J. HoZ. Bid. (1979) 131, 399407

LETTERS

TO THE EDITOR

Polyoma Virus Transcription Early During?Productive Infection of Mouse 3T6 Cells Nuclear and cytoplasmic RNAs were isolated from mouse 3T6 cells at different times early in lytic infection by polyoma virus. These RNAs were then annealed to the E and the L strands of 32P-labelled viral DNA prepared in. vitro by nicktranslation. RNAs complementary to both DNA strands were found in the nuclei and cytoplasm of the cells before viral DNA synthesis was detectable by DNADNA filter hybridization. Some of the initial E and L strand viral RNAs detected in t8he nuclei of the cells were longer than a complete transcript of the viral genome.

Productive infection of cells by papovaviruses is conventionally divided into two phases. The early phase, by definition, lasts from infection until the beginning of viral DNA replication, which with simian virus 40 and polyoma occurs 12 to 20 hours after infection. The late phase begins with the onset of viral DNA synthesis and ends with the production of mature progeny virions and cell death. Extremely small amounts of viral-specific RNA are synthesized during the early period of productive infections. This RNA can only be studied over a relatively short period in a mass culture since a population of infected cells enter DNA synthesis over several hours, the synchrony of viral-associated events within these cells becoming progressively lost after infection (Petursson & Weil, 1968; Fried & Pitts, 1968). The evidence for two phases of papovavirus infection has been obtained using DNA synthesis inhibitors. Cytosine arabinoside and 5-fluorodeoxyuridine both inhibit thymidine incorporation into DNA by more than 95%, reduce expression of late genes without strongly affecting the appearance of T-antigens (Rapp et al.. 1965 : Petursson & Weil, 1968) and thereby effectively prolong the early phase. Viral RNA synthesis during the early phase of infection is mostly from one strand of the viral DNA, the E strand (Khoury et al., 1972,1975; Sambrook et al., 1972; Weinberg et al., 1972; Weil et al., 1974; Kamen et al., 1974; Beard et al., 1976), socalled since it encodes the early messenger RNAs of SV40-/- and polyoma. There are at’ least, two species of early mRNA, with overlapping sequences and different spliced-out regions (Berk & Sharp, 1977; Favaloro et al., 1979) and they code for different Tantigens. Their sequences are complementary to just under one-half of the E DNA strand and map in the “early region” of the genome (Sambrook et al., 1972 ; Kamen et al.. 1974; Khoury et al., 1975; Beard et al.. 1976). During the late phase, i.e. after the time when viral DNA synthesis has begun, most of the viral RNA synthesis is from the other (L) strand of the DNA. These late transcripts are processed to mRNAs that map in the “late region” of the genome and which encode the viral capsid proteins. DNA synthesis inhibitors substantially reduce but do not completely abolish synthesis of the late RNAs (Kamen et al., 1974; Weil et al., 1974; Beard et aE., 1976; Rosenthal & Brown. 1977 ; Salomon et aZ., 1977). Certain non-permissive cells are non-productively t Abbreviations

usecl: 8V40, simian virus

40; l?dUrd.

,5-fluo~od~:oxZruridinr.

399 0022%2836/79/180399-09

$02.00/O

x> 1979 Acaclonlir

Press Inc. (London)

Ltd.

400

I'. w.

