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Regulation of polyoma virus early transcription in transformed cells by large T-antigen
VIROLOGY
121,
384-392
(1982)
Regulation
of Polyoma
in Transformed ROBERT Department
of Pathology,
Cells
G. FENTON New
Received
York
Virus
by Large
AND
University
March
23,
Early
Transcription T-Antigen
CLAUDIO
School
BASILICO’
of Medicine,
1982; accepted
May
New
York,
New
York
10016
28, 1982
We have studied the regulation of polyoma early gene transcription by viral large Tantigen in three rat cell lines transformed by the ts-a polyoma mutant (producing a thermolabile T-antigen), that contain a single nontandem insertion of viral DNA, including an intact early region. The viral integration of these lines is stable and the cells do not produce detectable amounts of free viral DNA at either permissive (33”) or nonpermissive temperature (39.5”) for large T-antigen function. In temperature shift experiments two of the three cell lines showed a 5- to lo-fold increase in the steady state levels of early mRNAs following growth at 39.5”, while in the third line the increase was about threefold. A polyoma-transformed cell line not capable of encoding a functional large T-antigen did not show this temperature effect, even though the early viral promoter appeared to be functional. The increase observed was very similar to that detected in mouse cell cultures lytically infected by polyoma ts-a virus following temperature shiftup, whereas wild-type infected cells failed to show this increase. Thus the results obtained strongly suggest that transcription of integrated viral DNA in polyoma-transformed cells is regulated by the early viral promoter and large T-antigen in a manner indistinguishable from lytic infection. The implication of these findings with respect to the evolution of polyoma-transformed cells in vitro is also discussed. INTRODUCTION
plexes (Khoury and May, 1977). Rio et al. (1980) have been able to demonstrate that the D2 protein, an analog of the SV40 large T-antigen, selectively decreases early transcription in vitro by binding to specific sequences near the SV40 origin of replication in a region overlapping the 5’ ends of the early mRNAs. Relatively little is known about the role polyoma large T-antigen plays in regulating transcription from the viral early region (Cogen, 1978), especially in cells transformed by this virus. Early in lytic infection with polyoma virus three mRNAs are produced that share common 5’ and 3’ ends but differ in the size of the intervening sequences removed during processing (Kamen et al., 1980). These RNAs, which encode the three polyoma T-antigens, arise by differential splicing of a common precursor, and are presumably regulated by the same early region promoter (Kamen et al., 1980; Treisman et aZ. 1981). RNAs
Ts-A mutants of SV40 and polyoma viruses produce thermolabile large T-antigens that are defective in the initiation of viral DNA replication at nonpermissive temperature (reviewed in Tooze, 1980). The observation that SV40 large T-antigen regulates its own synthesis by feeding back to inhibit early region transcription has been documented in several studies. (Reed et al., 1976; Alwine et al., 1977; Rio et ah, 1980; Myers et al, 1981). These results demonstrate that late during infection with ts-A mutants of SV40, cells produce 5- to lo-fold more early RNA following a shift to nonpermissive temperatures. This accumulation could be accounted for by an increase in the rate of early transcription and an increase in the number of actively transcribed templates which were isolated as viral transcription com’ To whom
reprint
requests
should
0042-6822/82/120384-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 384
POLYOMA
EARLY
TRANSCRIPTION
indistinguishable from the major early mRNAs can be identified in some polyoma transformed cells, and have been shown to arise from transcription of integrated viral DNA (Kamen et ah, 1980; Fenton and Basilica, 1981). However, the study of autoregulation in polyoma (and SV40) transformed cells is complicated by the fact that most cell lines producing a functional large T-antigen contain tandem head to tail repeats of integrated viral DNA (Basilico et al., 1979; Birg et al., 1979; Basilic0 et ah, 1980; Della Valle et al, 1981). These integrations are not stable under conditions permissive for large T antigen function; growth of ts-a transformants at 33” results in the formation of significant amounts of free viral DNA (Prasad et ah, 1976; Zouzias et al, 1977) which act as templates for early and late transcription. Integrated DNA is stable at 39.5” (excision requires the A gene function), (Zouzias et al, 1977; Basilic0 et aZ., 1979, 1980) so that comparisons at the two temperatures cannot be made due to the differences in struct.ure and number of transcriptional templates. To overcome this problem we have studied the role of large T-antigen in regulating early transcription in three ts-a transformants recently isolated by our lab (Colantuoni et ah, 1982). Each of these lines contains a single non-tandem insertion of viral DNA, including an intact early region; none of these cell lines produce detectable free viral DNA at 33” despite encoding functional large T-antigens. MATERIALS
AND
METHODS
Cells and viruses. The single-copy polyoma-transformed rat F2408 cell lines used in this work (R&l, tsa-1, and ts-a3) were produced by transfection with ts-a polyoma DNA at 39” as described (Della Valle et aZ., 1981) and isolated as agar colonies. The V3D cell line was also isolated as agar colony following transfection of F2408 cells with the purified HindIII-1 fragment of polyoma DNA. Infection of 3T3D mouse cells with polyoma wild-type or ts-a virus was carried out at m.o.i. of lo-20 PFU/cell
REGULATED
BY LARGE
385
T-ANTIGEN
