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HERPES SIMPLEX VIRUS! AMINO ACID
BEQUIREKWTB IN HUMAN SKIN CELL CULTURE

3!

Juno. x; Haxtnnnn

A THESIS

flaunt-d to
Michigan State Untvorcity
Mn partinl tnlfillnnnt of tho roqulrvnqntl
for the dogroo or

HASTEB 0? SCIENCE
Departnunt ot’Mteroblology and Public Health

19 67

(777/07
v- V. x“ ’3 I 1‘) 7’

BED IC 51‘ ICE

To I: puma”. thou support ma
guidance undo this work pouiuo.

imzwmm mam

nae apprecietim in extended to Dr. filter 5. neck
to: annealing end guidance reeeived throughout thie work.
The author in den indebted to Dr. Virginie no Hellman
end Dr. mm it. make for e eriticel review of the
unnecriyt. sine:- gratitude ie extended to fare. Bedeue
Bo Hinterten for techne‘iogieel eeeietenoe. The petience'
end mermaing at a: wife ie reeeivea with the «taut
eppmietiam ‘

11

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Influence of incubation timo on mount of
vine 331053006 in atticllnfi I‘dlte e e e e 0

Virus production in infected AU oene
incubeted for 1:3 noon in minimal eeeentiel
madman deficient in one eoino amid. e e e e e

time production by AU «no in been]. nedim
Eagle doflcltnt in one ”1210 ”16 e e e e o e

Amino eoid requirement: for time promotion
by the w cell in emeritus: to the 8539-2 cell

913CU331530 e e e e e e e e e e e e e e e e e e e

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1'83.

Effect of imbetioo tine on amount or vine
produced in exgeriaomtel ”die. e e e e e e e e e e 23

Amount et vine prodoeeo by w oeiie in minim
eeeentiei eedim deficient in one nino eoido e e e

womb of virue produced by no will in cmrserim

to that gyroduoed by Blip-2 oene in been}. medium
Bealeoericimtinoneeainoeoide e e e e ee e e 35

1V

L113? 01' FIGU’
Figure ;. 639

1. cmmmtive virue production by ii: cone in
woman free medium deficient in one mama acid. e 31o

2. cmztamtive time production by AH and EiEp-Z
none in ban]. sodium Eagle deficient in one
eoino acid e e e e e e e e e e e e e e e e e e e 3“

A“: “fit 4':- a «'- '1'
‘t 0 "Fee 4" ~- 1“"

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ii Erie-45.3 19322-463 .HI 3 I N iii. 5 3.3%: #3?" 3. ii C “Lei. €13 1-,4'1715 572' E

by Janos Hartmann

The eoino acid requiremente for yrofluction of herpee
virus in tieeue oeii onltnree were ltufiiefl. Each of thirteen
eeeentioi amino eoioe (L-ieomore) in minimuo.eeeootiei tediom
(HEM) or banal nefiium Eagle {333) were inflividualiy omitted
end the amount of virus prodooed eae assayed by plague
technique. rho requirements for the 53 {human akin epitheliuo)
were etufiied in gortiouleri Hip-2 (human epidorooid oeroinoaa)
nee investigated in comparieon to resuite with A3 ceiie. To
yrovide e comglete chemically derinefl mofiium mothyooiloloee
replaced strum.

.fiethionlne one the most and hietidine the ioeet required
to: in oolle in fififl. The methionine requirement lee ieee
marked with 833 than flfifi. Cyatine wee least required by both
an end Rap.2 when REE one eapioyed. Demonstration of en
erginino roguireoent to: viral replication degenéed on ite
concentration in the mediua eooioyed. either Box or aim.

AU cone and asp-2 oene had eioiiier requirxaente for
twelve of thirteen amino eoide in 3&3. In e.gioteoine deficient
median. Hog—2 cello grodooed one seventh the amount of virus
produceo by AH cello. whoa oomgarofl to the amount proéoeed in

coopiote mofiium.

James fiart‘mm

me amenity of deter-aiming the ogtimai time of enemy
for vine 93m infected eeiie in deficient eefiie. and the
impel-tome of mognizing met oofiie to analog were
oieoeeeed.

Ioraooucrxoa

1 Mel systole tor the etudy of human moor time and
tmor oen reletiGnehip wee needed. Since the art time
men I mar (vex-moo wiser“) in home, ieoletion em
ehereoterizetion of the vireo m e hoot cell eee highly
deeireble.

Beyeehi (22) deemetnted wort time intention by
inoefletion or pertieolete eert tieme onto cultured hm
ekin epithelium designated AD by wheeler et e1 (50). Twelve
other eeii lime were tented but only the A21 iine app-crud
north or the art time, detected by degerxeretion of the
eene. The vitae could be mtoultored only by peeeage or
inreoted oeue onto normal oeile, end it wee not ieoieted
tron cell-free tieme onitnre oediuo. Latent or nudged viral
growth mid motion occur. exhibiting inteotion only on
reputed peeoege. A biniaoiiy enriohed eediuo efforted but
growth or the agent.

the presence or ebeenoe or oertein amino eoide in tieme
emiture Iodine: hoe bean mm to markedly effect the mount
of vine produoeo (15) (36) (M). and the development of
11W! (29) (30) (36). The pertionler mino eoide required
by the AU oeii for wine production were unknown prior to thie
etudy. Eincidetion or theee requireomte may provide e eediue

for inoreeeed viroe production end perhepe eventual ieoietion
I at inreotive wart um: pmiolee from tieeue oultureo the
vitae could then be more readily studied in ite relation

1

2
to hoet «u infection and tumor (eert) production.

Since the wart virue no not reediiy ieoietea or
quontiteted on the my cell. line. mother related viroe,
hex-pee limplez. hed to he need. Another cell line no
teetea for emino eoid requiremente ee e oomperim to those
of the human akin epithelium. AU. A unique requirmmt by
the AU cell line eey indioete why it me the only one of
thirteen oeii iinee which wee eneceptibie to infection by
the eert virus.

    
 

REVIE? C? LITESA?URE

    

 

_Var oug_£ésgugweti‘figstennv_g
36.18 Izflafimfll

Investigatione on the role of amino ecide in viral
multiplication heve utilized enino ecid unloguee. en
eroeee oi' the notorel mino ecid. or e deficiency of the
amino mid in the median. Early etodiee employed
eneloggiee, chenicel oonpounde einilier to the notebolite
but with different mbetitnted ohenicel groups. It wee
eeeincd the analogue conpeted with. the natural enino acid
for utilization.

Thonpm (is). studied vaccinie virue growth in
chick embryo tieme cello. Nethoxinine. the analogue
of nethionine wee on: in varying concentration» Viral
multiplication vee inhibited. but it wee not determined
it nethorinine vee competing or toxic. ickemen (1)
found thet nethorinine vee not eeetroying virue or host
celle. nor did it prevent infection. Be unwanted the
mingue reducers the concentration of the essentiel
metabolite or caper-ed for enzymatic eitee.

Veccinie virue growth wee inhibited in chick abryonio
cell culture when Bozuthienyelenine wee edded without the
neturel metabolite phaiylalenine (56). Since inhibition
vee reverted by addition of phenylalanine. the enelogne
wee competitive. mother B-Znthimynlnnine ected directly

on the cell or on the virue wee not detemined.

3

Erna" lysine at one ills/E1 allowed very little
reprmmction of meiler'e virue in aimed mouse brain
(35) (3‘3). Eistidine and tmtophan had to be pros-amt
et three ng/nl te inhibitl other enino ecide were not
inhibitory even at this high concentration. The lack
of redionctive phosghete incorporation into phospholipid
. end protein-bound phocghnte fraction Suggested that en
. exoeee of nino eoide antagonized inoorporntion of
phoeohete into host ti ewe.

woe” «inc eeide inhibited amazon end. influonze
virue replication on entrioellnntoie continue “spender!
in We' balanced alt eolution (17). The basic amino
eoide erginine. lysine. ornithme and me others were
inhibitory in concentntione varying from 1 to 10 mgr/ml;
mino wide with tvo heio arcane were met native. and
the effect tree augmented by high pH.

Lysine urine end examine vere inhibitory at one
ua/nl for heiler’e vine in Ionee tieaie cell culture
(3M. Fhenylelenine mt! othere were inhibitory but at
3 Ila/ml concentratim. Lyeine wee found to be the most
inhibitor: in thie ”eta: even .5 35/131 ehoved activity.
Lysine inhibition N. reversed not only by methionine but
to lmcine end tyrosine ee tell.

fiflflfll or augmentation of iyeine imnibition by
other amino acids has been related to transport of lyaine
eoroae the cell membrane (13). tubbe 2 eeoitee tumor
nelle were chem en the ideel cell for thie study em!
infected! with influenza virus. when vine infected oe‘ile

5
were placed in e nininel Indie Iith an inhibitory concentra-
tion of lyeine. eddition of hietidine oeneeti increased.
uptake of lysine into the free mino ecid pool of the cell
and eloo increased lyeine inhibition of viral replication.
Other amino ecide decreeeed treneport of lyeine end reverend
ite inhibitory effect. However. it no leter doom (1?).
that reverml of inhibition one not releted to the prevention
of the nine acid from entering the cell. For inetence.

1hc phenylalanine

proline and elonine "6:;on treneport of
into Krebte 2 eeoitee tumor oelle end eleo reverted phenyla-
elenine inhibition of hmglntinntion (protein eoet) torna-
tion. oerine union eleo revereee phenyleline inhibition.
did not resinoe phenyleline treneport but increeeed it.

A possible node of action of lyoine. once ineide the
eell et inhibitory eoncentretione (18). rue e damaging
effect on oyntneeie of e virel precureor protein or enzyme
foned curing the first three hour- of infection. Lyeine
added et 15 eillieoler concentretion eeverel hoore efter
infection hed little effect on replication of virue protein
coat. if aided during the first three houre. it no
inhibitory. Lebeling lyeine with 1at: indiceted en intemtion
Iith the protein-nucleic ecio fraction of cello. pernerve the
virel precursor.

Viral replicetion inhibition by enino ecu eroeee hoe
been ertenied to include leneine. tryptOphene end phenyleline
(19). Their ebility to inhibit. with the exception of leucine.

could be revered by oddition of each of 13 other goino eoide

r-\
\

(6)
et non-irfixibitory concentretione. [Pei-hem the preeence of
exceee mine ecide 'in nature' any pley e role in reguleting
the mount of virne .1 cell can synthesize. If e cell wee
preeent in en organise et e eite where the eta-rounding
eedinn contained en exeeee of en mine ecid. thie cell would
be en unfavorable heet. The finding that D-enino ecide eere
Inch leee effective ee inhibitore er revereore inciceted e
epeeific cite cf ettecheent for the netnrel L-ieonere.

Uptake of 1’£'¢I.'-»lem:ine into cell protein den indicated that
exceee enino ecid e inhibited celluler ee veil ee viral
protein eyntheeie.

of the enino ecide discus-ed eboee. lysine end hietidine
eere related to time growth in eonee brain in vivo (#0).
inmate of then two «inc ecide eere reduced in time
infected tissue. indicating e role in cell eeteboliee during
virel prepegetion. further-core. virne infection etieuleted
incorporation of 1".6: tron glucoee into met enino ecide
except lysine ens: hietidine.

Perhcpe analogue were not indicetins e true require-
meat for en amino ecid. In the ceee of phenyelenine (3h).
enelegnee were inhibitory et 0.1 lug/ml. ehereee the eetcbolite
bed to be et thirty tinee thie concentretion.

