwN1mmUmmfiuv . . . . . . m$~hlh©umoh >az finma
Amos . . «flasmvmofi-e . . . . «Aweflvmmn ansmnumma m camuum->soa Hams
flNHV flmmfivme fiwmuws Asevmmfi sovmwm . . hawovmon >mm-mm ohms
H>4.“
flNHS hamm-omvme Asmvms Asqummfi “sovmwm . . flack-movmow-mh no“: >mm gasp” ohm”
any . . . . . . . . . . Awmmvmcoumuv nonN.H >42 Hozumnum momfl
fines ansfimvwe . . . . . . «fismvmom amwouvmmo >24 mood
nomv . . wonuoevmoflnv . . . . . . awoouonvwah >z< mom“
“Haw “eon-omvme . . . . . . . . “wow-onumeo >m¢ easum mom“
we moH-A me mum mmn mmn-om
oocouomom m5kw> use»

 

naauou mo xv unomoum <z¢ mo mofiuomw

 

.mow:hw> Hoasu <zx scum <zm o>wu¢2uu.m omnmp

 

.mpmw wonmfifinsm Eoym accuse an vopmasofimum

 

 

 

Ammv nsmmvme . . . . smugmwm om->mz mnmfi
hmaV whsomvmv . a somvmws . . ansomvmam >Sz->m2 mums
mflsmsvm4 m woonom-oH whsmfivmom .; 4N o:mm~m->mm
fimmv «Aswmvme . . a assumwfi a smfivmmm a smmumom CHE m mammad->mm mama
mmcoofime oofixev .
ANNHV wasnvmq . . a smvmmfi a soflvmmw a smhvmwm >4: Hams
fimev nsm-fiumv . . . . mmm >z< sums
may Asofivmofi-w . . . . somvmqe-mm >3: mama
w4 mofl-a mmfl mmm mmm
oocohomom msnfl> poo»

nfimpop mo wv ucomonm <zm mo mowoomm

 

.momsnw> noESu <zm Eoym <zm mmn-om mo mpficsnsmnu.m oHan

 

Native 48 RNA.--By the criteria of elution from methylated

 

albumin kieselguhr columns, mobility during polyacrylamide gel electro-
phoresis and degree of methylation, but not by the criterion of
nucleotide composition, the 4S RNA in RSV was identical to cellular

4S RNA (12)- Bishop, et al., suggested that viral 4S RNA is cellular
4S RNA somehow carried along into the virion, and that differences in
nucleotide composition between the two reflect population differences
perhaps due to a selection process operating during incorporation into
the virion (12).

The suggestion that 48 RNA is a breakdown product from 708 RNA
(7) is negated by base compositional differences between 48 RNA and
708 RNA, and the degree of methylation (12).

A number of laboratories have demonstrated that at least some
of the 4S RNA from AMV can undergo aminoacylation and is therefore
transfer RNA (38, 40, 93). When testing for amino acid accepting
activity of 4S RNA from AMV a non-random distribution of viral tRNA
was found by comparison to the tRNA population of normal liver or
from AMV infected leukemic myeloblasts (47). This suggested a
selective incorporation. There appeared to be no selectivity for iso-
accepting tRNA species, however, when the isoaccepting species were

analyzed by reversed-phase 5 chromatography.

Seventy_S—associated 4S RNA.--4S RNA molecules were reported

 

to be associated with the 60-708 RNA molecules of SR-RSV, AMV, and
MSV—MLV which dissociate under denaturing conditions of heat, DMSO or

formamide (39, 40, 45). It represents 2.5 to 4% of the 708 RNA, is

present at the rate of 4 to 5 molecules per 358 RNA molecule and is
structurally similar to tRNA by the criterion of base composition.

Rosenthal and Zamecnik established that in AMV, the 708-
associated 4S RNA as well as free 4S RNA contained tRNA on the basis
of ability to esterify amino acids and their minor base composition
(92, 93). Since the percentage of minor base composition was lower
in the 7OS-associated species than in cellular 4S RNA, they inferred
that an additional 48 molecule was also present in association with
708 RNA.

It was suggested early that 70S-associated 4S RNA possibly
functions as a linker molecule holding 35$ subunits together (39) or
as a primer molecule for DNA synthesis by the RNA-directed DNA
polymerase (20, 107, 118). Recent studies favor the hypothesis that
some, but probably not all, of the associated 4S RNA functions as a
primer (23). Canaani and Duesberg found that the Tm of 60-705 RNA in
RSV dissociating into subunits of 35S and 48 was 55°C, whereas the Tm
for loss of reverse transcriptase activity (due to loss of primer) was
70°C (40). They concluded from this that most of the associated 4S RNA
does not have primer activity, but that the primer is a small molecule
less than 108 in size. Dahlberg, Faras, and others have identified
the primer molecule for DNA transcription from 708 RSV RNA to be a 48
molecule approximately 75 nucleotides long, having pG at the 5'
terminus and CPCPAOH at the 3' terminus (23, 45). All primer molecules
had a common identity with respect to base composition, and as a class

differed in base composition from other tRNAs.

 

'\

10

Native 5S RNA.--Free SS RNA was reported in MSV (38, 68) but

 

no function was proposed for it.

Seventy_S-Associated SS RNA.--A SS RNA is associated with the

 

70S RNA of RSV. It is 120 nucleotides in length, constitutes 1% of the
708 RNA and is present at the rate of 3 to 4 molecules per 708 RNA
molecule. It has a nucleotide composition and structure identical to
the SS RNA associated with the large ribosomal subunit from uninfected
chicken cells. Since it melts from the 70S complex at the same
temperature required to dissociate 7OS RNA into 358 subunits it is

presumed to play a role in linking the 358 subunits together (45).

Native 78 (8S) RNA
Bishop, et al., first reported a 78 RNA species in RSV (13)

which was resolved using MAK chromatography. It is single-stranded
with a molecular weight of 80,000 daltons, constitutes 3-5% of the
total viral RNA, and is present at the rate of 5-10 molecules per
virion. It is not methylated and does not hybridize to 60-7OS RSV
genomic RNA. Its function is unknown. A species like it was not
found in cells. Erikson reported that oligonucleotides produced by
RNase T1 digestion of 7S RNA from AMV and from MSV yielded identical
fingerprints and suggested that there must be a conservation of
sequences among the viruses that replicate in different hosts (41).
The same molecule was found in infected host cells, and its function
in either case is not known. Two species of 8S RNA were resolved by

polyacrylamide-agarose gel electrophoresis from Maloney strain of MSV

(68). Its function is unknown.

 

11

Seventy S-Associated 78 (85) RNA
Emanoil-Ravicovitch, et al., report a 7OS—associated 8S RNA
in MSV (38). It has exactly the same electrophoretic mobility as
free 88 RNA from MSV and has a very similar base composition. It was

presumed to play a linker role among the subunits.

Native 18S and 28S RNA.--Bishop, et al., report the presence

 

of small amounts of 185 (approximately 4%) and 28S (approximately 6%)
in Bryan RSV + RAVI, and SR-RSV (12). They were resolved by gel
electrophoresis and were shown by mixing experiments to result from
cellular debris adventitiously purifying with virus.

The 18 and 28S RNA species in immature RSV Prague on the other
hand were concluded to be molecules destined for aggregation into the

68S molecule (22).

Seventy S-Associated 18S and 28S RNA.--These are discussed

 

below with 708 RNA and its subunits.

Native 3SS RNA.--In three instances, free 30-408 subunits of

 

the genomic RNA was reported (22, 42, 65). They were viewed as pre-

assembled genomic RNA subunits.

Seventy S-Associated 358 RNA.--This is discussed below with

 

70S RNA and its subunits.

Seventy S RNA and Its Subunits.—-The 50—75S RNA is believed to

 

be the genetic material of the virus but there is no direct evidence
for this since it has not been conclusively shown to be infectious.
It is predicted to be the same polarity (+) as the mRNA which codes

for viral proteins within the infected cell (9).

 

 

12

It can be seen from table 1 that a wide range in sizes exist
among the different virus species from 508 for FeLV-R, FeSV-R, and
MSV-SD (33, 34) to 75$ for Bryan RSV and RAV1 (12) for the high
molecular weight genomic RNA. This corresponds to a range of molecular
weights from 5.7 x 106 daltons to 13.4 x 106 daltons using Spirin's

equation (105):

M = 1550 x 52'1

Even within one virus group, such as the avian tumor viruses, a maximum
sedimentation coefficient difference corresponding to a molecular weight
difference of 1.3 x 106 daltons is found (15). The significance of
such a wide range in sizes is not yet known. The observed differences
in the avian viruses did not correlate with any biological function
such as transformation (15).

The 50 to 758 RNA from all oncornaviruses, when collected from
a neutral sucrose or glycerol gradient and heated or subjected to
agents known to destroy hydrogen bonding such as urea, formamide,
formaldehyde, or DMSO, is converted into two size classes, namely 48
and 28-358. Occasionally intermediate sizes are found. Data from
many sources are summarized in table 2. The 7OS-associated 45, SS,
and 7S RNA species are discussed above. McCain, et al., reported
that an 188 subunit was a consistent finding in MLV-MSV (79). Its
base composition was unlike 188 rRNA. An 188 peak appeared in addition
to the 36S RNA in MLV as reported by Watson (122), but no significance
was made of it. In most cases, RNA between 48 and 36S in size was
heterogeneous and its presence was attributed to degradation of higher

molecular weight RNA (7, 28) by nucleases present in the virion (62).

Bader and Steck demonstrated that the heterogeneous RNA increases with
the increasing time a virus spends at 37C and this correlates with a
decrease in virus infectivity (7).

In virions that are collected over short periods (1-4 h.) and
not allowed to age at 37°C, a majority of the 50-75S RNA dissociates
into 28-358 subunits. East, et a1 (32, 33), reported that the subunits
from MSV-SD, FeLV-R, and FeSV-R are 288 as determined by sucrose
gradient centrifugation, whereas, Bader and Steck (7), report the
subunits in MLV to be 3S—44S based on sedimentation data. These are
the two extreme estimates for sedimentation coefficients reported.

In general, it is estimated that 3 or 4 28—358 subunits each with a
molecular weight of approximately 2 or 3 x 106 daltons are linked
together to form a 50—758 molecule which exists at the rate of one

per virion. The 28-358 subunits are thought to be held together by
double-stranded regions of hydrogen bonding (28, 82). The possibilities
that protein or DNA bind the subunits together is ruled out by their
resistance to separation upon treatment with pronase or deoxyribonuclease
(7).

One notable feature is that the 50—758 molecule denatures to
28—358 molecules in a step-wise fashion. This was demonstrated under
controlled conditions by Travnicek and Riman (115). 60-708 AMV RNA
was converted to a 50-548 molecule and then to a 30-408 molecule when
using increasing formamide concentrations and detected by sedimentation
in glycerol gradients. This phenomenon is noted in other published
data although not extensively discussed (7, 20, 33, 42).

Cheung, et a1. (22), noted that an intermediate molecule of

55-6OS (intermediate between 368 and 60-708) was present in RSV

14

labeled for 5 minutes and East, et a1. (33, 34), noted that an
intermediate molecule of SOS (intermediate between 288 and 588) was
present in virions labeled for only 4 h. The common hypothesis is
that the molecule of intermediate size is an aggregate with fewer than
the maximum number of subunits (i.e., three rather than four or two
rather than three). The intermediate then can either be a product of
aggregation or disaggregation. Travnicek and Riman concluded that
different double stranded regions have different thermal stabilities
(115), Canaani and Duesberg found 30-408 subunits present in immature
(freshly released) RSV collected at 3 min intervals which assemble
to 60-708 molecules upon incubation, but no aggregates of intermediate
size were seen (21).

Duesberg and Vogt (30) report the interesting finding that the
30-408 subunits in avian RNA tumor viruses can be grouped into a and
b subunit classes based on electrophoretic mobility in polyacrylamide
gels. The a subunits are electrophoretically slower than b subunits
and are found in viruses which can transform cells in_vi££2’ whereas
b subunits are found in viruses which are unable to transform cells.
From oligonucleotide digest studies in which all oligonucleotides from
b can be found in a, but not vice versa, Duesberg hypothesises that b
subunits arise from a by deletion of a piece of genetic material (which
includes the gene for transformation) (31, 33, 85). That is, a = b +
x where x is the deleted genetic material. On denaturing formamide-
polyacrylamide gels, a subunits have a measured molecular weight of
2.4-3.4 x 106 daltons, and b subunits have a measured molecular weight

6

of 2.2-2.9 x 10 daltons (31). The a subunits are demonstrated to be

the cause and not the consequence of transformation by Martin and

15

Duesberg using mutants which are temperature sensitive for transfor-
mation but not temperature sensitive for virus production (77). In
these experiments, the a subunit was present in progeny virus produced
at the temperature restricting transformation. The above results hold
true for cloned stocks of virus (32). Sheele and Hanafusa were not
able to distinguish clearly between a and b subunits from transforming
and non-transforming avian oncornaviruses using polyacrylamide gel
electrophoresis (100). Distinct subunit classes have not been
distinguished in the mammalian RNA tumor viruses (33, 34, 123).

One fundamental question concerning the 28-358 subunits
remains to be answered. That is whether or not each of the 3 or 4
subunits possess identical genetic information (i.e., a haploid,
segmented 708 genome) or possesses unique genetic information (i.e.,

a polyploid genome) (121). At present, the polyploid model is favored
by Duesberg and Vogt (32) simply because it is easier to envision a
single deletion giving rise to b subunits from a than it is to
envision a multiple (3 or 4) deletion.

The 3' terminus of AMV is almost exclusively an unphosphory-
lated uridine residue as determined by periodate oxidation and
reduction with tritiated borohydride and analysis of the tritiated
base residues following hydrolysis (43). The 3' terminus of MLV
(AKR), FeLV, hamster leukemia virus, and viper leukemia virus 358 RNA
also contain uridine as shown by the same method (78).

The 5' terminus of RSV 358 RNA is 5'-0H adenosine as deter-
mined by radioactive phosphorylation with polynucleotide kinase and

analysis of the degraded nucleosides (103).

16

Poly (A).--At least some of the 358 subunits of the high
molecular weight RNA contain stretches of polyadenylic acid (poly (A))

as part of their primary structure (50. 55, 67. 95)- This is shown to

be true for all of the oncornaviruses looked at to date, i.e., the RSV,
AMV, FeLV, Mason-Pfizer agent, MMTV, MSV, and Rauscher MLV. The
tract of poly (A) can be selectively isolated and studied because of
its resistance to enzymatic degradation by pancreatic and T1 ribonu-
cleases in high (0.3M) salt (11). It is estimated to be 100 to 300
nucleotides in length from its sedimentation coefficient and electro-
phoretic behavior, with a corresponding molecular weight of 30,000-
60,000 daltons. It comprises roughly 1-3% of the total 708 RNA (50,
55), and is composed of 91-97% adenosine, % cytidine, and trace
amounts of guanosine and uridine (55). It is estimated to be present
in the amount of 1-8 stretches per 708 genome (55). It may not be a
part of all 358 subunits, however (55, 67). In one case (67) it was
reported to be present in the 4-128 RNA portion of RSV RNA but this
was thought to be derived from degraded 358 RNA.

Certain chemical properties of the poly (A) can be utilized
to purify it and the additional RNA molecule which is covalently
attached to it. (1) It is partitioned into the aqueous phase during
phenol extraction in pH 9.0 Tris buffer, and into the phenol phase
in pH 7.5 Tris buffer (17). A low ionic strength is necessary for
this phenomenon. (2) It binds to Millipore (nitrocellulose membrane)
filters in 0.5 M KCl and is eluted in low (no KCl) buffer (70). (3)
It binds by hydrogen bonding to oligo(dT)-cellulose in high salt

(0.5 M KCl) buffer, and is eluted in low salt (no KCl) buffer (3).