as previously described (Fenton and Basilico, 1981). Preparation of poly (A)+ RNA. Cells were fractionated into nuclear and cytoplasmic fractions using 1% NP40. RNA was then extracted and fractionated into poly(A)+ and poly(A)- RNA using oligo(dT) cellulose columns as previously described (Fenton and Basilica, 1981). Nuclease Sl analysis. Nuclease Sl analysis of poly(A)+ RNAs was performed by the technique of Favaloro et al. (1980). Hybridization (in DNA excess) to purified restriction fragments of viral DNA, Sl digestion, alkaline gel analysis, transfer to nitrocellulose filters, and identification of viral sequences by blot hybridization with “‘P-labeled, nick-translated polyoma DNA were all performed as previously described (Fenton and Basilica, 1981). Northern blot analysis. RNA gel analysis was performed as described by Alwine et al. (1977). RNA samples were run on 1.4% denaturing agarose gels containing methylmercury hydroxide; after electrophoresis, RNAs were transferred to DBM paper, and hybridized to 32P-labeled, nicktranslated polyoma DNA or DNA fragments as described (Fenton and Basilica, 1981). Filters were exposed at ~70” against XR-2 x-ray film with a screen intensifier. Markers used to estimate the size of the RNA species were 28 S and 18 S ribosomal RNA. RESULTS
Autoregulation
in Transformed
Cells
Three rat cell lines transformed by the ts-a mutant of polyoma virus were used in this study, each of which satisfied three requirements which allowed quantitative analysis of early mRNA regulation: (1) Each of these lines (R5-1, ts-al, and t.s-a3) contains a single non-tandem insertion of viral DNA, including an intact early region; integration into host DNA has occurred within the viral late region (Colantuoni et aZ., 1982). (2) In all cases, at least 500 base pairs of viral DNA including the presumed early promoters and T-antigen binding sites are present upstream from
386
FENTON
AND
BASILIC0
quences for excision to occur (Colantuoni et al., 1982). To demonstrate that the viral mRNAs produced in these cell lines were identical to early lytic mRNA and capable of en-
the intact early region. (3) None of these cell lines produce detectable free viral DNA at 33” despite encoding functional large T-antigen; this reflects the requirement for homology of viral DNA se-
m
1
2
3
4
5
6
m’
2140-
1127882702-
c
Haell
EcoRl
70 RS-1 EL
V3D
8’0 i =
90
III
10
?I
760
:' 1380 2140
760 850
:'
600
260 600
Hind
Hint
II
2'0
,
FIG. 1. Virus-specific mRNA present in the single-copy cell lines R5-1, m-al, and ts-a3. (A) Five micrograms of early lytic mRNA or 10 pg of polg (A)+ cptoplasmie RNA from each transformed cell line were electrophoresrd on a 1.4% agarose gel containing methylmercury, and transferred to DBM paper. Viral mRNAs were identified by hybridization to the mP-labeled P&I-l fragment of polyoma DNA. Lane 1: Early lytic mRNA extracted from 3T3 cells 16 hr after infection with wt polyoma virus at 37’ in the presence of 20 @g/ml of cytosine arabinoside. Lane 2: R5-1. Lane 3: m-al, Lane 4: ts-a3. The figures on the right indicate the approximate size (in nucleotides) of the main RNA species. Early lytic mRNA was run as a size control for full length early mRNAs. Quantitative comparisons with the concentrations of virus-specific RNAs in the transformed lines cannot be made since the frequency of infected cells was not determined and the cultures were grown under different conditions. (B) Cytoplasmic poly(A)’ RNAs from R5-1 and V3D were analyzed by the alkaline Sl nuclease procedure and compared to the early lytic patterns. Lanes 1-3: 5 ng of the HaeII-H&c11 fragment (m.u. 72-26) of the Py DNA hybridized to 5 pg of early lytic mRNA (lane l), 10 fig of R5-1 RNA (Z), or 10 fig of V3D RNA (3). Lanes 4-6: Early lytic (41, R5-1 (5), and V3D (6) cytoplasmic RNA hybridized to EcoRl-cleaved Py DNA. m and m’ are Py DNA marker fragments. (C) Schematic diagram aligning the DNA fragments protected from Sl nuclease by early lytic (E.L.), R5-1, and V3D RNAs with the physical map of the Py genome early region. The coordinates of the early region are given in map units and the lengths of the protected DNA fragments in nucleotides.
30
POLYOMA
RS-1 ‘33
EARLY
TRANSCRIPTION
ts-a3
tE.41 39’
‘33
39’
‘33
39’
REGULATED
V3D ‘33
39’
FIN. 2 Effect of growth temperature on the steady state levels of polyoma early region mRNAs in ts-atransformed cell lines. Poly (A)+ cytoplasmic RNAs were isolated from cells grown continuously at 33” or shifted to 39.5’ for 24 hr prior to harvesting. Ten micrograms of poly(A)+ cytoplasmic RNA from each sample was analyzed by the Northern blot technique and viral sequences identified by hybridization to the P.stI-I early region probe. The autoradiograms were quantitated by densitometry to obtain the relative levels of the early mRNAs at each temperature (summarized in Table 1). In most cases autoradiograms which had been exposed for shorter times than those shown here were used for this purpose.
coding the three Py T-antigens, cytoplasmic poly(A)+ RNA was isolated from each and subjected to Northern blot and Sl nuclease analysis. Poly(A)+ cytoplasmic RNA from each cell line was electrophoresed on denaturing methylmercury-agarose gels, transferred to DBM paper, and viral sequences identified by hybridization to the 32P-labeled P&I-1 (‘79.7-15.0 m.u.) fragment of Py DNA. As shown in Fig. lA, viral mRNAs produced in each of the single copy transformants (lanes 2-4) comigrate with mRNA produced early during lytic infection (lane 1). As previously described (Fenton and Basilico, 1981), the 2900n band corresponds to the small and middle T mRNAs (which differ in the length by only 14n at the splice acceptor sites) and the 2550n species to the large T-mRNA. Figure 1B shows Sl nuclease analysis performed on one of these lines (R5-1) compared with the early lytic pattern; the schematic diagram below (Fig. 1C) indicates the positions of the major Sl-resistant products. Poly(A)+ cytoplasmic RNAs were hybridized to puri-
BY LARGE