The firet evidence indicetive of eone of the linitetione
in the eee of eneloguee tee preeented by Bram (t). In
growth etndiee of policnyelitie time in monkey testicular
cell culture. five out of nine mine ecic mloguee were
inhibitory. Pour eere enelognee of eepnrtic. methionine or
phenylelanine and might indicate their eccontielity. but

7

nan: of these eene ennlcgnee inactivated virus in the
upenntnnt fluid. Thin indicated. the cmcpounde not only
on: not be useful in.oeternining enino acid requirencntc.
but In: eleo render the celle incapable of viral multiplica-
tion.

host of the work up to thie point enggeetec little
correlation between.enino ecid requiremente of e particular
time end ccrtein cell linee. Lysine end methionine. when
in.ereeee. eeeneo to be the prominent inhibitore. hcrgcn
(32) indicated cm neede wrong reriooe hoet-rirne carotene.
Kit finding thet the eneloguee n-zuthienyelenine. G-ncthyl
tryptogmen end ethionine inhibited peittecoeie rirue multiplica-
tion inguinceo chick embryo tieeue, correlated eith cannon
neede in other eyeteee. The analogue ethionine indicated e
need for methionine. proven.eeeentiel for veccinie (an).
' influenza (l). end uneina polionyelitie virueee (h) (30).
The requirement for chenylelenine eorreleted eith e.need by
Theiler'e on v11, nouee .noopnmti. (3h). vaccinie (as). end
noting polionyelitie rirneee (h). Morgan'e deteminetion of
the tryytochnn requirement for peittecoeie nnltiplicetion
wee not tiered with other viroeee in tieeie culture.

It thoulo be noted that the correlation.enon3 rirucee
for certain enino acid requirmnte wee not ebeolute. A
cannon neon. ee reflected by an entinetebolite, nay indicete
the metabolite nee note crucial for cell function end enrrirel
thenxrirel eyntheeie. The entinetebolite commonly need any

here canned tenporery or unnoticeeble {image to the cell.

3
Both these parameters We it diffioult to eesess how valuable
en emino said was in virel "graduation or general functioning:
of the cell.

The deteminstion of eiailer emino acid requimente
eoons vimoee booesne sore complicated by the use of different
oell tygseo. more use evidence thet en emino eoid pool
existeai within oelle end supplied mm some for virus
propagation (6). Therefore. so the intreoelluler pool. of e
sell line varied, so did its oepeoity for virus production.
Another varisble use introduced when different Indie eere
used to grow the eells beoeuee the intrecelluler oonoentre-
tion of amino eoioe was dependent on their concentration in
the extracelluler medium.

Almost three hundred mino eoide end amino eoio eneloguee
were testes}. for inhibition of influenza A e B viruses grown
in embryonic chick lung tissue (26). Toxicity of the compounds
wee determined by nioroeeopio erminetion of tieme imubeted
eitb the enslogue end vitae. If the cells were Altered or
their growth depressed. the empound use Judged. toxic.

The otur‘y mowed inhibition of influenza vine by
norleucine end other methionine eneloguee. The inhibition
use reversed coopetitively by methionine and rembled
inhibition by methoxine end ethionine dam by Aokemann (l).
thleerine. omavenine flevimte end onethylcyoteiae
lore eleo inhibitory em! were oompetitively reversed. by
pbmylelegnine. l-srginine end cysteine or oystino roepeotively.

Reversal of virel inhibition by «Mition of the mturel
metabolite was ”compound by e paralled reverwl‘ in toxicity

9
of the inhibitor to the tissue. The reletively lerge
concentrstions required to inhibit virus fomtion mangesteo
the ecologues were not directly inhibiting sites of virus
fornstion but me: heve been incorporated into virus protein.
The mounds found inhibitory to the viruses were tested
is nice infected with veccinis but none were effective.

Viral latency (e hidden infection) can be produced by
growing cells in deficient nefiis (23). when minced chick
mbryo roe placed in e selt plus gluooes union there was
cell growth. Hhen psittecoeis virus see coded to the culture.
no virel multiplication occurred. Both cells ens] virus were
vieble. but the virus reeeined letent or hidden. when mine
solos and vitamins eers edoeo to this letent systsn. virel
eultiplicetion use induced. This suggests that the cell con
obteiu eoino ecios necessary for survival fm either protein
degredstion or biosynthetio pathways. however. higher intro.
oelluler concentrstions of mino soids were required before
virel replicetion occurred. In this eyetera. the presence of
phenylalanine end tryptophen sore found to be the most essen-
tiel in rcectiveting the letent stete into viral replication.
These seas oino eoids ere eleo required for mmcelim cell
growth.

Eagle and Kobe]. (15) and Eamoll and Eagle (.5) found
little correlation between emino ecid requirements for sell
growth end those for virus production. than fleas cells.
sterved for 12 hours of ell componente but glucoso. sluteoins
end eelts. hers placed in e medium containing only theee

three components end infected with polioviruo type 1. virus

10

use synthesized. cell growth hsd been arrested and cell
injury her! occurred due to the sbeence of other amino acid s.
Vltsuins. and serum. without glutuuine. viral production
use delayed, suggestive oi‘ lstency. It only slutnine alone
were nosed. there was s tee thoumnd fold increase in virus
production. with only glucose alone there see s 170 fold
incresee and s delay in virus production. Decreased virus
production was not due to lee): of adsorption to host cells.
thus s cell line shieh needed 13 amino ecids for growth.
required none except slutmnins for virul synthesis. This
probably reflected eyntheeis or virel protein from arcino
soids prev-existing in the cell or from breakdown or the cells
on protein. The cell can not synthesize any of the other
12 omitted tron the medium

Beacons cell injury was evident it might be seemed that
absence or glucose and. glutsmine in connection with reduced
virus output reflected cell deeth. But since addition of
glucose and glutenine restored virus production. imaeired
cell function rather then desth use mggeeted. The obvious
corollary to thin obeervstion ties that glucose and gluteoine
sight be preserving cell integrity and, not be ectively
involved in viral synthesis. his use not true because there
was obvious cell degeneration. Another possibility was that
glucose end glutunine served either ss sources of energy.
precursors for synthesis of viral nucleic ucid or both.

Glucose wee slco deuonstrsted essential for herpeevirus
growth in the fists cell (2?). Unlike the remlts with

ll

poliovirus (5). glutenine ses not. then alutunine was
omitted from the ertrucelluler sediun there was en enhance-
sent of virus production. The reason for this is unknown.
It see not attributed to reuovel of glutenine es en inhibitor
because higher concentrations were not inhibitory. Serum
or en inhibitor in serum soy pley e role in this phenomenon
beceuse stiouletion occurs only in the ebsencs of serum.

foliovirus types I. 11 end in; had different require-
ment then see found with type I in HeLs cells (15). When
monkey kidney cells sere infected end e eediuu deficient in
cystine edged. cytopethie effect (cell degeneration) use
deleyei. Adsorption see elieinsted es the cause. A require-
sent for cystine which see not sham by Eagle end Rebel (15)
use indicated. This systn elso uggected e latent etste
since the virus had e longer eclipse phsee then see usuel.

Rappuport (kl) hed previously mosh that cystins repleced
ell other enino scids for the nultiplicetion of poliovirue
in sonny heart cells. tubes (7) therefore suggested thet
on entinetcbolite to cystine end/or cystine be used as e
therapeutic went eguinst poliovirus.

The requirement for L-cystine in virus-host cell eyetens
sas ertemfed by Tyndall end Ludwig (I37). Cormkie B, and
veocinie virus were found to require cystins when grain) in
rsbbit kidney. chick embryo. vssculor endotheliun. monkey
hesrt. and to sous extent in here cell culture. roliovirus

be 11 eleo required cystine when grown in monkey heart but

\bbit kidney only balenced salt solution. glucose. end

12
clutenins'eere needed. The inability of slutathicne. ascorbic
acid or thioglycollats to effectively replace the requirement
for systine indicated its function was not as a hydrogen
acceptor or environmental stabilizer. since theee compounds
function as such. It'eay seen that it see an actual struc-
tural unit in the synthesis of virus coat or in enzymes
involved in.production of new virus.

The eystine requireaent was highly specific (1:3).
Cystins analogues with various substituted chemical groups
would not effectively replace cystine. The analogues
dseonstrsted the specific need for the presence and spatial
orientation of the amino group. sulfur atoa. carbonyl group.
and the three carbon chain. If cells ears first starved for
twenty-four hours and than infected. the virus mained intent
or hidden. Addition.of cystine activated viral multiplicap
tion. No virus could be found during the latent stage.
pennaps it was present inside the cell as viral ribonucleic
acid and not intact particles.

Felncnt and norgnn (36) were the first to conclete a
coaprehencive study of the thirteen essential amino acids as
roqoimontc for herpes virus. may wloycd the I. strain of
mouse fibroblasts for virus growth. and titrated the virus by
counting lesions produced after inoculation of chick
charioallantoic neubrsnse

The cells and virus were gram in a synthetic nedln
couposed of the UL forms of the onino acids. not the natural

L-isooers. mission of phenylalanine. tryptophnn. arginine.

13
throonino. loucinc. valinc and hiutidino norkcdly dccronsci
virus growth. Isolcucinc. lysine. alanine and glycino wcro
removed without any effect on viral replication. The
romaining amino acids showed a slight decrease in viral
production when omitted.

It was also determined that hcrpco prolifcrotci in
a synthetic moiiun which did not allow growth of the L
cells. If cello were infected while in :arlc'o bclnncci
salt solution. no multiplication occurred. The virus was
activntci from this latent state when the salt solution
was replaced with a complote medium containing horse serum.
Latency was induced by the addition of othionlnc. tho
analogue of methionine. at 60 n;/nl concentration.

Tonkcrsloy (44) also studied the thirteen essential
amino ecido no rcquircncnts for hcrpos virus multiplication.
using natural L-iconoro of the amino acids. Another
improvement over Pclncnt end florann's work (35) not tho
replacement of the ocrum rcquircnont by mothylccllulooo.
allowing complete ohcoiccl definition of tho toot medium.

A more typical host cell than mouse fibroblasts. human
esophageal cpitholiuc. was ocplorod.

Kinimclly infectod cells were first plncoi in e
complete nciiun with corn: for zu-ca hours to assure infec-
tion. Infected shoots of colic were then riaood with salt
solution. and bncal moiiun Bugle deficient in one amino

acid was aided. After seventy-too hours further incubation

it
the supernatant fluii was diecerflefl uni the monoloycr
oesoycfi for vireo by the stoofierfl plaque method.

Eleven of the thirteen eeeentiel amino eciao and
glutemioo'eere required. Lysine was not nceiei and was
even pertielly inhibitory. Gmieeion of hietidine cancel
the greatest decrease in viral replication. In the ebeence
of arcinice and treeence of lysine. no virus was foreca
and no cytopethio effeot one detectoi.

ficizoeo. spring eel Scone (o ) grew a.mecroploque
strain of herpeevirua in human epidereoii carcinoma cells

31 confiroefl that omission of the essential amino acid
erginino markedly reduced replication of horoeevirue.
Arainine was not neefiei during the first four hours of
infection but one requirei six hours after infection. eel
taereefter. for virus multiplication. This woe shown by
colition of orginina from G—u hours after infection. with-
drawal the remaining h—lfi hours. out a euoeequent titer of
2.7 x 132 polyteryocyte forming unite per ml. If cr;inieo
res omittei 0-6 and eiied 6-15 houre after infection. the
titer wee 2.3 x 13h polykaryocyte forcing unite per ml.
the virus was oeecyci directly in the first series of
orperieente by count or polykaryocyte forming units.