17

(4) It binds by hydrogen bonding to poly(U) glass filters in high salt
buffer and is eluted in low salt (no salt) buffer (102). Each of these
properties can be utilized during the isolation and purification of
28-358 subunit RNA free of RNA which does not contain poly(A) such

as ribosomal RNA.

Remarkable similarities exist between the covalently attached
poly(A) found in the oncornavirus 28-358 subunits and those found in
eukaryotic messenger RNA, high molecular weight heterogeneons nuclear
RNA, and adenovirus-specific mRNA, the synthesis of which are all in
the nucleus of the cell. The poly(A) in these cases is 100-200
nucleotides long (25, 35, 70), resides on the 3'-OH terminus (81), is
added to heteogeneous nuclear RNA after transcription is completed
(2, 35, 87) and is transported from the nucleus to the cytoplasm.

This information alone raises the question of whether the oncornavirus
RNA is synthesized in the nucleus in the same fashion. In this
respect it would be interesting to know if 3' deoxyadenosine (cordy-
cepin) would inhibit the synthesis or transport of oncornavirus RNA,
as it does in the case of adenovirus specific mRNA. Such an effect
would doubtlessly manifest itself by affecting virus production and
should be seen at this level. It is interesting to note that
cordycepin inhibits transformation of mouse embryo fibroblasts by

MSV (73).

The full significance of poly (A) is not yet known.

Methylation.--Until recently only ribsomal 288 and 18S RNA

 

and transfer RNA were shown to be methylated (18, 24). Perry and

Kelly (86) have shown that mRNA from mouse cells is methylated in

18

both base and ribose moieties with approximately 2.2 methyl groups per
1000 nucleotides, one-sixth the level of ribosomal methylation. This
finding is confirmed and extended by Desrosiers, et a1. (26). This
raises the question of whether oncornavirus RNA is likewise methylated
in view of other similarities existing between viral RNA and mRNA,
namely, poly(A).

Erikson (42) labeled avian myeloblasts with (methyl-3H)-
methionine and found a high level of methyl labeling in 48 RNA with
19% of the total counts in 658 RNA. Too few counts were available for
analyzing the quality of the methylation. Also, the 658 RNA had not
been treated so as to remove the associated tRNA-like 4S RNA. McCain,
et a1. (79), found 15% of the methylated counts in 69S MSV-MLV RNA.
After heating all methyl counts sedimented with 4—108 RNA. Base-
specific methylase activity, namely NZ-guanine RNA methyltransferase,

is associated with AMV, RSV, and RAV and Nz-adenine RNA methyl-

1’
transferase is associated with RSV (48). This along with circum-
stantial evidence implicating a relationship between nucleic acid
hypermethylation and the malignant conversion of cells (106) suggest

that a close look should be taken at the methylation of oncornavirus

RNA.

Summary of Questions on the High Molecular Weight Subunits.--
In summuary, questions remaining to be answered about the 28-358
subunits are:
1. Exactly how many subunits exist for each 50-758 molecule?
2. How are the subunits held together in the 50-758 molecule?
3. Does each subunit have poly(A)? Where is the poly(A)

within the molecule? What is the function of poly(A)?

19

4. Are the molecules methylated? If so, what kind of methylation
(base or sugar)? On what nucleotides and where in the molecule
does it exist? What is its function?

5. Is each subunit in the 50-758 molecule identical (polyploidy)
or unique (haploid-segmented)?

6. What is the primary structure of the subunits?

7. Exactly what genetic information is present?

8. What are the secondary and tertiary structures of these

molecules?

3. Replication

 

Determining what biochemical events take place during oncorna-
virus replication from the time of infection to the time of progeny
virus release proves to be an arduous task since it requires detection
and quantitation of minute intracellular quantities of viral components
(DNA, RNA, and protein) (97)- There is no shut-off or even diminution
of cellular synthesis during oncornavirus infection (110).

Two approaches have been taken as a start toward elucidating
replication events: (1) to study the kinetics of virus replication
in permanently infected cells, and (2) to study the effects of the

cell cycle on virus replication in synchronized cells.

Kinetics of Labeling3--The interval between the addition of

 

radioactive RNA precursors (uridine) to infected cells and the
appearance of radioactive RNA in progeny virus has been measured for

RSV-RAV, MLV systems (6), MLV and AMV systems (10), and the MSV-SD

system (33).

20

Interpreting these results involves knowing the behavior of
uridine uptake and distribution by cells. Uridine is actively
accumulated in mammalian cells, becomes phosphorylated immediately
to UMP, UDP, and UTP and cannot escape from the cells (8). At a level
of 20uM radioactive uridine for 106 HeLa cells/ml, 80% of the radio-
active uridine is taken up by the cells from the medium (8). Intra-
cellular uridine pools are sufficiently large that they cannot be
diluted by added uridine within a chase period of 12 minutes (8).

A chase period of 4 hr. can effectively dilute the intracellular acid
soluble pool to 30% (6). Trichloracetic acid precipitable radioactive
cellular RNA is detected as early as 3-5 minutes after addition of
label (6, 10).

When infected cells were labeled with a continuous source of
radioactive uridine, radioactive RNA from progeny virus appeared as
early as 2 h. for RSV-RAV1 (6), 80 minutes for MLV (6), 30 minutes for
MSV-SD (33), and 1.5 hr. for AMV (10).

When infected cells were pulse-labeled 15 minutes with radio-
active uridine, radioactive RNA from progeny virus is released at a
maximum rate at 5 hr. for RSV-RAV, and MLV (6) and between 3 and 6 hr.
for AMV (10).

The above results are interpreted to mean that the minimum
interval between labeling of viral RNA intracellularly and its release
in progeny virus ranges from 30 min. to 2 hr., and the interval of
5 hr. is an average interval which viral RNA spends in an intracellular

pool before being incorporated into a virion (10).

21

Synchronization.--Early key experiments suggested that
oncornavirus replication was under the control of cellular events. RSV
had a specific requirement for DNA synthesis during the infections
cycle (5, 108) and DNA-dependent RNA synthesis was a requirement for
replication at all times after infection (4). Another experiment (60)
demonstrated that in synchronized cells, RSV production was seen at
early G1, only after cells had gone through mitosis, and regardless
of when cells were infected in the cell cycle. Non-oncogenic RNA
viruses show none of these metabolic dependencies for infection and
replication. The synthesis and release of RSV was further studied in
infected chick embryo fibroblasts synchronized in serum-free medium
(71). The synthesis of virus-specific RNA, quantitated by hybridization
to 12_vi££g_synthesized DNA, and of virus—specific protein, quantitated
after its appearance into the virion, occurred in early S phase.

Virus release occurred in early G1 phase. Leong postulated that a

controling factor was produced during early G which allowed subsequent

1
virus release, and a second factor was produced at the onset of DNA
synthesis which was important for RNA and protein synthesis. Kirsten
MLV and MSV-MLV production is synchronized in chronically infected
cells synchronized by a double thymidine block. Maximal virus release
coincided with the time of mitosis in both cell systems and is in
agreement with avian sarcoma-leukemia systems as reported by Temin
(110), Hobom-Schnegg, et a1. (60), and Leong, et a1. (71, 85).

The synthesis of cell products have been studied in synchronized
cells and appear to be under the control of cell cycle events. IgG

and IgM production was greatest during G and S phase and smallest

l

22

during mitosis in human lymphoid cells synchronized by thymidine and
colcemid, indicating that transcription of immunoglobulin genes occurs
during a limited portion of interphase (19).

The above methods for cell synchronization utilize chemically
modified environments (excess thymidine, isoleucine-free medium,
serum-free medium) to effect synchronization. One method which com-
pletely avoids chemical modifications is that of Shall and McClellan
(101) in which suspended cells from a heterogeneous population are
grouped on the basis of size (diameter) by velocity sedimentation
differences. The synthesis of cell products by cells synchronized in

this fashion was not studied.

23

MATERIALS AND METHODS
PART I

Source of Cells and Virus

The permanently infected feline thymus tumor cell line, F-422,
which produces the Richard strain of feline leukemia virus (FeLV-R)
was obtained from Dr. C. G. Rickard (Cornell University). It is
propagated in medium which is a mixture of 60% Leibovitz and 40%
McCoys and 15% fetal calf serum (Grand Island Biological Co. (GIBCO)).
Crandell (CCC) feline kidney cells were obtained from Dr. K. M. Lee
(Cornell University) and propagated in McCoys 5A medium and 15% FCS.

MOPC-21F cells were obtained from Dr. R. J. Patterson (Michigan State

University).

Determining Viable Cell Numbers
Viable cells exclude trypan blue dye and remain colorless.
Cells were counted using a hemocytometer in a 0.08% final concentration

of trypan blue dye obtained as a 0.4% solution (GIBCO).
Electronmicroscopy of F-422 Cells and FeLV-R

Embedding.--The fixation, agar infiltration and embedding

procedures of Howatson (61) were used. Twenty ml of cells (106/m1)
were fixed directly in situ with an equal volume of 6% glutaraldehyde.
Gleutaraldehyde was added slowly while cells were mixing. After

fixing for 24 h. at 4°C, cells were pelleted at 1000 r.p.m., 5 min.

24

in an International PR-6 centrifuge, washed 2X with PBS and pelleted.
The cell pellet was mixed with an equal volume of 2% agar at 50°C for
10 min., then cooled. The pellet was thinly sliced and fixed 1.5 h.
at 4°C in 1% osmium tetroxide made up in PBS. Slices were dehydrated
in sequential baths of 50%, 75%, 95%, and 100% ethanol (3X in 100%),
followed by 3 washes in propylene oxide. Slices were embedded in Dow
Epoxy Resin (DER)-812 (74), sectioned, and mounted on unfilmed copper
grids. Sections were stained with uranyl acetate and lead nitrate
before electronmicroscopic examination in a Hitachi-11 electron-

microscope.

Ngggtive Staining,—-Virus was purified by pelleting followed
by isopycnic centrifugation (see below). The viral band was collected
then dialyzed 20 h. against 1% ammonium acetate at 4°C. Five ul of
the virus preparation was dried onto carbon-coated, collodian-filmed
copper grid, then stained 2 min. in 1% aqueous solution of uranyl

acetate before electronmicroscopic examination.

Synchronization of F-22 Cells

 

Thymidine Excess.——The thymidine excess method as described by

 

Mueller and Kajiwara (83) was used. Cells at 0.8 x 106/m1 in a 25 ml
volume were treated with ZmM thymidine for 8 to 20 h. after which
cells were washed 1X with HBSS and resuspended in fresh medium
containing .01 mM levels of deoxyadenosine, deoxycytidine and deoxy-

guanosine. At times 0.1mM, 0.5mM, and 1.0mM thymidine were used in

place of 2.0mM thymidine.

 

25

Serum-Free Medium.--The serum-free medium method as described

 

by Leong, et a1. (71), was used. Cells at 0.5-0.8 x 106/m1 in a 25 ml
volume were grown in medium without serum for 24 h. Cells were then
resuspended into fresh medium containing 15% FCS or serum was added

to the already existing cultures to make 15% final concentration.

Duplicate cell counts were made at hourly intervals.

BSA Gradients.--Sterile continuous gradients of bovine serum

 

albumen were made by a modified method of Miller and Cudkowicz (80). A
35% W/W stock solution of BSA (fraction V power, nutritional Bio-
chemical Corp.) was made using T.N.E. 7.5 buffer (0.01 M Tris, pH 7.5,
0.1 M NaCl, 0.001 M EDTA), and filtered through a millipore (0.22 uM)
filter and stored at 4°C. Four ml 35-17% W/W gradients were made in

14 cm x 6.5 cm i.d. home-made test tubes using a gradient maker pre-
washed with 70% ethanol and dried with 90% ethanol in a laminar flow
hood. One to 10 million cells were suspended in 10% BSA solution and
centrifuged 2000 r.p.m. for 30 min. at 10°C in an International PR-G
centrifuge. Cell bands were collected using a sterile pasteur pipette.

This density gradient ranges from 1.0525 to 1.1004 g/cc.

Isoleucine deficient medium.--The method of Tobey and Ley

 

(112) was used. A mixture of Leibovitz-McCoys medium (60:40 respec-
tively) in powder form, without amino acids and without bactopeptone,
was obtained from GIBCO. Amino acids and bactopeptone in powder form
were obtained separately from the same source. The composition of
this medium is described in table 3. Bactopeptone was omitted from

the reconstituted medium with no ill consequences for cell viability.

26

Table 3.--Procedure for reconstituting amino acid-free and bactopeptone-
free Leibovitz-McCoys medium (GIBCO medium number 72154) for

isoleucine deficiency.

 

Constituent

Quantity

Solubilized in

 

Medium (GIBCO medium
number 72154, control

number 330150)3

NaHCOa
Penicillin
Streptomycin

 

 

Amino acids:

 

L-a alamine
L-arginine
L-asparagine
L-aspartic acid
L-cysteine
L-glutamic acid
L-glutamine
glycine
L-histidine
L-isoleucineb
L-leucine
L-lysine
L-methionine
L-phenylalamine
4-hydroxyproline
L-proline
L-serine
L-threonine
L-tryptophane
L-valine

L-tyrosine

powder for

5 liters
4.7 grams
500,000 units

500,000 ug
702.8 mg
1899.2 mg
840.0 mg
39.9 mg
613.4 mg
44.2 mg
1338.4 mg
615.0 mg
1055.0 mg
453.7 mg
453.7 mg
354.4 mg
254.8 mg
408.0 mg
39.4 mg
34.6 mg
652.6 mg
935.8 mg
66.2 mg
335.2 mg
936.2 mg

 

4.800 liters
H70

0.06 liters

H20

0.005 liters 5N HCl

 

aA mixture consisting of 60% L-15 (Leibovitz) medium without
amino acids and 40% McCoy's 5a medium without amino acids and bacto-

peptone.

b

Omitted when making isoleucine deficient medium.

27

Cells at 0.34-0.4 X 106/ml were grown 48 h. in medium deficient in
isoleucine. Isoleucine was then added to make a final concentration
of .608 mM, the concentration found in normal medium. Cells were

counted periodically.

Isotopic Labeling of Virus
FeLV-R was isotopically labeled by incubating cells with 3H
uridine (>40 Ci/m mole) or 14C uridine (57 mC/m mole) obtained from

New England Nuclear. The exact labeling conditions are described for

each experiment.

Purification of Feline Leukemia Virus, FeLV—R
FeLV-R was purified by any one of five methods, the exact
method being identified in each experiment. For each method, cell
supernatant was first clarified by pelleting cells at 1000 r.p.m.
(300xg) for 5 min. in an International PR-6 centrifuge, then further
clarified by pelleting cell debris at 10,000 r.p.m. (16,000xg) in

a Sorval GSA rotor.

1. Salt Precipitation Followed by Isopycnic Centrifugation.—-

 

Virus was precipitated by adding an equal volume of saturated

ammonium sulfate (pH 7.5) at 4°C (93). Precipitate was pelleted by
centrifuging 10,000 r.p.m. for 30 min. in a Sorvall GSA or 88-34 rotor,
resuspended in 1/100 original volume in T.N.E. 7.5 buffer, and
isopycnically centrifuged on a 40-15% w/w sucrose gradient for 4 h.

at 25,000 r.p.m. (84,000xg) in a SW 27 rotor or for 2.5 h. at 45,000
r.p.m. (190,000xg) in a SW 50.1 rotor. FeLV-R was recovered from the

fractionated gradient at the density of 1.14 g/cc.