T-ANTIGEN
387
fied viral DNA restriction fragments, and the resulting hybrids were digested with Sl endonuclease. The protected viral DNA segments were size fractionated on denaturing alkaline agarose gels, and their positions identified by blot hybridization. The R5-1 (lanes 2 and 5) and early lytic patterns are identical, whether hybridized to the HueII-Hi?zcII early probe (lanes 1, 2) or to EcoRl-cleaved Py DNA (lanes 4, 5). This is especially important for the 260 and 600n bands, which correspond to the 5’ exons of early mRNAs (Treisman et ah, 1981). The tsa-1 and tsa-3 cell lines produced Sl patterns indistinguishable from this. This finding indicates that the 5’ends of the mRNAs of the single-copy cell lines arise from positions identical to the early lytic RNAs and supports the notion that these RNAs are transcribed under the control of the viral early promoter rather than of an adjacent cellular promoter. The role of the A gene function in regulating early mRNA production was examined following growth of these cell lines at permissive or nonpermissive temperature for large T-antigen. R5-1, ts-al, and ts-a3 cells were grown at 33” and 24 hr prior to harvesting half the plates were shifted to 39.5”. Cytoplasmic RNAs were isolated and equal amounts of polyp’ RNA from each temperature were electrophoresed on denaturing methylmercury-agarose gels and analyzed by the Northern blot technique as described above. This technique allows quantitative comparisons of steady state mRNA abundances to be made even when the RNA species represent only a small fraction of total cellular RNA (Parker and Stark, 1979). As shown in Fig. 2, growth at 39.5” results in an increased level of steady state mRNAs as compared to growth at 33”. Densitometer tracings demonstrated that ts-al and ts-a3 contain 5- to lo-fold more early RNA at nonpermissive temperature, while R5-1 produces about 2.5-fold more (see Table 1). It is worth mentioning that the observed differences did not reflect changes in the modality of transcription, but only a higher amount of viral transcripts. Results obtained by Sl nuclease analysis as de-
388
FENTON
AND
scribed above showed that the mRNAs produced in these cells at 39” and 33” were indistinguishable with respect to their 5 and 3’ ends and splice junctions (data not shown). It was not possible to perform similar quantitative studies with wild-type transformants since these lines invariably contain tandem proviral insertions (and produce free viral DNA), or do not produce a functional large T-antigen (Lania et al., 1981; Dailey et ah, 1982). Therefore in order to rule out the possibility that these quantitative differences were due to nonspecific temperature effects, we studied viral RNA production in a polyoma-transformed rat cell line (V3D) that was not capable of encoding a functional large Tantigen. The V3D cell line contains a major insertion of viral DNA that is a partial tandem repeat of the HindIII-1 fragment (45-1.8 m.u.) and hence does not contain large T coding information between 1.8 and 26 map units (unpublished results). This was verified by Sl nuclease analysis of V3D cytoplasmic RNA, also shown in Fig. 1B (lanes 3 and 6). When V3D mRNA TABLE AUTOREGULATION
IN Py-TRANSFORMED Production functional TAg”
Virus
or cell line
ts-a infected Early times Late times w.t. infected Early times Late times R5-1 ts-al ts-a3 V3D
BASILIC0
is hybridized to the HaeII-HincII fragment (lane 3), the early mRNA body sequence of 2140n is no longer present, but is replaced by a 850n fragment (which maps from the 3’ splice sites near 85.6 m.u. to 1.8 m.u.). When EcoRl-cleaved DNA is used as the hybridization probe, the 840n band is shortened to 760n (mapping from 85.6 m.u. to the EcoRI site at 0 m.u.). In both cases, the 260 and 600n leader segments comigrate with corresponding bands protected by early lytic and R5-1 RNA. These results confirm that V3D transcripts cannot encode large T-Ag. Further Sl analysis using late region viral DNA probes (not shown) demonstrate that V3D mRNAs extend across the HindIII-l/2 junction of the tandem DNA repeat and extend about 450n into the late region. (This 450n band is observed in lane 6 of Fig. 1B.) Northern blot analysis of V3D cytoplasmic RNAs (Fig. 2) shows that these are about 1 kb longer than expected from the Sl analysis alone, and coincidentally migrated similarly to the early lytic mRNAs. These data indicate that following transcription of the anti-late se1
RAT CELLS AND DURING
LYTIC INFECTION
OF MOUSE
of large
39.5”
Relative abundance of early region RNAs* 39.5O:33”
-
5-1O:l 551O:l
-
+
+ +
+ +
I:1 1.5:1
-
+ + +
-
2.5:1 551O:l 5-1O:l I:1
33”
CELLS
+
Production of late mRNAs”
+
+ -
a In cell lines R5-1, ts-al, and ts-a3, this was determined by Colantuoni et al. (1982). * Autoradiograms were quantitated optically using a Joyce-Loebl recording densitometer. In control experiments, the band intensity was found to correlate with RNA concentration in a linear fashion. c The presence of late mRNA was determined by Northern blot analysis using as probe the HhaI-1 fragment (representing exclusively late sequences) or Py genomic DNA.
POLYOMA 1.8 -
EARLY
44.6
TRANSCRIPTION l.8
BarnHI
44.6
Hoell
FIN. 3. Schematic representation of the major proviral DNA insertion from V3D drawn above the virus-specific mRNAs produced in this cell line. V represent the deleted viral DNA segments (1.8-44.6 m.u.). The virus-host DNA junction on the right side (near 53 m.u.) was deduced by Sl mapping (see the text), and the length of the RNA segment transcribed from host DNA (1 kb) was determined from combined Sl and Northern blot data. The virus-host junction on the left side occurs between the EcoRl and H&d111 sites as deduced from Southern blot data (not shown). The numbers below the proviral insertion represent map units, while the size of RNA segments is given in nucleotides. (p) viral DNA; (~3) host DNA.