PIPER

infected by papovaviruses and a variable proportion of thescb Income stably t~r;msformed. In such cells the virus usually expresses only early viral funct’ions, notably t,hc: production of T-antigens. It is t#herefore of considerable intercd to understand ho\\ early genes are expressed in abortive infections early in lytic: infections, and in t,ransformed cells. There is evidence t)o indicate that a limited transcription of t,he 1, DNA &and can occur before the onset of DNA replication during productive infection of c?clls 1)~. SV40 (Ferninand et aZ., 1977) and that t,he two events ma)y not be as closely (!ouplcd as was previously believed. Also late SV40 transcripts can be deteot,ed in abortivelyinfected mouse cells and in cells infected with temperature-sensitive mutants of SV40 at the non-permissive temperature, both instances in the absence of viral DNA replication (Khoury et al., 1972,1974; Khoury & May, 1977). Since expression of Iat(L sequences may not require DNA synthesis, st’udies of viral t,ranscription early during productive infection must use hybridization probes t’hat distinguish between RLcIvaAh complementary to the E and the 1; strands of viral DNA. They must, also be conductt~ti before viral DNA synthesis can be detected and preferably in t,hr absenw of 1)SA synthesis inhibitors. In this study we investigated the viral sequences expressed during t’he early phaw of lytic infection of mouse 3T6 cells by polyoma virus, using as hybridization probes the separated strands of 32P-labelled polyoma DNA svnthesized in vitro. This DNA. prepared by nick-translation of viral DNA (Rigby 4t AZ.; 1977) and strand-separated according to Kamen et al. (1974), h as a much higher specific activity than the ill P+UO 32P-labelled DNA4 used in previous studies of polvoma early transcription (Kamen et aE., 1974; Beard et al., 1976). The higher specific activity exbends the lower limit of in hybridization experiments to one molecule per cell detection of viral transcripts or even less. The DNA is therefore a more sensitive probe for detecting early viral RNA sequences since these constitute an extremely small percentage (0.01 “.I, or 1~s) of the total RNA synthesis of the cell. However the size of t,he DNA probe diminishes with time through radioautolytic breakdown, and at any time after its synthesis thtb DNA contains a proportion of small single-strand fragments t,hat are unable t’o form S1 nuclease-resistant hybrids. The percentage of the probe capable of forming S, -stable hybrids can be determined by annealing L strand DNA to an excess of ilrl asymmetric transcript of the L strand of polyoma DNA. When hybridized t,o cornpletion with this asymmetric complementary RNA, X0 to go’:;, of the L st,ra,nd DX=2 probes used in this study were protected from S, nuclease digestion, as shown ill Figure 1. This percentage was taken as t,he total fract.ion of hhe DNA capabk ot annealing, a.nd in experiments conducted simultaneously with the cRNA annealingI: (Figs 2 and 3) the data presented a#re expressed rclatJivt: to t,his qua,nt,ity as IOO”,, of the annealable DNA. The E strand DNA probes did not snncal to asymmetric cRNl1 and therefore contained negligible L strand sequences (Fig. 1). Since t hey were prrpared in the same reactions in zCtro as the L strand probes, t,he E st,rand DNA was a~sumrtl to be capable of forming S-resistant hybrids to the samr: extent a,s L DNA. Another problem connect,ed with t)he presence of short DNA fragmentJs in the hybridization probe is the partial susceptibility of shorter hybrids to hydrolysis during prolonged incubation with nuclease S,. Digestion conditions were therefore chosen so as t80 give a.11apparent 80 to 90% protection of cRNA-L strand DNA hybrids and 4 to XC’{, of the tnta,l counts from Sr-nuclease-resistant single-stranded DNA, as measured 1)~. trichloroacet,ic acid precipitation. The experimrnt,al points in Figures I. 2 and 3 at’t’

LETTERS

TO

THE

EDITOR

A h”~.----------------~----------------------‘, --0.1

0.25

cRNA (0)

-----

0-I

o-5

------____

0.25

______________________

0.5

(ng) (b)