28

2. Polyethylene Glycol Precipitation Followed by_lsopycnic
Centrifugation.--A 50% solution of polyetylene glycol in T.N.E. 7.5
buffer was added to clarified cell supernatant to make a final con-
centration of 5% PEG at 4°C. After 2 to 16 h., the precipitate was
pelleted, and isopycnic centrifugation was carried out as described

above.

3. Pelleting_Followed by Isopycnic Centrifugation.--FeLV-R
was pelleted through a barrier of 20% w/w sucrose made up in T.N.E.
7.5 by centrifuging 3 h. at 25,000 r.p.m. in a SW 27 rotor at 4°C or
1.5 h. at 45,000 r.p.m. in a SW 50.1 rotor at 4°C. Pellet was
resuspended in 0.5 ml T.N.E. 7.5 and subjected to isopycnic centri-

fugation as described above.

4. Pelleting,—-FeLV—R was pelleted 2X through a barrier of

 

20% w/w sucrose as described above. This method is similar to one used
by Bader and Steck (7) and virus purified this way is called minimally

purified virus.

5. Pelleting Followed by Rate Zonal Centrifugation Followed

by_Isopycnic Centrifugation.--FeLV-R was pelleted once as described

 

above. The pellet was then suspended in 0.5 ml T.N.E. 7.5, by
sonicating 15 sec. in a Branson ultrasonic cleaner, and rate zonally
centrifuged in a 30-15% w/w sucrose gradient (22) for 10 min. at
45,000 r.p.m. in a SW 50.1 rotor at 4°C. Virus collected from the

fractionated gradient was concentrated by pelleting as described above.

29

‘RadioactivityAAssay_

 

Virus radioactively labeled with RNA precursors (3H uridine or
14C uridine) was detected and quantitated by totaling the number of
radioactive counts isopycnically banded at the density of FeLV-R
(1.14 g/cc). To accomplish this, 5 ml gradients (from the SW 50.1
rotor) were fractionated into approximately 0.19 ml fractions from the
bottom by dripping with a needle on to 2.3 cm Whatman paper filter
pads. The pads were then dried, precipitated 20 minutes in 5% TCA

at 4°C, washed 1X with acetone, dried and counted in toluene and POPOP

(52).

Infecting_Crandell (CCC) Cells

 

CCC cells were freshly grown to just less than a complete
monolayer in 75 cm2 Falcon plastic flasks. FeLV-R was prepared by
growing F-422 cells with greater than 90% viability in 100 ml medium
at 3-4x106 cells/ml. for 4 h. and purified under sterile conditions.
Virus was pellet from clarified cell supernatant at 30,000 r.p.m.
(75,000xg) for l h. in a type 30 rotor, using 33 ml screw capped
polycarbonate tubes. Virus pellets were resuspended in 1 m1 total
volume, and cells were absorbed with the virus suspension for 1 h. at
37°C, then washed 3X with 10 ml. HBSS. Cells were either assayed
immediately for virus production or passed with trypsin at 1:2 or 1:4
dilution and assayed at a later date.

A flask of cells was assayed for virus production by incubating
with 5 ml medium containing l6uCi 3H uridine/ml. Virus was purified

by PEG precipitation and isopycnic centrifugation and TCA precipitable

radioactivity was quantitated as described in the radioactivity assay.

30

Cordycepin Treatment

To determine the effect of cordycepin (3'-deoxyadenosine) on
the growth and viability of F-422 cells, 5x107 cells were incubated
at 1x106 cells/ml with 50ug/ml of cordycepin. Viable cell counts were
periodically made.

To determine the effect of cordycepin on viral synthesis,
6x107 cells in 1 ml were incubated with 50pg of cordycepin for 15 min.,
washed 2X with 4 ml medium, then pulsed for 15 min. with 50uCi
3H uridine in 1 ml. Cells were then diluted to 2 x 106/ml with medium
and incubated 6 h. Virus was purified by PEG precipitation and
isopycnic centrifugation and then assayed by radioactivity content as
described above. Control cells were handeled identically except for

cordycepin treatment.

31

RESULTS

Electronmicrographs of F-422 Cells and FeLV-R

Electronmicrographs of plastic-embedded, thin-section F-422
cells are shown in figures 1 and 2. The completed extracellular
virion and the budding virus are identified. They fit the description
for the FeLV-R by Rickard, et al. (89); the virions being 100mu in
diameter and having a darkly stained, centrally located nucleoid, and
the nucleoid material in the budding virus becoming apparent only as
the bud is assembling. No intracellular particles with this appearance
could be found. Figure 3 is a higher magnification of embedded virions
which were grouped and external to cells. Note that the diameter of
these particles is 100mm. Crescent shaped forms have been reported
(893- Figure 4 is a high magnification of virus purified by isopycnic
centrifugation and negatively stained. Note the non-spherical shapes
which is apparently an artifactual phenomenon resulting from the

uranyl acetate staining procedure (89).

Synchronization of F-422 Cells

 

Thymidine Excess.--The doubling time for feline thymus tumor

 

cells, F-422, is between 16 and 20 h. (Fig. 5) as measured by cell
numbers. When synchrony of cell DNA occurs as a result of thymidine

excess synchrony of cell division also occurs and cell numbers

32

Figure 1. Electronmicrograph of F-422 cell. F-422 cells were fixed
in situ with glutaraldehyde, embedded in DER-812, sectioned
and strained with uranyl acetate and lead nitrate. A budding
virion is noted (B).

 

34

Figure 2. Electronmicropgraph of F-422 cells and FeLV-R. Same
preparation as described in figure 1. Note virions (V) and
budding virions (B).

 

36

Figure 3. Electronmicrograph of FeLV-R. Same preparation as described
in figure 1.

 

38

Figure 4. Electronmicrograph of purified FeLV-R. FeLV-R purified by
isopycnic centrifugation was dialyzed against ammonium
acetate and negatively stained with uranyl acetate.

 

40

Figure 5. Growth curve for F-422 cells. Cells were seeded in fresh
medium at 0.3x106 cells/ml. Viable cell numbers were
determined by using trypan blue dye. The percentage of
viable cells is noted.

41

80

 

 

 

4+—

9b. x _E \ m44uu 4<Hoh

HOURS

42

double within a period of 8-10 h. (83). Synchrony was assayed by cell
numbers only. As shown in figure 6 thymidine at concentrations of
2 mM had an inhibitory effect on cell replication both while the
thymidine was with the cells (expected) and after it was removed (not
expected) for a period of 30 h. or greater. There was also a toxic
effect since cell viability decreased from 90% to 50% during the
experiment. The growth inhibition and toxicity were also seen (1)
when concentrations of 1 mM, 0.5 mM, and 0.1 mM were used rather than
2 mM thymidine, (2) when cells were washed free of thymidine and
placed in fresh medium or washed free of thymidine and placed in
medium supplemented with 1 mM each of deoxycytidine, deoxyadenosine,
and dexyguanosine (83), or (3) when cells were treated for periods of
14, 12, 10, and 8 h. rather than for 16 h. prior to washing and
resuspending. In each case, cells would not increase in number until
after a period of 20 h. and then at a rate of only 25% of controls.
To determine whether F—422 cells do incorporate thymidine an
experiment as described in figure 7 was performed. F—422 cells take

up thymidine at a rate equal to or faster than MOPC—ZlF cells.

Serum-Free Medium.-—After either (1) resuspending cells into
fresh medium with 15% FCS, or (2) adding FCS to a 15% final concen-
tration little cell growth was noted for a period of 10 h. After a
10 h. lag period, cell growth continued asynchronously with a curve
paralleling the curve of control cells (data not shown). Cells

therefore did not appear to be synchronized with serum-free medium.

 

Figure 6.

43

Growth curves for F-422 cells during and after excess
thymidine treatment. Cells were incubated for 20 h. in

0.4 mM, 0.2 mM, and 0.1 mM thymidine then (at arrow)
resuspended in fresh medium containing 0.01 mM each of
deoxyadenosine, deoxycytidine, and deoxyguanosine. Control
cell (handeled in parallel) growth curves are shown.

a. 0.4 mM b. 0.2 mM c. 0.1 mM.

TOTAL CELLS / ml x lo"

44

\

 

 

\-
\o_o\
L 1_\_l“"—’I
u/I
CONTROL

HOURS

Figure 7.

45

Incorporation of 3H thymidine into F-422 cells and MOPCI-24F
cells. F-422 and MOPCl-24F cells, 1.40x105 of each, wgre
incubated separately in .58 ml medium containing 8uCi H
thymidine (20 Ci/m mole). At hourly intervals triplicate
0.8 ml samples were taken and precipitated with 1/10 vol. of
50% TCA at 0°C. Precipitate was washed 2X with 5% TCA.

The washed precipitate was resuspended by sonication in a
Branson ultrasonic cleaner, solubilized in NCS tissue
solubilizer, and counted in 5 m1 Aquasol.

3H CPM x 10"

46

 

 

3r
2.—
F-422
O
0
Ir MOPC-24F
O
.. l l
1 2

HOURS

47

Isoleucine-Free Medium.--All experiments were done with special

 

medium formulated by GIBCO, MED-72154, control number 230628, which,
when completely reconstituted with all amino acids, failed to support
the normal growth of F-422 cells. It was later discovered, after
completion of these experiments, that the medium was improperly
formulated by GIBCO, and has since been replaced with MED-72154,
control number 330150 which does support the normal growth of cells.
Synchronization of F-422 cells was not attempted with MED-72154,

control number 330150.

BSA Gradients.--Populations of synchronized cells have been

 

obtained by fractionating an asychronous population of cells based on
sedimentation rate differences within the asynchronous population
(101).

Attempts were made to likewise fractionate a F—422 cell popu-
lation on density gradients of BSA (80). Conditions of these eXperi-
ments were such, however, that isopycnic centrifugation resulted rather
than rate-zonal centrifugation. Centrifugation times for longer than
30 min. gave the same results.

During centrifugation two cell bands resulted; a broad band
containing greater than 90% of the cells positioned near the middle
of the gradient, and a very narrow band resting on top of the 17% BSA.
Cells under the light microscope from the large, dense band, appeared
as cells from a heterogeneous population. From the upper band all cells
appeared to have distinctly visible nuclei. Cells from either band
when suspended in fresh medium grew with no detectable synchrony

(data not shown) but had a normal growth curve. Cells in the upper

 

48

band were presumed to be daughter cells or premitotic cells based on
the distinct appearance of the nuclei, but their asynchronous growth
did not support this presumption. F-422 cells were not synchronized

using this procedure.

Purification of FeLV—R

 

Because electronmicrographs of F-422 cells and of isopycnically
purified FeLV-R hinted at the existence of cell vesicular contamination
and because a trailing shoulder (less dense that the virus) was
frequently observed during isopycnic centrifugation of the virus, the
question was raised whether isopycnic centrifugation alone was enough
to purify FeLV-R from cell vesicular contamination, assuming cell
vesicles do exist and do have a density near 1.14 g/cc.

The experiment described in figure 8 was performed for the
purpose of determining if FeLV-R truely sedimented to its density (1.15
g/cc) in 2.5 h. (an established protocol for isopycnically centrifuging
virus in our laboratory). Three observations were made. (1) Yes,
the virus is at 1.14 g/cc in 2.5 hr. (2) What appears as a trailing
shoulder at 2.5 hr. is a resolved peak at 45 min. (3) No trailing
shoulder appears at 15 h., but rather a shoulder appears to be leading
toward the bottom of the tube.

Attention was focused on the resolved trailing peak at 45 min.
It was hypothesized to be the vesicular contaminant and a sedimentation
velocity experiment was performed as described in figure 9. The
purpose was to resolve more clearly the two peaks seen in figure 8 a.
Two observations were made. (1) Three peaks were resolved rather

than two. (2) The most slowly sedimenting peak (the presumed

 

49

Figure 8. Isopycnic centrignation of FeLV-R for varying periods of
time. F-422 cells (3.0x108) at 2x106/ml were incubated 16 h.
with 4uci/ml of 3H uridine. Virus was pelleted directly
(no sucrose barrier) from clarified cell supernatant,
resuspended by sonication in a Branson ultrasonic cleaner
in the TNE 7.5, and divided into 3 aliquots: a, b, and
c. Each was layered onto a 5.0 m1 gradient of 40 to 15%
w/w sucrose gradient and centrifuged in a SW 50.1 rotor for
(a) 45 min., (b) 2.5 h., and (c) 15 h., at 45,000 r.p.m.
The gradients were fractionated by dripping. Ten ul of
each fraction was TCA precipitated and assayed for radio-
activity as described in Materials and Methods.

‘-

3H CPM x 10‘3

45 min

50

 

 

1.165
1.150

1.130

 

 

20

FRACTION

W‘f;tfitL__J
30 TOP

DENSITY (g/cc)

 

 

Figure 9.

51

Velocity sedimentation of FeLV-R. F-422 cells from
experiment described in figure 8 were resuspended in fresh
medium with no isotope for a second 16 h. period. Virus
was pelleted directly (no sucrose barrier) from clarified
cell supernatant, resuspended by sonication in a Branson
ultrasonic cleaner in TNE 7.5 and centrifuged 15 min.
through a 35-15% w/w sucrose gradient. The gradient was
fractionated by dripping. Ten pl of each fraction was TCA
precipitated and assayed for radioactivity as described in
Materials and Methods. Peaks were numbered 1 (fastest
sedimenting), 2, and 3 (slowest sedimenting).

 

-2

H CPM X 10

3

52

15*-

IO-

 

 

 

 

10 20 30 TOP

FRACTION

 

53

vesicular contaminant) contained radioactivity roughly equivalent to
that in the combined first and second peaks.

The peaks were labeled 1, 2, and 3 and pooled separately. Two
further experiments were performed. (1) Aliquots from each peak were
isopycnically centrifuged 2.5 hr. (Fig. 10a,b,c). Radioactive
particles from peaks 1 and 2 sediment to a density of 1.14 g/cc whereas
material from peak 3 has a broad density range clearly less than
1.14 g/cc, peaking at approximately 1.125 g/cc. Note that the position
of the radioactive peak in figure 10c is such that if it were
proportionately integrated into either figure 10a or b, a prominant
trailing shoulder would be seen. (2) The RNA from each peak was
extracted and analyzed on 2.0% polyacylamide —0.5% agarose gels. The
results are summarized in table 4. Note that peaks 1 and 2 contain
50-608 viral RNA whereas peak 3 does not. Peak 3 contains very little

4S RNA.

Infection of Crandell CCC Cells

 

Jarrett (66) reported that feline embryonic cells can be
infected with a multiplicity of 103 particles/cell in a 2 h. period
and that progeny virus production peaked at 24-48 h. post—infection.
The rate of virus production by F-422 cells was unknown. The rate of
40 particles/cell/h. for MLV (10) was used as an approximation and
judging from the rate of viral protein production, 3-4 mg protein/
liter/24 h. (52) for F-422 cells, the rate of 40 particles/cell/h. for
F-422 cells did not seem to be an unreasonable estimation. Since

oncornavirus infectivity is known to decrease with the increasing age

Figure 10.

54

Isopycnic centrifugation of FeLV obtained from velocity
sedimentation gradient. Fractions comprising peaks 1, 2,
and 3 from the gradient described in figure 9 were pooled
separately. An aliquot from each pool was isopycnically
centrifuged on a 5.0 m1 40-15% w/w sucrose gradient for
2.5 h. at 45,000 r.p.m. in a sw 50.1 rotor. a. Material
from peak 1. b. Material from peak 2. c. Material from
peak 3. Gradients were fractionated and assayed for
radioactivity as described in Materials and Methods.