quences, these RNAs extend into flanking host DNA for about 1 kb. From these studies we were able to precisely diagram the proviral DNA insertion and transcription map of V3D (Fig. 3). Readthrough transcription into flanking host DNA has been previously described for other Py-transformed cell lines that lack the efficient early region poly(A) addition site at 26 m.u. (Fenton and Basilica, 1981). V3D cells were subjected to temperature shift experiments, and equal amounts of poly(A)+ cytoplasmic RNA were analyzed by the Northern technique (Fig. 2). Growth at high temperature was not accompanied by an increase in the steady state level of V3D mRNAs, in contrast to the transformed lines producing large T-antigen. These results (summarized in Table 1) indicate that the temperature-sensitive A gene function is responsible for controlling the steady state levels of early mRNAs present in R5-1, ts-al, and ts-a3. Autoregulation tion
during
Early
Lytic
Infec-
We wanted to determine whether the 5 to lo-fold increase in early mRNA levels observed at nonpermissive temperature in the single-copy transformants was similar in magnitude to that observed early dur-
REGULATED
BY LARGE
T-ANTIGEN
389
ing lytic infection. Also, previous studies of autoregulation (both in SV40 and polyoma) (Reed et al., 1976; Alwine et al., 1977; Cogen, 1978) have been performed at late times of infection, and it has not been demonstrated that large T-antigen plays a similar role at early times. Therefore, 3T3D fibroblasts were infected at 33” with 10 PFU/cell of either wild-type or ts-a Py virus in the presence of 20 pg/ml of cytosine arabinoside (to prevent viral DNA replication). Ten hours after infection, half of the cultures were shifted to 39.5” for an additional 6 hr, and poly(A)+ RNAs were isolated. Figure 4 shows the Northern blot analysis of cytoplasmic RNAs from wild-type or ts-a-infected cells with or without temperature shift. Equal amounts of poly(A)+ RNA were analyzed and early viral RNAs identified with “Plabeled PstI-1 fragment DNA. The qualitative pattern of transcription is indistinguishable in wild-type and ts-a-infected cultures. However, densitometer analysis shows that while equal amounts of the wild-type mRNAs were present at 33” or 39.5”, 5-10 times more early mRNA was
‘33w.i9
’
'33
ts-a 39’
2900-
2550-
FIG. 4. Relative levels of early mRNAs from cells infected with wild-type (W.T.) lor ts-a virus following shift to the nonpermissive temperature. Infected cells were grown for 16 hr (in the presence of cytosine arabinoside) with or without shift to the nonpermissive temperature (39.5”). F’oly (A)+ cytoplasmic RNAs were isolated and 5 Gg of each sample was analyzed by the Northern blot technique and hybridization to the PstI-1 probe. Quantitative comparison of early mRNA abundance was made by densitometric analysis of the autoradiograms (Table 1).
390
FENTON
AND
detected in ts-a-infected cultures shifted to 39.5” (see Table 1). This increase in the steady state level or viral mRNAs is similar to that observed in the transformed cell lines ts-al and ts-a3. DISCUSSION
The role of the A gene product in regulating early region transcription from integrated viral DNA has been studied under conditions in which no free viral DNA is produced. Three polyoma ts-a transformants were studied that contain an intact early region and synthesize functional large T-antigen, but do not produce detectable free viral DNA, even when grown at permissive temperatures for the A gene function. This reflects the requirement of sequence homology (present in tandem but not single-copy insertions) for the excision of integrated Py DNA from rat cell chromosomes (Colantuoni et ul., 1982). In temperature shift experiments, two of three cell lines (ts-al and ts-a3) showed a 5- to lo-fold increase in the steady state levels of early mRNAs following growth at 39.5”. A polyoma-transformed cell line (V3D) not capable of encoding a functional large T-antigen did not show this temperature-sensitive effect, even though the early viral promoter was functional, and by Sl analysis the 5’ ends of its early mRNAs mapped at a position indistinguishable from early lytic mRNAs. Cultures lytically infected with Py ts-a virus show a similar 5- to lo-fold increase in the steady state mRNA levels following temperature shiftup, whereas wild-typeinfected cells failed to show this increase. It is not known why mRNA levels in R51 were affected less by shiftup than in tsal or ts-a3. This could reflect an undetected mutation in R5-1 large T-antigen or in a regulatory site within the early promoter. Alternatively, the site of viral DNA insertion into host chromosomes may play a role in determining the interaction of large T-antigen with promoter sequences. It was not possible to study the regulation of transcription of integrated DNA in cells transformed by wild-type virus, since those cells invariably con-
BASILIC0
tained tandem insertions (and produce free viral DNA) or were unable to encode a functional large T-antigen (Lania et al., 1981; Dailey et ab, 1982). The evidence presented strongly suggests that transcription of integrated viral DNA in polyoma-transformed rat cells is regulated by the early viral promoter and large T-antigen in a manner indistinguishable from early lytic infection. In transformed cells, regulation of viral DNA expression could occur at an adjacent cellular promoter rather than at the viral promoter. This is unlikely to be the case for the transformed lines we studied. First, Southern blot analysis of the singlecopy cell lines (Colantuoni et al., 1982) shows that each contains viral DNA sequences that extend at least 500 base pairs upstream from the early mRNA cap sites (near position 73.3). This region of polyoma DNA includes the early region TATA box and the upstream control sequences required for in vivo expression of the early region. The presence of a functional viral promoter is further suggested by Sl nuclease analysis of mRNAs isolated from these cell lines which demonstrates that the 5’ ends map at the usual early cap site near 73.3 m.u. Recent evidence (Gaudray et al., 1981) indicates that polyoma large T-antigen binds to a region of polyoma DNA that overlaps with the 5’ ends of the early mRNAs, and supports the hypothesis that autoregulation of polyoma and SV40 early transcription occurs by similar mechanisms. These results may help to explain the selection against large T-antigen that has been shown to occur in Py-transformed cells both in vivo and in vitro (Lania et al., 1981; Dailey et al., 1982). Transformants that have lost the ability to encode a fullsize large T-antigen can be expected to overproduce the mRNAs encoding the viral transforming proteins and may have a selective growth advantage. Also, the presence of a functional large T-antigen causes instability (i.e., excision or amplification) of integrated proviral DNA (Basilico et al., 1979, 1980; Colantuoni et aZ., 1980). Transformed cell lines that have lost the capacity to produce large T-anti-
POLYOMA
EARLY
TRANSCRIPTION