Fm. 1. Annealing of asymmetric polyoma cRNA to in vitro-synthesized DNA probes. 321’labelled polyoma DNA (9 x lo7 cts/min per rg) prepared in vitro by nick translation (Rigby et (II., polymerase-mediated tran1977) and unlabelled asymmetric cRNA prepared by E. coli RNA gift from Dr scription of the L strand of polyoma DNA (Kamen et al., 1974) were a generous R. Kamen. The DNA was strand-separated according to Kamen et al. (1974) and used within 2 weeks of synthesis. Annealings, conducted in sealed Beckmann (Ref. 10123) microfuge tubes at 72°C for 5 days, were in 20 ~1 1 M-NaCl, 0.05 M-Tris.HCl (pH 7.5), 1 mM-EDTA, and contained sZP-labelled polyoma DNA and variable 250 pg yeast RNA carrier, 3 x 10e5 pg E or L strand amounts of asymmetric cRNA. After annealing, samples were transferred using a Pasteur pipette to tubes on ice containing in 2 ml 0.05 M-potassium acetate (pH 4.5), 0.2 M-N&I, 3.5 mM-zinc sulphate, 25 pg calf thymus DNA/ml, and sufficient S1 nuclease to render 92 to 96% of the nonhybridized DNA counts trichloroacetic acid-soluble during a 30-min incubation at 45°C. Total S, resistant radioactivity was determined by chilling the digests on ice, precipitating with loo/, trichloroacetic acid and filtering the precipitates through Whatman GFjC filters which were washed with 5% acid, dried and counted. (a) Hybridization of cRNA to the total E (--A-A~-) and L (-A-A-) strands of polyoma DNA; annealings that were performed in parallel with t,hose in Fig. 2 and which used the same DNA probes in the same quantity per annealing. (b) Annealings of cRNA to the E (--n--n--) or the L (-A-A-) strands of combined restriction endonuclease HpaII fragments I plus 3, and to the E (--O--O--) or the L (-m-m-) strands of combined HpaII fragments 2 plus 4. These hybridizations used t’he same quantity of DNA pel annealing as those in Figure 3(a) and were performed simultaneously under identical conditions.

shown without subtraction of this latter &-resistant component, which was a constant fraction of each DNA preparation under the conditions used. However, the,y are corrected for counter background (15 cts/min). Nuclear and cytoplasmic RNAs were prepared from mouse 3T6 cells 6, 9> 12, 15 and 27 hours after high multiplicity infection with polyoma virus, as described in the legend to Figure 2. These RNAs were then annealed to the separated E and L strands of 32P-labelled viral DNA synthesized in vitro (Fig. 2). Hybridization under conditions of DNA excess, represented by the initial slope of the annealing curves, gives the relative amount of RNA complementary to the E or L strands of polyoma DNA in each RNA sample. From this one can calculate the mean number of viral RNAs in the nucleus or cytoplasm of each cell, knowing the specific activity and amounts of the DNA used in the annealing (legend to Fig. 1) and the mass of nuclear (100 pg) or cytoplasmic (150 pg) RNA yielded by each dish of 2 x 10’ cells. The hybridization curves for nuclear RNA in Figures 2(c) and (d) show that E strand transcription is detectable by nine hours after infection, and appears to either