°H CPM x 10‘2

55

 

 

 

I—j

 

 

FRACTION

1.165
1.150

1.130

 

DENSITY (g/cc)

 

56

Table 4.-—RNA content in constituents resolved by velocity sedimenta-

 

 

 

 

 

 

tion.
Peak 1 Peak 2 Peak 3
RNA
Cpm % Cpm % Cpm %
Total 19,210 100 35,500 100 11,070 100
35-608 1,470 7.7 4,170 11.7 100 .9
10-358 7,290 37.9 13,230 37.3 8,300 75.0

4-108 10,450 54.4 18,100 51.0 2,670 24.1

 

57

of virus (7), virus was freshly produced and rapidly purified under
sterile conditions of infection.

To determine if CCC cells could be infected the experiment
as described in figure 11 was performed. Control (uninfected) flasks
were mock infected and handeled in parallel to infected flasks.

Flasks (control and infected) were assayed for virus production at
24-36 h., then after the first, fourth, and seventh passages corre—
sponding to 2, 11, and 31 days post infection. The results of the
fourth passage (11 day) assay are shown in figure 11. Results for

the 24-36 h. flask and the seventh passage flask are similar having
71% and 47% reSpectively the number of counts in the viral peak of the
fourth passage flask. Infected cells were frozen (52) and stored at
—80°C and in liquid nitrogen.

To determine the eclipse period for infection, i.e., the inter-
val between infection and first appearance of progeny virus, two
separate but identical experiments were done. CCC cells were infected
as described in Materials and Methods for two flasks. The first flask
was incubated with 80uci 3H uridine in 5 ml medium for 6 hr. periods
beginning at 12 h., 20 h., 28 h., 36 h., and 44 h. post-infection.

For each 6 hr. period, virus was purified, isopycnically centrifuged
and the gradient fractionated for radioactivity assay. The second
flask and an uninfected control flask were incubated with 80uci 3H
uridine in 10 ml medium from 12 to 44 hrs. post infection. From
these, virus was purified, isopycnically centrifuged, and the gradient
fractionated and assayed as described above. In no case was a

ratioactive peak at the density of 1.14 g/ml observed. Because all

 

 

 

Figure 11.

58

Infection of Crandell CCC cells with FeLV-R. Feline kidney
cells (Crandell CCC) in a monolayer were exposed to a
suspension of FeLV-R pelleted from the supernatant of
permanently infected feline thymus tumor F-422 cells (see
Materials and Methods). After 4 passages (11 days) a

75 cm2 monolayer of the infected Crandell cells was exposed
to 80uci 3H uridine in 5 m1 media for 12 h. Labeled virus
was precipitated from clarified supernatant fluid with 5%
PEG and isopyinically centrifuged 2.5 h. at 45,000 r.p.m.
in a 5 m1 gradient of 40-15% w/w sucrose. The gradient

was fractionated and assayed for radioactivity as described
in Materials and Methods.

 

59

_
6 4 2

 

Aoo\mv >P_mzmo

1
TOP

 

INFECTED

 

 

«-2 x 2.8 In

FRACTION

6O

infected cells were used for radioactive labeling, none was passaged,

and hence future testing for virus production could not be done.

Effect of Cordycepin on Cell Viability_
When F-422 cells are incubated continuously with 50ug/ml of
cordycepin cell viability decreases from 90% to 50% by 4 h. and to less

than 10% by 8 h. (data not shown).

Effect of Cordycepin on Virus Production

 

Cells were concentrated and pulsed with cordycepin at 50ug/ml,
washed and pulsed with 3H uridine and incubated in medium for 4.75 h.
This is essentially the procedure used by Philipson et a1. (87). As
demonstrated in Figure 12b no inhibition of virus production is
observed. In fact, radioactivity in virus from cordycepin cells

represents 109% of the radioactivity in virus from control cells.

Kinetics of Incorporation of Uridine Into FeLV-R

It was useful for several reasons to study the kinetics of
FeLV RNA synthesis and incorporation into released virions. (1) This
is a necessary parameter to know before one can study the intracellular
pulse-labeled viral RNA, part of a long—range study by this laboratory.
(2) It was important to determine what minimum interval was needed
after pulse-labeling cells before analytic quantities of viral RNA
could be obtained. (3) It was important to compare the feline leukemia
virus system with the murine and avian leukemia virus systems
previously studied (6, 10).

Permanently infected F-422 cells synthesize virus continuously

and presumably at a constant rate. Basic assumptions for these

 

 

 

|\

Figure 12.

61

Effects of cordycepin on FeLV-R production. Sixty million
F-422 cells were incubated with 50ug cordycepin in 1 m1 of
medium for 15 min., washed 2X with 4 ml medium, then pulsed
15 min. with 50uci 3H uridine in 1 m1, diluted to 2x106
cells/ml and incubated 4.75 h. Labeled virus was precipi-
tated from clarified supernatant fluid with 5% PEG and
isopycnically centrifuged 2.5 h. at 45,000 r.p.m. in a

5 ml gradiant of 40-15% w/w sucrose. The gradient was
fractionated and assayed for radioactivity as described in
Materials and Methods. Control cells were treated
identically except that cordycepin was omitted from the
first step.

3H CPM x lo”

 

62

 

 

  
 

CONTROL
r I I ”JE“HN*T° J
T-
o\ 1.165
— 11.150
I \° 1.130
CORDYCEPIN TREATED
Wm “fi‘m J

 

FRACTION

DENSITY (g/cc)

 

63

studies were that (1) acid precipitated uridine counts represent RNA,
(2) viral RNA was synthesized at a constant rate and transported into
released virions at a constant rate, and that this rate remained
constant during cell manipulations, (3) viral RNA is synthesized at
the same rate as cell RNA, and (4) viral RNA is synthesized from the
same precursor (soluble) pool as cell RNA.

Baluda and Nayak (10) and Bader (6) have measured two aspects
of viral RNA labeling kinetics: (l) a minimum interval between the
addition of labeled uridine into a cell culture and the release of

labeled RNA in virus, and (2) an average interval, i.e., the average

 

time (half-life) that labeled viral RNA remains in the cell before

being released in virus.

Continuous Labeling, Minimum Interval.--Baluda and Nayak (10)

 

reported a minimum interval for labeling AMV RNA in avian myeloblasts
to be within 2 h. of adding radioactive uridine to the cells, Bader
(6) reported this interval to be 2 h. for RSV-RAV, and 70 min. for
Rauscher MLV.

To determine this interval in the permanently infected feline
thymus tumor cell, F-422, for FeLV-R RNA, the method of Baluda and
Nayak (10) was used. F-422 cells, which normally grow to concentra-
tions of 2-3x106/m1, were concentrated to 10x106/ml and incubated in
the presence of 3H uridine. Periodically equivolume samples were
removed from the incubation mixture, the virus was purified, and the
RNA quantitated by the number of counts in the purified virus. Two
experiments were done as described in figure 13. A definite virus

peak, 2.4% of the 4 h. peak, was seen for virus purified 30 min. after

Figure 13.

64

Incorporation of 3H uridine into purified virions with
continuous labeling. F-422 cells at 106/m1 were incubated
with 4uci 3H uridine/ml. At 0, 0.5, 1, 2, 4, and 8 h.,

6.5 ml samples were taken. Virus was purified by

ammonuim sulfate precipitation and isopycnic centrifugation,
and the gradients were fractionated and assayed for radio-
activity as described in Materials and Methods.

3H CPM x10“

 

 

Oh

I‘
2 h
L
\-
WF 1 I ‘1‘

 

65

0.5 h

 

.- (N
#n‘m- _.“t‘;/_ -._. 1

4b

7*.

 

 

Chart 1 ‘f‘ f _.

I . "'1
IO 20 30 TOP

FRACTION

Ih

 

,Jicttfrn.__r__. “T _.._..1

8h

 

 

9+0- 44—4 A’fivo-c
u— w -.------.

I0 20 30 TOP

 

 

 

 

66

addition of labeled uridine. In both experiments, virus was detected
at 30 min. Making the assumptions stated above, this clearly
establishes a shorter minimum interval than those described for AMV,
RSV-RAV, or MLV (6, 10). A second observation was made in this
experiment. As noted in figure 13, virus counts decrease after 4 h.,
down to 5% at 8 h. of what is seen at 4 h. This suggested that the
conditions of this experiment, perhaps, the artificially high cell
density, resulted in something destroying viral integrity such that
either (1) virus no longer purified at its normal density, or (2) virus
no longer contained acid precipitable RNA. The fact that no radio-
active peaks were seen in any other position in the gradient argues
against the first possibility.

To test the second possibility, 14C uridine labeled virus was
purified from another source and added exogenously to a culture
mocking the labeling conditions described in figure 13. The results
are shown in table 5. The total counts in progressive aliquots
surprisingly did not stay constant, as one would expect if viral
integrity were being pneserved, or decrease, as one would expect if
viral integrity were being destroyed. While no cause for the vanishing
labeled virus could be found, it was presumed to result from the
artificially high cell concentration. Further experiments were done
by keeping the cells at their normal, growing concentrations (2-3x106/
ml) except for brief periods of pulse labeling.

To determine what portion of the minimum interval measured
(30 min.) for FeLV-R is due either to (l) a delay of 3H uridine into

the cell, or (2) a delay of RNA synthesis from the cellular precursor

 

III-II

67

Table 5.--Behavior of exogenously added 14C uridine labeled FeLV-R in

tissue culture.

 

O

6 of maximum peak
h. after adding

 

 

 

exogenously labeled virus experiment 1 experiment 2

0 60 23
1 88 A 40
2 100 40
4 97

5 5 40
6 88

8 . . 100

10.5 70 . .

 

68

pool (uridine and its phosphorylated products) the following experi-
ment was done (Fig. 14). Cells at 2-3 x 106/ml were incubated with
3H uridine, and periodically the total amount of intracellular uridine
and total TCA precipitable material were measured. The results indi-
cate that as early as can be detected (<5 min.) both radioactive
uridine and precipitable counts are seen, and by 15 min. after the
beginning of labeling 69% of the maximum intracellular radioactivity
is seen. Thus, if any delay into released virion RNA results from

a delay of 3H uridine uptake into the cell or from a delay during 3H

 

uridine to the site of synthesis, it can be no greater than 5 min.

Q

Additional observations are made from this experiment. (1) By 1 h.
74% of the total uridine introduced into the cell culture becomes
taken up by the cells. (2) The TCA soluble (precursor) pool is maximum

at 1 hr., representing 42% of the intracellular radioactivity.

Pulse Labeling, Averagg Interval.--Baluda and Nayak (10)
measure the average interval for AMV RNA to be between 3 and 4 h. For
the purpose of determining the average interval that viral RNA remains
intracellular before being released in a virion, certain labeling
conditions had to be established. (1) Cell concentrations must be such
that labeled, released virions could be assuredly quantitated. (2)
Cells must survive a short period (15 min.) of concentration for pulse-
labeling for the purpose of maximum isotopic utilization. (3) Cells
must survive repeated pelleting and resuspension for the purpose of
monitoring virion (and hence virion RNA) release during the chase
period. The following experiments confirmed that these conditions

could be established.

Figure 14.

69

Incorporation of 3H uridine into the acid soluble pool and
into RNA in F-422 cells. F-422 cells were incubated at

3 x 106/ml with 30 uCi/ml 3H uridine. At 0, 0.25, 0.5, l, 2,
4, and 8 h. after addition of the isotope, 0.1 ml samples
were taken in triplicate. For each sample (1) the total
radioactivity was obtained after solubilization in NCS
tissue solubilizer, (2) the radioactivity in washed cells
was obtained after solubilization and (3) the radio-
activity in washed TCA precipitate was obtained after
solubilization. NCS solutions were counted in 5.0 ml
Aquasol.

       

-5

H CPM X I0

3

 

 

7O

TQAL V

WASHED CELLS

0

TCA PRECIPITATE

 

71

Cells were concentrated and pulse labeled 15 minutes then
diluted to 2-3x106/ml and grown in medium without label (Fig. 15).
At time intervals of constant size, equivolume aliquots were removed,
and the total counts in the purified virus was measured. The results
from this experiment show (1) a small number of counts is produced by
0.5 h. following the beginning of the pulse, a result which agrees with
the continuous labeling experiment described above, and (2) counts in
the virus peak continue to increase through the 6 h. aliquot. The
experiment was repeated (Fig 16) except that aliquots were taken

over a 24 h. period. These results show virus labeling levels at

 

about 11 h.

Since viral integrity did not appear to be destroyed, the above
experimental conditions were used for pulse-chase experiments in which
cells were pulse labeled and pelleted at constant intervals in order to
quantitate the virus produced in the entire cell supernatant during the
chase period. The experiment is described in figure 17. A parallel
experiment was performed (Fig. 18) in which cells were mock pulsed
at a high density, and pelleted periodically but resuspended in medium
containing 3H uridine. The results of these experiments in combination,
demonstrate that from pulse labeled cells, labeled virus is produced
maximally between 4 and 5 h. following the beginning of the pulse.

From continuously labeled cells, however, labeled virus production
continues to increase over a 10 hr. period.

To determine the behavior of the soluble (RNA precusor)
nucleotide pool in pulse-labeled cells, an experiment as described in
figure 19 was performed. From this it can be seen that by 2 h. 56%

of the TCA soluble pool is depleted.

 

Figure 15.

72

Incorporation of 3H uridine into purified virions after
pulse labeling. F-422 cells, 468x106, were incubated in
4 ml medium containing zoouci 3H uridine, washed 2x with

5 ml medium containing lOuM uridine then incubated in

200 ml medium containing luM uridine. At 0.5, 1.0, 1.5,
2.5, 4.5, and 6.5 h. following the beginning of the pulse,
33 ml samples were taken. Virus was purified by PEG
precipitation and isopycnic centrifugation, and the
gratients were fractionated and assayed for radioactivity
as described in Materials and Methods.

-2

H CPMX IO

3

 

 

0.5h

AAA‘A

2.5h

Ak‘A AAA‘
.5‘.

1

 

1
30

T
TOP

73

 

1.011
3..
4..
l:d=121::ttf??graunfnanu_1
4.5h
8—
4H

 

 

T
IO 20 30

FRACTION

1
“DP

I.5h

 

 

 

 

T—
6.5h
8 h—
4 .—
Il—w 2‘ 3' T2)?

74

Figure 16. Incorporation of 3H uridine into virions after pulse
labeling. Experiment was performed identically to that
described in figure 14 except that samples were taken at
4, 6, 9, 11, 12.5, and 24 h. following the beginning of

the pulse.

H CPM x lo“3

3

fl

 

4h

 

 

 

 

Nb
4
I
r r i
To 20 30

75

6h

 

 

12.5h

 

 

 

 

 

 

 

 

 

 

T
9 h ’
1 j I 1
F‘
p I
24 h
I
H Tfi I I ‘1
IO 20 30 70"

 

Figure 17.

76

Incorporation of 3H uridine into purified virions after
pulse labeling. F-422 cells, 66 x 106, were incubated in
1 m1 of medium with 50 uCi 3H uridine for 15 min., washed
2X with 5 ml medium containing 10 uM uridine. Cells were
then incubated at 2 x 106 cells/ml in medium containing

1 uM uridine. At hourly intervals, cells were pelleted
and resuspended to 2 x 106 cells/ml in fresh medium.
Virus was purified by PEG precipitation and isopycnic
centrifugation, and the gradients were fractionated and
assayed for radioactivity as described in Materials and
Methods.

77

 

203.32...

 

 

 

 

 

 

 

 

0

 

 

doe can o.~. m: doe an o.~. M: a9 mm mm m. mg on mu m. d9 an om. m.
“I - \Ltlril ql fig} 1 o If q N17}
,.
l u.
“
IA 1
.3 ,5 is fan :3.
a l u
a n c .r _ .I L _ T. _ u _ M L T. L 4
.fl V n I y‘tixla t
1 _ - -
, \
Lnd fflu 1m:— EEOm EEm—

 

 

 

 

 

 

z_01 x we IIc

 

rh

Figure 18.