gen (but continue to synthesize middle Tantigen) would have stable proviral insertions and may be selected on this basis. Selection favoring cells that no longer produce a functional large T-antigen could reflect both the loss of transcriptional inhibition and the increased stability of the retained proviral DNA. Our results indicate that autoregulation of polyoma early region transcription occurs to a similar quantitative extent in transformed cell lines and during lytic infection. Autoregulation has also been reported to occur in SV40-transformed hamster cells (Alwine et ah, 19’77), although to a much lesser extent than observed during the SV40 lytic cycle. The steady state levels of the SV40 early mRNAs increased 5to l&fold following shift of SV40-tsa-infected cells to the nonpermissive temperature. Shiftup of SV40 ts-A-transformed hamster cells resulted in a maximal threefold increase and in some cell lines no detectable effect was observed. These experiments probably underestimated the quantitative effect of autoregulation for the following reasons: (1) hamster cells are semipermissive for SV40 DNA replication, and free viral DNA was probably produced at the permissive temperature. This would increase the number of viral DNA templates present at the low temperature, and consequently the amount of viral RNA transcribed; (2) the transformed hamster cell lines studied by Alwine et ah, (1977) lose the phenotypic indicators of transformation when grown at the nonpermissive temperature (Martin and Chou, 1975), and it is likely that the cell populations from which RNA was isolated at high temperature contained a sizable proportion of cells which had stopped cycling. Basilic0 and Zouzias (1976) have shown that Gl arrest of SV40-transformed mouse cells results in the disappearance of detectable large T-Ag and viral mRNA. A similar decrease in SV40 transcription could be occurring in the tsA-transformed hamster cells at high temperature, leading to an underestimation of the effect of large T-antigen. This problem is not encountered in the polyoma transformants we have studied since these
REGULATED
BY LARGE
T-ANTIGEN
391
cells are fully transformed at permissive and nonpermissive temperatures. Most previous studies of autoregulation have been performed at late times of infection with SV40 or polyoma (Reed et al., 1976; Alwine et al., 1977; Khoury and May, 1977; Cogen, 1978). We have shown that autoregulation occurs at early times of infection (and in transformed cells) in the absence of autonomous viral DNA replication. This rules out the possibility that a special class of replicated progeny DNA molecules is specifically derepressed at nonpermissive temperatures. The data presented also demonstrate that autoregulation occurs to a similar extent at early and late times of infection. This seems to be in disagreement with the hypothesis that repression of early transcription by T-antigen plays a major role in the increased production of the late mRNAs (Rio et al., 1980). Polyoma large T-antigen represses viral mRNA production at early times of infection, in the absence of detectable late mRNA. Also, as described earlier (Fenton and Basilica, 1981), late mRNAs are not produced in Py-transformed cells under conditions in which free viral DNA molecules are not produced. This cannot be explained solely by the early promoter repression model, but rather suggests that the most important function of large T-Ag in the production of late mRNAs is its role in the initiation of viral DNA replication. ACKNOWLEDGMENTS This investigation was supported by PHS Grant CA 16239 from the National Cancer Institute. R.G.F. was supported by NIH Training Grant 5T32GM07308. REFERENCES ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels analyzed by diazobenzyloxymethyl-paper and hybridization with DNA probes. froc Nat. Acad. Sci.
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ALWINE, J. C., REED, S. I., Characterization of the virus 40 gene A. J. Viral BASILICO, C., GATTONI, S., VALLE, G. (1979). Loss
and STARK, autoregulation
G. R. (1977). of simian
24, 22-27.
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392
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AND
sequences in polyoma-transformed cells is associated with an active viral A function. Cell 17, 645659. BASILICO, C., and ZOUZIAS, D. (1976). Regulation of viral transcription and tumor antigen expression in cells transformed by simian virus 40. Proc. Nut. Acad Sci. USA 73, 1931-1935. BASILICO, C., ZOUZIAS, D., DELLA VALLE, G., GATTONI, S., COLANTUONI, V., FENTON, R., AND DAILEY, L. (1980). Integration and excision of polyoma virus genomes. Cold Spring Harbor Symp. @ant. Biol. 44,611-620. BIRG, F., DULBECCO, R., FRIED, (1979). State and organization DNA sequences in transformed ViroL 29, 633-648.
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in 3T6 virus.
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DELLA VALLE, G., FENTON, R. G., and BASILICO, C. (1981). Polyoma large T antigen regulates the integration of viral DNA sequences into the genome of transformed cells. Cell 23, 347-355. FAVALORO, J., TREISMAN, R., and KAMEN, R. (1980). Transcription maps of polyoma virus-specific RNA: Analysis by two-dimensional Sl gel mapping. In “Methods in Enzymology” (L. Grossman and K. Moldave, eds.), Vol. 65, pp. 718-749. Academic Press, New York. FENTON, R. G., and BASILICO, C. (1981). Viral gene expression in polyoma transformed rat cells and their cured revertants. J. ViroL 40, 150-163. GAUDRAY, P., TYNDALL, C., KAMEN, R., and CUZIN, F. (1981). The high affinity binding site on polyoma virus DNA for the viral large-T protein. Nucleic Acids Res. 9, 5697-5710.
BASILIC0
KAMEN, R., FAVALORO, J., PARKER, J., TREISMAN, R., LANIA, L., FRIED, M., and MELLOR, M. (1980). Comparison of polyoma virus transcription in productively infected mouse cells and transformed rodent cell lines. Cold Spring Harbor Symp. Qua&. BioL 44,63-75.
KHOURY, G., and MAY, E. (1977). Regulation of early and late simian virus 40 transcription: Overproduction of early viral DNA in the absence of a functional T-antigen. J. Viral. 23, 167-176. LANIA, L., HAYDAY, A., and FRIED, M. (1981). LOSS of functional large T-antigen and free genomes from cells transformed in vitro by polyoma virus after passage in vivo as tumor cells. J. ViroL 39, 422-431. MARTIN, R. G., and CHOIJ, J. Y. (1975). SV40 functions required for establishment and maintenance of malignant transformation. J. ViroL 15, 599-612. MYERS, R. M., RIO, D. C., ROBBINS, A. K., and TJIAN, R. (1981). SV40 gene expression is modulated by the cooperative binding of T antigen to DNA. Cell 25,373-384.
PARKER, 8. A., and STARK, G. R. (1970). Regulation of simian virus 40 transcription: Sensitive analysis of RNA species present early in infections by virus or viral DNA. J. Viral. 31, 360-369. PRASAD, I., ZO~JZIAS, D., and BASILICO, C. (1976). State of the viral DNA in rat cells transformed by polyoma virus. I. Virus rescue and the presence of nonintegrated viral DNA molecules. J. ViroL 18, 436444.