The seconfl eeriee of experiments was performed with
the knooieigo that ur;ioino was required 6-15 hours after
infection. Cells grown on corerelipo were or inioe starved
for 0-6 hours one then infectei. Cells on one half the

coverelipe received erhiniee. the other half dii not.

II.

15
Eighteen hours after infection cells wars stainad with
fluorescent human IF-gloTulin (denonsfirating cytoplasmic
virus) and rabbit hyperimmuna Earn: nQainst boilad infec-
tai cells (ficzonatrnting nuclear virus). Aiiitian of
staining causafi a 30-fold increase in the numbar of
calla containini cytoplasmic and nuclear fluorescent

granules respectively.

'9 4mg 9,
4L'hL-n

f" '51 .'

     
 

  

'“' fl“"’"“‘n ‘. ‘1
a. I. t... [‘1‘ -." '

 
 

 

  

sari; davslovnental madia far th growth or tissue

cells in vibro ”are usually oxtracts from tissue: or
other acurcea rich in grawth factara generally ch33-
icall: undefinei. To stufiy th effect or certain
nutrilitaa on cell metfiboiigm. ezgacially virus infea-
tion.ana unitipliaatian. each growth factar prasont in
the medium ahauld be known. Such cheniaally dcfinai
mails have been deweiopefl wifih tho deflania of the call
far each nutrilito eatinntai (12). It was thus poasibia
to precisely vary the meiium nonposition uni obaerve
the subsequent effect on cell function. Enfortunately
it was still nacasaary to ad} an unfiafincd component.
serum. in order far the calls to proliferate rapidly
(10). Kathylccllulosa may raplaeo aerun in yratectin;
calls and heaping than viah-a. but it dii not permit rapid
growth (2;).

A tyyisnl ehfiniaally defined mciium cantainn a

balanced salt sciatica. a au;ar. vitagiaa anfi amino acids.

16
The aging acids uaai have been 295% exfiaqsively stuiiad
ainoa they are tha builiia: b10331 a? call pvotain.
Established cell lines utilize ani lite?a$e amino nails in
a characteriatio way. It was poasible ta differentiate
cell llama on this basis (23).

T39 amino acia raquirczanta for a particular cell typ
have beea diviiel into essential and nan-essential. ?ho
eaaential amino acifls war» thoao Which mug: be provide! in
the maiium far the cell to urow'uhila nan-essential amino
acifla can be synthesized by the cell.

ragla (11) (12) (2.3) found t'nat thirteen 12331510 acme
of the natural twantyuona are essential for trouth of human
carcinoma and mange fibroblast cells. fiziasion of any of
these thirteen from the medium. cause! micrascagic changnz
in the cells within two to threa aaya. The chanjoa reflectei
cell" r injury an! differed accorflin; to particular amino
acifls. probably inflicative of thgir cellular functicn.

The requirement of thirteen essential axino acifia by
most call linaa was correlated with the cell's intracellular
amino aoii pool (37). float essential amino acifis. were
actively transported int: the cell gal concentratei five to
eleven timaa greater than in he extracallular maiium.
Lyaina gna arginina were consentrmtai only two ta three
timaa. cystiaa was not concentratoi. rfie nan-esaential
amino nails had a very hi:h intramellular cancefitrntion
ratio since they wars ayathesizad internally by the cell.

The free amino aciis or the intracellular pool wara aenarally

17
the coma in most cell lines. although there were signif-
icant differences (3?).

The role of the intracellular amino scii pool in viral
replication has boon demonstrate: by labeling experiments.
Qarnell and stintov (5) have shown that the protein cost
of poliovirus was msio from tsslvo.aoino sciis in the intro-
ccllulsr pool. lhuo in order to study tho amino acid
requirements for proiuction of virus protein cost. the
meoiuo the cells were grown is could be depicted of so
osscntisl amino acid (25). This depiction was reflected
in the intracellular pool. and the protein cost might either
be deficient in the amino acid or not aynthcsizsi at all.
The cell could compsnssto for this deficiency by breakdown
of its own protein or use of dipsptiiss.

An amino acii deficiency can probably offset viral
replication in many other ways. floor are unknown. Boos may
be so intimately sssocistsfl with cell function that they
are inseparable. Viral cirsctci synthesis of early cnz"scs
needed for tho biosynthotic machinery involve! in replica-
tion can be siverscl: affected. The studies of amino acid
deficiencics rcly on.s decrease in tho scount of virus
procucsi. The spocirio effect at the oolsculsr level is

unknown.

2a 37:133.; ma ”m #13

I. fiaterials

A.

{I}

t:( 1'???
Herpes simplex virus (I3!) HcIntyre strain.
Aaerican type culture 31:. H39 obtained from

Richigan state health Laboratory. East Lansing.

fiichignn.

 

1. AU. a.normal human skin type. derived by wheeler
at. 81. (SO).

2. fifibi} normal rabbit kidney. meals at. al- (3)
was procured from Henry ?ord Hospital. Detrcitu
fiichiaan. courtesy of G. La @rippo. H.3.

3o KTP-Z. human epiiarmoifi carcin0wa of the larynx.
fiooro at. 51. (31) :as obtainei from $103

Laboratories. Eockville. Earyland.

 

The procedure for the propagation of as? was
a moiification of Eaplan’a (2%). :abbit kidney
cells wars grown to oonfluency in a 150 ml milk
dilution battle with a final medium volumo of 10
ml. Hinimal essential meiiun with .33 mathooel
(mathylcelluiose. 15 ops. prenium. now Chamical
20.. x Aland. Kichignn) supplementing serum was
anpioyai. One tenth m1 of untitratai virus atack
was inoculatei onto a fifi-i) call monal&yar ani

1$

330

1?

allowefi to aésnrb to the galls tar one hour. The
inaculating fluid was fihcn dacanbad.

arm;- a. total h?! hours lnnumemn at. 37°C. the
culture nan frozen, and thawed three times. mnfi
mantrirugefi at 550 maximum roiativo neutritugal
torao (Internatiunai contritngn modal Vania: 2) far
three minutes. Fiva ml aliquots at the aupcrnatant
fiaifi, with a virus titar or 105 pfu/hi, were placed
intn snreu-¢ap tubes. analod with plastia tag», ang
trczan.’ *ho stock was re-eitered Just haters use
to obiain tho dacirad inoculum.
gaifiafiefinfigig_fioiutiaq
1. flarie'n balanced salt aoiutian (5333-1ox) (16).

 

 

figfiwgpfing Amannt $re§arfizinn
Unit $1 $391 $8.00 gm viauoiva in

900 ml water*
as; $.00 gm
fiafiayah'xzc 1.25 gm
fiafiau’7fizfi 2.30 gm
glucoaa 19.00 gm
rhencl Rafi 9.20 gm
fini: g2 cafllz 2.00 gm Eissoivo in
130 ml unto!
Two mi of chiarorata was aéand to unit £1 and
bath sagging refrigarated. Far 3 ix sniutian. 9 ml

or unit 31 and 1 mi of aux: $2 were ‘5536 to 93 mi

“-“L

“ ‘ w o
' Ail materialu'uaro frozen at ~20 C.
* $11 water usea was giann‘ciatilloa twice.

   

of 22ter.ari steriliza. by.aatoa 1321

52‘) a
J ' .927

2. Laals‘ 52122092 2212 30122132 (5221-132) (21).

cu- T‘
knit -l
”"0. I!

he: u:.i: ii 222 002212tciy 21320120 . unit ;

:23;

15 min‘* was.

9" J. ‘

 

22:1

yaw.

full
«r . -. O a

121:3 ‘EEZG
5122922
""“?

I- 2: 31‘
Phenol r22

31' at 15 153.

«6

fluenqgt—L ri‘fif-thg‘fi“! *4

   
 

22.3 .2 Sieaolva in
230 mi 22322

1.2 gm 31222122 in
50 ml

223 22 222 to it. an2 the {inn voluna brought to

1 liter.

rotriuemt

9!“ ‘.‘!fi {'9‘- _

22221 $221

T23 21 of 3:1:

630

22 :2;10 (12).

 

Biotin

20110 acid
$221122 ?:L
flicotinaflifla

Cara-Iantot hm

233 21122 232 the stock

 

0.001
0.831
0.081
ate 0.021

(902212222 on next page)

rd

F0

21
iyrldoxal 36L
121 amine 2:3.
flibcflavln
Inanltal

h I“
{.12 H

0.001
0.001
0.0891
0.0013

{urchasad frozen an 103x tram microbiological

Assaclataa, netheafln. ”.3.

The solution was

tha2efi. dispensed into 5 n1 quantitics. and

refrnzen an ~20°c until “:92.

£333?_2212 22003

30122192: tor’mtnimal Decantlal meaium (222) and
basal mcdium Earl. (22%).

Unit #1

$21: $2

 

burg-31:11:10 iiCL

Lpeyamlnu

Lav-hi at!!! inc mu;

L-lanleuclno
L-Ioucina
h—lysina HQL

inmathionino

anhenylalanlne

L-threonlne
buryptmzhm
L-tyrsalna
Lovnlina

Luglutamine

   

0.126 0.021
0.026 0.012
0.032 0.0093
0.0525 0.023
0.052% 0.025
0.973 0.0365
0.315 0.0675
0.033 0.0165
0.023 0.02%
0.010 0.00h
0.035 0.018
0.027 0.6235
0.292 0.292

5.

 

22

All of the amino acli uaai were purchased :3
the natural L-forn from flutrltional Riochcmlcal
Company. Clevelant. 09:10. Unit 591 was preparad as
535 and sterilized by nutcolaving at 123°: for
15 min. Cyatine. tyrosine. and mafihionlno ware
firat dissolved aegaratoly in .25 3 321. than
aidei :04: water aolntlon of the remaining amino
aalfin. The stack solution was held at ~20°C until
unai-

Uni: £2 was praparod as 100x in water. sterilizai
by Hillipora'menhrane filtration. «A1 frozen (-20°C)

until uaafl.

&*+‘k‘afi95 "* eh.

 

A final concentration of 133 units ponlcillln

and 130 u: streytcmycin per ml of tissua culture

391133 was enplayefi.

 

   

1. 3131331 essential maiiua (333-13) (11).
Pfi???*fif9 Féfivjfi
Hanks. R33-1X or 93.5 ml
Karla's aflé-lx.as
aaairoi
Vitamin stockblcox 1 m1
- Amino 3311 atockp?2f~50i 2 m1
L-filutanina-lcax 1 m1
Stock antibiotics 1 ml
mm} 44.23..

00 ml

3o

23

The balanoad salt solution was otarilizoa by
autoolaving at 133°C. 15 min. uni the remaining
atarilo in;rofiionta aidad ascytically. The amount
of 5&3303 varied 1.5 ml. dopenfiin; on the desirofi
p230
Basal rtoiim 1331::19 (23.27%13) (12).

Ibo conpooition and proooration uaa identical
to that for fififl except R33 amino acid atock was
nooplonoazod for the K33 amino acid stock.

Experimental E13 and 333 media floficient in one
amino acid.

fhoso'woro mafia iiootiool to tho preparations
givon abovo exoopt for substitution or a stock
amino acid solution 505. main up from indiviiuai
anino acids (o2 describe: in part F) with one amino
ooii ooittoé.

xothooel (301 Company. xidloni. nichigan) at
o3§ concentration. 15 centipoiso viscosity was added
to mofiia as a suppiomont for the usual serum roquir0-
moat (23). This allowed complete chemical definition
of a moiiun. Tho material woo aided to boiling
water. mode infio a slurry. and autoclavefl at 120°:
for 15 minutes. ?ina1 hyfirotion was aocomplionod
by first cooling at room temperature for two hours.
thon refrigerating (-h°;) ovornito. The components

of the medium were than.ad£oi aseptically.