78

Incorporation of 3H uridine into purified virions, with
continuous labeling. F-422 cells, 66 x 106, were
incubated in 1 ml medium for 15 min. (to simulate pulse
labeling described in figure 17), washed 2X with 5 ml
medium, then resuspended to 2 x 106 cells/m1 with l uCi/ml
3H uridine. At hourly intervals, cells were pelleted and
resuspended to 2 x 106/ml fresh medium containing 1 uCi/ml
3H uridine. Virus was purified by PEG precipitation and
isopycnic centrifugation, and the gradients were
fractionated and assayed for radioactivity as described

in Materials and Methods.

3H CPM x I0‘2

2h

 

 

79

4h 6“

 

H?

 

 

 

 

Ix

IO 20 30 TOP

 

 

 

8h 10b
12 — -
6 ,_ _.
\/ A...
J
I I T J I T
TO 20 30 TOP '0 20 30 TOP

FRACTION

Figure 19.

80

Acid soluble pool in F-422 cells after pulse labeling with
H uridine. F—422 cells, 50 x 10 , were incubated 15 min.
in 1 ml medium containing 10 uCi 3H uridine. Cells were
then washed 2X with medium containing 10 uM uridine, and
resuspended to a concentration of 2 x 106 cells/ml in
medium containing 1 uM uridine. 0.2 m1 aliquots were taken
in duplicate at 0.5, 0.75, 1.0, 1.5, 2.5, 4.5, and 5.5 h.
following the beginning of pulse labeling. Radioactivity
was measured in (l) washed whole cells after NCS tissue
solubilization, and (2) washed TCA precipitate after
solubilization. NCS solutions were counted in 5 ml
Aquasol. The TCA soluble pool was determined by sub-
traction.

°H CPM x 10“

81

 

 

   
 

8L
P—K\
°\L\1‘o WASHED CELLS
6~ 0 NO“
O-—— 0
TCA PRECIPITATE
4_
'- 0
\N
0
2~ ‘\\\\\\TCA SOLUBLE POOL
0\
.— O\
U
1 l I L

 

HOURS

82

Incorporation of 3H uridine into FeLV during pulse-chase
and continuous labeling. Total counts in the 7 peak
fractions for each gradient depicted in Figures 17 and 18
was plotted against time. Time is measured from the

beginning of labeling in each experiment.

Figure 20.

 

83

   

 

10'-
8r—
I.
CONTINUOUS
6r—
7
O
X L—
z
m
U
”I
4..
I.
2 0 PULSE-CHASE
O
O
I“ \
O
I J L L .l J
4 6 8

84

DISCUSSION

Electron Micrographs of F-422 Cells and FeLV-R

 

Electron micrographs of thin-sectioned feline thymus tumor
cells reveal particles external to the cell as well as budding from the
cell which fit the size and qualitative descriptions of the FeLV-R
particles (89). High magnification of the particles from a preparation
by thin sectioning have the same dimensions (100 mu diameter) as
particles negatively stained and prepared from an isopycnically
centrifuged preparation having a density of 1.14 grams/cc. This argues
that particles purified isopycnically are the same particles identified
as virus in the thin section. Particles not fitting the exact
description for FeLV were sometimes seen and this leaves open the
possibility of cell vesicular contamination. Aggregates of virus
particles were seen but this may be an artifact developing during the

purification process.

Synchronization of F-422 Cells

 

All attempts to synchronize F-422 cells proved unsuccessful.

1. Thymidine Excess.--Cells appeared to be toxic to levels

 

of thymidine ranging from 2 mM to 0.2 mM by the criteria of cell

viability and growth. Altering the blocking period within a range of

85

8 to 18 h. did not alter the toxic effect. F-422 cells take up
thymidine as well or better than MOPC-ZlF cells. The cause of the

toxicity is not known.

2. Serum-Free Medium.--F-422 cells did not grow in serum-free

 

medium, and when serum was added or cells were suspended into fresh
medium after 10-16 h. of incubation in serum—free medium, a lag period

of 10 h. was observed before cells grew asynchonously.

3. Isoleucine-Free Medium.--Attempts to synchronize F-422

 

cells in isoleucine-free medium were hampered by an improperly formu-
lated amino acid-free medium mixture from GIBCO. Experiments were not

repeated with the corrected medium mixture now available.

4. BSA Gradients.-—F-422 cells were isopycnically centrifuged

 

for the purpose of fractionating a population into groups of cells,
each group being at a specific stage in the cell cycle. Two groups
were resolved, neither of which grew synchronously. Fractionating the
cells by velocity sedimentation might have yielded more fruitful
results. This experiment raises the question of what the upper band

cells were.

Purification of FeLV-R

 

In view of the fact that mammalian cells produce vesicles (91)
the question persists as to what extent cell vesicles contaminate
purified virus preparations. It is conceivable that vesicles would
contain cytoplasmic RNA thus giving rise to a particle having nearly
the same density as virus. Usually a two-step procedure is used for

purifying RNA tumor viruses. (1) Salt precipitation using ammonium

86

sulfate (27, 90) or potassium tartrate (14), followed by isopycnic
centrifugation, or (2) pelleting (6, 17, 22, 33) followed by isopycnic
centrifugation. At times velocity sedimentation is used followed by
isopycnic centrifugation (22). In spite of this, apprehension that
cell vesicles contaminate virus preparations persists.

Because electronmicrographs of the feline thymus tumor cell
(F—422) hint at the existence of vesicular material and because of a
trailing shoulder on iscopycnically purified virus a series of experi-
ments as described in figures 9 and 10 and table 4 were performed.

From the results in figures 9, 10, and table 4 the following
conclusions are made.

1. Peaks 1 and 2 of figure 9 are virus particles since each
contains 50-608 viral RNA. Perhaps the leading peak is
aggregated virus.

2. Peak 3 contains no 50-608 RNA, and is therefore not virus.

It does contain RNA which may possibly be ribosomal RNA
although no distinct 18S and 288 peaks were seen. Peak 3
which sediments much slower than virus is a good candidate for
cell vesicles. However, no 48 RNA was seen either, which is
surprising if peak 3 does represent cell vesicles.

3. Material in peak 3 (vesicle particles?) partially contaminates
the virus peak when the preparation is isopycnically centrifuged
2.5 h. (the condition described in figure 10). From figure 10,
52% of the peak 3 counts would be in the 1.14 g/ml density
region which would be collected as purified virus (fractions

11-21).

87

4. Material in peak 3 is the source of the trailing shoulder
normally seen in 2.5 h. isopycnically centrifuged virus
preparations (under the conditions described).

5. A velocity sedimentation purification step is recommended to
purify virus from particulate contamination which is presumed
to be cell vesicular material.

It will be noted that in part II of this thesis that FeLV-R for
RNA studies was purified simply by pelleting the virus twice through a
20% w/w sucrose solution. Under conditions in which RNA degradation
can be ruled out, no 188 or 288 RNA is ever seen in these preparations
and indeed very little RNA in the 108 to 408 region is seen. Con-
tamination from the putative cell vesicle as described above does not
seem to exist on the basis of RNA present in the purified virion.
Perhaps vesicles sediment through the sucrose too slowly to pellet
under the conditions used.

The kinetic studies, discussed later, in which the rates of
virus production were measured, were done without knowledge of the
putative cellular contaminant. The effects of such a contaminant,
however, were minimized since radioactive counts in the trailing

shoulders were ignored.

Infection of Crandell CCC Cells

 

While FeLV, like other leukemia viruses, does not transform
the cell it infects and replicates in and hence is not a virus with
which to study 12_vitro transformation, it is of interest for studying

replication. It does cause leukemia, a malignancy. And in this broad

sense it does transform normal cells to malignant cells. In the

88

narrow common usage of the term, "transformation" refers to the visible
alteration of a fibroblast (or other cell type) 12.yitrg_brought on by
infection with a virus.

There were two reasons for infecting a normal cell line with
FeLV. (1) It was not possible to obtain an uninfected thymocyte
(thymic lymphocyte) cell line to use as control cells during experi-
mentation either from (a) primary cultures of fetal thymus tissue in
our laboratory, or (b) an established cell line from another source.
And hence infecting another established cell line (ca. Crandell-CCC)
would be an alternative solution. There was the unfortunate possibility
that infected CCC-H cells would not prOduce FeLV at the same high rate
as F-422 if infected. This would be a disadvantage. (2) It was of
great interest to have an oncornavirus system in which the latent
period between infection and appearance of progeny virus was extremely
short, for example, less than 8 h., in order to study the obscure
events (e.g., the necessity for DNA synthesis) during the infection
process.

At the time our laboratory received the Crandall-CCC cells
(1971) they had been grown for over 5 years in tissue culture at
Cornell. During this time there was close monitoring by electron
microscopy for virus production. Efforts to induce an endogenous
virus with thymidine analogues were unsuccessful. It appeared to be
a good candidate for an uninfected control cell.

Since this time, RD-ll4 has been induced from CCC-A cells

(46, 72). These data open the possibility that endogenous virus was

89

induced by infection with FeLV-R. Possibly a mixture of RD-114 and
Rickard-FeLV are being produced in the ”infected" cell.

There is no apparent reason why cells did not become infected
during the experiments to determine the eclipse period. It was an
error not to prepare a simultaneous parallel flask for continuous

passage and for future assays of virus production.

Effects of Cordycepin on Virus Production

 

Philipson, et al. (87), demonstrated that cordycepin (3'
deoxyadenosine) inhibits almost totally the appearance of mRNA into
cellular cytoplasmic polysomes, and in adenovirus infected cells,
cordycepin prevents the accumulation of 3H adenosine into polysomal
RNA late in the infection by 85-90%. Since cordycepin does not
inhibit the synthesis of large heterogeneous nuclear RNA (HnRNA)
(thought to be messenger precursor RNA), and since adenovirus mRNA
is synthesized in the nucleus, and possesses poly(A) apparently
identical to cellular mRNA, it was concluded that poly(A) is necessary
for the processing of HnRNA into mRNA or for the transport of mRNA
to the cytoplasm.

Because oncornavarius RNA possesses poly(A) resembling very

closely the poly(A) found on mammalian mRNA, an assumption was made

that FeLV RNA synthesis and transport might be similarly affected by
cordycepin. That is, it was predicted that FeLV RNA synthesis and/or
transport would be inhibited by cordycepin, and the manifestation of
this would be an inhibition in the production of labeled progeny virus.
The effects of cordycepin were surprisingly not as predicted.

Cells incubated continuously in 50 ug/ml of cordycepin suffered mildly

90

at 4 h. a 50% viability when compared to 90% viability in control
cells. By 8 h. cells were only 10% viable. Cordycepin is clearly
toxic to cells at 50 ug/ml.

After 4.75 h. of incubation, as much radioactivity could be
,found in virus from cordycepin treated cells as from control cells.
While this was not predicted, possible explanations might include: (1)
Cellular polysomal and adenovirus-specific polysomal inhibition as
reported by Philipson, et a1. (87). was measured within 45 min. after
cordycepin treatment, whereas the FeLV detected in these experiments
was labeled over a 5 h. period. The effects of cordycepin might have
been reversed by this time. One would still expect a depressed
production of virus, but a minor depression may not be detected within
the sensitivites of this assay system. (2) Viral RNA, while possessing
poly(A) may not be affected in the same way as adenovirus mRNA and
cell mRNA. Poly(A) may serve an entirely different function in
oncorna viral RNA. Controls on processing and transport may not be
the same. (3) Viral RNA may, during cordycepin treatment, he synthe-
sized, processed and transported without acquiring a poly(A) segment.

It is known that some of the viral RNA does not possess poly(A)

(55, 67).

Kinetics of Incorporation of Uridine Into FeLV-R

 

For FeLV-R it was concluded that the minimum interval between
the uptake of radioactively labeled uridine and the appearance of
radioactive viral RNA in the released virion is 30 min. or less.

’This is considerably less than the 70 min. measured for MLV (6) or the

2 h. measured for AMV (10) and RSV-RAV1 (6). Since 3H uridine

 

91

concentrations were similar in all experiments (AMV, MLV, RSV-RAVI,
FeLV) the shorter interval for FeLV may relfect a more efficient
labeling system or possibly a more rapid viral synthesizing system.

It is interesting to note that East, et al., reported a 30 min. minimum
labeling interval for MLV-SD subsequent to the above studies (33).

The average interval for FeLV-R was measured to be between 4
and 5 h. This is longer than the 3-4 h. half-life interval measured
:fbr AMV (10), but the methods differed between the two determinations.

An exogenously added, differentially labeled FeLV in the
(experiments described in figures 15 and 16 would have demonstrated more

<:onclusively that viral integrity was preserved under the conditions

of the experiment .

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1C).

93

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Properties of Feline Leukemia Virus:

III. Analysis of the Ribonucleic Acid

David A. Brian and Leland F. Velicer

Department of Microbiology and Public Health
and
Department of Biochemistry
Michigan State University

East Lansing, Michigan 48824

1Article No. 6987 from the Michigan Agricultural Experiment
Station. Most of this work was submitted by D. A. Brian in
partial fulfillment of the requirements for the degree of Doctor
of Philosophy. Presented in part at the 75th annual meeting of
the American Society for Microbiology, 13-17 May 1974, Chicago, Illinois.

105

ABSTRACT

The interval between the uptake of radioactive uridine into
feline thymus tumor cells (Rickard, F—422) and its release into
virions (Rickard strain of feline leukemia virus, FeLV) was measured.
Labeled virus is found within 30 min after adding radioactive
uridine to the cells. Production of labeled virus reaches a maximum
at 4 to 5 h following pulse labeling. RNA from FeLV resolves into
three size classes when analyzed by electr0phoresis on 2.0% poly-
acrylamide-0.5% agarose gels: a 6.2 x 106 to 6.8 x 106 dalton mol wt
(50 to 608) class, a l x 105 dalton mol wt (85) class, and a 2 x
104 dalton mol wt (4 t0 5&3) class. These respectively make up
52 to 76%, 2 to 5% and 7 to 21% of the total RNA and respectively
represent 1, 3 to 10, and 23 to 122 molecules per virion. The 85
RNA in FeLV has not previously been reported. The 50 to 60$ RNA
from virus harvested after 4 h of labeling electrophoretically
migrates faster and sediments more slowly than the same RNA harvested
after 20 h of labeling. This was concluded to be the result of con-
formational changes in the molecule. Fifty to 60$ RNA dissociates
into subunits cosedimenting with 288 ribosomal RNA when analyzed
by velocity sedimentation through 99% dimethyl sulfoxide (DMSO).
Aggregates intermediate between 50 to 608 (825, DMSO= 7.09) and

28$ (3 = 4.22) suggest that conditions other than 99% DMSO

25, DMSO
alone are needed for complete dissociation of 50 to 60$ RNA.

106

INTRODUCTION

The RNA tumor viruses from all species studied to date possess
in common a high molecular weight, single-stranded RNA which is an
aggregate of smaller molecules thought to be held together by
hydrogen bonding (2,14,15,l8,l9,21,29,37,38). This aggregate can
be denatured into its subunits by heat, urea, formamide, formaldehyde
or DMSO, (2,14,15,17,18,21,22,29,37,38) and subunits of two major
sizes result: a large 28 to 35$ subunit which possesses stretches
of polyadenylic acid [poly(A)] showing characteristics similar to
the poly(A) tract on mammalian messenger RNA (25,27,32,42), and
small tRNA-like subunits which can be aminoacylated (20,22). In
addition to the aggregate there are smaller amounts of 368, 288,
18S, and 4 to 105 RNA present in the virion (10,11,14,15,18,l9,21,
29,33,37,41). In immature virus these may be preassembled subunits
which eventually become aggregated (10,11).