REEL), S. I., STARK, G. R., and ALWINE, J. C. (1976). Autoregulation of simian virus gene A by T antigen. Proc. Nat. Acad. Sci. USA 73, 3083-3087. RIO, D., ROBBINS, A., MYERS, R. M., and TJIAN, R. (1980). Regulation of simian virus 40 early transcription in vitro by a purified tumor antigen. Proc. Nat. Acad. Sci. IJSA 77, 5706-5710. TOOZE, J. (1980). “DNA tumor viruses: Molecular biology of tumor viruses,” Part 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. TREISMAN, R., COWIE, A., FAVALORO, J., JAT, P., and KAMEN, R. (1981). The structures of the spliced mRNAs encoding polyoma virus early region proteins. J. Mol. A&. Genet. 1, 83-92. ZOUZIAS, D., PRASAD, I., and BASILICO, C. (1977). State of viral DNA in rat cells transformed by polyoma virus. II. Identification of cells containing nonintegrated viral DNA and the effect of viral mutations. J. ViroL 24, 142-150.
121,
384-392
(1982)
Regulation
of Polyoma
in Transformed ROBERT Department
of Pathology,
Cells
G. FENTON New
Received
York
Virus
by Large
AND
University
March
23,
Early
Transcription T-Antigen
CLAUDIO
School
BASILICO’
of Medicine,
1982; accepted
May
New
York,
New
York
10016
28, 1982
We have studied the regulation of polyoma early gene transcription by viral large Tantigen in three rat cell lines transformed by the ts-a polyoma mutant (producing a thermolabile T-antigen), that contain a single nontandem insertion of viral DNA, including an intact early region. The viral integration of these lines is stable and the cells do not produce detectable amounts of free viral DNA at either permissive (33”) or nonpermissive temperature (39.5”) for large T-antigen function. In temperature shift experiments two of the three cell lines showed a 5- to lo-fold increase in the steady state levels of early mRNAs following growth at 39.5”, while in the third line the increase was about threefold. A polyoma-transformed cell line not capable of encoding a functional large T-antigen did not show this temperature effect, even though the early viral promoter appeared to be functional. The increase observed was very similar to that detected in mouse cell cultures lytically infected by polyoma ts-a virus following temperature shiftup, whereas wild-type infected cells failed to show this increase. Thus the results obtained strongly suggest that transcription of integrated viral DNA in polyoma-transformed cells is regulated by the early viral promoter and large T-antigen in a manner indistinguishable from lytic infection. The implication of these findings with respect to the evolution of polyoma-transformed cells in vitro is also discussed. INTRODUCTION
plexes (Khoury and May, 1977). Rio et al. (1980) have been able to demonstrate that the D2 protein, an analog of the SV40 large T-antigen, selectively decreases early transcription in vitro by binding to specific sequences near the SV40 origin of replication in a region overlapping the 5’ ends of the early mRNAs. Relatively little is known about the role polyoma large T-antigen plays in regulating transcription from the viral early region (Cogen, 1978), especially in cells transformed by this virus. Early in lytic infection with polyoma virus three mRNAs are produced that share common 5’ and 3’ ends but differ in the size of the intervening sequences removed during processing (Kamen et al., 1980). These RNAs, which encode the three polyoma T-antigens, arise by differential splicing of a common precursor, and are presumably regulated by the same early region promoter (Kamen et al., 1980; Treisman et aZ. 1981). RNAs
Ts-A mutants of SV40 and polyoma viruses produce thermolabile large T-antigens that are defective in the initiation of viral DNA replication at nonpermissive temperature (reviewed in Tooze, 1980). The observation that SV40 large T-antigen regulates its own synthesis by feeding back to inhibit early region transcription has been documented in several studies. (Reed et al., 1976; Alwine et al., 1977; Rio et ah, 1980; Myers et al, 1981). These results demonstrate that late during infection with ts-A mutants of SV40, cells produce 5- to lo-fold more early RNA following a shift to nonpermissive temperatures. This accumulation could be accounted for by an increase in the rate of early transcription and an increase in the number of actively transcribed templates which were isolated as viral transcription com’ To whom
reprint
requests
should
0042-6822/82/120384-09$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 384
POLYOMA
EARLY
TRANSCRIPTION
indistinguishable from the major early mRNAs can be identified in some polyoma transformed cells, and have been shown to arise from transcription of integrated viral DNA (Kamen et ah, 1980; Fenton and Basilica, 1981). However, the study of autoregulation in polyoma (and SV40) transformed cells is complicated by the fact that most cell lines producing a functional large T-antigen contain tandem head to tail repeats of integrated viral DNA (Basilico et al., 1979; Birg et al., 1979; Basilic0 et ah, 1980; Della Valle et al, 1981). These integrations are not stable under conditions permissive for large T antigen function; growth of ts-a transformants at 33” results in the formation of significant amounts of free viral DNA (Prasad et ah, 1976; Zouzias et al, 1977) which act as templates for early and late transcription. Integrated DNA is stable at 39.5” (excision requires the A gene function), (Zouzias et al, 1977; Basilic0 et aZ., 1979, 1980) so that comparisons at the two temperatures cannot be made due to the differences in struct.ure and number of transcriptional templates. To overcome this problem we have studied the role of large T-antigen in regulating early transcription in three ts-a transformants recently isolated by our lab (Colantuoni et ah, 1982). Each of these lines contains a single non-tandem insertion of viral DNA, including an intact early region; none of these cell lines produce detectable free viral DNA at 33” despite encoding functional large T-antigens. MATERIALS