Io

J.

9
2-:

Eigguo‘isaag;fivorlav (521.

Hothooel. 2% concentration. b.080 oentipoiso
viscosity. was includoi in Hi3 moiiufl With Hanks'
£33. The prooadure for hyfirntin: the proinct was
identical to that given for etvorimeutnl mofiio part
G section 3. Agar was not employed since it

inhibits herpes virus (#9).

 

A .256 solution in fianka' 333 was achievoi by
warming at 37° for & hour. sterilization was
atroctod by salt: filtration.

5“?“ A ‘614-3“*Yfi%¢
“414' u ”A -*

 

Crystal violet 0.1 go and citric acii 2.1 53

were adiod to 100 ml water and autoclavei at 129°C

for 15 minutes.

 

T.e particular coll tyre usoi in each experiment
was first grown to a monolayer in a 150 ml milk
dilution bottle. and tho manolnyev movoi by either
trypoinizotion or use of a rubber tipped rod. The
cells wore counted in a homgcytozotor and proporly
diluted to 105 cells/a1 with mofiiua containing 20;
hoot inactivated. storila calf serum (?low.Lab-
oratories. fiockville. 3.9.).

All tests were done in aorow cap standari tissue

culture tubes which were sooiei with 1 ml or the

25
cell suspension. The cells were crown at 37°C and-
the medium replaced with fresh medium plulefl
serum ovary 24 hours. when a monolayer had formed.-
uaually 2-3 days. the mediun.waa decanted and 0.1 ml
or the desired titer of virus oddcd. After udocrp~
tion for one hour at 37°C the inoculating; fluid was
decanted. Hedi: deficient in one amino ncid was
added at this time unless otherwise indicated. Each
test was done in triplicate.

After the desired incubation time. 1.0 ml or
Hanka' balanced salt solution. containing 51 serum
was added to each tube as a stabilizer for virus
activity. and to neutralize thoracic p3. The tube:
were sealed with plastic tape and immediately frozen:

with the medium covering the monolayer until the

time of assay.

 

The procedure was aimiliar to the method used
by Kaplcn (23) with modifications given here. The
cells were ruptured to release the virus by freezing
and thawing three times. While thawing. the ice
sheet was shaken inside the tubes to free the cells
from the surface of the tube.

the triplicate tubes for each deficiency test
were then pooled. and the cell debris centrifuged at
550 max. R.C.F. (International centrifuge model V

25
size - 2) for three minutes. ?he virus conteinin:
supernatant fluid was withdrawn and diluted through
four tenfold dilutions by edling 0.5 ml to h.5 ml
or 3333.

One tenth ml of each dilution was inoculated
onto e rabbit kidney monolayer grown inns 1.0 oz.
‘French square bottle. The virus was adsorbed for
one hour at 37°C with rotation of the bottle every
15 minutes to secure even distribution. The inoculun
was decanted and three ml of s 2; nethocel overlay
medic: we: added.

Following incubation for ?2 hours. 6 m1 of water
(22°C) was added and gently nixed with the nethccel
no it would become less viscous and enciiy decanted.
The cell nonclnycr was stained sith citric acide
crystal violet solution for two minutes. rineci with

enter and the plaques counts}.

R 733 ULT3

 

Preliminary studies have. indicated that the human

akin epithelial cells (At) uniorwont do:encrativo changes
within seventy-too hours when grown in a motion deficient
in one essential amino acid. This degeneration had been
otoervoi microscopically. out the subsequent effect on
viral replication within those cells was unknown.
:xperioant l was perforooi to determine the
noproprioto time tho virus infoctei coll: snouli remain
in deficient medium in orflor that amino acid require-
ments for viral replication would bo best demonstrated.
All coll.mooolayors were simultaneously infected
with 500 plaque forming unite. and deficient medium
containing .31 nethocol afifloi one hour otter infection.
After incubation for 2%. no. and 72 hours. the cultures
were frozoo.until time of assay.
tho rooulto in table 1 are in plaque forming units
(pfu) per milliliter. Ono plaquo forming unit was shown
by Dulbccco to ropraoont an area of cell lysis caused
by one virus particle (9).
The data revealed replication in the amino ocii
deficient media until oovooty-two hours. At this time
a marked drop in the number of plaques was observcio
In complete moiium. containing servo or mothocol. viral

27

Tabla 10

to
£1

Effect of incubation time on amount

of virus produccfl in experimental mciio

 

 

 

ficdium 25 hrs. o3 hrs. 72 hrs.
Arguing" 31o ' 23:39 72
erotica 6&0 1700 70
olutaminc 50 150 O
filatldino 330 1930 125
Icoloucinc 575 2333 163
Leucino h53 isoc 9h
Lyolnc 225 129 h
Kathionino 92 no 2
icnylalaninc #90 2000 72
Throonina ‘33 2753 23
fryptopnan 512 2559 130
Tyrooino 6&5 2529 So
"an” 1:53» 12:39 go
ijgoamothocol 3&5 2630 t.n.t.c.
Koo-oncrum $53 3330 t.n.t.c.

 

" flinging amino acid.

b' Pro per ml at 16'1 dilution.

°‘ t.n.t.o. - too humorous to count.

2?
multiplication continuoi: col at the dilution assayed
toe yloquoe were too numerous to count (t.n.t.c.).

The extreme roiuction in.cmount of virus at seventy
too hours one best explained by dc;enoretion of the
cello and dovcloyoeot of lesions. as previously shown
by Eagle (12). Set only would viral multiplication
cease. but intracelli‘ r virus wouli be releoooi or
left unrrotoctoa by rupture of the cello.

Since herpeeviruo woo shown to be an extremely
thormlebilo viruo at 3?°C (3:3) (23). both the extra.
cellular viruo normally present and that release! from
injure! cells could be inactivatei. Thus the cmo'nt
of virus uouli not to loss. but the number of infectious
perticloe surviving 37° would aivo en.cpperent reduction
in number of viruo particles as only infectious particles
wore assayed.

Another explanation one that the amino acid pool
was copletoi. especially lo the particular amino acid
omitted..cnd that Viral multiplication could not occur

thout those present. This was likely to account for
port of the reduction in titer: but if multiplication
stooped without degeneration. ouch virus must have
regained within the cell. flowevor. this virus should
hove reneinoi infectious cod detected during eoooy.
eioco the cello were ruptured by freezing one thawing

gust prior to nosey.

II.

 

30

It woo cpparoco froo.nicroocopio ovidooco and
:oorkod roioction in infectious virus that grooo coll
insole occurred from.hS-72 hours after infection.
Forty-ei;ht hour: woo than chosen as too apfiropriato
time to nooay all future oxoorioonto no oulfiiplicotion
was at a pooh. amino acii pool daylotion cviicntly
ooourroi. col Boocific omdno acid requirements wore boat
donoootrotol.

Tho 2% hour ozoorioont woo not done in triplicoto
a: this time. but lotor exporioonto ohoaod all deficient
moiia with loss virus than complete. not all too amino
acid roqcirononto Kora eviicnt at 2o hours. many being
idooticol at this Ciao. 80 b3 hours woo chooon as tho

iool timo for incubation and aoooy.

\1'.

finnnrim ‘a‘§v-
u..»,-.J.alm —;~A Asl-

 

 

Sinco it hoi boon concluioi from exocrimoot on
that to hours woo on appropriate time for assay. tho
next throo exporioooto wore performed with ooooyo at
thio time. Each oxporimcnt was conicctod in.triolicoto
for coon amino acid. Otherwise the proociuroa wore
idcntical to cxpcrizont Ono.

Tho ovoro;c nuaocr of plaques for tho throo
czoooioooto are aivoo in table 2. Virus proiuction in
deficient media was also calculated in terms or porcont

14

, flucod in comploto ocflium. Cozolcto oodium woo lOOfl.

$3310 2:

in not of virus producol by AU

cello in minimal essential moiiuo dofioiont in

coo ooioo acid.

7—w—

5 q. \ *-
‘ 0 it": 9- I”

Dru/m1, 3f ooh. _AV§¢

 

 

fiofiiua on dovion
‘coxogozo,m§;oac
armiimno 7.1 a: is” :32 *7
cyatiao 5.6 : 10$ b9 1%
Glutomino 1.3 x 103 15 :5
Kiotifiino 8.5 2 1o“ 73 :35
Iooloucio 1.0 I 10“ $.S t3
Louoino 1.5 x 133’ 13 :2
L321"; 2.5 x 103 2.5 3 .5
E‘ot‘lioni'zo 5.7 '3 oz 0.5 3 .2
E'horwlalanlna 5.5 x 1"!" 3&3 3 .10
Throonimo 6.1 x 13' 5% $2
Tryotoonao 3.0 x 10. 25 :5
Tyrooioo 3.5 x 10“ 31 32.6
mm. 1.1 1: io‘!‘ 10 :3

1.1% x 195 100

i
Avoro;o of three oxoorioont aooo in tripliooto.

Aoooago dovi

$1.1"; 4' Kai-"‘2 + XII-P

3

otioo.fl 3

._ ‘3}. "’ Fa. * P Git
chore 1: II 3.3. ”A '3 3:11 P .- n‘vo‘oor of pfu.

*o

 

I.“

J

fironoloy. may C.. Quantitati?o Analysis. ”.rooo Sable.

Inc.. How York. 1939. p. 2%.

318

AHGININE
CYS’I'INE
GLUTAMINE
HISTIDINE
ISOLEUCINE
LEUCINE
LYSINE
METHIONINE
PHENYLALANINE
THREONINE
THYPTOPHAN
TYHOSINE
VALINE

 

Percent pfu in complete medium

Fig. 1. Comparative virus production by

AU cells in serum free medium deficient in one

amino acid.

n
50

1
100

IIIe

32
ee here the greeted; mount of wine “I creamed. The
peremtage on e deficient eediun indicated that propor-
tion of the vine I'MROOQ on complete medium was produced
men men pertinent amine eeid was «nuts. The ensue:
the percent. the were required the name «it! tree for
Viral replieetien.

The unite are an graphically in figure one. elm
en e pereentiie heele- n nee tame that am minim:
at methionine from the medium lent viral multiplication
occurred, only 0.51 of the mat produced in each”
Iodine

Lyeine wee the next met required. the remaining
nine eeide requires in varying Meme. without
hittiéine. 73:3 of the name). vine output eee eehieeea.
It mould be noted. in "hum to tankereley'e work (his)
the: erginine tree the eeeond but requires! mum»
in thie mien!” eyetem.

  
  

159: ixcéuctio _
:efielaat inwing Am no

medium w‘25‘3agile

The real:- in the purine expel-menu differed
green: from the requiremente (am by Werner (6h).
For thie mean experience eere reputed using e
procedure emitter to hie.

Rem! medium angle (m3) wee enbetitnted tor minim
eeeential ”sum. The eelle Iere green in eoavpiete
medium. then eterved of the pertienler nine acid in

question for :15 been. After 21! been in the deficient

IVe

33

eeéiu'a. eeeh mortality” nae infected eith 109 plaque rem-
in3 unite. Incubation nee terminated at Iii-3 houre although
femoral” unmanned ineuhation for 72 Mare. The renultn
from ewe-titan!» one demon-trend the dimeventate incurred
by institution at thin tell line in entitient medium for
72 hours.