When this study was initiated, characterization of FeLV RNA
was limited to one report (29). Based primarily on velocity
sedimentation studies, it was shown that FeLV possesses a uniquely
large proportion of native 358 and 4 to 10$ RNA molecules when
compared to other oncornaviruses. This discrepancy made it necessary
to further characterize FeLV RNA under conditions which minimize de-
gradation with an analytic system giving superior resolution (gel

electrophoresis). These experiments were undertaken l) to characterize

the interval between the uptake of radioactive uridine into infected

cells and the release of radioactive virus, 2) to characterize the

107

native RNA molecules in FeLV of varying ages with respect to
molecular weight and percentage composition, and, 3) to determine

the molecular weight of the denatured high molecular weight subunit.

108

MATERIALS AND METHODS

Source of cells and viruses. The permanently infected feline
thymus tumor line, F-422, which produces the Rickard strain of
feline leukemia virus (FeLV) (40), and grows in suspension
culture, was obtained from Dr. C. G. Rickard (Cornell University).
Cells were propagated as previously described ( 26). The Kansas-
Manhattan strain of Newcastle disease virus (NDV) was obtained from

Dr. R. P. Hanson (University of Wisconsin).

 

Adaptation of F-422 cells for monolayer growth. Cells which
adhere to a flask surface were: selected by periodically
replacing media on a stationary flask of cells. A monolayer of
adhering thymocytes is detached by vigorous shaking. Progeny cells
can either form new monolayers in a flask or roller bottle, or be
grown in spinner suspension culture.

Isotopic labeling of FeLV RNA. Cells were incubated in 36 to
200 ml volumes in medium containing 10% fetal calf serum and either

3
H uridine ( 40 Ci/nM). 14

C uridine (57 m Ci/nM) or both as described
in the Results. New England Nuclear Corp. (NEN), Boston, Mass. was
the source for all isotopes.

Purification of FeLV. For studying kinetics of viral labeling,
virus was purified as follows. Cells were pelleted at 1000 rpm
(300 x g) for 5 min in an International PR-6 centrifuge, and cellular
debris was pelleted from cell supernatant at 10,000 rpm (16,000 x g)

for 10 min in a Sorvall GSA rotor. Virus was precipitated from

clarified supernatant in 50% (wt/vol) ammonium sulfate or 5% (wt/vol)

109

polyethylene glycol for 16 h at 4 C, and the precipitate was pelleted

at 10,000 rpm.for 20 min in a Sorvall GSA rotor and resuspended in

0.5 m1 TNE-7.5 buffer (0.01 M Tris, pH 7.5, 0.1 M NaCl, 0.001 M EDTA).
Virus was layered onto a 5.0 ml gradient of 15 to 40% (wt/wt) sucrose
made up in TNE-7.5 and isopycnically centrifuged for 2.5 h at 50,000 rpm
(240,000 x g) in a SW 50.1 rotor. Gradients were fractionated by drip-
ping through the bottom of the tube.

For studying viral RNA, FeLV was purified as follows. Clarified
cellular supernatant was layered over 8 ml of 20% (wt/tn;) sucrose made
up in TNE-7.5, and centrifuged at 25,000 rpm (84,000 x g) in 3 SW 27
rotor for 3 h. The viral pellet was resuspended into 1 ml of TNE-7.5
by 15 sec of sonication in a 150 Watt Branson ultrasonic cleaner (Branson
Instruments Company, Stamford, Conn.), layered onto 4.5 ml of 20% (wt/Wt)
sucrose in TNE-7.5 and pelleted at 45,000 rpm (190,000 x g) in a SW
50.1 rotor for 1.5 h.

RNA extraction. The viral pellet was resuspended in 0.3 ml
TNE-9 buffer (0.1 M Tris, pH 9.0, 0.1 M NaCl, 0.001 M EDTA) by
15 sec of sonication. The suspension was made 1.0% SDS by adding
10% SDS in TNE-9, and an equal volume of pronase (Calbiochem) at
SOO‘pg/ml in TNE-9 (self-digested 2 h at 37 C) was added to make
a final concentration of 250‘pg/ml. The solution was incubated
5 min at 37 C before extracting 3 times with an equal volume of TNE-

9 saturated phenol. When necessary, RNA was precipitated with or
without carrier (5 A26O/ml Torula grade B RNA, Calbiochem) in 67%

ethanol for 16 h at -20 C.

110

Preparation of cytoplasmic RNA markerg. Cytoplasmic RNA was
prepared from.3H uridine labeled feline thymus tumor cells by the
method of Erikson (22 ). Murine cytoplasmic RNA was a gift from
Ron Desrosiers (Michigan State University).

Preparation of 32P-NDV RNA. The lyophilized source of NDV was
passaged once in 9-day-old chick embryos. Allantoic fluid after
48 h contained 5 x 109 pfu/ml as assayed on baby hamster kidney
cells ( 12). Radioactive labeling, virus purification and RNA

extraction methods were modified procedures of Duesberg (16).

32
3

into the allantoic cavity. After 24 h incubation at 38 C, 10 3 to

Eight-day-old chick embryos were inoculated with H P94 (1 mc/egg)
104 pfu of NDV were inoculated via the same route. After 48 h
incubation (embryos were usually dead) allantoic fluid was collected,
frozen, thawed, and clarified at 10,000 rpm, 10 min.in a Sorvall GSA
rotor. Virus was pelleted through 8 ml 20% (wt/wt) sucrose made up
in TNE-7.5 buffer at 25,000 rpm for 4 h in a SW 27 rotor at 4 C.

The viral pellet was resuspended into 0.5 ml TNE-7.5 buffer by

15 sec of sonication and layered onto a 5.0 ml gradient of 65%

(Wt/Wt) sucrose in D O to 20% (Wt/Wt) sucrose in TNE-7.5. Virus was

2
isOpycnically centrifuged 15 h at 35,000 rpm (110,000 x g) in a

SW 50.1 rotor at 4 C. The virus was collected visually from a
dripping needle, diluted to 5 ml in TNE~7.5, pelleted at 45,000

rpm in a SW 50.1 rotor at 4C, and frozen at -76 C. RNA was extracted

as described for FeLV RNA. Twenty to 30% of the trichloracetic acid

(TCA) precipitable counts sedimented as SOS (30).

111

Polyacrylamide-agarose gel electrophoresis. The method of Peacock
and Dingman (39), as described by Bunting (9) was modified for use
in tubes to facilitate accurate fractionation of low percentage gels
in a Gilson gel fractionator. Ten tubes, 18 cm x 0.5 cm inside
diameter, each containing 3.0 m1 of 2.0% polyacrylamide-0.5% agarose
were prepared as follows. 0.16 grams of agarose (Biorad) was
refluxed 15 min with stirring in 22.6 ml water and cooled to 48 C.
Simultaneously, 3.2 ml of 20% acrylamide-bis acrylamide solution
[20%(wt/vol)water solution of cyanogum 41, Fisher] was combined
with 3.2 ml of Peacock's lO-fold concentrated electrophoresis buffer
(0.89 M Tris, 0.89 M boric acid, and 0.025 M EDTA in water) and
warmed to 48 C. Within 1 min, 1) 1.0 ml of a 1.6% (wt/vol) ammonium
persulfate solution was mixed into the agarose solution, 2) 2.0 m1
of a 6.4%(wt/voD 3-dimethylaminOpropionitrile solution was mixed
into the acrylamide-bisacrylamide buffer solution, 3) all solutions
were mixed well together and dispensed with a syringe and needle
into vertical tubes that had been treated with Photoflo (Kodak),
and dried. Gels were allowed to polymerize for 1 h at room temperature,
capped with parafilm and stored at 4 C. Storage for up to 6 months
seemingly caused no detrimental consequences. Just prior to use,
gels were slightly displaced with a Gilson gel piston and sliced
transversely with a razor blade, forming a flat surface for the RNA
sample. The gel was then retracted into the tube. Parafilm was
used to cap the bottom end of the tube and 12 to 15 holes were made

with a needle to allow for current flow.

112

Gels were me—electrophoresed 1 h at 150 V. Electrophoresis was
done at 150 V constant voltage at 4 C for approximately 3.0 h. Gels
were fractionated into 2 mm fractions with the Gilson gel fractionator.

Velocity sedimentation of RNA through_aggeous sucrose gradients.
RNA was centrifuged through a 5.0 m1 linear gradient of 20 to 50% (wt/wt)
sucrose (ribonuclease free, Schwarz—Mann) made up in TNE-7.5. Centrif-
ugation was for 80 min, at 45,000 rpm and 4 C, or 70 min at 45,000 rpm
and 20 C when the sucrose contained 0.1% SDS, in a SW 50.1 rotor.
Gradients were fractionated by dripping.

Velocity sedimentation of RNA through 99% dimethyl sulfoxide.

RNA was centrifuged through a 5.0 ml linear gradient of 5 to 20% (Wt/wt)
sucrose, ribonuclease free, made up in 99% dimethyl sulfoxide (DMSO).
Ninety-nine percent DMSO, 0.001 M EDTA, was prepared using reagent

grade 99.2% DMSO (Fisher),TNE'7.5 and a().2M.EDTA stock solution. The
pH of the final DMSO solution was brought to pH 8 using 1.0 N HCl.
Centrifugation was for 13 to 16 h (exact number of revolutions given

in the data) at 45,000 rpm in a SW 50.1 rotor at 25 C. Gradients

were fractionated by dripping.

Oligo (dT)-cellulose chromatography. Oligo (dT)-cellulose was
prepared essentially as described by Gilham (24) and the chromatographic
method was essentially that of Aviv and Leder (1). RNA sedimenting
with a 525’ DMSO of 4.22 S on DMSO gradients was precipitated in 67%
ethanol after making the pooled fractions 0.1 M NaCl. The precipitate
was dried in a nitrogen stream, redissolved in high salt buffer (0.0]

M Tris, pH 7.4, 0.5 M NaCl, 0.001 M EDTA, and 0.2% SDS) and applied to

a column (0.5 x 6 cm) previously equilibrated with this buffer. Poly(A)(-)

 

113

RNA was eluted by washing the column with high salt buffer. Poly (A) (+)
RNA was eluted with low salt buffer (0.01 M Tris, pH 7.4, 0.001 M EDTA,
and 0.2% SDS).

Radioactivity assay. Entire fractions or samples of fractions from
isoPycnic gradients were dripped or spotted, respectively, on 2.3 cm
Whatman paper filter discs, air dried precipitated 20 min at CC in
5% TCA, washed one min with 5% TCA, and one min with acetone, air dried,
and counted in toluene-POPOP in a Packard Tricarb liquid scintillation
spectrometer. Entire fractions or partial fractions from velocity sedi-
mentation gradients were assayed as described above, or counted directly
in 5 m1 Aquasol (NEN). Gel fractions were digested 1 h at 50 C in 0.1 ml

NCS tissue solubilizer (Amersham/Searle) then counted in 5‘m1 Aquasol.

 

I\

 

114

RESULTS

Kinetics of viral labeling. When cells infected with
oncornaviruses are labeled with a continuous source of radioactive
uridine, radioactive RNA from progeny virus appear as early as 2 h
for Rous sarcoma virus-Rous associated virus, (RSV-RAVI) (3 ),

1.5 h for avian myeloblastosis virus (AMV) (4), 80 min for Rauscher
murine leukemia virus (MLV) (3) and 30 min for Sohner-Dmochowski
murine sarcoma virus (SD-MSV) (18). Continuous labeling exPeriments
using F-422 feline thymus tumor cells (Fig. 1) demonstrate that
radioactive virus is detected as early as 30 min after addition of
the radioactive RNA precursor. This minimum interval is similar to
the 30 min interval for SD-MSV (18).

When infected cells are pulse-labeled 15 min with radioactive
uridine, radioactive RNA is release into progeny virus at a
maximum rate of 5 h for RSV-RAV1 and MLV (3), and between 3 and
6 h for AMV (4). Pulse-chase experiments during which 56% of the
intracellular nucleotide precursor pool disappears by 2 h (D. Brian,
data not shown) demonstrate that production of labeled virus reaches
a maximum at 4 or 5 h following the beginning of the pulse (Fig. 2).
These results with the feline leukemia virus are similar to those

for RSV-RAV MLV (3) and AMV (4).

1’

 

 

Figure l.

115

Incorporation of 3H uridine into purified virions during
continuous labeling. F-422 cells at 107 cells/ml were
incubated with 4 uCi H uridine/ml. At 0, 0.5, l, and

2 h, 6.5 ml samples were taken. Virus was purified by
ammonuim sulfate precipitation and isopycnic centrifugation,
and the gradients were fractionated and assayed for radio-
activity as described in Materials and Methods.

3H CPM X IO"4

116

Oh

 

lh

_

 

 

LWI/¥ I

IO 20 30 TOP

0.511

 

 

2h

 

 

I 1

l l
IO 20 30 TO

FRACTION

 

 

Figure 2.

117

Incorporation of 3H uridine into FeLV during pulse-chase
and continuous éabeling. Pulse-chase labeling (o). F-422
cells (6.6 x 10 ) were pulse-labeled 15 min in 2 ml medium
with 50 uCi H uridine, washed twice with 5 ml medium
containing 10 uM uridine, and resuspended in 33 ml medium
containing 1 uM uridine. Cells were periodically pelleted
and resuspended in fresh medium containing 1 uM uridine.
Virus from each collection was purified and its radioactive
content was assayed as described in Materials 3 d Methods.
Continuous labeling (o). F-422 cells (6 6 x 106) were
incubated in 33 ml medium with l uCi/ml 3H uridine.
Periodically cells were pelleted and resuspended in 33 ml
fresh medium containing 1 uCi/m1 H uridine. Virus from
each collection was purified and its radioactivity content
was assayed as described in Materials and Methods.

 

3H CPM x I0"3

'0‘

118

 

CONTINUOUS

PULSE-CHASE

 

 

119

Ge1_glectrophoresisA§pd velocity sedimentatiopgof native viral
‘RNA, Velocity sedimentation of FeLV RNA in aqueous sucrose gradients
does not resolve the 4 to 108 species (19,29). A recent gel electio-
phoretic study of FeLV RNA focuses only on the high molecular weight

species and its subunits (46).

In an effort to resolve the entire RNA content of FeLV into
size classes and to obtain an approximation of corresponding molecular
weights and percentage composition, electrophoresis was done on

combination gels of 2.0% polyacrylamide-0.5% agarose (39). Figure 3

illustrates the results obtained when virus is uridine labeled for
4 h or 20 h and the RNA is extracted and electrOphoresed. In each

case three molecular weight size classes are identified: a 6.2 x 106

to 6.8 x 106 dalton mol wt class, a l x 105 dalton mol wt class and

a 2.5 x 104 dalton mol wt class. These make up 52 to 76%, 2 to 5%

and 6 to 12% of the total RNA respectively, with the remainder being
heterogeneously dispersed throughout the gel, and they represent respec—
tively l, 3 to 10, and 23 to 122 molecules per virion. From sediment-
ation data, these correspond respectively to 50 to 605, 83 and 4 to SS
classes. The sedimentation coefficient of the 1 x 105 dalton mol

wt class was not measured, but its electrOphoretic mobility appears
identical to the 8S Species reported in the murine sarcoma virus
(20,33), and is therefore called 88. Molecular weights were determined
by reference to feline cellular 4S tRNA, and 18S and 288 rRNA run

in parallel gels under identical conditions, and to differentially

labeled SOS NDV RNA (30) included in all gels with FeLV RNA. Under

Figure 3.