60 -

Cytoplasmlc RNA (pg) (Cl

i

I 0

IO

20

30

40

50

60

70

0

IO

20

30

40

50

60

70-

Nuclear RNA Cpgl FIG. 2. Hybridization of HNAa oxtractetl at tliffcrent tunes during infoct,wn to E or I, ~LY~~I,I polyoma DN;Z. r\ total of 100 SO-mm dishes of mouse ST6 cells were grown to confluencr it] Dulbecco’s modified Eagle’s (DME) medium supplemented wit,h lo:/, calf serum. The cells wcr’,~ infected with plaque-purified polyoma virus (strain A2) at 100 plaque-forming units/cell in 1 ml DME containing 5% foetal calf serum for 90 min. Addit,ional medium (5 ml) was then added wr~ti the infected cells kept, at 37°C. At different times (6 h (-a-@--); 9 h (----**); 12 h (--i\ -;‘,-); 15 h (-m-m-) and 27 h (- A--A--)) after< virus had been plated on t,he cells, 20 dishes were removed from the incubat,or. The cells on these dishes were washed wit,h icrcold TD buffer (Fried & Pittn, 1968) and d&ached using a silicone rubber policeman. The rt~ll suspensions wew then centrifuged at, 100 g for 10 min at O”C, and tho cells resuspended in 6 ml ice-cold Iso-HipH (10 mm-Trix, pH 8.5, 0.14 mNaCI, I.5 mmMgCI,, 0.5°$J NP40 containing 5”,, macaloid gel as ribonuclease inhibitor. Xuclri U’CI’O pc~lletrtl by cont,rifugatiorl :+t 10.000 g I’r,r 5 min, and the upper cytoplasmic fraction decantctl and mixetl with an eyus.1 volume of a buffs wntjaining 2% sodium dodocyl sulphat,c, 10 mm’l’ris (pH 7.5). 10 rn~-EDTA and 200 pg prot,rinaw Ii/ml. The nuclei were mixed in 5 ml of t,hr same buffw, dilutetl with 5 ml water. and resuspcv~tl~~l by repeated passage t,hrough a 19-gauge hypodermic needle. After 2 h at :17”C the solut’ions ww vxtractetl with an equal volume of l~henol/chlo~oform/isoamyl alcohol (50: 50: I). Nucleic acul~ were precipitated from the aqueous phase by precipit,ation wit,h ethanol. DNA was hydrolyzed t)j tligestion for 2 h at 37°C in 5 ml 10 mwr-Tris (pH 7.5), 10 miwMgCl,, 50 pg deoxyribonurleaac, I/ml (Worthingt,on ribonuclease-free grade). After addit#ion of sodium dodwyl xulphate t,o O.l’:;, and EDT-4 to 20 mm the exbraction and ethanol precipit&ion were repeated and the RNA pallot, redissolved in 1 ml 20 mmsodium acet,ate (pH 5.0). After addition of 4 ml 5 iv-NaC’l, high moltwular weight RNA was precipi&t,etl overnight at ~ 20°C. The final RNA pellets were resuspended a.t it concentrat’ion of 20 mg/ml in O.lq/, sodium dodecyl sulphate. Hybridizations were performotl as described in t,he legend to Fig. 1, t,he total amount of RNA per 0.02 ml annealing being adjustetl t,o 250 pg using yeast carrier RNA. Aft)er 5 days at, 727; the S,-nuolease-resistant hybrid was measured. For hybridizations shown in A and C!, :j x lo-” pg E strand 32P DN.4 was used pet annealing. B and D show hybridizat,ion of the sarncb RN.4 samples to an ident,ical quantity of 1, strand DNA.

LETTERS

* QJ

TO

THE

EDTTOR

403

Nuclear RNA lpg)

Fraction no Prc,. 3. (a) Hybridizat,ion of E (-----) and L (-----) strand l)iFAs from t,he wly ant1 late regions of t,he polyoma genome to total nuclear RXA of mouse XT6 tolls II h after high multiplicit,y infection. Cells were infected and nuclear RNA isolated 11 h post-infection as tleacribwl in t,hn legend to Fig. 2. This RNA was then annealed to the E ( A) or the 1, ( A) strands of combined restriction endonuclease HpaII fragments 1 plus 3, which together const,itut.e most of the squences expressed as late mRNAs (Griffin et nl., 1974; Kamen & Shure, 1976). It was also annealed to t,he E (111) and the L (w) strands of combined HpaII fragment,* 2 plus 4. which toget,her comprise mow than 70% of sequences expressed as early mRNAs. (b) Hybridization of E (-----) and L (--------) strand DNAs from the complete viral grnome to nuclear RNA from cells 11 h post-infection sedimented on a sucrose gradient. A sample of the RNA sample used for the annealings in (a) was dissolved in 0.25 ml 10 mnr-EDTA, O.l?,, rc~lium dodeql sulphate, combined with 0.5 ml dimethylformamide and 0.25 ml dimethylsulphoxide, and heated 2 min at 63°C to disrupt RNA associations (Dubroff L Nemer, 1975). It, was thrn wtiiment,ed on a 10% to 30% (w/v) sucrose gradient prepared in 10 mM-Tris (pH 7.5), 2 mw-ED’I’A, O~dO~, odium dodecyl sulphate, in the SW27 rotor at 23,000 revs/min for 15 h at 25°C’. l-ml fractions were collected, and groups of 2 or 3 consecut~ivc fractions were precipitated with ethanol. ‘L’hc precipittttes wwc redissolvotl and half of the total placed in each of 2 Hcckmann microfugv i’ubes. Thee samples were then annealed t,o oit,her t,hc F: or t,hv 1, st,mnds of polyoma 1)K.i w(l hylrricl forrnat~ion ~letert,cxl by rvsirit.n.nw to S, rructwse :rs rlwwitwtl irr tho lcgr~rcl 1,~) Fig. 1.