The. nee or n different media; and procedure did
elter me metronome for the it cell line. en recorded
in table 3. Arginine non appeared en much of e require.
neat en methionine. The date indicated valine me the
nut essential. but einoe the reunite were very clone to
enthionine and lyninn requimentn, it cannot be data
initely mid mien nee mare emeinl for virel mtheeie.
A requirement tor hintidine wen more merited in thin ‘
"Item name nit}: warm wanting it even the tenet required

amino acid.

   
   

£32134, fr" ”P‘*g1_tfl“~,.z t for Vin t 1?: 1 .
" ' . dammit , ‘ ~ ‘

Tee mine eeia maxim" for the at cell in these

 

exgerir mite were ditremt m than or the team
emphaatal epithelium experinmtn done by maternity (m

It nee contended that the dinememien use: been been

due to dirterentee in tell typee rather than experimental
pmwure. i‘o tent thin hypethenie mother cell line.

human widen-maid carcinoma. ene upland. Thin cell line
had been need. by Heisman (52) Ibex: he confirmed Tmzkersley'n
findin3 (hit) for en erg; ginine requirecent.

34

 

Medium —AU cells =HEp-2 cells

ARG IN IN E

 

1% E3?

_-_— 4375

CYSTINE
GLUTAMINE
HISTIDINE
ISOLEUCINE
LEUCINE
LYSINE
METHIONINE
PHENYLALANINE
THREONINE
TRYPTOPHAN
TYBOSINE

|||||"|I||TF

VALINE

 

 

If #4-”: 100%

COMPLETE ENE ?__-— 100%
5% 10% 15% 2575
Fig. 2. Comparative virus production by AU and HEp-2

cells in basal medium Eagle deficient in one amino acid.

35

Table 3. kammt cf vine produced by AS}
cclln in camel-icon to that produced
by 5332-2 celln in canal martian Eagle

deficient in one amino ccide

A“ A. w _g

mam A33 celle w cal-2 celle

 

ginine 250‘ 1:36b
Cystine 5200 £1000
Glctcaine 3999 i113
El etidine 600 313
.I mlcucine 390 191
Maine 112'! 210
Lysine 2&3 lfal
iiethicnine 220 £23
Itczzyiclncine 550 2&3
machine 500 3:30
Eryttophnn 600 355
Wm cine . 31 2635
Vance 132' 226

ammo 12:13 9900 53:39

A A 4 A... _.. .._.

———— Y. _,'_

temitn of one experiamt done inbipliente
‘g-m/nl c 20 erg. eta. Gee.
t{am/cl r 19"} a 25 avg. etc. an.

36

the H33»? cell line can tattcé ciaultanccucly with
the an cell line. The procedure was identical for both
cell lines and has been given unficr part III at the rcaultc.

The resultn of the erycriccnt are given in table 3 and
figure 2. the amino acid requirements wcre cicilicr for
both cell lines crccgt for one amino acid. Glutamine was
requirci to a.mnch greater extent by the KEQ¢2 cells. it it
was emitted curing viral proliferation in the AU cell. kji
or the casuat replicated in coaylctc medium was crofiuced.
uncover. it glatacine was omitted during replicatian in the

33p.2 cell. only 6; of the unreal accent was produced.

1313313331023

fimimtion of tho ”all" indicated that authianino
un- tho moat roquirud amino said {or thoruultiplication of
harp“ maple: vino in An all: main minimal umtial
Indium us and. taunt-lay (Ml) "ported that minim
no nut toquind for harps-viru- growth in human onrw-hagogl
epithelium. an cyst» dittorod in chm ways-vim: stain.
host «11. and Indian. rho strain of harp“ viru- an
isolated in his laboratory. The Minty" strain of tactical:
typo cultnrc :1: In. and in thin study. pitta-ant amino
acid mum-nu can be duo to ditramu vim. two: and
meant: no mu mom with polioviml (8 l (51).

Tanacralay used banal uofiiua Eagle. I.Iynthotie uofiium
timing-«i for optimum. 5mm of mu fibroblast-o Thu
study raportod hero originally cnployod ninimal onscntinl
medium. aptimal {or growth or'huaan auroinama (303:) calls.
arm A3 «11 an: optimally in ma. much contained twice tn.
amount of such Iaino acid in Egg. oxenpt can: Eintaal
oniontial ncéiun contained nix ciao. the caoune ot’crginino
in BEE.

When minim umtial noéiua mu and. tho maimmt
far nothioniao mu wry max-Ind in campa‘rioon to abhor ”quin-
uontl (fig. 1). whun 335 III Imployod in following exgorimantl.
five nfiéitiannl rtquiramcnts. including arginino. were as
‘marRCd an the methionino roquirnmcnto Thee: may‘bu requiron
uyntl for gancral functioning at the eoll rather than for

Virnl "illusion. 37

33

A deficiency of individual mine wide wee reflected
in virel mltiplieetim et 118 home (table 1). Addition].
imbetion caused eel]. (Regeneration end did not mantel:
dancers“ specific virel remix-amino Thin me effect
my here been eehieved then BEER wee need. eepeeiell: eith
prolonged inwbetien. Beietien er en nine eoia from e
eeain mien timer hm! e mil nine eeid concentratimi
ue econ reflected in viral replication. ee the nine acid
pool ie engined within 12-23 Mare (15) (37)- iime the
requimtmte inundated by 2m could have been due it: e
ueee mine mid, depletion end abutment eel]. aegeneretion.
A timed may mid be eeeeeeery to eee if e tingle defieieney
bed the me effeei on viral mitiplieetien both early and
lete in infection.

Werner" deaaneeretion thee lyeine inhibited
minimisation of herp-eevime can not be expleined by nee
of e different nedium Both 52%;; end my teen with the AU
cell indigent! it use «can! to teethionine ee e maiment
for viral mlicetien. Lynne bee definitely been exam to
be en eemiiel amino acid for units). end smut of mm.-
lien oelle (ll) (12). It inhibited certain vimeee. but ml:
rhea: present in exceee (13) (3h). 1: ie I constituent in
the protein can of metal vine” (6).

5:01er ee e1. (32), axing e different eel]. line.
emfimezi Imkereley'e finding (MI) fer en urginine require-
eeni in hex-genital multiplication. lack of erginine during
mltiplicetien realized in deereeeed vitae ”Mutation. Tfie

3’}

relative regulation“ for other amino eoifie were not
determinate finleee oonparetive etudiee ere mafia. the emim
acid which in most required in not known.

i‘ho dimrogvanoiee bate-eon fi‘anzereley'e work and the
study here could have been one to use of different cell
lines. To toot thie poesibility the SEEP-2 cell line now by
iioimn mo compared to the in cell. The pmeduree. newt
for e charter incubation period. end, media: were like the:
of Martin“. mie aoriifioetion nee neoeeeery ee notm
in the results on the effect of incuhetion time on production
of eirue in deficient Mom (27). Each of the thirteen
mine aoid deficiencies. except glutamme. had e eioilier
effect on viral multiplication in both the AU and}, sap-2
cell. claiming: no more neoeeeery for the £3sz oell than
in cell. Cray one month of the mutant of wine proéocmi
by the A23 cell was produced by min-2 in alumina oefioient
area into.

the varying; ”quit-moat: for replicatim of the some
virue do not amour to depend on differing mint: eoiai. noteb-
oliu mom: hoot «119. In monomer: cello. the thirteen
eeeontial Amino home. ercegt glutmaine. ere metabolized to
e mini-ital dogma: they are primarily inoorwmtozi into oeil
protein (13). (mission of mm eeeontiel amino mm probably
linite the inoorgmretion of that mine: acid into viral mmee
and viral protein coat.

iii-zen one win. emino acid was deleted rm tho extracellular

meriium, host- oelle Maintained! the contamination of the rmoining

1:)

«11n9 111531111 11111: c1 ange far twenty-four agar: (37).
131911111 :21: V1. 151113111111 1:315.- rmzlzmm$s x. a: net: 111.110 to
filtrate? 1: 1:11: 1.31:" 19111301112. mm different air-11m) 10121
requirérmnm 11:111. have 1111115119 ta the mmmtmum at an
0.1an M151 1:: the 12111111111111! poo). am: 11a erreat en mantra].
111311111113 0:? the hunt can. men was 9. 11.4111 infirm 111111”
42:11:19 191:! (9211;911:1311-2311. 11111112» to each. mama 12.111 . that
ma axe-11.11111 my a c.2111. ta 132111-110 12101111; synthesis {135).
mu may 1111:: eanamzom to vim]. protein 93211211113. 11' the
9301191111111 :1 0! 1m @11an m1! drcr m1 helm n 1111811111 thresh-
o-m. vim}. 12111-111 9M thus «2.11;;1910 virus. WM mt 1311111312312.

.1 11111111 amnmztmtmu or an cams: cam my 1112: 32-11}
a 1:910 1:1 11.11.1111 mm synthesis. 1: has Man mam that n
313111;! at 1111:: mm a was nwmmr‘y for mania 80113-1 (anthems
111 mm 111112111: 3:. 11111 (33): A £11111: genera in 11111-
1111 162115 1111 not 'men émmatmtedu If one 91111911. 11‘-
might 121:? 11.119 mam riaquimmta far rag-9114111102: of viral.
nu11111 1113.

“Eh: 111212111111: M110 1131:! cancer trauma 5133113 (m
the ear11111311111: 11 1511:: axe-1:3 tom 111211103. em 123911111
31121:: 11125.- 11: 12m 1111 11:19. 121 1113 1:21:13; 1310 run :16
{means were assistant only 1"- m (2111 lines flirtama. There-
fore. 1:13 1111121911 11121:: 1111 amnimmnta for 919 "1-2
m! :11: 1111: was: mflecu 1111: 0.1130113?” to 10211211111th a
particular 111-1210 acid, 121 £113 cam glutammm

311:: 11:11: 3117-3143 (3?) hEIVfi 11101:: that 1116 free 11.11119 1.01:1

p991: at mmmlian «all: do as» filffa! sslstnitlcmttlyo {me

I21
32:12-12) 2122122, 2.4212221222112212, mm 11-31: $321212? 1:: the free 32.22.2223 M123
5222221 211" 111212: 2122119 3:213 my {222.2221 12.: tha 2511222 (2211. I!
22212222121221.2213 1222243 22:11 nmmntmtm! 12211122211112.1111? by 1.220 .12!
cells, 11:21:: 27:22:22.2! 21922021221: for the: 2122213722192: 1213 12:21:: 12:2 2:22:11:
(aunt! 121, 122122 $121.53. 2.112221%, 1222.11122221223 1:22.52 not 1212.23 been
nu mart-2212* a rmmirwent 121 um «1121.1 11m}. 222222.21 by rant-231611;:
12.32.221.112: 1:222: can. {223211.21 emaentmto 11m 221112222 «22121 12112122222221-
lularly.