120

Coelectrophoresis of native FeLV RNA, feline thymus tumor
cell rRNA and tRNA, and NDV RNA on 2 0% polyacrylamide-0.5%
agarose gels. 4F-422 cells at 2 x 106/m1 were incubated 20 h
with 2.5 uCi c uridine and 4 h (the lagt 4 h of the 14c
uridine labeling period) with 20 uCi/m1 H uridine to provide
doubly labeled virus as a source of FeLV RNA. Fifty micro-
liters of phenol-SDS-pronase extracted RNA from tgs purified
FeLV was used to solubilize ethanol precipitated P NDV
RNA marker (see Materials and Methods). Final solution was
then made approximately 10% sucrose and QQOOSZ bromophengl
blue and electrophoresed 3 h. Insert: P NDV RNA and H
uridine labeled feline thymus tumor cell 283, 188 ribosomal
and 4S tRNA were coelectrOphoresed on parallel gels. Four

S tRNA and SOS NDV RNA positions on the two gels were super-
imposed.

121

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(“T-'1’) z-O' X WdO Hg

 

 

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IIIIII I I I IIIITIIT I W'+ A r I
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DISTANCE MOVED ( mm 1

122

these electrophoretic conditions a straight line relationship existed
for migration distances versus molecular weights for all marker
molecules (see insert, Fig. 3). The electron microsc0pically determined
molecular weight of 5.2 x 106 to 5.6 x 106 daltons (31) was used
for NDV. To use this graph for molecular weight estimates of 50 to
608 viral RNA mol wt one must assume that a straight line can be
projected for up to 3 fractions beyond NDV (insert, Fig. 3). Shoulders
on the 4S and 8S peaks suggest that other minority species might be
present which would require gels with a smaller pore size for resolution.
Velocity sedimentation in sucrose gradients (Fig. 4) resolves
FeLV RNA into a broadly sedimenting 50 to 608 class and a group
of unresolved molecules sedimenting betwen 4 and 108. The sedimen-
tation coefficient of 50 to 605 is determined by the method of
Martin and Ames (36) in reference to differentially labeled SOS
NDV RNA included in the gradient.
One constant observation is the existence of much (25 to 50%)
TCA precipitable RNA in the 4 to 10$ region of velocity sedimentation
gradients (Fig. 4). This percentage is similar to the findings of
Jarrett, et. al., in which phenol extracted FeLV RNA in analyzed on
sucrose gradients (29). By contrast, 4 to 108 RNA on gels represents
less than 20% of the total (Fig. 3). Making the sucrose solution 0.1%
SDS did not decrease the occurrence of small molecular weight RNA in

the gradients.

 

Figure 4.

123

Velocity sedimentation of FeLV RNA and NDV RNA. FeLV

RNA was labels? and prepared as described in Fig. 3. Ethanol
precipitated P NDV RNA was solubilized with FeLV in
extraction buffer before sedimenting. Sedimentation was

as described in Materials and Methods. Fractions were
counted directly in Aquasol.

124

 

AIV N|O_ X 2&0 U!

 

 

 

 

30 TOP

 

 

 

Ti To. x .28 1»

2‘0
FRACTION

Io

125

Effects of labeling time on viral RNA. In three out of three
experiments in which virus was doubly labeled, 4 h with 3H uridine
and 20 h with 14C uridine, the 50 to 608 RNA of 20 h RNA electro-
phoretically migrated 0.5 to 1.0 fraction slower than the 50 to 60$
RNA from 4 h virus, giving an average difference of 0.7 fractions(Table 1).
This represents an apparent difference in molecular weights of 0.5 to
1 million daltons in this region of the gel (insert, Fig. 3). By

comparison to reference RNA molecules, mean molecular weight estimates

 

of 6.2 x 106 daltons and 6.8 x 106 daltons are derived for the 4 h and
20 h high molecular weight molecules respectively (Table 1).

This apparent size difference in the 50 to 60$ RNA between 4 h and
20 h labeled virus is confirmed and measured by velocity sedimentation
in aqueous sucrose gradients (Fig. 4). The results of two experiments
are summarized in Table l. The mean sedimentation coefficients are 50.48
for 4 h viral RNA and 59.68 for 20 h viral RNA. 32P NDV SOS RNA was an
internal marker for each gradient.

These results confirm the findings by East, et. a1. (19) in which
FeLV RNA collected after 2 h of labeling had a sedimentation coefficient
of 50S while after 20 h of labeling had a coefficient of 588. The
explanation given by East, et.a1. (18), is that a structural modfi-
Cation of the RNA, either by a joining of subunits, or an altered
secondary structure to an already assembled molecule, gives the older
form of the viral RNA (20 h) a higher sedimentation coefficient. If

this explanation is correct, then one should see a more slowly sedi-

menting 50 to 60$ molecule for virus produced over any 2 (19) to 4 h

126

 

 

 

 

 

 

 

 

 

 

 

 

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mucmaflummxm q no mmmum>m m
muaoaflummxo m mo ommum>m .M
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OH x N.O OH x N.O OH x N.O OH x w.O OOHUHOOO Hum scum OmumaHuwu AmaouHmOO
o o o o uswaos HMH=omHoE mwmuo>m udmummm< .m
n. w. n. I <zm >Aom .u: 0N amnu Hoummm woumnmwa
mzm >Aom mfioauomuw mo nonasz .N
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<zm >Aom wdoauomum mo uwnasz .H
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Amunozv mafiamnma mDHH> mo Hm>umuaH
«mm mom >Oz ou w>HumHmu
<zm momlom >Awm mo coaumHaoEHcmm uHooao> cam aOHumu Ma oHuouon ouuumam .H mqm<a

 

127

period, not just the first period following addition of isotope. To
test this, cells which had undergone 20 h of labeling with 140 uridine,
and 4 h with 3H uridine, were resuspended in fresh medium (with no
isotopes) for an additional 4 h period. Hence, 14C uridine labeled
viral RNA becomes incorporated into the virion during the 20 to 24 h
interval post-labeling from an intracellular pool of l4C-labeled viral
RNA. Likewise the 3H—labeled RNA becomes incorporated during the 4 to 8
h interval post-labeling.

The 50 to 608 RNA incorporated during the 4 to 8 h and 20 to 24 h
post-labeling periods electrophoretically comigrate (Fig. 5). Averages
of several experiments show them respectively migrating 0.8 and 0.7
fractions faster than 0 to 20 h RNA (Table 1). Sedimentation analysis
appears to confirm the electrophoretic data since an average of 50.78
and 56.58 are found for 4 to 8 h and 20 to 24 h RNA respectively as

compared to 59.68 for 0 to 20 h RNA (Table 1).

Recent studies with RSV demonstrate the existence of unassembled
358 subunits in newly formed virus particles, particles 3 min to 1 h
in age (10,11). Such subunits were sought in l h FeLV. F-422 cells
adapted for adherence to flasks were used to facilitate rapid medium
changes. Adapted cells were grown to concentrations of l x 106 ml
in spinner culture and then placed in a roller bottle to which most of
the cells adhered forming a near-monolayer in 8 h. Unattached cells
were removed by pipetting, and adhered cells were incubated with tritiated

uridine for 16 h, following which, collections of cell supernatant were

made at 1 h intervals. Figure 6 demonstrates the coelectrophoresis of

 

Figure 5.

128

Coelectrophoresis of FeLV RNA and NDV RNA. Cells from the
experiment described in Fig. 3 were resuspended in fresh
medium (no labeled uridine) for 4 h. All other steps were
carried out as described in Fig. 3 except that FeLV RNA
was ethanol precipitated with carrigr before dissolving in
electrophoresis buffer along with 3 P NDV RNA.

129

 

AEEV ow>0_2 moz<._.m_o

 

 

ON. Om O? I
q ..

 

 

 

 

mv

 

 

26 1 ON
mM. mum 52.: momIOm

 

 

(-——-) ._Ol x was H,

 

 

Figure 6.

130

Coelectrophoresis of FeLV RNA and NDV RNA. FeLV labeled
1 h wi h H uridine was prepared separately by incubating
4 x 10 F-422 cells in monolayer with 200 uCi uridine for
16 h, after which a series of 8 l-h collections were made.
Cell supernatant was kept at O C until all collections
were made. Virus was purified immediat§%y. Virus puri-
fication and RNA extraction as well as P NDV RNA
preparation was as described in Materials and Methods.
Electrophoresis was as described in Fig. 3.

131

(-—~) .-ou x was s,,

v

N

 

AEEV

om>os_ moz<._.m_o

 

 

 

ON. m w
.. A _ q fi —
66 A_..o\e..e.o.e.e.e. .o.o. .o.o.o.o.o.o.o.o/o.o. .o
.o/ O _ o 1»
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,, Ho ,1.
Poi 0
mm
5¢NIONIV
mu.
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(om-o) 9-0: x was H.E

132

3H—labeled l h viral RNA prepared from monolayer cells with 32F NDV

RNA. No 358 subunits were found in the l h virus (Fig. 6). The 48,
88 and 50-608 RNA species were present in the amounts of 21, 3 and 58%
respectively.

Velocity sedimentgtion of viral RNA through 99% DMSO. To
determine whether differences in size (length) or integrity of the
50 to 608 RNA subunits might explain the observed differences in

sedimentation and migration behavior of the 4 h and 20 h 50 to 608

 

molecules, the following experiment was done. Total RNA from virus
double-labeled 20 h with 14C uridine and 4 h with 3H uridine was
denatured inSM)% DMSO, a treatment known to completely denature
single-stranded and double-stranded RNA (44) and to dissociate

high molecular weight RNA from oncornaviruses into its subunits (2,14).
Denatured viral RNA was then analyzed by velocity sedimentation in
sucrose gradients made up in 99% DMSO. The rationale was that

1) if subunits are identical in length then superimposible
sedimentation profiles will be seen since the sedimentation coef-
ficient of RNA in DMSO is a direct function of its molecular weight
(44) and 2) if subunits contain discontinuities, for example from
ribonuclease nicks, its fractured integrity will become apparent

by a heterogeneous sedimentation pattern (L4). Twenty—eight 8 RNA
with a molecular weight of 1.8 x 106 daltons (35) which sediments in
99% DMSO at 25 C with a sedimentation coefficient of 4.228 (14) was
used as a marker to determine the s for FeLV RNA subunits

25, DMSO
by the method of Martin and Ames. The molecular weight was calculated

133

from the sedimentation coefficient using Strauss' equation:

325, DMSO = 0.052 M 0'31
Three observations were made. 1) Under conditions of complete
denaturation (Fig. 7A), 4 h viral RNA sediments as two major peaks:

a small molecular weight peak coincident with cellular tRNA, and

a large molecular weight peak nearly coincident with 4.228 (25,

DMSO) rRNA. (4.38),with a corresponding mol wt of 1.9 x 106

daltons. The 1.9 x 106 dalton mol wt species is known

to be the large subunits of 50 to 608 RNA since the latter RNA
purified on aqueous velocity sedimentation gradients denatured on DMSO
gradients to molecules primarily this size (D. Brian, data not shown)
and because they contain poly(A) (Fig. 8) a property of oncornavirus

large RNA subunits (27,32,42). It was not determined what fraction

of the 48 peak is contributed by 50 to 608 denaturation products. The 43

and 8S RNA species are not resolved by these sedimentation conditions.
2) Under conditions of complete denaturation (Fig. 7A) the high

mol wt 20 h RNA differs from the high mol wt 4 h RNA in two respects.
a) It sediments more slowly, 3.98 (25, DMSO) as compared to 4.38

(25, DMSO) with a corresponding mol wt of 1.2 x 106
daltons. b) It is broader, presumably containing a population of
molecules more heterogeneous in size. The slower sedimenting peak
comprises 50% of the 20 h RNA as compared to 20% for the 4 h RNA.
3) When ethanol precipitated viral RNA is dissolved and heat
treated (56 C, 15 min) in anything less thanEN)% DMSO, complete

dissociation may not occur (Fig. 7B , C , D). Fig. 7D demonstrates

the resolution of two RNA peaks in RNA treated 56 C, 5 min in 80%

 

Figure 7.

134

Velocity sedimentation of FeLV RNA and murine cytoplasmic
RNA through 99% DMSO. Gradients were made, centrifuged,
fractionated and assayed as described in Materials and
Methods. The position of 28S rRNA (4.228 in 99% DMSO

at 25 C) was determined in separate gradients run under
identical conditions. l4C-labeled 20 h FeLV RNA and
3H—labeled 4 h FeLV RNA were prepared as described in
Fig. 3 and ethanol precipitated with carrier. Pelleted
precipitate was dried in a nitrogen stream, solubilized
in water with 0.001 M EDTA, 0.1% SDS, made 90% DMSO,

and treated 5 min at 58 C before layering onto the gradient.
Centrifuged 43.192 x 10 revolutions. B. Same as A
except that RNA was heated 56 C, 5 min in 80% DMSO.
Centrifuged 40.510 x 106 revolutions. C. Same as A
except that RNA was heated 56 C, 5 min in 84% DMSO.
Centrifuged 33.879 x 106 revolutions. D. Same as A
except that RNA was heated 56 C, 5 min in 80% DMSO.
Centrifuged 42.517 x 106 revolutions.

135

(°—°) ..01 x was 0,,

 

4.22 S

E

 

 

 

 

 

 

 

 

 

 

 

 

4.22 S

 

 

 

 

4.22 S

 

 

4.22 S

 

 

 

 

 

 

 

 

 

 

 

(-----) 3-0I x was H.,2

l0 20 TOP IO 20 TOP IO 20 TOP

20 TOP

FRACTION

 

 

Figure 8.

136

Oligo(dT)-cellulose chromatography. F-422 cells in
suspension at 3 x 106/ml were incubated 16 h with 4 uCi/ml

H uridine. Virus was purified and RNA was extracted as
described in Materials and Methods. RNA was ethanol
precipitated with carrier, dissolved in water with 0.001 M
EDTA, and 0.1% SDS, made 90% with 99% DMSO, heat dissociated
at 56 C for 5 min then sedimented through 99% DMSO, 40.113
x 106 revolutions. The 4.228 peak (profile was very similar
to Fig. 7A, data not shown) was collected, made 0.1 M NaCl,
and ethanol precipitated with carrier. The resultant
precipitate was dried in a nitrogen stream, dissolved in high
salt buffer and chromatographed on oligo(dT)—cellulose as
described in Materials and Methods. Elution with low salt
buffer started at fraction 16.

2H CPM x I0"2

 

 

 

137

POLY (A) (-) POLY (A) (+)

 

IO 20
FRACTION

 

138

DMSO prior to centrifugation. The faster peak sediments at 7.098

(25, DMSO) with a corresponding molecular weight of 7 x 106 daltons
using Strauss' equation. This assumes Strauss' equation holds

true for molecules considerably larger than 2 x 106 dalton mol wt
tobacco mosaic virus RNA (44). The 7.098 (25, DMSO) molecule

therefore appears to be undenatured 50 to 608 viral RNA. At

times when RNA from the same extract as sedimented in Fig. 7A and 7D is

heat treated 56 C, 5 min in 80% (Fig. 7B) or 85% (Fig. 7C) a peak

 

corresponding to the 1.2 to 1.9 dalton subunit peak begins to
appear. Peaks sedimenting faster than 4.38 (25, DMSO) are not
seen with heat treatments (56 C, 5 min) in 90% DMSO, and dissocia-
tion is assumed complete under these conditions (A. Thomason, data
not shown).

