The Trasnscription Herpes Cacu

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1973;33:1402-1416.Cancer Res

Bernard Roizman and Niza FrenkelProductive Infection and in Human Cervical Cancer TissueThe Transcription and State of Herpes Simplex Virus DNA in

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[C AN CER R ESE AR CH 33. 1402 1416. June 1973]The Transcription and State of Herpes Simplex Virus DNA inProductive Infection and in H um an Cervical Cancer Tissue1Bernard Roizm an2 and N iza FrenkelDep ar tmen ts o f M icro bio lo g y [R K \ F ] a n d Biop h ys ic s [B /? ] The Un iversi ty o f Ch ica go Ch ica go I ll in o is 6 0 63 7

SUMMARYW e are reporting on the transcriptional program ofherpes sim plex viruses 1 and 2 (HSV-1 and HSV-2) inproductive infection in hum an epiderm oid (H Ep-2) cellsand on the transcription and the state of HSV-2 DNAin a hum an cervical tum or. Our results m ay be sum m ariz ed a s fo llo ws .In productive infection, a total of 48% of HSV-1 DNAis transcribed. A nalysis of the transcripts show s tw o k indsof controls. O ff-on control of transcrip tion is evide nt

from the fact that early, before the onset of viral DNAsynthesis, the transcripts arise from 44% of the DNAw hereas late in infection the transcripts present in the infected cell arise from 48% of the D NA . The extent of transcription of the HSV-1 DNA early in infection is not affe cte d b y c yc lo he xim id e, a n in hib ito r o f p ro te in sy nth esis .C ontro l o f R NA ab undance is evident fro m the observ ationthat the transcripts present both early and late in infection form tw o classes differing in m olar ratios. The abundant R NA is com plem entary to 14 and 19% of viral DNAe arly a nd la te in in fe ctio n, re sp ec tiv ely , w he re as th e sc arc eR NA is com plem entary to 30 and 28% , respectively, ofthe DNA at the sam e tim e intervals. Several lines of evidence suggest that th e abund ant R NA specifies structuralp ro te in s o f th e v iru s.The transcriptional program of HSV-2 DNA differsfro m that of H SV -1 D NA in tw o respec ts. F irst, the am ounto f D NA tra ns crib ed e arly a nd la te in in fe ctio n c orre sp on dsto 21 and 50% , respectively. S econd, w hile the viral R NApresent late in infection also form s tw o classes differing inabundance, that present early in infection form s only onea bu nd an ce c la ss. C yc lo he xim id e d oe s a ffe ct tra nsc rip tio nof HSV-2 DNA in that in the presence of the drug 45% ofviral D NA is transcribed by 2 hr postinfection indicatingthat a t least o ne off-on control of transcription is m ed ia te d b y p ro te in s yn th esis.HSV -1 and H SV-2 D NA's share in com m on approxim ately 50% of their sequences w ith good m atching of basepa irs. A nalysis of the transcription o f the D NA seque nces

' P re sen ted at th e A me rica n C an ce r S oc ie ty C on fere nc e o n H erp es-v irus a nd C erv ic al C anc er, D ec em be r 8 to 1 0. 1 97 2, K ey B isc ay ne. F la .T hes e stud ies w ere a id ed by g ran ts C A-08 49 4 fro m the N ation al C anc erIn stitute. G ran t V C 1 03 H from th e A m eric an C an ce r S oc iety, an d G ra ntG B 2 73 56 f ro m t he N a ti on al S c ie nc e F o un da ti on .2 P re se nt ed b y.3 P os td oc to ra l T ra in ee s up po rt ed b y a g ra nt f ro m th e W h ite ha ll F ou nda tio n to D r. R oi/m an .

shared in com mon indicates that they are about evenlydistributed am ong the tem plates for abundant and scarceRNA. However, the com m on sequences form 71% of thetotal sequences specifying abundant RN A and only 39%o f th e se qu en ce s sp ec ify in g sc arc e R NA .A cervical tum or free of virus or viral antigen wasanalyzed by D NA -R NA hybridization techniques for thepresence of viral RNA transcripts. The cervical tum orcontained viral RNA transcripts com plem entary to 5%of HSV-2 DNA , and prelim inary studies show that theyc orre sp on d to b oth e arly a nd la te tra ns crip ts.A nalysis of the cervical D NA for the presence of H SV -2D NA sequences led to three conclusions, i.e., first, onlya fragm ent representing 40% of H SV -2 D NA w as present;second, the fragm ent was present at concentrations of 1m ole/m ole of cell DNA and, third, at least parts of thisfragm ent are c ovalently linked to ho st D NA .INTRODUCTION

T he ProblemH S V-1 4 a nd H S V-2 a re b io lo gic ally a nd im m un olo gic ally

related and are am ong the m ost com mon viruses infectingm an (25, 40). A lthough both viruses are transm ittedb y c lo se p erso na l c on ta ct, th ey u su ally in fe ct d iffe re nt b od ysites (25). N um erou s studies indicate that H SV -2 is potentially if not actually oncog enic. B rie fly, th e associa tion ofH SV-2 w ith hum an cervical cancer is based on viral andseroepidem iological studies and on the presence of viralantigen in exfoliated cells from p atients w ith cervical dys-plasias and cancers (25. 31, 41). The oncogenic potentialof the virus also em erges from reports of production oftum ors in ham sters and m ice (25, 27) and from the experim ents of Duff and Rapp (6) which resulted in the transform ation of baby ham ster fibroblasts into highly m alignant, m etastatic cells. H SV -1, alb eit genetically rela ted,a pp ea rs to b e m uc h le ss o nc og en ic in e xp erim en ta l stu die s.The above recital of facts and observations leads to 2 obvio us q ue stio ns. T he 1 st q ue stio n is w he th er th e re pro du ctiv ecycles of H SV-1 and HSV-2 differ in som e specific property which m ight account for the apparently greateroncogenic potential of HSV-2. The 2nd question relates

Thebbreviations used are: H SV -1. herpes sim plex virus type I;H SV -2. herpes sim plex virus type 2: H Ep-2. hum an epiderm oid carcinoma.

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Tr anscr ip tion of H er pes Simplex Vir us D NAs pe cific ally to the as so ciatio n o f H SV -2 to hum an c erv ic alcancer. If indeed this virus causes the tumor, it could bepredicted that the tumo r w ould co ntain v iral D N A and atleast some products specified by the virus. This reports um marize s o ur atte mpts to de al w ith the se que stions .The Background

Pertinent to this report are several fac ts and observationsregarding the virus and the infected cell. Since much ofthis information is published it may be summarized asfollows.HSV -1 and HSV -2 consists of proteins, lipids, poly-a.nine s, and D N A . The se s truc tural e le me nts are arrang edinto 3 arc hi te ctural c om po ne nts , i .e ., a D N A c ore , a c aps id,and an env elo pe (3 9). The HS V-1 v irio n has been s tudiedm os t tho ro ug hl y to date and c ons is ts o f at le as t 2 4 pro te insnumbered co nsecutiv ely 1 thro ug h 2 4 and rang ing in siz efro m 2 75 ,0 00 to 2 5,0 00 dallo ns in m ole cular w eig ht (4 4).The envelope of the virion is derived from a modifiednuc le ar m em brane . In the e le ctro n m ic ro sc ope it appe arsas a trilaminar mem brane w ith s ho rt spikes pro trudingfro m its surface. The env elo pe co ntains sperm idine (1 5)and lipids, presumably derived from the host, and Proteins 7, 8, 1 1, 12 , 13, 14, 15, 16, 17, and 18, a ll o f w hich areglycosylated (18) .Three kinds o f caps ids hav e been analy zed to date (1 6).Capsid Forms A and B are found in the nucleus of the infected cell. Form A lacks the core and consists of 4 proteins (Pro teins 5 . 1 9, 2 3, and 2 4) arrang ed in 2 lay ers. Theouter layer consis ts of 162 subunits or capsomeres arrangedin the fo rm of an icosahedron. Fo rm B contains the D NAcore and consists of 6 proteins (Proteins 5, 19, 22A , 23,and 24). Of the proteins present in Form B but not inForm A , Protein 22A appears to be situated on the surfac e o f the c aps id le av ing o nly Pro te in 2 1 as the c andidatefo r the co re pro te ins. Capsid Fo rm C is o btained by deterg ent treatm ent o f the env elo pe v irus. It co ntains sperm -ine, trace amo unts o f v irus g ly co pro teins , and Pro teins1 to 4 , 5, 19, 21, 23 , and 24. A nalysis of the structural proteins of HSV -1 and their assembly indicates that in theco urse of envelopment the B capsid binds Proteins 1 to 4and at the same time Protein 22A is cleaved to yield Prote in 2 2. The deterg ents treatm ent remo ves Pro tein 2 2 butleaves Proteins 1 to 4 and the rest of the capsid proteinss ti ll attac he d to the c aps id.The core of the virion consists of the D NA spooled on acylindrical body probably consisting of proteins. Thespooled D NA has the shape of a toro id w ith the cylindrical m ass bo dy passing thro ug h its ho le (1 2).HSV -1 and HSV -2 D NA 's differ in base composition(2 , 1 7, 1 9). A t le as t 9 5% o f the s eque nc es c ontaine d in H SV -1 D N A are unique (8 ). B y c ontras t, as re po rte d he re , H SV -2 D N A appe ars to hav e re pe ti tiv e s eque nc es am ounting toabout 16% of its sequences. The DNA's of both HSV-1and HS V-2 are linear do uble-strande d mo lecules [M .W .(9 9 5 ) x IO 6dal to ns ] (1 7) and bo th c ontain alkali-l abilebo nds at unique sites (9 ). The nature o f the alkali-labile

bonds is no t know n. A nalysis of the D NA extracted fro mnuclei o f infected cells after labeling w ith thy midine-3 Hfo r inte rv als rang ing f ro m 3 to 1 20 m in s ug ge sts that H SV -DNA immediate ly af ter synthesis contains numerous alkal i-labile bo nds w hich are re paired and/o r lig ated after sy nthesis. The alkali-labile bo nds in v iral D NA co ntained inv irio ns mig ht be the co nsequence o f inco mplete repair atunique sites (9 ). Hy bridizatio n studies indicate that theHSV -1 and HSV -2 D NA 's share in common 47% of theirsequences w ith good (85%) matching of base pairs (20).A nalyses of the kinetic complexity of HSV -1 D NA showthat 1 v iral D NA molecule (M.W . 10 daltons) containsall the g ene tic info rm atio n nec essary fo r the multiplic ation o f thi s v irus ( 8) .The bio sy nthe sis o f v iral s truc tural c om po ne nts is c ompartmentalized. V iral D NA is made and transcribed inthe nuc le us . V iral pro te ins are m ade in the c yto plas m. Pe rtine nt to this re po rt are s ev eral fe ature s o f the trans criptio n of the D NA . First, at least a portion of the viral D NAis trans cri be d in the fo rm o f hi gh-m ole cular-w eig ht RN Aw hich contains the same sequences but is larger than theRN A fo und o n c yto plas mic po ly ribo so me s. The se finding simply that the RN A transcripts are cleaved at somepo int betw een sy nthesis and func tio n o n po ly ribo so mes(35, 45). Second, at least a fraction of viral RNA trans cri pts is ade ny late d. The ade ny latio n o f the RN A i s a po st-transcriptio nal ev ent taking place during a 1 5- to 2 0-m ininterv al betw een sy nthesis o f the RN A in the nuc leus andits appearance in the cytoplasm (2) .The co nsequence o f pro ductiv e infectio n is inev itablycell death (34). As has been stated before (32, 34), thisc onc lus io n im plie s that if the infe cte d c ell is to s urv iv e thee xpressio n o f the v irus in these cells must be less than thatin productively infected ones.M AT ER I AL S AND M ET H ODSC ells and V ir us