If 1221.27.21 122211 2222219 23:? 1:22 9 M 222221 22.12:»: (2:21.123 new: vary
51211151. as 2.22m 1:11:32 1:52:22. 11122::- sn-f! cmgzmnnm (37), $219
durum: 51-21219 3:122 arguments my ham been (no 1-: 1.2222112
ability 1:9 retain 2222 2222221210 iatmnanulmly 2mm 11 has beam
2122112422 12121211122122.1213. A affirm: moan: at 191.12.32.23» or
1932: has 12222121 9512an with 2:22: 2.21 am! llvar naus- ( 3?).

ma en... 2.222? 2.22 29 host cell 02322992211311!” M1210 112232235

1.21 its 12211221221211.1111? 1:221:11 may 2111:) e122,:lain 1:123 1222211118 111 $22.13
32.212332 921.1221 03222121215 dariaient; 2222122121222. 1222112131- 12: er 321-?
mu 1222121112211 eye's-21219 {29.1- flml regucamon 12:; the 22:21:22,223

of 8.1.1. 21-27223: 1322:1120 212112.51 101.13. 31:22:29 2232221221: 12021121 21.23: be
Guam-1125. 1n Em 12211232122134.2111: 32:11:12: M132 {.0211 (37 ), 212211
nynthcsis 2221.221: not: 1mm deg-21212112221 on th—n 3.2001 far 1E8 mums
at wanna 1:21 1229 121122221 2212221221212 c.2321:- Cygsti'm (22.12.2121 1229 521223211-
6»?! by £212.21- 223212191211 2211123221225 122. the 12022:; c311. 122.112.111.221; 512
ram) 2.232222132222113 (13). $322222: (:22 232.2; *1 bioasynfimms 212.2222: n91:
mrflm 1‘21? 52122122122: at 1:229 hos-.1 c2211, 11 2122.32 be eamgt‘a 1'22!-
flral 3222;211:2111921. .21 13 223.119 2221213111121 1.21211: c2222 1M2; 2.2.2.223 1123:

a cMnmmi’ at 32221-2 we 21.2113 ant" was flat :21: all news-1! 122

3 «s:
u ‘2.

synthaglan Cchifia 333 5 30343333: of Ether viruses (5)
33133 313 3334133 1: IEE EanEEEEs (7).
I33 EE'iI 1133: first Eet1i 3113 e In: tnBE AJ an? L“ 2

cells 333 E3 EEIEEEE t3 E33 1n33111Ey a! 23333 cells ta

eff33t13313 . EEEIEEA 97L§L119n1f cymtlna 3333 E cGEEonEE.
3t 3E3 virus 33¢ requirafi r33 Eta synthczia. $33 3311 33_

EJEEEEEIEEc :33E133 £233 3333133133. It 3333 a zatEtmy EEEEE E.

1% maulE eE E31. tfixc :31uizewrat fcr'mathtaalna 333 1333 of
3 13331192333 :33 3333133.

3333:3E333133 tfia 33:3 similarities @3333 cell 1133: may
E3 3033 Imyartamt tEan an elucEfation of ElfftEant w inn
3313 requirEEEEtEo Thu 313ml r rezulEe Eats b3 3333 £33 33
333 ""~ ? {c113 far tEelva or t or :E3 thirtasn G$fl&£t1&1
amino 33133 313! at eErEaiwta with $131: 313339» €1“torauvea
as cell ty;33. TEE r‘-E call was a cancer333 3311 2133 a
rueh {3323. ”Enerativa Elma than the "301E313 A3 c311.

It 333 3333 33333 tEat mast 3311 11333 have 523113?
groth 1333333333: 3 (13) 333 as inc 3:13 33313 (W V). 3331
c311 lines a! Mttarent ortEln are 1n“13t*p,Eiahable
mar330133§33113. Thia'may he 3333333 they hava 3333 13313333
331 maintEiEEE unfinr 313119 E envirawrental 3n3 nuE xitinnal

caniittona.

1.

2.

30

he

5.

~01 '3'" ‘9‘”
""J. ‘-v ‘r‘r l" It

or the thirtmn amino acid: in alnlaal csmtlal median.
miasmn cf methionine caumd tha smug: raiuatian in
tha mount at hemowlms precinct! by the w 1201.13.
gunman or human. caused. :21: least mnemon-

uhen basal mains: mg)... no 0219103942, additional mat-no
aclé :eaqulmaenu including ”amino worn as. marked as
am vacuuming raqulmmz hm! been 1mm minimal cam-
um. manta: was «3:210:06.

A1: @5119 am: 3732?:ng can! has! 5125111“ ”quits—gents for
twelve out: of thirteen mama new. tn basal medium angle.
In a glutmzlno deficient medium 11:ng can: profiucoé
Mesa-seventh the Mann: 0! virus undue-96 by Av «110 when
amp-area to tha mama produced In ample“ and ma.
6:31:an éeficiency least “faucet! viral replication in
basal manna Eagle by both the» A1) and Hag-2f c.1180
Incubatian time in a «flaunt «am: and (turnout

ms! in affected tau anpmt amino mid requlrmmts.

‘33

2.

3o

‘0

5o

6.

7o

3.

9o

10.

11.

12.

BIRLISGFIAT‘HI

Aek’emm. at. H. 1951. B016 or mathianinu in virus
grog-muon- J. Exptl. Hod. flan-3'43. ,

Buéer. J. r. and H. B. Horgan. 1953. Latent viral
infection of calls in name culture. VI. Role
of amino “1&8. glumino and glucose in psittaoosil
zigugiggropmpum in 1.. «11:. .1. E19“. mod. 17....3'

3&1. 8- Jo. G. C. ChflICOMI. W I. Go 30 “Ringgit.
i963. Rabbit mn- mmtiblc to when. virus.
mus. flash-0.61;}.

81mm. 6. Co 1952. Th. influence at ohmic.“ on th.
propagation orhEolicnniiul vital in tinn- culture.
J. Mano. £2: 1-H}.

23de. J. E. and E. “2310. 1953. Glace-o uni guanine
gr: rgiéoviml ”Mutation b: not: «11-. Virology 5'31
5;)-

Unmon. J. B. and L. min”. 1950. Poliovirua
protein: source. or mine acid. and time count or
aynthocilo J. 810. Ch“. £21 930. 1.7’k77o

Duties. Guru 3. 1956. Cry-tin. maimmt for normal.
poliovirus nation on tame: kidney new. culture».
35mm 5000 2.31331. 8101. EM. 211129'1320

mine. a. 5.. and Chapin. no 1953. rational. mutants
119231335.“ "upon." to ”tum. J. Gen. microbial.
’J " O

1mm». 8.. 1952. meditation of plaque: in nonolayor
new. culture. by singi- particles of an animal.
virus. 1m. Hat. Am. 801. 11:787-752-

Dupreo. Lo Ta. 8.. K. Snafu”. a. B. Wotan. and A. E.
navaloa'gy. 1962. Innuenoc of «ma protein: on
«incomimtim of misritioml muirmmts 9! (nut
in cultun. up. can Bu. flu33hhoso

Ear-31.0. R. 1355. Thu maniac mine acid muirmmtn

of 3 human car-aim e011 (cumin flora) in than
culture. J. Efxptlo Ned. mir‘b9c

Eagle. E. 1955. nutrition new: of manna cell. in
than calm". anions“ Lawn-50h.

Mi

13.

13.

15.

16.

1?.

13.

19e

Zle

2'2.

23.

’15

Eagle. E1. 1959. Mina ecid uetebolien In emanate
cell cultures. aelmee. mange-1:37.

£8318. He. He Re 31323, and He We lgt’éle Th.
intracellular amino new eomxmtmtione requiem!
tar proteln synthesls ln cultured manna cells.
«’0 Bible Chem. m:2039-20h2e

£3618. He and He Rebel. 1955e Th. nutritmnel
requirement" for the propagation of pone-Venue
time by the Item cell. J. Exptl. not}. lawn-237.

Earle. 51. 3. 19h). Erodeotim of malignancy lg; 1133.
H. The mouse fibrablest culturee end chugea'aeea
111 living celle. J. Eatl. Camer- Inst. 3.155.212.

mto'n. E. 13.. B. Emmix. m. E. Ferry. and n. Zambian.
1:351. Inhibition of influenza end new: virus in
tissue culture by halo amino cattle. Fm. Eon-e
mptle 35101. Ewe 211505-503e

23:03. E. DI. 30813. A. He. and E. 31171,... 1955.
Lysine inhibition or cell protein and Viral
syzxtheei a. ween-eel by other .31an wide. Free.
509. firtle Elel. Efide Ml993-10930

57.33031. E. De. he E‘e 3031‘. 0115 lo 3e We 195-230
mm» 301:! itabelsmoe and incomplete viral replies-
non. Arch. Gemte Vimafereoh Elissa-593.

taxman. A. 8.. and A. A. ”More. 1959. me effect of
awe emirmeneal factors on hers.“ Vine groin
In 1456?... eelle. VITOIGfiye 13113394610

13ml. Je fie. and fie Se Hallwfle 19h9e 1'31!th Of
affirm ma. tamer-azure 1n the preservation of
tum" hy refrigeration. Em. soc. Emu. Biol.
lied. 11:196-230.

fimafizl. R. 1961. Studiee on the viral etiology
vormce vulgar-1e in ti ewe cull culture. meals,
Kid-amen State U'nlverelcy.

Elm-35:19, A. 21.. and a. R. Mots-zen. 1955. :3th viral
infectian of «III in tieme culture. Hall or
cwtain “1119 ”It!“ Free. We EJ‘gtle Eiel. Ewe
H1536-539e

Eur-lax. e. a. 195?. e study of the herpes simplex
rims-rabbit kimey cell system by the plaque
technlauee Viz-clay facials-1657.

i3

339

39o

‘30.

file

52.

333.

b3.

1.5.

1:6.

#7.

“7

£168. .11.. em 31. I.” :1e. 1:153. 'me free amino amid
mnl 9r matured mm 0611!. J. E191. Chm. 2’11:
533*5 45e

$11.31!“?V' 50. “I“ Be 11.191518. 1.39450 ixer-nimtivnum
at new” cinder vitae end nytmegtelovime. J.
Bfifge ”11471.3?4e

Enfn‘imn. M. $1.. Jr", H. E. Harm. and II. J. 131213191“.
1351. he Minute of certain axino uni-ate and
zwtn 10113 mbemlfi‘l mi :Irorawntian at i‘hnller's
TJII virus and wage by minced one-day-nlfi
nbnne bxein. Arc-h. bitumen. 27H: 9-

Enfelnm. 1:. 52.. E. 3. Uinzler mid H.131. Emma. 1351.
A viral effect on the uptake at c from glueaae
in vitro by amine: wide in mouse brain. J. Biol.
Chm. 13:295-117.

Enwawrt. C. 1955. 16110111er cultures of trypsinized
new-Iraq kidney cells in synthetic medium. Applica-
Mon to poliovime ammonia. Iran. 309. Exptl.
1:491. rated. fichéfl-WO.

Rahflafi. 30' 3. El 5 71M" .3123 PO Ffi 36331.. J“. 11:37.
1111111131! emvnnnmtelixntim a! hernee 71 ‘13 _
daring; viral mtglidetinn. J. Vimloé: la]. 1-132.

Wow, E. K... Ii". 1‘. Hc-zfidilkin, II. I}. Flomonti.
V. J. Evans. and In”. I. Earle. 195:3. Study at
amino mid requimente tdr increase in cell no saile-
tion of HIS": “I": elme 929 (3118111 1.). 4’. flat. Cancer
infite‘§23775”735e

Tnnbersley. R. w... 1.1%.. 1961!. Mina mid requimente
Of hgrazzafhnp ex'viree lnIhunnn.eells.. Je Babb.
“I ”L'L 3.

{3105335531, II. L. 19347. The effect of metabolites.
mtnbelite mtamiete and mme inhibitors on
the bronchi: or the anlnie virus in finitlmad byze
.r ileum nature. J. 1113321191. ‘3: 36.351.