Olig9_(dT) cellulose chromatography. The 1.2 x 106 to 1.9 x
106 dalton mol wt subunits of viral RNA were confirmed to be 50 to
608 RNA subunits on the basis that 69.5% bound to oligo (dT)
cellulose in 0.5 M NaCl (Fig. 8). Under these conditions rRNA and
tRNA do not bind (1). Recovery from oligo (dT) cellulose columns

is complete.

139

DISCUSSION

The minimum interval for labeling FeLV in F—422 cells with an RNA
precursor (uridine) is 30 min. This short interval of virus production
is similar to the SD-MSV system described by East et a1. (18) but
shorter than the 80 min interval for MLV described by Bader (3) and for
the 1.5 and 2 h interval for AMV and RSV-RAV, and MLV (3) and AMV (4).
It remains to be determined whether the shorter interval for FeLV is

a reflection of a higher rate of virus production, more efficient uridine

 

incorporation, or merely a reflection of total cellular metabolic rate
differences. Too few counts were present to determine what RNA species
were present in the 30 min virus.

Analysis of FeLV RNA by gel electrOphoresis yields three size classes

6 dalton mol wt class sedimenting in

of RNA: 1) a 6.2 x 106 to 6.8 x 10
0.1 M NaCl with a 50 to 608 sedimentation coefficient and comprising 52

to 76% of the total viral RNA, 2) a l x 105 dalton mol wt class not yet
reported in feline leukemia or sarcoma viral RNA, corresponding to the 8S
RNA reported in MSV RNA (20,33), comprising 2 to 5% of the total RNA,

and 3) a 2.5 x 104 dalton molecular weight class sedimenting with 48
cellular tRNA in 0.1 M NaCl and comprising 6 to 12% of the total viral
RNA. Assuming that the 50 to 608 molecule exists at the rate of one per
virion, then the 88 molecule is present at the rate of 3 to 10 per virion,
and the 4 to SS molecule is present at the rate of 23 to 122 per virion.
Species sedimenting as 188, 288 or 348 are not detectable and in this
respect the Rickard strain of FeLV differs from that used by Jarrett et.

al., (29). The absence of 18S and 288 RNA argues that there is no cell

vesicular contamination. Perhaps the cellular material does not traverse

140

the sucrose barrier rapidly enough to be pelleted during virus
purification.

The apparent molecular weight of 6.2 x 106 to 6.8 x 106 daltons for
the 50 to 608 RNA is based on the demonstration by Peacock and Dingman
that a straight line relationship exists between the electrophoretic
migration distance of a single-stranded RNA molecule and the log of
its molecular weight (39). It was measured under conditions in
which a straight line was found among 5.2 to 5.6 x 106 dalton mol
wt NDV RNA, 1.8 x 106 dalton mol wt 288 feline rRNA, 0.5 x 106 dalton
mol wt 188 feline rRNA, and 0.2 x 105 dalton mol wt tRNA, but assumes
that a straight line can be projected up to 3 fractions beyond the NDV
marker RNA (insert, Fig.3 ). 'Using Spirin's equation (43):

M = 1550 x 52'1

the molecular weight for a 50 to 608 molecule is 9.7 to 11.7 x 106
daltons. Applying this equation to an aggregate molecule may lack
validity since the equation was derived from data using single-
stranded molecules ranging from 0.3 to 2.1 x 106 daltons. The
measured sedimentation coefficient of 50 to 608 agrees closely with
50 to 588 for FeLV-R measured by East et a1. (19). It does not
agree with the 74S measured by Jarrett, et. al. (29). It appears
therefore that a significant different exists between Rickard strain of
FeLV and other (A, B and C) strains which have been studied (46).

RNA from virions purified isopycnically yields larger proportions
of both 88 and 4S RNA (16 and 35% respectively) and a relatively

smaller proportion of 50 to 608 RNA (17%) (D. Brian, data not shown).

Presumably intravirion RNA degradation occurred during the lengthy

 

141

purification process (2). While 88 and 48 species were minimally
present in virus purified only by pelleting, the question remains
whether 88 and 4S RNA species present are degradation products of
50 to 608 RNA. Nucleotide analysis (7) may answer this question.
Notable is that small mol wt RNA species are absent in MLV prepared
similarly (2). Pelleting virus a second time through sucrose pre-

sumably purified them from ribonucleases present in the serum (R.

Patterson, personal communication) since this step improved the yield of

 

undegraded RNA. Noteworthy also is that 4 to 88 RNA routinely composes

 

a larger fraction (25 to 60%) of the total RNA when total RNA is
analyzed by sucrose gradient Centrifugation as compared to gel
analysis (8 to 17%). Recovery of counts on both gels and gradients
is equivalent. It appears, therefore, that degradation occurs
during sucrose gradient centrifugation, a process that is not
inhibited by making the gradients 0.1% SDS.

The electrophoretic mobility and the sedimentation coefficient
of the 50 to 608 molecule are variable depending upon the length of
time over which the virus was labeled. From virus labeled for 20 h,
high molecular weight RNA electrophoretically migrates with a mean
molecular weight of 6.8 x 106 daltons, and sediments in 0.1 M NaCl
with a mean sedimentation coefficient of 59.68. From virus labeled
for 4 h the corresponding mol wt and sedimentation coefficient are
6.2 x 106 daltons and 50,4s,These data confirm and extend findings
of East et al., in which the sedimentation coefficients for the

Rickard strain of FeLV were measured to be 58 and SOS respectively.

A similar phenomenon was observed in RSV (10,11). In

142

agreement with the maturational hypothesis, i.e., that the mod-
ification giving rise to a faster sedimenting molecule occurs within
the virion after budding, our data demonstrate that virus labeled
over any 4 h period, not just the first, possesses the faster
electrOphoretically migrating (slower sedimenting) molecule. Virus
labeled from 4 to 8 h, and from 20 to 24 h following addition of
label to the cells have electrophoretically estimated molecular weights
of 6.2 x 106 daltons in each case, but mean sedimentation coefficients
of 50.78 and 56.58 respectively. Electrophoretic data alone suggest,
and sedimentation data does not rule out that RNA in virus labeled during
the first 4 h is identical or very similar in size and arrangement to RNA
from.virus labeled over any 4 h period. The hypothesis favored by
others (10,11,18) to explain the maturational modification says that
subunit structures assemble into a larger molecule. In immature
(60 min) RSV, subunits from 158 to 608 are found which apparently
assemble into the larger aggregate of 688 (11). No subunits within
the range of 88 and 508 are found in FeLV collected at 60 min intervals.
Any assembly within FeLV would therefore involve two or more 528
aggregates to form a 598 aggregate (18). This would yield a molecule
of 21 x 106 using Spirin's equation, with a resultant sw of 1088. This
is a molecule far larger than any found in FeLV, and therefore the
above hypothesis seems implausible. An alternative explanation was
sought by observing FeLV subunits under complete denaturation.

The 60 to 708 molecules of RSV apparently retain their aggregate

structure while possessing nicks which are revealed only after the

 

143

aggregate has been denatured (2). Conceivably, nicks within the loops
of an aggregate molecule could allow the ends to fold more tightly by
way of secondary bonds and thereby create a slower electrophoretically
migrating (faster sedimenting) molecule compared to the unnicked
aggregate. Such a model would explain the faster sedimenting older
(and presumably more nicked) 50 to 608 viral aggregate structure.
Sedimentation of completely denatured FeLV RNA through gradients

of 99% DMSO suggests such a qualitative difference. Large subunit

 

RNA from 20 h virus sediments more slowly and more heterogeneously
than subunits from 4 h RNA (Fig. 7A) suggesting it harbors nicks
before denaturation. The proposed hypothesis is therefore a favorable

one and the apparent difference in molecular weights between 4 h and 20 h

50 to 608 RNA more likely represents a conformational difference.

Sixty-nine percent of the 4.228 (25, DMSO) RNA binds to poly
(dT) cellulose under conditions which will bond only poly(A) containing
RNA, and will not bind rRNA or tRNA (1). This confirms these are
subunits of the large 50 to 608 previously shown to possess poly (A)
stretches (27,32,42). It also argues that most of the 4.228 (25, DMSO)
subunits are intact. Finally, it establishes a procedure whereby
large subunit RNA can be prepared free of small subunit (4S RNA) and
rRNA contamination for analytic purposes.

It is noteworthy that denatured FeLV sediments in DMSO at the
same rate as 288 rRNA. Its molecular weight therefore is approximately
1.9 x 106 daltons, data which agrees with the results of East et al.,
in which heat denatured FeLV 50 to 608 RNA subunits sediment in aqueous

sucrose gradients with 28S rRNA. This contrasts sharply with the 5.358

144

(25, DMSO) subunit found in RSV (14) for which the corresponding
molecular weight is 3.1 x 106 daltons. It also contrasts sharply
with the results of Whaley and Jarrett et. al., (29,46) in which subunits
of 74S FeLV RNA sediment as 348 molecules, and had electrophoretically
determined molecular weights of 2.2 to 2.6 x 106 daltons. This further
substantiates a possible difference between Rickard's strain of FeLV
and other strains (A, B and C) studied (46).

From our data the 50 to 608, 6.2 x 106 to 6.8 x 106 dalton mol wt,
RNA molecule would possess 3 to 4 1.9 x 106 dalton mol wt subunits.
This agrees with the number of subunits calculated for RSV (14).

Strong forces apparently form the aggregate 50 to 608 molecule
since heat treatment in combination with 90% DMSO seems to be required
for complete denaturation. Ninety-nine percent DMSO at 25 C alone
seems ineffective for complete RNA dissociation since these are the
conditions under which the RNA is subjected during centrifugation and
in certain cases (Fig. 7D) no dissociated subunits are seen. Aggregate
structures intermediate between 60 to 708 and 34S occur in AMV RNA
during controlled denaturing conditions (45) and therefore it is not
surprising to find a similar phenomenon for FeLV RNA. What is surprising

is that any aggregate at all survives the 99% DMSO gradient (44).

 

  

145

ACKNOWLEDGEMENTS

We thank Alice 1. Swanson for excellent technical assistance.
This research was supported by Public Health Service Grants CA-lZlOl
and CA-l3l75 from the National Cancer Institute and also by Grant
DRG-123O from the Damon Runyon Memorial Fund for Cancer Research, Inc.
D. A. Brian was supported by Public Health Service Training Grant

GM-Ol9ll from the National Institute for General Medical Sciences.

 

146

LITERATURE CITED

Aviv, H., and P. Leder. 1972. Purification of biologically
active globin mRNA by chromatography on oligothymidylic acid-
cellulose. Proc. Nat. Acad. Sci. U.S.A. 69:1408—1412.

Bader, J. P., and T. L. Steck. 1969. Analysis of the ribo-
nucleic acid of murine leukemia virus. J. Virol. 4:454-459.
Bader, J. 1970. Synthesis of the RNA of RNA-containing tumor
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APPENDIX A

METHOD FOR POLYACRYLAMIDE-AGAROSE GEL ELECTROPHORESIS

(9) for
for use

1.

153

APPENDIX A1

METHOD FOR POLYACRYLAMIDE-AGAROSE GEL ELECTROPHORESIS

The method of Peacock and Dingman (39) as described by Bunting
making combination polyacrylamide-agarose gels was modified
in gel tubes. Reagents used were made up as follows.
Acrylamide--N,N'-Methylenebisacrylamide (Cyanogum 41, Fisher)

200 g. Cyanogum 41

water to 1 liter
Filtered. Stored in brown glass bottle at 4°C.
Tris-EDTA-Borate Buffer, pH 8.3 (Peacock's 10X Buffer).

216 g Tris-(hydroxymethy1)-amino methane

110 g boric acid

18.6 g disodium ethylenediaminetetraacetate

water to 2 liters
Solution is filtered and stored in a glass bottle. Solution
is ten times working concentration.
DMAPN, 6.5% (catalyst).

16 m1 3-dimethylaminopropionitri1e concentrated reagent

(Eastman Organic Chemicals)

water to 250 ml

Stored in brown bottle at 4°C.

 

1List of references are on page 146.

 

 

154

Ammonium Persulfate, 1.6% (catalyst).
0.16 g ammonium persulfate
water to 10 ml
Prepared fresh just before using.
Agarose, (Biorad) electrophoresis grade.
40% sucrose -1 mM EDTA-Bromphenol Blue Dye Solution (for
sample dilution).
20 g sucrose

0.5 ml 0.1 M Na EDTA

2
0.05 g bromphenol blue dye

 

water to almost 100 ml Adjust pH to 6.2 with 5N NaOH.
Water to 100 m1. Freeze in 0.3-0.4 ml aliquots.

Gels were made by the following protocol.

Protocol for making polyacrylamide-agarose gels.

155

 

 

20% Acryl-Bis 6.4% 10X AmmogiZm
Gel Agarose Water Solution(19:l) DMAPN Buffer Persulfate
l.5%-0.5% 0.16 g 23.4 ml 2.4 ml 2.0 ml 3.2 ml 1.0 ml
1.75%-0.5% 0.16 g 23.0 ml 2.8 ml 2.0 ml 3.2 ml 1.0 m1
2.0-0.5% 0.16 g 22.6 ml 3.2 ml 2.0 ml 3.2 ml 1.0 ml
2 5-0.5% 0.16 g 21.8 ml 4.0 ml 2.0 ml 3.2 ml 1.0 ml
3 0-0.5% 0.16 g 21.0 ml 4.8 ml 2.0 ml 3.2 ml 1.0 m1
3 5—0.5% 0.16 g 20.2 ml 5.6 ml 2.0 ml 3.2 ml 1.0 ml

 

l. Reflux agarose in water 15 min.
(stirring bar).

2. Combine 20% Acryl-Bis solution

3
to 48°C.

Cool agarose solution to 48°C;

4. Within one minute:

a.
b.

C.

Add ammonium persulfate to

Add DMAPN to Acryl—Bis-buffer solution.

agarose solution.

(19:1) with 10X buffer.

Combine all together, mix thoroughly, pour gels.

5. Allow to polymerize at 20°C for 1 h.

with constant stirring

warm Acryl-Bis and buffer

156

Gel tubes [18 cm. x 0.8 cm. outside diameter (0.5 cm inside
diameter)] were rinsed with Photoflo (Kodak), dried, stoppered at one
end with parafilm, and mounted vertically. A 30 ml syringe with a
5 inch cannula was used to rapidly fill the tubes. The quantity in
the above protocol (32 m1) will fill 10 tubes. Filled tubes were
stored for longer than 6 months at 4°C with no noticeable detrimental
consequences.

Just prior to use gels were slightly (2-3 mm) displaced with a
Gilson gel piston and sliced transversley with a razor blade forming
a flat surface for the RNA sample. The gel was then retracted into the
tube. Parafilm was used to cap the bottom end of the tube and 12-15
holes were made with a needle to allow for current flow. Gels were
preelectrophoresed for l h. at 150 volts.

A11 RNA analyzed by electrophoresis was radioactively labeled.
The concentrations of RNA preparations were estimated to be well within
the limits of 500 ug RNA/ml, and sample sizes within the limit of
10 ug RNA as advised by Bunting (9). RNA samples were dissolved in
either extraction buffer or electrophoresis buffer, mixed 1:1
(sometimes 10:1, in which case a small sucrose crystal was also added)
with bromphenol blue dye solution, and loaded onto the gel in 50 ul or
less. Resolution between 88 and 4S RNA species was not obtained when
sample sizes approached 100 pl.

Electrophoresis was carried out at 150 volts constant voltage
at 4°C using a Polyanalyst (Buchler Instruments). Gels were fractionated

into 2 mm fractions with the Gilson gel fractionator. Each fraction

 

157

was digested l h. at 50°C in 0.1 m1 NCS tissue solubilizer (New

England Nuclear) then counted in 5 ml Aquasol.

 

 

 

   
           
   

  
   

 

     

 

 

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