H SV -1 and H SV -2 pro to ty pe s us ed in the se s tudie s w erepre pare d in HEp- 2 c el ls . The pro ce dure s f or m ainte nanc eand infe ctio n o f the se c ells hav e be en de sc ribe d e ls ew he re(38). A n isolate from a facial infection passaged at lowmultiplicity a limited number o f times and desig nated atthe F strain served as a prototype of HSV-1. An isolatefro m a g enital infectio n passag ed in a similar fashio n anddesig nated as the G strain se rv ed as a pro to ty pe o f HS V-2 .In additio n, w e also tested strain 1 74 o f HS V-2 iso lated byD r. M auri zio Te rni at the U niv ers ity o f Fe rrara, Fe rrara.Italy . The G and 1 74 s trains c anno t be diff ere ntiate d w ithre spe ct to any o f the pro pe rtie s re le vant to the re sults pres ented here. The g eneral pro perties o f H SV -1 and HS V-2prototypes have been reviewed e lsewhere (40).Pr epar ation of V ir al D N A

The pro ce dure s fo r in v iv o labe ling , e xtrac tio n, and thecharacteristics of herpes simplex D NA w ere publishedJUNE 1973 1403



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Ber na rd Roizma n a nd Niza F r enkelels ew here (8 , 9 , 1 9, 2 0). The pro cedure fo r preparatio n o fin vir o-la be led vir a l D NA wa s tha t of Non a ya ma a ndPagano (28) as described in detai l e lsewhere (11) .E xtr action of R N A fr om I nfected C ells

The RNA was extracted by the method of Lee et al.(2 1) to e ns ure re co ve ry o f ade ny late d v irus -s pe cif ic R NA(2).The Cervical Tumor

The cerv ical tumo r, w eig hing appro xim ately 7 0 g , w assurgically removed from an untreated patient and w asfurnished by Dr. AndrNahmias from Emory Univers ity .A ppro ximately 8 0% o f the tumo r m ass co nsis ted o f larg e,w ell-diffe re ntiate d c anc er c ells . It w as e xte ns iv ely te ste dat Em ory U nive rs ity f or inf ec tious v irus and v iral antige nsw ith ne gativ e res ults (1 1). The tumo r RN A and D N A w ereextracted as previously described (11).Experimental Design

The s tudie s de sc ribe d in this pape r de al w ith analy se s o fthe kine ti cs o f hy bridi zation o f puri fi ed labe le d v iral D N Aw ith excess unlabeled RN A fro m productiv ely infectedHEp-2 cells and with excess unlabeled D NA and RNAextracted from a cervical tumor.The D N A-RN A hy bridizatio ns inv olv ed incubatio n inliquid o f s mall am ounts o f in v /v o-l abe le d D N A w ith e xc es sRN A in 0.05 M Tris (pH 8.05)-0 .07 M N aCl as described inde tail e lsew here (1 0). The amo unts o f D NA -RN A hy bridsw e re de te rm ine d f ol lo w ing dige stion o f the hy bridi zationm ix ture w ith nuc le as es ( N euro spo ra c ro ss a nuc le as e aloneo r after treatment w ith shark liv er e ndo nucleas e) hig hlyspecific for single-stranded D NA . The objectives of theanaly ses w ere to determine: (a) the total amount of viralD NA transcribed; (6 ) the number o f clas ses o f v iral RN Adiffering in abundance: (c) the molar ratios of the RN Adiffering in abundance; and (d) the amount o f D NA template trans cribe d trans cribe d at diff ere nt tim es in the repro duc tiv e c yc le . The re sults o btaine d by o ur te chnique sreadi ly permit calculation of these parameters . Speci fical ly ,let D + R - D - R be the reaction of single-stranded D NAfrag me nts w ith ho mo lo go us RN A ; furthe r, le t the c onc entration of D NA be such that the reassociation of D NA isnegligible . It follows that:

D t/D , = e -k R,-l (B )

dD /dt = k-R-D (A )w here / is the length of hybridization, D is the molar conc entrati on o f s ing le -s trande d D N A at tim e /, R is the m olarconcentration of single-stranded RN A, and k is the rateco nstant. In hig h RN A ex cess, w here the co ncentratio n o fRNA in hybrid is small compared to that of the single-strande d RN A left, R can be assumed co nstant and equalsR0 the imput RNA co ncentr a tio n). E qua tion A upon integration yields:

w here D t is the co ncentratio n o f sing le-stranded D N A attime / and >0s the initial concentration of single-strandedDNA.Equation B assumes that all the D NA is transcribed toy ield RN A w ith a sing le abundance R0. The equation canbe applied to the m ore g eneral case w ith n classes o f RN Ae ac h appe aring in m olar c onc entrati on Rn. S uc h re ac ti onc an be ac tually v is ual iz ed as the s um to tal o f the inde pe ndent reactions of each RNA class with the DNA homologous to it. For each such class the fraction of the DNAremaining s ingle stranded is:

D ln/D on = (C )How ever, D on equals D 0-an w here an is the fraction ofthe total viral DNA serving as a template for this RNAc las s, and > 0s the to tal D N A input. If fo llo w s the re fo rethat:

D tn/D 0-a n = e kR l (D )The o bs erv ed frac tio n o f s ing le -s trande d D N A w ould the nbe:>, //>(Dtl/D0) + + D tn /D 0) + 1 - a , + + a )where 1 - (a, + - a)s the fraction of the D NAthat is no t transcribed and w ill therefore remain singlestranded throughout the hybridization. Hence:

The calculatio n of the parameters and R requires thenumerical value of the hybridization rate constants inadditio n to the technique fo r quantitativ e separatio n o fD NA in duplex from single-stranded DNA describede arl ie r in the te xt. Whe n /?, /? ,he c onc entratio n o fRNA in Equation E, are expressed in moles RNA perliter, the numerical values of the rate constants are inde pe nde nt o f the c om ple xity (s um o f unique s eque nc es ) o fthe RN A species re presented in the different terms o f theequation. If w e assume that the base composition of thevarious RNA species do not vary appreciably, the numerical v alues o f rate co nstant kt- kappearing in thedifferent term s o f the equatio n sho uld be abo ut the same.The rate constant, k, de fined by the equation

dD /dt = -kD 2w here D is the concentration of D NA ex pressed as molesDNA per liter was calculated from the reassociationkinetics of the same batch of DNA as the one used in theD NA -RN A hy bridizatio ns o n the assum ptio n that the hybridization rate constant for RNA -DNA is the same asthat of D NA -D NA reassociatio n. The reassociation w asmonitored by digesting the hybridization mixture w ithnucleases as described earlier in the text. The experimentally determined value of k for the DNA-D NA hy-

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bridization in 0.07 M m onovalent cation concentrationand for sonically disrupted D NA fragm ents with anaverage sedim entation constant of 5 S was found to be1.12 x IO 5-liters-m or1-sec'1 (10).The rate constant for DN A-DNA reassociation m ightbe different from that for DNA-RN A hybridization. Indeed M elli et al. (24) on the basis of hybrid ization of ex cessEscherichia coli D NA to co mplementa ry RNA prepa redi n vi tro cal cul ated tha t the o bs erved ra te consta nts d ifferby a factor of 0.43, but the applicability of this quotientto our analyses is not entirely clear. G elderm an et al. (13)also assum ed that the DNA-DNA and DNA-R NA hybridiza tion constants are the sam e. H ow ever, the absolutenum erical value of the hybridization rate constant is required for calculations of the absolute concentrations ofthe herpesvirus RNA in the cell. It is not required for thecalculation of the relative concentrations of the R NAspecies or for the determ ination of the size of tem platef ro m w h ich t hey a re t rans cr ib ed .The purpose of the DNA-D NA hybridizations w as todeterm ine the am ounts and size of the DNA in the cervical tum or. Except w here stated otherw ise, all hybridizations were done at 79in 1.06 M NaCl-0.05 M Tris (pH8.05). It is convenient to discuss the theoretical basis ofthe D NA -D NA hybridization later in the text.RESULTST he T ranscr iptional Pr ogr am of I I SV I in Pr oductivelyInfected Cel ls

The Program Analyses of vir al RNA transcr ipts inproductively infected cells w ere done on R NA extractedat 2 and 8 hr postinfection. The tim e intervals were selected on the basis of observations that viral DNA synth esis beg ins rou ghly at 3 hr postinfection (33, 37) and th atinfectious progeny appears at 6 hr postinfection and ispresent in appreciable levels (33). The 2-hr RNA shouldtherefore contain the bulk of the R NA m ade before viralD NA synthesis begins w hereas the 8-hr R NA should contain all of the species m ade late in infection and requiredfo r th e sy nth esis o f in fe ctio us p ro ge ny .The results of the hybridization tests for HSV-1 RNAare shown in Chart 1 and m ay be sum m arized as follow s.The transcripts present in R NA extracted from H SV -1-infected cells at 2 and 8 hr postinfection were com ple-

Tra ns criptio n o f H erpe s Simple x Virus D NAm entary to 44 and 48% of the total HSV-1 DNA, respectively (Table 1). Assum ing that the RNA arisesfrom asym metrical transcription of viral D NA , it follow s

2 h r

2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 5 0 0K 0 t m o le s n u c l e o tid e s - s e c - lit e r- ) 2 0 0 0

C hart 1. H ybridization of H SV -1 D NA w ith excess R NA extractedfro m H Ep -2 c ells at 2 an d 8 h r p ostin fec tio n. S inc e v ariab le a mo unts o fR NA w ere hybridized for different tim e intervals as described in thetext, the data are presented as a plot of the fraction of single-strandedDNA as a function of the input concentration of RNA (Ra) in molesn ucleo tid es p er liter tim es th e tim e o f hy brid iza tio n (/). O , e xp erim entally determ ined points. The line is a com puter plot calculated acc or di ng to E qu ati on E a nd f itt ed b y n on li ne ar l ea st -s qu ar es m et ho d. T hec om pu te r a na ly si s i nv ol ve d t he d et erm in ati on o f p ara me te rs < * nd R in E quation E in w hich the num ber of classes of R NA (n) w as assum edto be I, 2, 3, 4, etc. The fit for n = 2 was better than that for n = I.W hen n w as greater than 2, the results w ere m eaningless. T hus for thehybridization of R NA extracted at 2 hr postinfection, for n = 3, a, w asless than 0.001 w hereas a and os retained the same values as o,and a2 in the case of n = 2. The sam e result w as obtained w ith RN A ext rac ted f ro m 8 -h r -infec ted ce ll s.