Tfififlfiflflfi. Re Lee and fie Le wilkln. 1953. Inhibition
of growth of he meninia vim! by B-Z-thiniylalnnlne
and it: reversal by: t‘Iwwvlanmne. Woo. ma.
bfiele Elflle bad. 51I434w431e

Tyndall. a. L. and 11 H. Ludwig. 1959. nutritional
rnqairmnta for the prmtuction or paliovimnee
t.» e 111. 3313.1ch and vmcinln time by
mtinuoue animal 141.12 culture» J. Eng-It. mt
93-133e

1&3.

500

510

333

Tyndall. I”. L. and 3:. 21. mama. 1953. emu“,
"filament rd).- mw‘fmnm or coxmakzo 231 vim:
in guitar!!! maxim-y ham-1; calla. J. FMSKE‘?
133‘3'13‘459 '

T115211. A. A. mi 11;. 3.. IT‘WJMI. 1953. A my? 111:: free
or war. ear-Inn. mad pqttme. tar plaque mum: of
hfit‘r‘qa Mable: flrua. Iran. 300. 11:511. Biol. 210d.
.,,.1§3...,;u ans-346.

htlafilflr’ C. Em. C. 3‘3. €115.23? and E. 1“. CEHIG‘y‘o 195-7.
immobem 113mb culture or wit’mllaL-liko cell:
rum taxman mm. J. Invest. Heme. fi13‘33-332.

Imwfiwr. J. 3. and J. V. 31311131. 19:53. Cystlnaa
immanent thumamustmt mutants of 130110711113.
3. 3313‘. 312’55-255.

3?
"lat!" ”quiz-menus for othor amino new: war. my:
detaminefi. aimless cmmmuva studio: are mac‘s. tho amino
acid thick: 1: most required 13 not known.

The discrepancies bah-man I‘mfimrsley'l work am: the
stud: her-o could. but been due to use of different cell
nun. To teat this paambnuy tha Him}! «11 line used by
Rolmm mm emanated to the AU 3011. The gyroemiuna, weep:
for a mortar incubation ported . am medium warn 113:. that
of tankerslay' I. This moMfloauon Ital necessary as unseat
in tha results on tho affect of incubation time on prm'uctton
or virus 1:: annual: momma (27). Each of tho thirteen
amino acid. deflumun. cxcopc alutmalne, had 1 similar
ct‘tece on vim]. aultiyllcation in both the AU and 52332-2
ecu. alumina my mare amount: for the 3:39.; can than
m «11. $11.13 an: «math of tho mount of virus graduate
by tho 1.: cell was ”Manet! by 21331-2 11: glutamina ancient.
”film.

The varying; nqulrmmua far repllaamm of the awn
virus fin not 11:23:“: a: gamma on (turning min: new metab-
olism mung, has: «119. In Manning ”110. cm thirteen
essential amino Acids. snags-t glutmlns. an annualized ta
a mini-m1 diagram the: are primarily Incormmtefi later cell
protein “-3). emission of out essential amino mm probably
11m:- the Inaorpouuon or that amino new into viral mum"
and viral premix: out.

Bataan on: mine who ace-ad was deleted true; the extract-annals:

madman, hoe: can: maintained the concentration Of tho malnlng

123

212221 22:21:22 221221 1111111 (2222.12.22: far twenty-£2222 1* 22:12:11 (37 )2
13122222122 12*a 722 25121:? 2222. 12121 2222121 rmflrmeuu 2212!. 22:21 22212212 121
2122222: 1 2221213 22221:" 22222122 122211.131, 112221 diffemnt 3:13.220 2.22121
11222212222221 2.12221. 22.2.29 1222121 25.2.19 1:9 1519 22221222211121.1221 121‘ 2221
32121120 5212'. 1.22 2.219 12212122222112.2122r 2:20:11 am! 113* 12f {“2211 2222 4221211261
22122222211222: 221‘ the 222311 2.2211. ”21211 22:2: :2 2212111221 1:212:22: 111211211-
11121: 2.2212! 9***e**21m113.., 1212211122: 10 M811. 5221219 221122. 122-1:
1213 2222222232223 1222: a. 2:211 14-22 122111.21: 32:91:11.2 2322212122213 (13).
This my 221223 eatery: 121 21221312212121 22:21:22: 22.22122. 11‘ 1219
commtmtim 21* 9.21 @1222) 2.2.2.12 amped balm n 2221112221 112222512
1m, 1:12:21 221122121 12222 1222123 1212:1912 1121222. 1.212: 22:21: 22:22 2221221": 2

.1 2212112131. 9.9212231212211621 at 2211 3221110 2222121! 22223 2.2114: :12}
a role 1:: 22:22.11121 12:21:! anthers”. It. has bean #2222221 1:21:21 3
mgxgfly 01’ 22:21:21: 2222122; 1.22.22 2221211222125: for 2212211012: 122121 2232113229213
121 1221 22121221222: 31;. £3.13. (33):- 1 2:12:11 1221* 3222112222 1222222111121-
11122 2222-1123 12:25 not 1212:: 21321921221221.1222. If 0221 2212122101, 11
22122.1 1’1‘11’ 21% 21:21:20 twflmwmts far :11 21192211021 a? flu).
nucla12 2211.

Tim 12* .1. 221112211: mine 32.1122 (20222131212221 113:: 2222;223:222 22*:
1210 a12112<2r1221122182 2222121232 22421:?! 11502131312311.2221. the 12:22:11.1:
22221222: 522122 2222.22. 212: 222.211 112.129. 1:1 12:13 221225;! 1213 ram: 1219
{321121121 22222:: 2223212122221. 0:21....y the (22:11 11222143 212221223. The!»
fora. 1.219 221?? 4:229:15. 3:221:20 21:21.21 mmurmnuta far 1212: 22:. wk
and 2123 12119 2.12121: rafl....t- 121211? 02.51“”? to 22021224221122.2129 a
warty-2.21.3: 22:21:22! 3121:” 2. 121 12213 case 9.112211211221212

2122:: 222222 22:12:11 (3?) haw: 25102222 121221 12123 free. 22:21:19 22221:!

5111211: 22! 22222212211122: mils m: not 211551: Sammie-many. 222m

9:1

amino 51:71:, mathmnmm, 1:13.: mt. fauna”: 111 £312 free: 3:11:13 0,3115
pan-1 :11" liver 31:11:: 321:2; 1:33 {mam in the: 331?..3 (3311. .2!
11131211331213 1:11:14: m: 10311111211134: 11112131111111.1113; by the 1::
cans, 1311:: 1:11:11 amen-:1: m:- the: mctfitmzlne 111.511.1111. "zen.
(mm! 111 £11.13 $111651. 1111;111:1111, 31111113111113: 11:11: mt 1:31:19 1:21:12:
am 31112131111 :1 11:111.:1rr1-9nt m the can 113:1 113111 1:131 11:11:111113:
beenusa 1213 31313.. mania eonnentm .m the: mum and 11:11:21.1.-
lulnrlro

If ¢.+:1:::'::.3 11:11:: 11:11:15 of 1:31.11 .11! mafi- 122:3»: 333.13 11111:: vary
al.3113131, as mam: 1:11:31 1:15:41, 111m:- snfi cmmmtim (37), the
“Hermit- 5:11 121.49 a: 11:. 111111111 11111111111: may have; been 61m $3 2:11:11:
ability {:3 1161153111 51:: 5:211:16) intrana’tlulnrly 11110.11 1% has ham
mines: cflmmllularly. 1 4913111121: may: i; of 3.11131: 011
1.033 has b11111 051111-1111? with :5133 1.1.1:: 1111111 3:11.15 (3?).

Tie baud: t a? b3“:- 3 11:15:13 (2311 «om-11:1 tra11n1 3.121110 31:21:14!
In its an: 1113‘). 11:1 11331 :113 111:: 1:11-3:21 the 1111:1211: in 11:13
51.211131 9:112: 11111121219 11:11:31.1: 1113511111. :135.:11r- 12:73 or 1:31:13
@0113 11:11:11,191 oyctino- far vim]. reg; 1133.111: ts: $214: ext-111:1:
of all. @1113: £313.12. name)- 11:13. 311134: 1:323:13. 1031:! 2111:: he
(11:1:th 1x: the: intranellulnr 9:11:11: 9.91:3 1:031 {37). 11122:}.
synthwu 1:112 mt have (1135:1131 on this 31661 1‘31- 119 3111111130
or 1313:1111 1:1 1:13 vim}. 1121019111 111:. {2:121:13 30:11:: :33 1:131:31-
(3011 by £111.” gm metal 1:12:11. 1:31.13- tn the hast; cell, incluflag 1;:
{19:19 3111:11‘191159. (13}. 3.121711% €12 0331: m1.)- 11111105113 (“was um;
suffice $21.11 3:133:21 or Me host call, it. 11:13! 159 12151111: :1:
firs-.11 2111;111:111911. it 13 also 11123111113 tan 1: (2:15:11: 1: was: no:

a (tangy-.1111: 0f ham-111.11: virus and 11:13 net at: 3.11 33311-16 in

ntheais. Cyzzifia ”11 a £113: 11$ 0? 911111311111 ($)
11111:: CW1 131111 it £11 synthesis (?).
$31 11311L 111% f3: 1111111135 {11*tflfih AU and £7p~2
01113 111 11 zfijatef ta £11 1113 1111 c! 1111 @1119 t:

.

eff11civfl‘y 11111131111 1311111. if cyetlna 11a 1 r11'0111*

:1:
:1
:4
1
‘, '3

11;

at :1: Virus 115 11311113 {11 1: E11111,
tjfith£1111 (151111 {19'11233131 if 3311 1 3121111 1111131.

1% maul? cr~1115 £11 r1qu111111t far 1at1131111 an! 1311 of

a 11111114111 €91 111E111.

3131111111113 t1: 1113 £11111 tttea 11511 (111 111111113
ta more layattamt than an elucL Ha ha: at flffsrent amine
311d regulrfiwents. I3 @111 H 1611111‘1nt9 hr wean thm 13
aha E19»? {1’115 far t1 1311 011 cf :31 thirtgen 111112111
13119 11111 511 111 c311111.11 with £111: 1111111 differanaea
as cell 2:;11. 111 113~2 c111 was 1 112911113 1211 with
ENflh {11111 3*1crat111 £111 $3.1 the "normal’ 13 call.

11 113 3111 c:1arn t1 1 mast cell lines 1311 5111111
grewt3 1131$1111Jn (13) 133 amine aria 10613 (373- 1333
cell lines 1! 11111131: crigln are infliatiggnxahable
meryhelagic1ily. @113'111 ha heaxuca they have teen 11 latai
11* rai1t1*w~‘ an er 111 liar envir1wmc1t11 and nu’zit11113

oanfiltiana.

10

20

3.

’h

5.

.‘v‘§awi a "V
d- . M $.- a

01' the thuwm mama mm: In minimal “annual meal-am.
61515514323 or watt/11011130 mama um mafia” mfluction 1n
the mmunb at hazy-antral firmmceé by me Ail wells.
mum-1m or hinldlna mm the least. reduction.

unaa baml 2mm Eagle was employed. Mammal mine
mm remumnmts including; alt-25mm. no" as marked as
tha meta-alanine mama-amt had been. when 51:11:25.3. cam-
tml me. has ms mmoyed.

as cells was} flan-2 c.0111 hm! $131113: requirements for
twelve an: at thin-m amino acid. in ham]. grandma Sing)...
In a glutmino defiaimt mounts 13929-2 can: pm‘ueod
anew-seventh the mount cf vim: pmfiuaw by M! 00110 when
tamper”. :0 ma amount produced in ammo“ and mm
Crating (influenza: 1mm affected viral replicatlan in
13:29:31 madam Eagle by both um A1) and H23ng calla.
lawman-an cm. in a deflated: medium and “throat

mafia affected tn: wpamt mama mm requirements.