AND
METHODS
Cells and viruses. The single-copy polyoma-transformed rat F2408 cell lines used in this work (R&l, tsa-1, and ts-a3) were produced by transfection with ts-a polyoma DNA at 39” as described (Della Valle et aZ., 1981) and isolated as agar colonies. The V3D cell line was also isolated as agar colony following transfection of F2408 cells with the purified HindIII-1 fragment of polyoma DNA. Infection of 3T3D mouse cells with polyoma wild-type or ts-a virus was carried out at m.o.i. of lo-20 PFU/cell
REGULATED
BY LARGE
385
T-ANTIGEN
as previously described (Fenton and Basilico, 1981). Preparation of poly (A)+ RNA. Cells were fractionated into nuclear and cytoplasmic fractions using 1% NP40. RNA was then extracted and fractionated into poly(A)+ and poly(A)- RNA using oligo(dT) cellulose columns as previously described (Fenton and Basilica, 1981). Nuclease Sl analysis. Nuclease Sl analysis of poly(A)+ RNAs was performed by the technique of Favaloro et al. (1980). Hybridization (in DNA excess) to purified restriction fragments of viral DNA, Sl digestion, alkaline gel analysis, transfer to nitrocellulose filters, and identification of viral sequences by blot hybridization with “‘P-labeled, nick-translated polyoma DNA were all performed as previously described (Fenton and Basilica, 1981). Northern blot analysis. RNA gel analysis was performed as described by Alwine et al. (1977). RNA samples were run on 1.4% denaturing agarose gels containing methylmercury hydroxide; after electrophoresis, RNAs were transferred to DBM paper, and hybridized to 32P-labeled, nicktranslated polyoma DNA or DNA fragments as described (Fenton and Basilica, 1981). Filters were exposed at ~70” against XR-2 x-ray film with a screen intensifier. Markers used to estimate the size of the RNA species were 28 S and 18 S ribosomal RNA. RESULTS
Autoregulation
in Transformed
Cells
Three rat cell lines transformed by the ts-a mutant of polyoma virus were used in this study, each of which satisfied three requirements which allowed quantitative analysis of early mRNA regulation: (1) Each of these lines (R5-1, ts-al, and t.s-a3) contains a single non-tandem insertion of viral DNA, including an intact early region; integration into host DNA has occurred within the viral late region (Colantuoni et aZ., 1982). (2) In all cases, at least 500 base pairs of viral DNA including the presumed early promoters and T-antigen binding sites are present upstream from
386
FENTON
AND
BASILIC0
quences for excision to occur (Colantuoni et al., 1982). To demonstrate that the viral mRNAs produced in these cell lines were identical to early lytic mRNA and capable of en-
the intact early region. (3) None of these cell lines produce detectable free viral DNA at 33” despite encoding functional large T-antigen; this reflects the requirement for homology of viral DNA se-
m
1
2
3
4
5
6
m’
2140-
1127882702-
c
Haell
EcoRl
70 RS-1 EL
V3D
8’0 i =
90
III
10
?I
760
:' 1380 2140
760 850
:'
600
260 600
Hind
Hint
II
2'0
,
FIG. 1. Virus-specific mRNA present in the single-copy cell lines R5-1, m-al, and ts-a3. (A) Five micrograms of early lytic mRNA or 10 pg of polg (A)+ cptoplasmie RNA from each transformed cell line were electrophoresrd on a 1.4% agarose gel containing methylmercury, and transferred to DBM paper. Viral mRNAs were identified by hybridization to the mP-labeled P&I-l fragment of polyoma DNA. Lane 1: Early lytic mRNA extracted from 3T3 cells 16 hr after infection with wt polyoma virus at 37’ in the presence of 20 @g/ml of cytosine arabinoside. Lane 2: R5-1. Lane 3: m-al, Lane 4: ts-a3. The figures on the right indicate the approximate size (in nucleotides) of the main RNA species. Early lytic mRNA was run as a size control for full length early mRNAs. Quantitative comparisons with the concentrations of virus-specific RNAs in the transformed lines cannot be made since the frequency of infected cells was not determined and the cultures were grown under different conditions. (B) Cytoplasmic poly(A)’ RNAs from R5-1 and V3D were analyzed by the alkaline Sl nuclease procedure and compared to the early lytic patterns. Lanes 1-3: 5 ng of the HaeII-H&c11 fragment (m.u. 72-26) of the Py DNA hybridized to 5 pg of early lytic mRNA (lane l), 10 fig of R5-1 RNA (Z), or 10 fig of V3D RNA (3). Lanes 4-6: Early lytic (41, R5-1 (5), and V3D (6) cytoplasmic RNA hybridized to EcoRl-cleaved Py DNA. m and m’ are Py DNA marker fragments. (C) Schematic diagram aligning the DNA fragments protected from Sl nuclease by early lytic (E.L.), R5-1, and V3D RNAs with the physical map of the Py genome early region. The coordinates of the early region are given in map units and the lengths of the protected DNA fragments in nucleotides.
30
POLYOMA
RS-1 ‘33
EARLY
TRANSCRIPTION
ts-a3
tE.41 39’
‘33
39’
‘33
39’
REGULATED
V3D ‘33
39’
FIN. 2 Effect of growth temperature on the steady state levels of polyoma early region mRNAs in ts-atransformed cell lines. Poly (A)+ cytoplasmic RNAs were isolated from cells grown continuously at 33” or shifted to 39.5’ for 24 hr prior to harvesting. Ten micrograms of poly(A)+ cytoplasmic RNA from each sample was analyzed by the Northern blot technique and viral sequences identified by hybridization to the P.stI-I early region probe. The autoradiograms were quantitated by densitometry to obtain the relative levels of the early mRNAs at each temperature (summarized in Table 1). In most cases autoradiograms which had been exposed for shorter times than those shown here were used for this purpose.