Table IAna lys is o f the tra ns cripts o f H SV 1 a nd H SV 2 D NA in pro duc tive ly i nfe cte d H Ep 2 c el ls a t 2 a nd 8 hr po sti nfe ctio n

VirusHSV-1HSV-2(postinfection)(hr)2828Fraction

o f v iral D NAtranscribedi0.140.190.210.3120.300.280.000.19Total0.440.480.210.50R,(nmoles/1)7.97.10.512.2bundanceR,(nmoles/l)0.0580.1830.00.28R

M ole s o f vira l R NA fra g m en ts. M .W . 0 .9 9 x IO allons.

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Ber na rd Ro izma n a nd Niza F ren kelthat 88% of the to tal geno me w as already transcribed by2 hr postinfec tion.Analysis of the kinetics of hybridization of HSV-1transcripts indicates the presence of at least 2 classes o fRN A differing in abundance (Table 1 ). A t 2 hr po stinfec-ti on the abundant c las s w as c om ple me ntary to 1 4% o f v iralD NA and w as present in molar concentrations 136-foldgreater than the scarce class. A t 8 hr postinfection theabundant class w as co mplementary to 1 9% o f v iral D N Aand w as prese nt in mo lar co ncentratio ns 4 0-fo ld g reaterthan the s carc e c las s.The Requirement for Protein Synthesis The differencebetw een the amo unt o f HSV -1 D NA transcribed at 2 and8 hr postinfection is so small as to raise the questionwhether the RNA extracted from 2-hr infected cells isindeed early RN A, i.e., RN A made before the onset ofviral D NA synthesis. The problem arises from the factthat determinations of the onset of D NA synthesis areim pre cis e: the e xpe ri me ntal de te rm inatio n hing es o n theability to de te ct trac e am ounts o f v iral D N A . The alte rnatives open to us w ere to test the transcripts made in thepre se nc e o f inhibito rs o f e ithe r D N A o r pro te in s ynthe sis .The latter alternativ e is based o n o bserv atio ns that v iralD NA synthesis requires prior protein synthesis and isnot initiated in cells treated w ith inhibitors o f proteinsy nthesis from the time of ex posure of virus to cells (37).Of the 2 alternatives w e choose the inhibition of proteinsy nthesis larg ely bec ause in o ur hands m ost inhibito rs o fDNA synthesis reduce but do not completely inhibitD N A s ynthe sis , w he re as the inhibitio n o f pro te in s ynthes is appe ars to be alm os t c om pl ete .In 1 series of experiments HEp-2 cells w ere infectedw ith HSV -1 in the presence of 100 /ig of cycloheximideper ml of medium and maintained in the presence of thedrug fo r the entire duratio n o f the ex periment. The RN Aw as extracted at 2 hr postinfection and hybridized w ithv iral D N A. The results o f the hy bridiz atio n tests sho wedthat the amount of HSV -1 D NA transcribed by 2 hr

po stinfectio n in the presenc e o f cy clo hex imide w as 4 4%,i.e., exa ctly the sa me a s in un tr ea ted cells. We concludefrom these experiments that (a) the transcription of atleast 44% of HSV -1 D NA is independent of protein synthe sis and (b) the trans cri pts ac cum ulating duri ng the 1 st2 hr po stex po sure o f cells to v irus co nsists o f trans criptsmade befo re the o nset o f v iral D NA sy nthe sis.The Identity of Abundant RNA Sequences Ear ly andLate in Infect ion The presence of 2 dist inct abundanceclasses in 2-hr RNA raised the question whether thenucleotide sequences in the most abundant class at 2 hrare ide ntical to tho se in the mo st abundant class presentat 8 hr postinfection. To answ er this question, we perfo rm ed (1 0) a s erie s o f abundanc e c om pe ti tio n te sts . Thistype of analysis is designed to answ er the general questio n o f w hether any c lass o f RN A w ith a g iv en abundancec ontains the s am e nuc le otide s eque nc es as any o the r c las sof RN A w ith the same or different abundance. A s show nin Table 2, 2 sets of hybridization tests w ere done. In thecontrol set the 2- and 8-hr RN A w ere each hybridized tosingle-stranded D NA at a concentration (R0) and time(/) such that all of the single-stranded D NA fragmentscomplementary to the most abundant RN A w ere driveninto D NA -RN A hybrids. In the abundance competitionset, the 8- and 2-hr RNA 's were mixed. However, theconcentration o f each RN A species w as adjusted so thatfo r a co mmo n time o f incubatio n tc the pro duct R0 -tc w asexactly the same as the product R0-t for each of theRN A's in the co ntro l set.Table 2 show s the amount o f single-stranded D NA expected to be driven into D NA-RNA hybrid if the mostabundant s eque nc es at 2 and 8 hr w ere ide ntic al and if the yw ere different. The data clearly demo nstrate that the sequences in the most abundant 2-hr RN A are also presentin the mo st abundant RN A at 8 hr postinfection, i.e., themost abundant RNA at 2 hr is a subset of the abundantclass of late RN A.T he N atur e of the Pr oteins Specified by the A bundant

T abl e 2Ab un da nc e c om pe tit io n b etw ee n 2 -h r m os t a bu nd a nt s pe cie s a n d 8 -h r RN ACompetitionexperiment1RNAsource2

hr alone8 hr alo ne8 hr alo ne8 hr + R0 t 71 .672.5144.972.5Observed

% D N Ainhybrid18.824.828.8Predicted

%Allabundant sequences in2 -hr R NA are pre se nt incompeting 8 -hr RNADNA

inybridMostabundantRNA dif ferentcompetingspecies

i n 2 -h rf ro m th os e i n8 -h r R N A

2hr2 hr al one8 hr al one8 hr alo ne8 hr +2hr

7 1 . 671.6289.9362.4289.97 1 . 6

22.618.832.635.635.6

24.8 28.8 43.6

32.6 35.6 51.4 E xpre ss ed as m ole s nuc le otide s- se c/l ite r. Tw o- hr R NA , 8 -hr R NA , o r bo th w ere i nc ubate d w ith l abe le d D N A to Ral s pe cif ie d. D ata f ro m th e w o rk o f F re nke l an d R oi /m an ( 10 ).




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Tr anscr iption of H er pes Simplex Vir us D NAH SV 1 RNA Present in Productively Infected C ells inH igh Abundance One hypothes is c once rn ing the functionof viral RNA present in high abundance is that it specif ies structural proteins. This hypothesis is based not onlyon the expectation that the most abundant RNA shouldspecify the most abundant viral proteins but also on the estimated size of the DNA template requi red to speci fy theamino acid sequence of vi ral structural proteins. As reported elsewhere in detai l (18, 40, 44) , the herpesvirionconsists of 24 structural proteins. On the basis of the-aggregate molecular weights of the proteins, i t was previously estimated that 23.5 of viral DNA was requiredto speci fy thei r amino acid sequences. This f igure is anoverestimate since one-half of the proteins are glycosyl-ated and the polysaccharide moieties, while contributingto the molecular weight, should be excluded from thecalculation. Nevertheless even this amount of DNA template (23.5 ) is in good agreement with the DNA templatefor the abundant RNA (19 ). A direct test for the information content of the abundant viral RNA would require in vitro synthesis of proteins using that RNA asmessenger. The experimental question that we wouldlike to ask is what proportion of the 24 viral structuralproteins is made early after infection particularly since, aswe have shown earl ier in the text, a subset of high-abundance RNA corresponding to 14 of the DNA is al readypresent at that time.In these experiments 4 x 10scells were labeled between0.5 and 2 hr postexposure to virus with 10 ^Ci of 14C-labeled amino acid mixture (reconstituted protein hydroly-sate, New England Nuclear, Boston. Mass.) per ml ofEagle s minimal essential medium lacking amino acids. Atthe end of the pulse the cells were washed and replenishedwith M i xture 199 supplemented with 1 calf serum.Replenishment with medium containing unlabeled aminoacids precludes further incorporation of labeled aminoacids into proteins (7, 43). An infected cell culture labeled continuously beginning 4 hr postinfection served ascontrol. Both the pulsed and the control cultures wereharvested 20 hr postinfection. The vi rus was puri fied,solubilized, and subjected to electrophoresis on polyacryla-mide gels. The absorbance profiles of the autoradiogramare shown in Chart 2. The data show the following, (a)Structural proteins are already made between 0.5 and 2 hrpostinfection; however, only Proteins 5 through 24 become labeled at that time, (b) A ll viral proteins are labeled and therefore synthesized after 4 hr postinfection. c) O n the ba sis of the mo lecula r weigh ts of the p r oteins,we calculated that as much as 68 of the estimated DNAtemplate for structural proteins is expressed between 0.5and 2 hr postinfection. This isin comparatively good agreement with the proportion of the DNA template for abundant RNA transcribed at that time (74 ) .There is independent evidence that synthesis of moststructural proteins is an early function. Thus in additionto the evidence presented in this paper that most vi ralstructural proteins are made between 0.5 and 2 hr post-infection, i t has also been shown by us and by others (14,29, 34) that inhibi tors of DNA synthesis do not block the

0 .5 -2 h r

4 2 0 h .