’43

2.

3.

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5.

6.

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9.

10.

11.

12.

BIBLIWRAT‘HY

Aekamwm. ‘2. if. 1951. P010 or mothimino in viral;

:mpagatim. J.
3333". J. r. and! H. R.

EIptlo Eifio 218333‘3h3o
Morgan. 1953. Latent viral

infection of cells in name culture. V1. 33:31.
of amino new a. glutamino and slam” in p811: moni-
Virus propagatim in i. «11-. J. Earp“. #305. #33:

617-6300

3031.. A. JO. Go Co Chriltaf131I. and I. Go 30 Farmingcr.
1-9.3. Rabbit coil.- nmtibio h when. rims.
Lancet. 13: 61.0.6145.

13mm. 6. C. 1952. The inflame of ahmieal: on the
pronngation orufoiiomyclitiu virus in tin“ culture.

J. IQEHROO 598

Damn“. J. 3. cm! H.

hbh3.
333.10. 1753. Glace-o and giumino

in renown-u produetiou by Horn «11.. Virology 35.

555s566.
Damon. J. 3. and L.

wins”. 1963. reliant-u-

protein: Ham-co of amino acid- and tine ammo of
aynthonin. J. €310. €131”. z}: 2300 1875‘770

Buzzes. Heart. a. 14355. Cyctinn roquimmt tor mama}.
paliovimn action on make: kiémy new. culture.
2:93. we. Exp“. Biol. 130:1. 23.12.39-132.

was. “. EC. and @8313. H. 1953. Fonbflmi u‘lthfit.
with altered rowan-u to entino. J. Gen. Pic-rabid.

13.320-329.
Buiueaco. 3.. 1952.

Production of plaqudn in 1:07:01.er

new. culture. h: singi- particles of an animal
virus. rm. Hat. Acad. Sci. 11:737-752.

Cameo. L. '1'" K. K. Sanford. B. B. Homttsil. and A. *3.

rovalwizy. 1952.

111(1qu of «ma proteins on

Hoeoraimcim or mtritiomi "gunmen” of «3.11!
in culture. 81p. Cali Ron. 2%.33L-h95.

3851., no 1955. Th.

ermine amino acid ”quiz-nont-

or 3 human carcinoma can (strain mm) in name
culturo. Jo Siptlo Fed. 122I37-h90

&;II. a. 13955. Hun-tum and: or manual: can. in

ti nan nulturo.

sumo. £25.50).- 591;.
Ma

13¢

15'.

15.

16.

170

13.

1?.

20.

21.

22.

23.

250.

‘15

Rm]... *1. 1959. Mina acid "ammo. in when
cal). mutant. Sciatica. mange-.1237.

mile. E... K. i. tier-s, and R. Levy. 1961. Thu
intracellular amino acid commutation. roquirod
for protein synthesis in culturod malim 00113.
J. 33101. Chan. muggy-202:2.

Eagle, F5. and K. and. 1356. ’1'in nutritional
requirements for the imagination or poliam olitin
virus by in. mm mi .N J. Emmi. Mod. 1042271437.

Eat]... H. R. 19113. rmdaotion of miimcy in 333313.
I'll. Th. was “trams: eult’anl and changes mag:
in living cells. J. Mil. Cancer Inst. 31:155-212.

mtan. ‘3'. m. 5. Emma. H. B. Ferry. and D. gunman.
Uffil. inhibition of inflating; and mm» virus in
new. (sultan by basic amino acids. Em. soc.
Lirtlo 33101. 33%. 223535-5593.

Eaton. a“. D... “can. A. $1.. amd H. Bimio. $55.
Lysin- inhibition or «ii protein and 71ml
writhui a. finer»). by other amino 11216.3. Fm.
30G. Marti. 3161. EM. miggklao'flo

Eatflfi. No D..- M E. 3681.. and I. R. m. 196-90 ‘
Mina mid 1.5.1.3.. and imam-plot. vinl uglier
tion. Arch. Gem” Vimsforaoh Ens-33.5921.

mm. A. 3.. and A. A. Newton. 1959. m. often: of
at». unfit-mental factors on hart-ml Vim! gram
in MM cells. Vining. ZlhhMSIO

Eanzu. J. 3.. and H. E. Ufillwfio 191390 5013th at
arr-ran and tumor-amt. in tn. preservation of f {
$13310! by refrigeration. ”£53000 30¢. uptlu 31010
91:31. 11:195-230.

M53521. H. 1361. Stain! on tho vim]. Wining-z?
warning vulgar-it in Home can Galvin. Thosi a.
:aichisaa Stan cairn-city.

magi-7:19. i. 11.. and H. B. mfgz'm. 1952’». Lani viral
infection of call. in than. ouitnn. Ital. or
certain mini: midi. PM. 304:. Emu. Eiel. m3.
231536-5090

Karim. A. .5. 1-957. A My of tho, heme. nimble!
virus-rabbit kimay cell cyst” by the plaque
tacimiauo. Viroiagy awn-1457.

25.

25.

27.

230

30.

31.

320

33.-

33*.

350

36.

136

mom”. R. J. 19.53:. m. tiniblo until?! at the min»
acid :ml in L strain fibmblgata. firms. 30¢.
Era-91:1. 3101. Fiat!- wiZQ-ZSO

Eunéin. U. 13.. F—f. L. Eobbina. and E. K. mun. 1959.
lmibition of inflnma virus multiplication in
clam. culture by unnatural analog- of amino
mic a. Virgina: lib-2?.

Lflfilfig 7. JD. Jr..-und L. V. 360:3. 1352. HQtflthfifll
manimmts far it» proéuntion at hem" simian:
virus. I. Influmo or glucose and glutmina on
homes timplax virus straduction by 11.3.. 61511..

3. 232398. 333%75432.

M¢rfihant. U. Jog E. E» Hallman. Ru fiahnoider. £35 3- E.
wink-neat}. 1962. Protection of animal calla rim
minylcolinlon. Bacterial. Elmo... p. lhi.

magi-t7, K. H. 1%2. saloon" utilization of minor
acids by Win 0011 animal. in. Call. fies.
221239—230.

Hanger, .5. and J. Sabina. 1966. The :01. of methionine
deficiency in poliavimn rag-lingual: in than cul-
tnri. J. Emil. ma. min-21%.

mean. A. E... L. mmahusmy. and a. H. Toolan. 1955.
(3leer characteristics of {our permanent line:
a! human. men: «110. (“armor is”. 53:593-632.

Eflrflflflv E. R0 1955. Factor! tainted to Ch. growth Of
peittaoonic virus. 17. certain amino cal-:19,
vitwinn'md other mam... J. lazy-t1. mad.
gzfihslnbeO

rat—«Me,- Arm? 3.. am! ITES‘XflEfiQ-p mu. 3. 1955.
1'31. dopenémco or nucleic acid synthesis on 1:33.
presume of mint) acias in Escher! 9511A wig.

Jo flant.‘250677-633.

Emma, a. 2'... n. L. macrborg and R. J. winzler.
1-,.3fi2. Effects of certain amine: acids and related
www.m- on propagation of nmu meet-shunti-
Virus. iron. Soc. mu. Biol. and. 2213409411.

I'garm. ‘51. 3.. mm B. J. wimlnr. 1950. Amino acid
mummies and mailer“ 6:“).le virus in none.» brain
ties... culture. Pod. Fm. £12339. 1 2.339-396.

ransom. J.. and H. R. ifiorgan. 1959. Pactmrn nutrirs
inf’laoncan: la 12:913ch du vim. do l‘hcr‘nu em:
in macho do nannies L c. Eula. Ann. Inst.
immu- afizhha-Iajla.

3-39

39.

3&0.

b1.

132.

333.

1.5.

126.

'47.

1*7"

3133, @- A0. £33 fl. Eflfilfic 1953. Th. f?‘fl naina 301d
5:41:31 9: mutant-.1 hmm cells. J. Eiol. (than. 2‘31:
533“5“3~

F13”fi9?, 30. flfid Bo Lflfllflu 1955c Theraninactivalion
cat new” 1:123:19: Virus and cflmaxaloflma. J.
Ratl.‘fl2:fi7l.&?h.

Iiiafelmn, H. 31.. In. R. E. Pearson. ill-Id 5?. .3. E13131”.
195?. TF1.» crust! of certain amino sci-:51. and
metabfilio mtagani ,I on wr¢*r>&:x;ation of ’i‘haiicr's
[c.(,'J'-';Ii virus and i; 113::sz by minccd one-carols!
£52293 mam. Arch. Eioohm. £3.69-

Esfelam. FE. 51.. R. J. Hinzlor and 3.15%. Farm. 1-351.
A virus effect on :21. uptake of c ' from glue”.
in vitm by amino acids in nous: brain. J. Biol.
Chm. miEQ5-2170

Eagraahart. C. 1955. itmolayor cultures of trypainizod
ammo: kidney calls in mthctio again. Applica-
tirm to poliovims synthesis. 43m. 396. anti.
1.5.1331. filed. flihw79.

Romam. 8.. 3. 3. 8:13.35?) and r. 2. Emma. Jr. 1367.
Callular omr~artramtallzatiun or honms v1.71.” ,
daring viral rot-gliution. J. Virology lull-192.

SOMO'M, 51'. K" 'w'. T. Hefiailkin. K. C‘. Flora-ion“.
V. J. Evans. and E”. F. Earls. l?5’§. Study of
who acid ”quiz-manta m increase in cell pea-mala-
”an of mm elmo 92*} (strain 1.). J. Rat..- Cancer
inst. gays-.735.

mammal”, :2. w... Jr" 196% Amino acid maximum.
9! batman Mp1” 71m! in hwm cells. J. Rant.
21:59:30“).

“amazing 1?. L. 19117. n. “he: of notabolitu.
mmmlitn mama“. and «mm. inhibitors on
we 35:1”th or we vaacinia virus in Hamlin-id in.
Of iisaua culture. J. immunal. £§£355*3510

Thawrmfi. n. I... may 3.3. L. mum. 195%. Inhibition ‘
of firm?! at no meninia virus by B-zuthimylalanino
6&2 it: reversal by phenylalanim. hm. ma.
kflyila £191. fled. éll§3#*3350

Tym‘iall. E. L. and E. H. Ludwig. 19:30. :Eutritimml
raqairmnta for tha pmductian or poliwimeen
ti... III. ammonia 33 and vmninia. virus bg
ameinuaua animal «:91 guitar”. J. met. .. m
96-133. ’

w

3.3.

543.

50.

51.

343

Tméall, R. L. and E. a. mania. 1963. Cystitis
requimant tar yrfluctimi of coxmckio f3. vima
in eulturfl 9.01226}! ham-1'. (212115. J. Mctfiifg
13.3?“1335.

T?t%ll. A. A. «M Ii. 5;. Emma‘s. 1.953. A meéim free
of mmr, sea-mm. and regime. for plaque! assay or
hex-gm! single: Virus. Iran. 300. Ezptl. Biol. flied.
.13.}. Jul-43%.

maelar. C. fine. Fa. (tasty and £1. 1‘. lecy. 195W.
Lama-tam tissue (sultan of at mental-1m. cells
tram human Mia. J. Inn-st. llama. 33:333-392.

Immmr. J. 8. and J. V. Milan. 1953:. Cysti'na-
im.g:mccns chomontiatant nut-ants at poliovirus.
3. Iii-13%. 3325502553

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