commence before, or initially exceed, L strand transcription. Xine hours post-infection the E strand viral RNA per cell is, on average, equivalent to no more than five copies of the E DNA strand. However L strand transcription rapidly overtakes E strand transcription approximately 12 hours post-infection at a time when there are, on average, about 20 to 40 viral RNA molecules per cell. After 12 hours there is an apparent decrease in the amount of E strand RNA (Fig. 2(c)), since these sequences become sequestered as RNA-RNA hybrids by the excess of L strand sequences and are thereby prevented from annealing to t,he labelled DNA. The data show that this

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PIPER

1, strand RNA is complementary to thrl entire 1, DNA strand late in infection (Wig. 2(d)), although in Figure 2(c) the E strand probe is not saturated hy any of thr nuclear RNA samples and the data do not indicate what fraction of the E strand is being expressed. E strand sequences have also appeared in the cytoplasm by nine hours after infection (Fig. 2(a)). At this time cytoplasmic L strand sequences are not detected, but they are in evidence soon afterwards, at 32 hours post-infection (Fig. 2(b)). Since the stable polyoma mRNA species synthesized from the E and L strands have extremely little self-complementary sequence (Kamen & Shure, 1976: Smith et aZ., 1976; Favaloro et al., 1979) the hybridization curves in Figure 2(a) and (10) arf’ not, appreciably affected by competitive RNA--RNA annealings. The saturation levels of E and L strand DNAs by the 15hour and 27-hour cytoplasmic RP;As reflect the fact that, the stable mRNAs are complementary to slightly less than one-half of the E and the L DNA strands late in infection (Kamen & Shure, 1976; Beard et (AZ., 1976: Favaloro et al., 1979). The higher saturation level of L strand DNA with the 27-hour sample (Fig. 2(b)) may represent a, small leakage of nuclear RNA into the cytoplasm. TABLE

Tim,e-course of the onset of viral DNA

--.

-)NA extracted ,I ;rrcnn+;,Yr. 1”)

8 9 10 11 12 13 14 15 15 h, mock infection 15 h + 10 mM-hydroxyurea Purified form 1 Polyoma DNA

1

synthesis after high multiplicity

Total DNA added

Cts/min retained by

to filters

1)NA

w’snk filters

,infectiorr o;, Total counts binding to polyomil I)NA filters

28,92 I 36,172 34,234 38,154 39,817 60,963

19 13 32 26 41 3203

11 4 27 29 8 ‘6

04J6 WO3 0.09 0.07 0.10 5.”

109,536 129,398 53,743 32,976

12,635 15,994 62 182

34 6 24 3

11.5 12.4 (i.11 0.55

286,331 28,633 2863

31,843 2921 381

12 5 :!

1 I.1 IO.2 IX.5

Mouse 3T6 cells were infected as described in t,he legend to Fig. 2, and labelled 3 h after infectjion with 20 PCi [3H]thymidine/ml (47 Ci/mmol, Radiochemical Centre, Amersham). At l-h intervals beginning 6 h after infection, viral DNA on a single SO-mm dish of cells was extracted as described by Hirt (1967), and then digested with pancreatic ribonuclease and annealed to minifilters cow taining an excess of polyoma DNA or blanks containing no DNA as described by Rosent,hal & Brown (1977). d mock-infected dish, and an infected dish maintainetl in medium containing 10 mwhydroxyurea were processed together with t,hr dish harvested 15 h post,-infection.

Viral DNA synthesis first becomes detectable by DNA-DNA filter hybridization 12 to 13 hours after infection as shown by the experiment described in Table 1. The efficiency of retention of labelled DNA by the DNA filters was no more than 15 to 20y0. This, together with the necessity to measure DNA synthesis by incorporation of radioisotope in vivo, resulted in the assay for DNA synthesis in infected cultures being an order of magnitude less sensitive than that used to measure transcription.