10 30 50 70D is t an c e (mm )

90

Chart 2. Absorbance tracing of autoradiograms of structural proteinsin purified HSV-I virions subjected to electrophoresis in polyacrylamidegels. Top, proteins labeled between 0.5 to 2 hr postinfection: bottom.4 to 20 hr postinfection. The purification of the virus, the polyacrylamidegel electrophoresis of the proteins, and the autoradiography were doneaccording to the procedures of Spear and Roi/.man (44).

synthesis of viral proteins, the assembly of proteins intocapsids, or the appearance of the structural componentsof the virus on the surface of the cells. I n this respect,there is a substantial difference between the transcriptionalprogram of herpesvirus and that of other DNA virusesinfecting animal cells.If our suspicions concerning the function of abundantRNA are correct, i t fol lows that the scarce RNA specifiesnonstructural proteins, i .e., enzymes involved in the synthesis and processing of vi ral structural components(DNA, proteins, glycoproteins, l ipids, and polyamines)and in the modification of the host that involves inhibitionof host DNA and protein synthesis, alteration of thesynthesis, processing and transport of host RNA and ofthe structure and function of cellular membranes.Most of the viral functions listed here are known to beearly functions, expressed independently of viral DNAsynthesis (33-35).On the basis of comparisons between total 2- and 8-hrRNA, it would seem that the difference between earlyand late functions consists of transcription of a small percentage of DNA template at high abundance. The functionof this RNA could be to specify maturation proteins, etc.We cannot exclude the possibi li ty that the temporal programming of viral functions is also determined at the levelof processing and transport of the RNA from nucleus tothe cytoplasm of the infected cell.T he T ran scrip tio nal P rogram of H SV 2 in P rod uctivelyInf ec ted HEp 2 Ce ll s

The Program The design of these experim ents wassimilar to those used in analyses of the transcriptional

JUNE 1973 1407



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Ber na rd Ro izma n a nd Niza F re nkelprogram of HSV -1. The results (Chart I; Table I) showthat the RN A trans cripts pre se nt in H SV -2 -infe cte d c ellsat 2 and 8 hr postinfection were complementary to 21and 50% of HSV -2 D NA , respectively. Thus, unlike theRNA extracted from HSV-1 infected cells, the earlyHSV -2 transcripts w ere complementary to only 21% ofviral D NA . A nalysis of the kinetics of hybridization ofthe HS V-2 RN A trans cripts present early in infectio n indicates that they co nstitute only 1 abundance class. Onthe o ther hand, the RN A transcripts prese nt in the cell at8 hr after infection formed 2 classes of abundance diff ering o nly 8 -f old in m olar ratios and w e re c om plem entaryto 31 and 19% of the viral DN A, respectively. In otherexperiments, w e fo und that the difference in the extentof HSV-1 and HSV-2 DNA transcribed by 2 hr postin-fection is obliterated in cells treated w ith 100 /gofcycloheximide per ml of medium. Thus RN A extractedfrom treated cells at 2 hr postinfection hybridized w ith45% of viral D NA compared w ith RN A from untreatedcells, w hich hybridized w ith 21% of viral D NA .The Transcr ipt ion of DNA Sequences Shared in Common by HSV 1 and HSV 2 A pr evious r epor t fr om ourlabo rato ry sho wed that H SV -1 and HS V-2 D N A's share incommon at least 47% of their base sequences w ith goodmatching of base pairs (19). It w as of interest to determine (a) the ex tent o f transcriptio n o f the co mm on sequences and (b) the distribution of the templates forabundant and scarce viral RN A species among uniqueand co mmo n D NA sequences. In the se e xperiments RN Aextracted from 8-hr HSV -1-infected cells w as hybridized w ith labeled HSV -2 D NA . The results may be summ arize d as f ol lo w s.Co mpariso ns o f the hy bridizatio n o f RN A ex tracted at8 hr from HSV-1-infected cells with homologous andheterologous D NA (Chart 3) indicate that HSV -1 RN Ais complementary to 24% of the HSV -2 DN A, i.e., thefrac tio n o f H SV -1 and H SV -2 D N A's that are trans cribe dis homologous to the extent of 50%. Since nearly 50%o f the D N A , i.e ., the e quiv ale nt o f 1 s trand, is trans cribe d,it follows that the extent of homology betw een HSV-1and HSV -2 D NA 's is 50%, i.e., the same value as that obtained fro m D N A-D NA hy bridizatio ns (2 0).The calculation of the distribution of D NA templatesfo r abundant and scarce viral RN A species betw een theunique and common DNA sequences of HSV-1 andHSV -2 D NA 's is based on 2 considerations. First, RN Atrans cribe d o ff c om mo n s eque nc es m us t ne ce ss arily hav ethe same molar ratios vis- -v is the complementary regions of both HSV -1 and HSV-2 DNA's. Second, thethermal elutio n pro files o f she ared HS V-1 D NA -HS V-2DNA heterohybrids show a very uniform matching ofbase pairs even though it is somew hat low er (85 %) thanthose of homohybirds. We conclude from this findingthat the matching of base pairs is uniform for all D NA -D N A he te ro hy brids that the hy bridiz atio n rate c ons tantfor all of the DNA-RN A heterohybrids should be unifo rm. On the basis o f these co nsideratio ns w e c alculated(Table 3, Legend) that 13% of HSV-DNA is complementary to a corresponding amount of HSV-1 DN A

1 . 0 0

UHSV-2DNA

5 0.50 600 1200R g t (m o le s n u cl eo ti des -s ec -l it er )Chart 3 . Hy bridizatio n o f HS V-2 D NA w ith e xce ss RN A ex trac te df ro m c el ls 8 h r po sti nf ec ti on w i th H SV - 1. T he p ro ce du re f or th e h yb ri diz ati on, e tc ., w ere the s am e as de sc ribe d in the l eg end to C hart I e xc eptthat th e hy bri di zati on w as c arri ed o ut at a te mp erature 3 3 b el o th eTm of the H SV-2 D NA. , computer plot for homologous D NA-RNAhy bridiz ation sho wn in Chart I. , co mputer plo t lo r t he he te rolo goushybridization calcula ted according to Equation E except that the values o f

/?, and R2 w ere taken to be the same as those for 8-hr RN A reactivei n the h om ol og ou s D N A -R N A h yb ri di zati on ( Ch art I) .

T abl e 3C om ple me nt ar y o f H SV- l- HSV-2 D NA s s er vin g a s te mp la te s fo ra b un da n t a nd s ca r ce R NA s pe cie sHybridiza tion was done a t incubation tempera ture o f 62(30belowthe Tm o f HS V-2 D NA ).

Fraction ofotalDNAhybridizingw ith 8 -hr H S V- 1RNA,jTotalHSV-1DNA192848HSV-2DNA131124HSV-2SV-10.710

Region of D NA serving as template or reacting w ith RN A of highabundance.6 R egion of D NA serving as template or reacting w ith RN A of lowabundance.

serving as template for RN A of high abundance and that11% is complementary to the template specifying RN Aof low abundance. The data also show that the region ofthe HSV-1 DNA serving as a template for RNA of highabundanc e c ontains a larg er f rac tion o f s eque nc es c om plementary to HSV-2 DNA (71%) than does the templatefo r the lo w-abundanc e RN A (3 9%).T he T ranscr iptional Program of H SV 2 in CervicalTumor Cells

The hy bridiz atio n o f labe le d D N A w ith unlabe le d RN Ae xtrac te d f ro m pro duc tive ly inf ec te d c el ls w as de signe d to

1408 CA NCER RESEARCH VOL. 33



9/16

Tr an scr iption of H er pes Simplex Vir us D NApreclude the reassociation of D NA . On the other hand,because the amounts of RN A recovered from the tumorwere small, the formation of DNA-DNA hybrids couldno t be pre ve nte d. Fo r pre ci se de te rm ination o f the am ountof DN A-RNA hybrid, the labeled DNA was also hybridized w ith equal concentrations of RN A extractedfrom uninfected HEp-2 cells. The net amount o f labeledD N A-tumo r RN A hy brid determined by subtracting thepe rc entag e o f hy brid fo rm ed in the pre se nc e o f uninfe cte dRN A from that fo rmed in the presence of cervical tumo rRN A is show n in Chart 4 along w ith the transcriptionalpro gram o f HS V-2 in pro ductiv ely infected ce lls. The fo ll ow ing po ints , ho w ev er, be ar o n the se re sul ts .Tw o features of the experimental design excluded thepossibility that the 5% difference at the plateau level(Chart 4) between the amount of hybrid formed in thepresence of cervical tumor RNA and in the presence ofHEp-2 RNA are due to DNA-DNA hybridization or toe xpe rime ntal e rro rs . F irs t, ide nti cal ne t D N A -R N A hy brid( 5% abo ve c ontro l) w as o btaine d w i th 3 di ff ere nt pre paratio ns o f purified in v /v o-labeled D NA differing w idely ins pe ci fi c ac tivi ty and the re fo re inc ubate d at di ff ere nt D N Aconcentrations (C0 ) to reach the same Rat values.Second, the same net amount of DNA-RNA hybridformed w hen R0t and D NA concentration were keptc ons tant, but the duratio ns o f hy bridiz atio n (?) and R NAconcentration (R0) w ere varied in a fashion designed tov ary the D NA co ncentratio n and therefo re the am ount o fviral D NA -RN A hybrid formed. W e co nclude thereforethat the net amount of DN A-RNA hybrid obtained inthese ex periments w as independent o f the D NA pre paration of the DNA C0t and dependent only on the R0t towhich the hybridizations were carried.

1l*wv ^^^B*Oi a 9 0 ^ . E . ?

0.80- '5

0 .70 -s 'S0 .60 -O

W k \ o \ 0 _ l v -0 ^ ,

Cerv ica lumor*2hr

In fec tedHEp-2ells j>.^_8hrjnfected

H Ep- 2el l s10002 0 0 0 3 0 0 0 7 0 0 0R g t ( m o le s n u c le o t id e s - s e c - lit e r- ')

Chart 4 . Hy bridizatio n of labeled HS V-2 D NA w ith e xc ess RN A extrac te d f ro m 2 - and 8 -hr- infe cte d H Ep- 2 c ell s and fro m the c erv ic al tumor. O. 3 . 2 - and 8-hr- infec ted ce ll s, re spec tive ly . The l ines are computerplots calculated according to Equation E and fitted by a nonlinearleas t- squares method as described in the legend to chart I. The net amountof D NA hy bridiz ing w ith c erv ical tumo r RN A w as calc ulate d by s ubtracting the amount of D NA -D NA hybrid formed in the presence ofHEp-2 RNA from the amount of hybrid (DNA-DNA and DNA-RN A) formed in the presence of cervical tumor RN A. D ata from thew ork o f Fre nke l e t al. ( 1 1) .

A ddi tional and m ore dire ct e vide nc e that the di ff ere nc ebetw een hy brid fo rmed in the presence o f cerv ical tumo rRN A and that f orm ed in the pre se nc e o f uninfe cte d H Ep-2RNA was due to D NA-RNA hybrid emerged from ananaly sis of the hybrid formed in the presence of cerv icaltumor RNA. Briefly 4.6 /ug of DNA with a specificactiv ity o f 1 2,0 00 cpm/V g w ere hy bridized w ith c erv icaltumor RN A to a R0t of 1000. The same amount ofD NA w as hybridized w ith an identical concentration ofuninfected HEp-2 RNA for the same interval. Theamount of hybrid formed in the presence of uninfectedHRp-2 RNA indicated that of the total DNA hybridformed in the presence of cervical tumor RN A (45% ofinput), 88% was in D NA-DNA hybrid and 12% was inD NA -RN A hybrid. The hybrid formed in the presenceof cervical tumor RNA was digested with N. crossanuclease, denatured w ith 0.3 M N aOH for 10 hr to hy dro-lyze the RNA, and then dialyzed against 0.07 M NaCl-0.05 M Tris (pH 8.05). The DNA recovered after alkalitreatment w as then rehybridized w ith RN A extractedfro m c erv ic al tum or and fro m uninfe cte d H Ep-2 c el ls . Theresults are show n in Table 4. Since this DNA was enriched w ith viral D NA sequences transcribed in the cervical tumor it could be predicted Table 2, (legend) thatthe diffe re nc e be tw ee n the am ount o f hy brid fo rm ed in thepresence of cervical tumor RNA and in the presence ofHEp-2 RN A should be 14.6%. The actual difference w as1 4.4 %. W e co nclude that the amo unt o f v iral D N A transcribed in the tumor is 5%, i.e., considerably less than inproductively infected cells .In this and earlier (10, 36) publications we have repo rte d o n di ff ere nc es in the e arly and late trans criptionalpro grams o f HS V-1 and HS V-2 and o n the transcriptio no f D N A se quences shared in co mmo n betw een HS V-1 andHS V-2 . It w as o f interest to dete rmine therefo re w hetherthe D NA template transcribed in the tumor represents asubset o f templates transcribed early o r late and w hetherit is shared in common betw een HSV -1 and HSV-2.Preliminary experiments indicate that a fraction of thev iral D N A te mplate trans cribe d in the c erv ic al tum or c el lsis als o trans cribe d e arly in pro duc tiv e infe ctio n o f H Ep-2cells w ith HSV -2 and that appro ximately o ne-half of thev iral D NA tem plate transcribe d in the cerv ical tumo r belo ng s to the set o f sequences w hich are shared in co mmo nbetw een HSV -1 and HSV -2 (20, 36).T h e A m ou nt an d G en et i c C om p l exi t y of t he D N A Sequ ences Pr esen t i n t he C er vi cal T um or an d C om pl em en t ar y t o H SV -2 D N A

In the pre ce ding s ec tio n w e hav e s ho wn that the c erv ic altum or c ontains v iral R N A trans cripts c om plem entary to 5 %o f v iral D N A. It w as o f interest therefo re to dete rmine theamo unt o f v iral D NA present in the cerv ical tumo r and itsgenetic complexity . The design of the experiments describedin this sectio n w as to co mpare the rates o f reasso ciatio n o fin v/A-0 -la be le dH SV-2 D NA in th e p re se nc e o f va r ia bleamo unts o f c erv ical tumo r and HEp-2 D NA . Tw o s eries o fexperiments w ere done. In the 1st series, labeled HSV -2

JU NE 1973 1409



10/16

Ber na rd Roizma n a nd Niza F renkelT abl e 4Rehybr idza li on of N hybr id f ormed in the presence of cervi cal tumor RNT he e xpe ri me ntal de tail s are g iv en i n the te xt. T he c al cul atio ns are bas ed o n the f oll ow ing . T he D N A i nthe hybrid obtained by hybridization o f m v/vo -labeled DNA with cerv ical RNA cons is ts o f 88 .4% DNA-DNAh yb ri d an d 1 1. 6% D N A - RN A hy bri d. T he c al cul ati on i s b as ed o n th e am ou nt o f D N A i n hy bri d f orm ed i n th epre senc e of HEp-2 RN A. In the 2 nd hy bridization c arried o ut to abo ut the same R0 t as the first, but to ad if fe re nt D N A C 0l b ec aus e o f d ec re as ed D N A c on ce ntrati on , i t c o ul d b e e xpe cte d th at al l 1 1. 6% o f D N A th athy bri diz ed to c erv ic al tum or RN A w oul d e nd up i n D N A- RN A hy brid. In additio n, the D N A o ri ginall y i nthe D NA -D NA hybrid w ould end up in part (12.3 of 89%) in D NA -D NA hybrid and in part (5 of 89%) inD N A -R N A hy bri d. T he p re di cte d to tal D N A h ybri di zi ng i n th e p re se nc e o f c erv ic al tu mo r s ho ul d the re fo re

b e 1 4. 6% g re ate r th an th at h yb ri di zi ng i n the p re se nc e o f H Ep -2 R N A. D ata f ro m th e p ap er o f F re nk el e t al .(M).

S o urc e o fNA1sthybridizationCervical tumorUninfected HEp-2DNA

RNA % ofC0f Rat DNA inybrid,. 1000 45.01000 39.8%

o f D NA in hy bridaboveontrolExpectedObserved5.0

5.22nd hybridizationCervical tumorUninfected HEp-2 0.21 1 1 0 0 2 6 .7

In moles nuc leo tides sec- li ter ' and for0 .07 MNaCl.1 4 .6 1 4 .4

D N A w as hy bridi ze d w i th a s ing le c onc entration o f c erv ic altum or D NA and w ith an equiv alent am ount o f H Ep-2 D NAfor variable time intervals. In the 2nd series of experiments ,cervical tumor D NA and HEp-2 D NA w ere mixed in variable pro po rtio ns so that the final c oncentratio n o f D NAw as co nstant. These w ere then mixed w ith labeled D NAand allow ed to hybridize for variable leng ths of time. Itis convenient to present the results in the framew ork ofthe kinetics governing DNA-DNA reassoc iations . Ingeneraithe reassociation of DNA follows 2nd-order reactionkinetics and is predicted by the relationship of B ritten(4 )D ,/D 0 - (F)w he re D , / ) s the fraction of single-stranded D NA remaining after hybridization for time /, C0 is the concentratio n o f D N A in m ole s nuc le otide /li te r and K is a c ons tantA plot of D 0/D , ag ainst / should yield a straig ht line w ithan intercept of 1 and w ith a slope proportio nal to C0. Thel ine representing the reassoc iation of DNA without repeti tives eque nc es s ho uld hav e a s lo pe o f 1 .The results of our hybridization studies are show n inChart 5 in the form of a series of plots of D0/Dt against/ fo r e ac h c onc entratio n o f c erv ic al tum or D N A te ste d. Thein v/ir o-la beled D NA in the p r esence of H Ep-2 D NA a lonehy bridi ze s at a c ons tant rate c ons is te nt w ith the kine ti c c omplexity of HSV D NA (8) except that, unlike HSV -1 D NA ,HSV -2 D NA has a small amount of repetitive sequencesw hich cause the intercept (Chart 5) to be 1.198. This appears to be a general characteristic of HSV -2 D NA sinces im ilar kine tic s w ere o btaine d w ith D N A pre paratio ns labe led in v iv o o f 2 different H SV -2 s trains. The salie nt feature of o ur results is that the points for the labeled D NA ,hybridized in the presence of cerv ical tumor D NA , forminitially a straig ht line w ith the same intercept but laterlevel off and ultimately appear to form a line parallel or

M 20 01.81.6UUL 100t i m e h r 20 0Chart 5. Hybridization of in vi/ro-labeled D NA in the presence ofD NA from cervical tumor cells and from uninfected HEp-2 cells. A ,HSV-2 DNA-'H (0.046 ^g/ml) was hybridized in the presence ofH Ep-2 D NA (3 .0 7 mg /ml) ( )o r c erv ic al tumo r D NA (3 .26 mg /m l)(O) for the time intervals shown. The plot for HSV-2 DNA inth e pre se nc e o f the c erv ic al tu mo r D N A b eg in s to d ev iate f ro m th e i ni ti alrate afte r 4 0.2 % o f the D N A w as i n the hy bri d fo rm . T he plo t f or H SV -2D N A i n the pre se nc e o f D N A c ontinue s to inc re as e l ine arly unti l at le as t4 88 hr at w hic h point 6 5.7 % o f the D NA w as in hy brid form , indic atingthat the in w /ro -lube le d D N A re ass oc iate s no rmally w ith 2 nd-o rde rkinetics. B . HSV -2 D NA -3H (0.012 ^g/ml) w as hybridized in the

presence of cervical tumor D NA in concentrations of 5 mg/ml (D ).3.26 mg/ml (y). 1.74 mg/ml (A). 1.09 mg/ml (O) or HEp-2 DNA,O ). T he f inal c onc entrati on o f D N A in the hy bri diz atio n w as adjus te dw ith HEp-2 D NA to the hig hes t co nce ntratio n o f tumo r D N A in o rde rto m in im iz e th e v ari ati on i n v is co si ty o f th e h yb ri di zati on m ix ture . D ataf ro m th e w o rk o f K re nke l e t al . ( 1 1) .inte rc epting the l ine re pre se nting the hy bridi zation o f thelabeled D NA in the presence of HEp-2 D NA alone. The results imply that o nly a po rtion of the viral D NA is present

1410 C A N C E R R E SE A R C H V O L . 33



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Tr anscr ip tion of H er pes Simplex Vir us D NAin the tumor cells and that after this portion of the D NAwas exhausted the hybridization of labe led DNA dece leratedto that observed in the control hybridization, i.e., in thepre sence o f HEp-2 D N A alo ne.The amount of viral DNA in the tumor may be calculate d by s ubs ti tuting in the g ene ral e quation f or D N A -D N Ahybridizations the observed 1.198 intercept and by rewritingit as

[(Do/0,) - 1.1981-r1 - KC0S inc e the v iral D N A in the hy bridiz atio n te st c om es fro m2 sources, i.e., the labeled D NA and that fraction of thec erv ic al tum or D N A w hich i s v iral , the e quation be co me s

[(/> / ,) 1 .1 98 ]-r1 = KC + KfC0 cw here / is the ratio of moles nucleotides of viral D NA tomoles nucleotides of cellular D NA in the cervical tumorand Co and Cac are the c onc entratio ns o f in v /'/ro -labe le dviral DNA and cervical tumor DNA , respectively, inthe hybridization mixture. / may be calculated by dividing the slope of a plot of [( >/ >,)1.198]-r1against C0C by the value of K calculated from the intercept KCa . The plot based on initial, ascending points inChart 5 B y ie lde d a s traig ht line ; the v alue o f/c alc ulate dfrom this plot (Chart 6) is (1.02 0.17) x IO'5 Thecalculation of the number of viral DN A molecules percell hinges on the content of DNA in human cells. Thisvalue ranges from 6 x 10 6 /gper diploid cell (22) to17 x 10~6 tg/heteroploid HeLa cell (23), which orig inated fro m a cerv ical tumo r. On the basis o f the mo lecular weight of HSV-2 D NA [(99 5) x IO6] and thecorrectio n factor (0 .8) fo r the fraction of tumor cells inthe tumor tissue, w e calculated that the number of viralD N A m ole cule s pe r tum or c ell rang es fro m 1 (he te ro plo idcell) to 3.5 (diploid cell).