LETTERS

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EnnoR

406

There is very little published data concerning the sequence composition of the initial transcripts synthesized early in productive polyoma infection. Kamen et al. (1974) found the entire E DNA strand expressed in the nuclei of polyoma-infected cells maintained for 20 hours in the presence of cytosine arabinoside, as well as smaller amounts of L strand transcripts. Also using in vivo 32P-labelled viral DNA strands as probes, Beard et al. (1976) found early nuclear RNA hybridized to saturation with only 40 to 45% of the sequences of the E strand and that no more than 5 to 10% of the viral nuclear RNA sequences could be complementary to other portions of the E strand. This data was obtained using RNA from cells which had been maintained in FdUrd, although the authors mention having obtained similar data with RNA from cells minus FdUrd in the true early phase of infection. The results could be an indication that only those sequences present in early mRNAs are transcribed early in infection (Beard et al., 1976; Acheson, 1976). Alternatively, those E strand sequences not destined to become mRNA may be rapidly broken down and have a very low steady-state concentration in nuclear RNA. The initiation of E strand transcription probably occurs near the beginning of the early region since a defective polyoma genome that contains tandemly-repeated copies of a 17% region of the DNA around the replication origin is an elIicient template for E strand RNA synthesis in vivo (Condit et al., 1977). In an attempt to determine the sequence composition of early transcripts in the absence of inhibitors of DNA synthesis we isolated nuclear RNA from cells 11 hours after high multiplicity infection, before viral DNA synthesis could be detected (Table 1). This RNA was annealed to E and L strands of restriction endonuclease HpaII digestion fragments from the early and late regions of the polyoma genome (Fig. 3(a)). The results show that this preparation of nuclear RNA contains sequences complementary to both DNA strands in both the early and late regions of the viral genome. Although E strand transcripts are present in slightly greater abundance than L strand transcripts, the relative proportion of the latter is nevertheless appreciable. Those sequences represented in stable mRNA species, present in HpaII - 1 + 3L and 2 + 4E, appear to predominate in the nuclear RNS over those sequences not export,ed to the cytoplasm. However this may be partly an apparent effect due to competitive RNA-RNA annealing. Although there is not enough viral RNA to saturate all the probes except HpaII - 2 -+ 4E in Figure 3(a), the results show that a substantial fraction of the E strand from the late region is transcribed early in infection, a situation also found during the late phase (Kamen et al., 1974; Aloni, 1974). Acheson & Mieville (1979) have also detected, in pulse-labelled early nuclear RNA, E strand transcripts from the late region of polyoma DNA. It also appears from Figure 3(a) that the L strand transcripts synthesized towards the end of the early phase include L strand sequences from the early region, a situation that resembles that found late in infection (Kamen et al., 1974: Birg et al., 1977) when L strand transcription was found to represent more than 90% of total viral RNA synthesis (Flavell & Kamen, 1977). Other experiments (data not shown) have indicated that those E strand sequences from the late region and L strand sequences from the early region present in the nucleus towards the end of the early phase are substantially in non-polyadenylated RNAs. Early XV40 or polyoma-specific RNA constitutes an extremely small proportion of the total RNA synthesis of the cell, and it is difficult to pulse-label this fraction in order to determine the size of the initial transcript. Tonegawa et al. (1970) and Weil