The genetic Co mplexity of the D NA fragment presentin the tumo r emerges from the reg ion of the plot at w hichthe hy bridiz atio n be gi ns to de viate fro m the s traig ht line srepresenting the initial rates. The data summarized inTable 5 indicate that the tumor contains a unique fragment constituting approximately 39%, i.e., 39 x IO6daltons, of the HS V-2 D NA molecule.T he S tate of th e DN A Seq u en ces of th e C erv ical T um orC om p lem en tar y to H SV 2 DN A

The purpose of this series of experiments was to determine whether viral DNA and host DN A's are co-v ale ntly linke d. The c ho ic e o f e xpe rim ental te chnique w asbased o n the observ ation that HSV -1 and HSV -2 containalkali-labile bo nds ( 9, 1 9). S inc e the nature o f the se bo ndsis not clear, w e chose an alternative to the classical technique of looking for virus-specific sequences in high-mo lecular-w eig ht D NA o btaine d by s edimentatio n o f tumo r D NA in alkaline dens ity g radients. The alternativ ewas to look for viral DN A sequences in tumor cell DNAreassociated under conditions designed to exclude by aw ide m arg in the re as so ciatio n o f the v iral D N A.

1. 5(G) ^

1- 0

(H)

0.5 O2-Ci; 5Chart. 6. Calculation of the ratio of HS V-2 D NA to cervical D NA

i n the c erv ic al tum or. The data i n the ini ti al , l ine ar po rtio ns o f the pl otsshown in Chart 5 B were plotted as [(DJD,) - 1.198]-1 ' versusthe c onc entrati on o f the c erv ic al tum or D N A i n the hy bridi zati on m ixture . The l ine i s a c om pute r plo t f itte d by a l ine ar re gre ss io n. T he ratio(/) of viral D NA to tumor cell D NA w as calculated from EquationH. The calculated intercept for the line is (4.5 0.5)-10 7-sec~ 'S inc e C 0' is 4 .1 1 x 1 0 ' m ole nuc le oti de /li te r and the c al cul ate d v al ue sf or K S .D . i s ( 10 .9 7 1. 07 ) l ite r- mo le s n uc le oti de s- ' e c~ ' an d th eslope of the line S.D . is (1.12 - 0.08)-10 ', the value of/is (1.02 0 .1 7). IO 5 . The v alue s fo r the inte rce pt and the s lo pe are sig nific antw ithi n 9 5% c onf ide nc e l ev els . T he appare nt s catte r o f the po ints in theplo t is due to the ex tre me se nsitivity o f this type o f plo t. D ata fro m thew ork o f Fre nke l e t al . ( 11 ).

T abl e 5G en etic co mp lexity o f H SV-2 D NA in th e c er vic al tu mo rThe po ints us ed in the se c alc ulati ons are the 1 st 2 po ints in the re gi onw he re the curv e de viate s fro m the initial rate . D ata fro m the w ork o fFrenkel e t al . (11) .

Concentration (mg/ml) o fcervical tumor DNA in Time ih yb ri di zati on m ix tu re ( hr) Ch an53.26 43.483.2Chart

SB5.0046.378.13.2678.1119.11.74

96119.1Av.% of v ira l genome in tumor5o //> ,9

ID.67.82.61.67.61.65.56.62%ofDNA inhybrid40.

1644.9938.0440.

The e xpe rim ents c ons is te d o f 3 s te ps . The firs t inv olv edpartial reassociation of the cervical tumor D NA ; it w asdesigned to permit recovery of reassociated sequencesand viral D NA covalently linked to these sequences butto exclude viral DNA that is not integrated, i.e., notJU NE 1 973 1411



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Ber na r d Roiima n a nd Niza F renkelcovalently bound to host D NA . The cervical tumor D NAin concentrations of 3.26 mg of buffer per ml containing0.07 M N aCl-0.05 M Tris (pH 8.05) w as denatured byhe ating at 1 10 o r 1 0 m in and the n allo we d to re as so ciateat 53 for 1.6 hr. The calculations w ere based on thefo llo wi ng c ons ide ratio ns , (a) The e quiv ale nt (i.e ., at 0 .1 8M Na+ concentration and for fragments 400 nucleo-tides long,) C0t of 50 viral D NA is 0.4 mole nucleotide-sec-liter ' (8). At C0t of 10, the DNA is completelyre as so ciate d. O n the as sum ptio n that c erv ic al tum or D N Acontains 10 moles of viral D NA per mole of cellular D NAand that viral DNA strands are intact, i.e., i .6 x 10snuc le oti de s lo ng and no t inte grate d, i t c ould be c alc ulate dthat viral D NA w ould require a minimum of 1600 hr completely to reasso ciate but that little or no reassociationw ould take place if the D NA w ere allow ed to hybridizefo r 1 .6 hr. i.e ., to an e quiv ale nt C 01 o f 0 .0 1. The re as so cia-tio n of viral sequences w as expected to be low er since thehybridization w as done at 41 below the Tm of HSV -2DNA. (b) In calculating the C01 to which the DNA wasreassociated, we assumed that the average size of thedenatured cervical tumor D NA consisted of fragmentswith an average molecular weight of 5 x IO7 dallons,i.e., the sa me a s th a t of inta ct single str ands o f vir al D NA.The calculated equivalent C0 /, i.e., after correction forsize [from the assumed 1.6 x IO5 nucleotides long to 400nuc le oti de s lo ng (4 6)] and fo r m olarity o f the m ono vale ntcations [from 0.07 M Na+ to 0.18 M Na+ (5)] in the hybridi zation buf fe r, w as 9 1. 8. Hum an c el l D N A re as so ciate dto this C0 i co nsists larg ely o f repetitiv e seque nces (3 , 4 2).The average size of the DNA was probably smaller andthe re fo re the e quival ent C 0 / to w hich the re as so ciation w ascarried out is probably low er. The 2 nd step in the ex perim ental s eque nc e w as to f rac tio nate the D N A o n hy dro xy -apatite. The design o f the fractio nation experiment w asbased on the follow ing considerations. HSV -2 D NA contains 69 guanine plus cytosine moles per 100 ml. In a rec ons truc tion e xpe rime nt in w hich no ns he are d HSV -2 D N Ain a concentration equivalent to that of 10 DNA molecules per D N A cell equiv alent w as allo wed to reass ociatefo r 1 .6 hr and w as then fractio nated o n hy dro xy apatite,the v iral D NA that remained sing le stranded w as quantitatively eluted fro m hydroxyapatite columns w ith 0.19M phosphate but not with 0.12 M phosphate. Consequently, after the partial reassociation, the cervical tumor D NA w as diluted w ith sodium phosphate buffer to afinal co ncentratio n o f 0 .1 2 M pho sphate buffer and put o nhydroxyapatite in a w ater-jacketed column heated to50 .The absorbed D NA w as eluted first w ith 0.19 andsubsequently with 0.4 vi phosphate buffer. The DNAco ntained in the 0 .1 2 M effluent and that eluted w ith 0 .1 9and 0.4 M phosphate buffer w ere concentrated and di-alyzed against the hybridization buffer. The results ofthe re as so ciatio n and frac tio natio n w ere as fo llo ws . O f thetotal, 63% w as recovered in the 0.12 M effluent and w astherefore single stranded, 26% eluted in 0.19 M phosphate, and 1 1% eluted in 0 .4 M pho sphate.In the 3rd step of the experimental sequence the re-associated cervical tumor D NA eluted w ith 0.4 M phos-

,,0

IO 20 30 40t im e h r )C hart 7 . In iti al rate s o f h yb ri di zati on o f i n v /'f ro -l ab el ed H S V- 2 D N A

i n th e p re se nc e o f a f as t- re as so ci ati ng f rac ti on o f tu mo r c el l D N A . H S V- 2D NA -3 H (0 .0 09 iig /ml) w as hy bridize d in 0.0 5 M Tris -HCl (pH 8 .05 )-1 .0 6 M N aCl in the pre senc e o f so nic ally disrupted tumo r D NA (0 .4 2m g/ ml ) re as so ci ate d an d e lu te d f ro m hy dro xy ap ati te as de sc ri be d i n th ete xt ( O) an d i n th e p re se nc e o f s on ic al ly d is rup te d u nf rac ti on ate d c erv ic al tum or D N A (0 .4 2 m g/ ml ) ( ) .D ata f ro m the w ork o f F re nke l e l al .(ID.phate buffer w as hy bridized w ith in v //ro -labeled D NA .The design of the experiment w as limited by the amountof the cervical tumor D NA recovered in the 0.4 M phosphate buf fe r f rac tion. B ri ef ly , in v /'/ ro -labe le d v iral D N Aw as mix ed w ith the re asso ciated cerv ical tum or D NA andw ith ex actly the s ame amo unt o f unfractio nated cerv icaltumor D NA and hybridized to relatively low C0/ levels.The results w ere as follow s, (a) A t low C01 levels show nin Chart 7 the amount of hybrid formed in the presenceof reassociated tumor cell D NA w as consistently higherthan in the presence of cervical tumor D NA indicatingboth the presence and some enrichment of viral D NA sequences in the reassociated DNA. (b) At 400 hr (notshow n) the amount o f the hybrid formed in the presenceo f reasso ciated cerv ical D NA w as the same as that fo rme din the pre se nc e o f unf rac tionate d c erv ic al D N A , indicatingno enrichment. The enriched sequences arise from atmost 20% of total HSV-2 DNA.D I SC U SSI ONT he D N A s of H SV -1 and H SV -2