406

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r’IPli:H

et al. (1974) have studied the size dixtrihutiorl of papovavirus RNA ~~~~Istr-l;r~b~~llr:tl during the early phase and detacttd a major I9 to 20 S peak togethar with scmu~ viral-specific RNA sedimenting heterogeneously between 30 S and 50 S. These studies did not use strand-specific hybridization probes and I therefore examined the size of nuclear RNAs annealing to the E and L strands of polyoma DNA during the early phase of 3T6 cell infection. The major SV40 and polyoma early mlLNAs sediment at 19 to 20 S (Weinberg et ul., 1972; Weil et al.. 1974; Kamen & Shure, 1976). Nuclear RNA from cells 11 hours post-infection: t$he same sample as that annealed in Figure 3(a)> was denatured and sedimented on a 150;, to 3O’j;, sucrose gradient (Fig. 3(b)). Different regions of the gradient were then annealed to tlither the E 01’ the L strands of polyoma DNA. Figure 3(b) shows the presence of E strand transcriptIs that sedimented as a peak in approximately thfh 19 to 20 S position, as well ati much larger E strand RNAs. Early nuclear RNA also contains L strand t,ranscripts mart’ than genome length and, like those in late nuclear RNA (Kamen et al.: 1974; Birg et al., 1977 ; Lev & Manor, 1977), these proba,bly contain all L strand srqut~nces. Many of the larger E strand transcripts sediment faster than t,ht? marker 28 S rihosotnal RNA and are therefore considerably larger t,han a full-length transcript of’ the E DNA strand. It is therefore probable that they colltain tandem repeats of the nucleotide sequence of the entire viral E DNA strand, and resemble E strand tjra8nscripts synthesized late in infection (Kamen et cc2.. 19’74) in possessing mosi or all of thcx E strand sequences (Fig. 3(a)). The time-course experiment in Figure 2 shows tIllat t,herta is a comparativel>~ ~irtlall difference between the times that E and L strand transcripts first hecomc dctectahl~~ E strand transcription ih by hybridization in a culture of infected cells. ,\lthough initially greater t.han L. it is possible that remova,l of the block t’o t,ranscription of’ the viral DNA immediat’ely after infection leads to transcription of ltotlt DNi\ &%tlater times in infection tKL?lstrands in the absence of DNA synthesis inhibitors. scription of the L strand rapidly overtakes that. of the E st,rand, probably as the hovel of T-antigen increases above a threshold level in each individual cell. A substantial fraction of the initial E and L strand transcripts synthesized in normal lytic inf&ion are of greater than genome length (Fig. 3): at least, as soon as t,heir level becomrh detectable by the hybridization technique employed here. Alt,hough L strand transcription could be demonstrated before vira.1 DNA synthesis became detectable b,v filter hybridization (Fig. 2 and Table l), the assay for DNA synthesis rcliod upotl incorpora,tion of radioisotope in vi~voand was less srnsitivc t,han the det,et:tiotl of’ viral RNAs by an in vitro-synthesized hyhridizat’ion probe. Because of this it. is st,ill conceivable that the L st,rand transcript,ion observed before 13 hours post-infi&otl ustis viral DNA molecules that bavca replicated as a t~ctmplatt~. Similarly it might, by ill.gutbtl that E strand t)ranscription may bc limited to the early region or1 DriA tcmplatt+ which have not replicat.ed, as observcld in the presence of TIIYA synthesis iIlhi~JitW~S (Beard et al., 1976). In this case some of the I9 to 20 S E strand nuclear ItIC& ilk Figure 3(b) might represent primary transcripts. It is probable thatJ these objections cannot be resolved by observing the temporal sppearance of’ viral RNAs in a mass culture of lytically-infected cells, and that the conventionally defined early and la&b phases of infection cannot be precisely delineated in the absence of DNA synt,hesi:: inhibitors. However it will be possible to extend t’hese results using the S1 nucleast’ mapping technique recentlp developed 1)~ Berk & Sha,rp (1977) and to rna~) t~ht~ initial polyoma transcripts on the viral genome.

LETTERS

TO THE

EDITOR

4oi

Note added in. proof: B. A. Parker and G. R. Stark ((1979) J. Viral. in the press) have recently exa,mined the viral RNAs present very early in infection of monkey cells by SV40. Although their results strongly suggest an active role for T antigens in the enhancement of lat,e RNA synthesis, late RNAs were nevertheless detectable 9.5 h post-infection although at a, lower level than early mRNA. Furthermore, synthesis of these late mRNAs was not abolished by the DNA synthesis inhibitor, cytosine arabinoside. I thank Dr L. V. Crawford assistance.

R. Kamen for their

for his help with preliminary comments on tho manuscript,

experiments, and Frances

Drs Kamen and Crew for technical

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19 December

1978

PETER W. PIPER

Polyoma virus transcription early during productive infection of mouse 3T6 cells.

J. HoZ. Bid. (1979) 131, 399407 LETTERS TO THE EDITOR Polyoma Virus Transcription Early During?Productive Infection of Mouse 3T6 Cells Nuclear and...
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