A s s um marize d unde r Intro duc tion, the HSV -1 andH SV -2 D N A's c anno t be diffe re ntiate d w ith re spe ct to s iz e[(99 + 5) x IO6 dalto ns] or the presence and distributionof alkali-labile bonds (9, 19). They differ, how ever, inbas e co mpo sitio n [6 7 and 6 9 g uanine plus cy to sine mo le sper 100 ml for HSV -1 and HSV -2 (17, 19)] and, perhapsmore strikingly, in genetic complexity. Thus w e previously reported that HSV -1 D NA sequences are at least95% unique (8), and indeed a plot of reassociation ofHSV-1 DNA according to Equation (F) yields a linew ith the intercept at 1 indicating absence o f apprec iablere pe titi ve s eque nc e. O n the o the r hand, the re as so ciatio nof HSV -2 DNA plotted in a similar fashion yielded an

1412 CANCER RESEARCH V OL. 33



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Tr anscr ip tion of H er pes Simple x Vir us D NAintercept o f 1 .1 89 indicating that appro ximately 1 6% o fHSV-2 DNA consists of repetitive sequences. The signific anc e o f this o bs erv atio n partic ul arly i n re gard to the 1bilo gic al diffe re nc e be tw ee n H SV -1 and H SV -2 o f inte re stto us here (the o nco genic po tential o f HS V-2 as co mparedto that of HSV -2) is not clear. It w ould be interesting tode te rm ine no t o nly w hat g ene tic info rm atio n is re pe titiv ein HSV-2 DNA but also w hat information present inHSV -1 DN A has been replaced by the repetitive sequences in HSV -2 D NA .T he T ranscr iptional Programs of H SV 1 and H SV 2in Product ively Infected Cel ls

The transcriptional programs of HSV -1 and HSV -2 inproductively infected cells have been analyzed from 4points of view. These are (a) the fraction of viral DNAcomplementary to viral RN A transcripts present in theinfected cells at 2 and 8 hr postinfection, i.e., pre- andpo stv iral D N A s ynthe si s; ( b) the e ff ec t o f pro te in s ynthe si so n the ex tent o f v iral D NA transcribed early in infectio n; c) the number of cla sses of vir a l RNA tr a nscr ipts differing in abundance and the size of the correspondingviral D NA templates from w hich they are transcribed;and (d) the distributio n o f D N A sequences s hared in co mmon by HSV -1 and HSV-2 DNA's among the templatesf or abundant and s carc e R NA 's , re spe ctive ly .A s s um marize d in Table 6 , the trans criptional pro gram sof HSV -1 and HSV-2 share in common 2 features. Bothe xhibit o ff-o n c ontro ls o f trans cripti on and c ontro ls

of RNA abundance, described in a previous paper ofthis se ries (1 0). They diffe r, ho wev er, in detail and mo respecifically in 4 majo r respects as fo llo ws: (a) the ex tento f D NA transcribed at 2 hr po stex po sure o f cells to v irus,i.e ., pr ior to the onset o f D NA synth esis; b) the compo sition of RN A transcripts and, more specifically, thenumber of classes of RN A templates differing in molarratio s; (c) the size o f the templates s pecify ing abundantRNA; and d the extent of DNA transcribed in 2-hr infected cells treated w ith cycloheximide. Several pointssho uld be made in co nnectio n w ith these data.HSV-2 D NA is transcribed to only 21% early in infection. The RNA made early forms only 1 abundanceclass. Late in infectio n, the RN A is transcribed fro m 5 0%of the DNA and forms from 2 classes, abundant ands carc e, aris ing fro m 3 1 and 1 9% o f the D N A , re spe ctiv ely .By contrast, both the abundant and scarce RNA's aremade early in cells infected w ith HSV -1. It w ould seem,there fore , than an off -on control of transcription presentin HSV -2 D NA is either muted or expressed much earlierin HSV -1 -inf ec te d c el ls . The s ig ni fi canc e o f the se f inding semerg es fro m the o bserv atio n that amo ng the HS V-1 templates transc ribed early are tho se specify ing at least 2 0 o fthe 24 structural proteins. If the oncog enic potential ofherpes viruses is the consequence of an expression of an e arly f unc tion in the abs enc e o f e xpre ss io n o f f unc tionsc onc erne d w ith the s ynthe sis and as se mbl y o f the v irus andcell death, the v irus ex pressing the least functio ns o therthan those concerned with cel l transformation is more l ike ly

Tabl e 6Th e t ra n sc ri pii on a l p ro gr a ms o f H S V- 1 a n d H SV- 2 i n p ro du cti ve lyi nfe ct ed H E p- 2 c el lsFeatures o f the program HSV-1 HSV-2

E a r l y 2 h r p o st in fe c ti on )i of D NA tr a nscr ibed 44 21N o. of RNA classes differing in abun- 2 Idance% of D NA specifying abundant class 14 21% of DNA specifying s carce class 30) o f DNA transcribed in the presence of 44 45cycloheximide L a te 8 h r p os ti nfe ct io n)) of DNA transcribed 48 50No. o f RNA classes differing i n abun- 2 2dance% of D NA specifying abundant class 19 31% of DNA specifying scarce class 28 19

to be oncogenic. From this point of view HSV -2 might bein certain situations potentially more oncogenic thanHSV-1.In the absence of protein synthesis inhibited by cycloheximide, the HSV -2 D NA early in infection is transcribed to 45%, i.e., to the same level as HSV-1 DNA.Thes e o bserv atio ns sug gest that the transcriptio n o f 2 4%o f HS V-2 D N A (the difference betw een 4 5% transc ribedin the treated cells and 2 1% transcribed in the untreatedcells) is regulated by a negative control w hich involvespro te ins m ade afte r i nfe ctio n. A m ore triv ial hy po the sis isthat 2 4% o f H SV -2 D N A is trans cribe d but the trans criptsare rapidly de grade d. Pare nthe ti cal ly , the e ff ec ts o f c yc lohe xim ide no te d in o ur s tudie s diffe r m ate rially fro m tho sededuced by Rakusano va et al. (3 0) w ho co ncluded that theRN A m ade in the presence o f cy clo hex imide w as co mplem entary to a s mal le r te mpl ate than that m ade e arly . W ec anno t ac co unt f or the di ff ere nc es in o ur re spe ctive c onc lusions except to point out that our techniques involvinghybridization in l iquid of excess unlabe led RNA with labe ledD NA is free of errors due to ribonucleotide po ol size andm ore am enable to analyti cal tre atm ent than hy bridi zationtests involving labeled RNA in liquid to DNA bound tof il ter, particularly s ince the kinetics of such hybridizationsis no t kno wn.HSV -D NA 's may contain more than 1 site for off-onco ntro l o f trans criptio n. This c onclusio n is based o n thefact that early in infection HSV -1 is transcribed to only44%, w hereas late in infection 48% of the D NA is transcribed. The difference is significant on the basis of 3 o bs erv atio ns , i.e ., the f inding is re pro duc ible , s ev eral s tructural pro te ins o f the H SV -1 are m ade o nly late in infe ctio n(10), and in the presence of cycloheximide both HSV -1and HSV -2 are transcribed to appro ximately 4 4 to 45%.N othing is y et kno wn c onc erning this o ff-o n c ontro l o ftranscription.The abundant HS V-1 RN A extracted late in infectionis transcribed off 19% of HSV-1 D NA. Several lines ofevidence cited elsew here in detail (10) led us to suggestthat the abundant RN A specifies structural proteins.The co nclusio ns w ere in part base d o n the siz e o f the D N Atemplate required for structural prote ins , the ir abundance

JU NE 1973 1413



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Ber na r d Roizma n a nd Niza F renkelrelative to nonstructural proteins, and analy ses of thestructural pro teins made at diffe rent times in the reproduc tiv e c yc le . B y c ontras t, the late abundant H SV -2 RN Ais complementary to 31% of HSV-2 DNA. Since HSV-1and HSV-2 DNA are closely related, it is not likelythat HSV -1 structural proteins require 50% more D NAtemplate (31% o f total) than the HSV -1 structural proteins (19% of total). One possible explanation of thisdis cre panc y aris es fro m the o bs erv atio n that H SV -1 D N Aconsists larg ely (at least 95%) of unique D NA sequences(18). By contrast, both the G and 174 prototypes ofHSV -2 isolated and propagated a continent apart contained repetitive sequences, amounting to 16% of theD NA (Ref. 11; N . Frenkel and B . Roizman, unpublishedstudies). The nature of the functions specified by there pe titiv e s eque nc es are no t kno wn; the y c ould be a s ubs etof the sequences specifying the abundant RNA late ininfection.A s summ arized in this paper, earlier studie s (1 0) hav edemo nstrated the presence in the infected cells of RN Atranscripts differing in abundance. This could be predicted on the basis of the fact that structural proteinsare m ade in m uc h larg er am ounts than no ns truc tural prote ins . The o bv io us and v ery re le vant que stion i s m ec hani smby w hich RN A abundance is reg ulated. Preliminary studie s in o ur l abo rato ry (S . S ilv ers te in, S . L. B ac he nhe im er,N . Fre nke l, and B . Ro iz man, m anus cript in pre paratio n)indicate that abundant D NA species in both nuclei andpo ly ribo so me s are ade ny late d w he re as the s carc e s pe cie swhich are also present in polyribosomes are not adenylated.The data presented in this paper show that the regionof HSV-1 DNA serving as a template for RN A of highabundance co ntains a larg er fractio n (7 1%) o f sequenc escomplementary to HSV-2 DNA than the template forthe low -abundance RNA (39%). The significance ofthis finding arise s fro m the nature o f functio ns spec ifiedby the D NA templates for early and late RNA. Specific all y, e arlie r in the te xt w e l is te d s ev eral line s o f e vide nc ethat suggested that the HSV -1 template for abundantRNA specifies the structural proteins of the virus. Inthis paper we have shown that, although the shared seque nc es are div ide d am ong the te mplate s fo r abundant ands carc e RN A , tho se c ontaine d i n the te mplate fo r abundantRN A constitute 71% of that set. If our interpretation ofthe function of the abundant RN A species is correct, itwould follow that, in the course of evolution of HSV-1and H SV -2 fro m a c om mo n anc es to r, the s eque nc es c odingstructural pro teins diverg ed less, on the average, thanthose speci fy ing nonstructural prote ins .T he Significance of the V ir us specific R NA Sequencesin the C er vical Tumor

The tum or c ontains trans cripts aris ing fro m 5 % o f v iralDNA. This amount of DN A is considerably less thanthat trans cribe d in pro duc tive ly inf ec te d HEp- 2 c el ls e ithe re arly (2 1%) o r late (5 0%) i n infe ctio n and by a w ide m arg inles s than that required to specify structural pro teins (1 0,44). Tw o points bear on these data. The first arises from

the fact that HS V-2 like all o ther herpesv iruses no rm allykills the productively infected cell (34). Most of the cy-to ly tic functio ns o f the v irus, i.e., inhibitio n o f ho st D N Aand pro tein sy nthes is and alteratio n o f ho st RN A m etabo li sm , e tc ., are e arly f unc tions . C le arly, i f the inf ec te d ho stis to s urv iv e, the func tio ns e xpre ss ed by the v irus s ho uld befe we r than tho se e xpre ss ed e arl y in pro duc tiv e i nfe ctio n.A s w e pointed out in the text, w e do not know w hat functions are expressed in the tumor or w hether this amounto f transcriptio n inv ariably acco mpanies the presence o fviral D NA in tumor cells.Character ist ics of V i r al DNA Present in the Tumor

D N A -D N A hy bridiz atio ns indic ate that o nly 3 9% o f theviral genome is present in the tumor cells. The findingsare significant from 2 points of view . First, since only afragment of the D NA is present in the tumor tissue inratios of 1 to a maximum of 3.5 fragments per cell, itseems reaso nable to co nc lude that the frag ment aro se as aconsequence of a single event of infectio n. The 2nd pointconcerns the significance of the finding of a fragmentrather than o f an entire g eno me. A s indicated elsew here,infection of human cells cultivated in vitro w ith HSV -2i s inv ariably pro duc tive ( 34 ) and in ac ute inf ec tion the v irusre adily m ultiplie s in c erv ic al tis sue s. A no npro duc tio n infe ctio n c ould aris e in 2 w ay s, as a c ons eque nc e o f i nfe ctio nby a defective virus or as a result of infection of a non-pe rm is siv e c ell w ith c om pe te nt v irus . W hile w e c anno t e xclude the po ssibility that the cerv ix co ntains no nperm issive cells w hich preclude the expression of many viralf unc tions and s urv iv e, an attrac tive al te rnative i s that no nproductive infections in w hich the viral genome is perpetuated w itho ut cell destructio n arises also fro m infection w ith parti cl es c arry ing de fe ctive g eno me s. The transformation of susceptible cells in vitro (6) by HSV -2 w asaccomplished only w ith U V-irradiated virus and, to o urkno wle dg e, infe ctio us v irus c anno t be is olate d ro utine lyand w ith e as e fro m c erv ic al tum or tis sue s o r c el l c ulture s.T he A ssociation of V ir al D NA Sequences with R epetitiveSequences of C er vical Tumor D N A

As indicated in the text, these experiments were desig ned to determine w hether v iral D NA present in the tum or is c ov ale ntly linke d to ho st D N A . The re sults i ndic atethat the viral sequences are linked to host DNA in theproximity of host repetitive sequences. The amounts ofviral D NA sequences recovered in association w ith therepetitiv e se quences appear to be sm aller than tho se calculated to be present in unfractionated cerv ical tumorDNA. A trivial explanation of the data is that viral sequences are fragmented and that only some of these seque nc es are c ov alently l inke d to ho st re pe ti ti ve s eque nc es .A more likely explanation is that (a) fragmentation oftumor cell D NA did occur, particularly during heat de-naturation of the D NA at 115 for 7 minutes; (b) viralsequences that remained linked to repetitiv e sequenc es




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Tr ansc ription of H er pes Simplex Vir us D NAwere those near to the ends of the viral DNA molecule;and (c) the probability of viral sequences remaining co-valently linked to host D NA decreased in proportion tothe ir di stanc e f ro m the e nds . We c anno t pre se ntly di sc riminate be tw een these 2 po ssibilitie s, but the data do arg uethat at least some of the viral sequences are covalentlylinked to repe titiv e se quences o f ho st D NA .T he R elationship between I I SV 2 and C er vical T umor s

This is the 1st demonstration of viral DNA, viralRN A, and of covalent linkage of viral D NA to cellD NA in a cervical tumor. These data w ould be predictedif the virus causes the tumor but in themselves do notexclude the possibility that the presence of the viralg eno me is unrelated to the transfo rming ev ent that led tothe tumor. A s suggested elsew here (11), w hile more tum ors bo th natural and ex perimental sho uld be analy zed,direct pro of o f the causal re latio n may ultim ately em erg eo nly fro m analy sis o f po pulatio ns pro te cted fro m acquiring infection w ith HSV -2 (26).Acknowledgments

T he s tu di es o n th e c erv ic al tu mo r w e re do ne i n c ol lab orati on w ith D r.A nd r N ah mi as at E mo ry U ni ve rs ity w ho c ontri bu te d th e tum ors an danaly ze d them fo r the pre se nc e o f infec tio us v irus and v iral antige nsand w ith D r. Enz o Cassai w ho ex tracted the nuc leic ac ids . W e w ish toacknowledge the participa tion o f Dr. Saul Si lvers te in in the experimentso n the effec t o f cy clo hex imide o n the transcriptio n o f v iral D NA andfor the prepara tion o f the N. crossa nuc lease .References

1 . B ac henheime r, S . L., Kieff. E. D ., Le e, L., and Ro iz man, B . Co mparative S tudies of D NA 's of M arek's D isease and Herpes S implex virus. In: G. de Th ,P. M. Biggs, and L. N. Payne (eds.),Onco genes is and He rpesv irus es, pp. 7 4 8 1. Ly on, France : Intern ati on al A g en cy f or R es earc h o n C an ce r. 1 97 1.2. B achenheimer, S. L.. and Roizman, B . RN A Synthesis in CellsInfected w ith Herpes Simplex V irus. V I. Poly A S equences inV iral mRN A. J. V irol., 10: 875 879, 1972.3. Britten, R. J. Repeated Sequences in Human DNA. CarnegieIns t. W as hi ng to n Y ear B oo k, 6 7: 3 27 3 30 , 1 96 9.4. B ritten, R. J. The A rithmetic of N ucleic A cid Reassociation.C arn eg ie In st. W as hi ng to n Y e ar B o ok, 6 7: 3 32 -3 35 , 1 96 9.5. B ritten, R. J., and Smith, J. A Bovine Genome. Carnegie Inst.W as hi ng to n Y ear B oo k, 6 8: 3 78 3 89 . 1 97 0.6 . D uf f. R .. and R app, F. Pro pe rti es o f H am ste r E mbry o Fibro blas tsTrans fo rm ed i n V itro af te r Ex po sure to U ltrav io le t- Irradi ate dH erpe s S im pl ex V irus Ty pe 2 . J. V iro l., 8 : 4 69 -4 77 , 1 97 1.

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1 8. H ei ne , J. W ., S pe ar, P. G ., and Ro iz man, B . The Pro te ins S pe ci fie dby Herpes Simplex Virus. VI. Vira l Pro te in in the Plasma Membrane.J. V iro l., 9:4 31 4 39 , 1 972 .19 . Kie ff, E. D ., B ache nhe im er, S . L., and Ro izman. B . S ize , Co mposition and Structure of the DNA of Subtypes I and 2 of HerpesS im ple x V irus es . J. V iro l., 8 : 1 25 1 32 , 1 97 1.20. Kieff. E. D ., Hoyer, B ., B achenheimer, S. L., and Roizman, B .G ene tic R el ate dne ss o f T ype I and Ty pe 2 H erpe s S im pl ex V irus es .J. V iro l., 9 : 7 38 -7 45 . 1 97 2.2 1. Le e, S . Y ., M ende cki , J.. and B raw erm an, G . A Po ly nuc le otide S egm ent ric h i n A de ny li c A ci d i n the R api dly L abe le d P ol yribo so malR NA C om po ne nt o f M ous e S arc om a 1 80 A sc ite s C el ls . Pro c. N ati .A cad. S ci. U .S ., 6 8: 1 33 1 1 33 5.1 97 1.2 2. Le uchte nberg er, C., Le uchte nberg er, R., and D av is , A . A M ic ro-s pe ctro ph oto me tri c S tud y o f th e D e ox yri bo se N uc le ic A c id ( D NA )Content in Cells of N ormal and Malignant Human Tissues. J.P ath ol ., 3 0: 6 5- 85 , 1 95 4.2 3. Lin, H. J., and Charg off, E. M etaphase Chrom oso mes as a S ourceo f D NA . B io chim. B iophy s. A cta, 9 /:6 9l 6 94 , 1 96 4.2 4. M el li , M ., W hi tf ie ld. C .. R ao . K . V .. R ic hards on. M .. and B is ho p, J.O. D NA -RN A Hybridization in V ast D NA Excess. N ature N ewBiol .. 2 .J/ .-8 -12 , 1971 .2 5. N ahm ias, A . J., N aib, Z. M .. and Jo se y, W . E. He rpesv irus hom inisT ype 2 A s so ci ati on w ith C erv ic al C an ce r an d P eri natal D i se as e.In: M . P olla rd ed.) P er spectives in Vir ology, Vol. 7. pp. 73-89.

Academic Press, Inc. , New York: 1970 .26. N ahmias, A . J., N aib, Z. M ., and Josey, W . E. Genital Herpes andC erv ic al C an ce r C an a C aus al R el ati on b e P ro ve n? In : G . d e T h P. W. B iggs, and L. N . Payne (eds.). Oncogenesis and Herpes-v irus es , pp. 4 03 -4 08 , Ly on, Franc e: Inte rnati onal A ge nc y f or R es earc h o n C an ce r, 1 97 2.27. Nahmias, A . J., Naib, Z. M.. Josey. W. E., Murphy. F. A., andL uc e, C . F. S arc om as af te r Ino cul atio n o f N ew bo rn H am ste rs w ithH er pe svir us h omin is Typ e 2 Str a in s. P r oc. So c. E xp tl. B o l. M ed .,1 34 : 1 06 5 1 06 9, 1 97 0.2 8. N onay am a. M ., and Pag ano , J. S . H om ol og y be tw ee n E ps te in- BarrV irus D NA and V iral D NA fro m B urkitt's Lym phom a and N aso -

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