coding the three Py T-antigens, cytoplasmic poly(A)+ RNA was isolated from each and subjected to Northern blot and Sl nuclease analysis. Poly(A)+ cytoplasmic RNA from each cell line was electrophoresed on denaturing methylmercury-agarose gels, transferred to DBM paper, and viral sequences identified by hybridization to the 32P-labeled P&I-1 (‘79.7-15.0 m.u.) fragment of Py DNA. As shown in Fig. lA, viral mRNAs produced in each of the single copy transformants (lanes 2-4) comigrate with mRNA produced early during lytic infection (lane 1). As previously described (Fenton and Basilico, 1981), the 2900n band corresponds to the small and middle T mRNAs (which differ in the length by only 14n at the splice acceptor sites) and the 2550n species to the large T-mRNA. Figure 1B shows Sl nuclease analysis performed on one of these lines (R5-1) compared with the early lytic pattern; the schematic diagram below (Fig. 1C) indicates the positions of the major Sl-resistant products. Poly(A)+ cytoplasmic RNAs were hybridized to puri-
BY LARGE
T-ANTIGEN
387
fied viral DNA restriction fragments, and the resulting hybrids were digested with Sl endonuclease. The protected viral DNA segments were size fractionated on denaturing alkaline agarose gels, and their positions identified by blot hybridization. The R5-1 (lanes 2 and 5) and early lytic patterns are identical, whether hybridized to the HueII-Hi?zcII early probe (lanes 1, 2) or to EcoRl-cleaved Py DNA (lanes 4, 5). This is especially important for the 260 and 600n bands, which correspond to the 5’ exons of early mRNAs (Treisman et ah, 1981). The tsa-1 and tsa-3 cell lines produced Sl patterns indistinguishable from this. This finding indicates that the 5’ends of the mRNAs of the single-copy cell lines arise from positions identical to the early lytic RNAs and supports the notion that these RNAs are transcribed under the control of the viral early promoter rather than of an adjacent cellular promoter. The role of the A gene function in regulating early mRNA production was examined following growth of these cell lines at permissive or nonpermissive temperature for large T-antigen. R5-1, ts-al, and ts-a3 cells were grown at 33” and 24 hr prior to harvesting half the plates were shifted to 39.5”. Cytoplasmic RNAs were isolated and equal amounts of polyp’ RNA from each temperature were electrophoresed on denaturing methylmercury-agarose gels and analyzed by the Northern blot technique as described above. This technique allows quantitative comparisons of steady state mRNA abundances to be made even when the RNA species represent only a small fraction of total cellular RNA (Parker and Stark, 1979). As shown in Fig. 2, growth at 39.5” results in an increased level of steady state mRNAs as compared to growth at 33”. Densitometer tracings demonstrated that ts-al and ts-a3 contain 5- to lo-fold more early RNA at nonpermissive temperature, while R5-1 produces about 2.5-fold more (see Table 1). It is worth mentioning that the observed differences did not reflect changes in the modality of transcription, but only a higher amount of viral transcripts. Results obtained by Sl nuclease analysis as de-
388
FENTON
AND
scribed above showed that the mRNAs produced in these cells at 39” and 33” were indistinguishable with respect to their 5 and 3’ ends and splice junctions (data not shown). It was not possible to perform similar quantitative studies with wild-type transformants since these lines invariably contain tandem proviral insertions (and produce free viral DNA), or do not produce a functional large T-antigen (Lania et al., 1981; Dailey et ah, 1982). Therefore in order to rule out the possibility that these quantitative differences were due to nonspecific temperature effects, we studied viral RNA production in a polyoma-transformed rat cell line (V3D) that was not capable of encoding a functional large Tantigen. The V3D cell line contains a major insertion of viral DNA that is a partial tandem repeat of the HindIII-1 fragment (45-1.8 m.u.) and hence does not contain large T coding information between 1.8 and 26 map units (unpublished results). This was verified by Sl nuclease analysis of V3D cytoplasmic RNA, also shown in Fig. 1B (lanes 3 and 6). When V3D mRNA TABLE AUTOREGULATION
IN Py-TRANSFORMED Production functional TAg”
Virus
or cell line
ts-a infected Early times Late times w.t. infected Early times Late times R5-1 ts-al ts-a3 V3D
BASILIC0
is hybridized to the HaeII-HincII fragment (lane 3), the early mRNA body sequence of 2140n is no longer present, but is replaced by a 850n fragment (which maps from the 3’ splice sites near 85.6 m.u. to 1.8 m.u.). When EcoRl-cleaved DNA is used as the hybridization probe, the 840n band is shortened to 760n (mapping from 85.6 m.u. to the EcoRI site at 0 m.u.). In both cases, the 260 and 600n leader segments comigrate with corresponding bands protected by early lytic and R5-1 RNA. These results confirm that V3D transcripts cannot encode large T-Ag. Further Sl analysis using late region viral DNA probes (not shown) demonstrate that V3D mRNAs extend across the HindIII-l/2 junction of the tandem DNA repeat and extend about 450n into the late region. (This 450n band is observed in lane 6 of Fig. 1B.) Northern blot analysis of V3D cytoplasmic RNAs (Fig. 2) shows that these are about 1 kb longer than expected from the Sl analysis alone, and coincidentally migrated similarly to the early lytic mRNAs. These data indicate that following transcription of the anti-late se1
RAT CELLS AND DURING
LYTIC INFECTION
OF MOUSE
of large
39.5”
Relative abundance of early region RNAs* 39.5O:33”
-
5-1O:l 551O:l
-
+
+ +
+ +
I:1 1.5:1
-
+ + +
-
2.5:1 551O:l 5-1O:l I:1
33”
CELLS
+
Production of late mRNAs”
+
+ -
a In cell lines R5-1, ts-al, and ts-a3, this was determined by Colantuoni et al. (1982). * Autoradiograms were quantitated optically using a Joyce-Loebl recording densitometer. In control experiments, the band intensity was found to correlate with RNA concentration in a linear fashion. c The presence of late mRNA was determined by Northern blot analysis using as probe the HhaI-1 fragment (representing exclusively late sequences) or Py genomic DNA.
POLYOMA 1.8 -
EARLY
44.6
TRANSCRIPTION l.8
BarnHI
44.6
Hoell
FIN. 3. Schematic representation of the major proviral DNA insertion from V3D drawn above the virus-specific mRNAs produced in this cell line. V represent the deleted viral DNA segments (1.8-44.6 m.u.). The virus-host DNA junction on the right side (near 53 m.u.) was deduced by Sl mapping (see the text), and the length of the RNA segment transcribed from host DNA (1 kb) was determined from combined Sl and Northern blot data. The virus-host junction on the left side occurs between the EcoRl and H&d111 sites as deduced from Southern blot data (not shown). The numbers below the proviral insertion represent map units, while the size of RNA segments is given in nucleotides. (p) viral DNA; (~3) host DNA.
quences, these RNAs extend into flanking host DNA for about 1 kb. From these studies we were able to precisely diagram the proviral DNA insertion and transcription map of V3D (Fig. 3). Readthrough transcription into flanking host DNA has been previously described for other Py-transformed cell lines that lack the efficient early region poly(A) addition site at 26 m.u. (Fenton and Basilica, 1981). V3D cells were subjected to temperature shift experiments, and equal amounts of poly(A)+ cytoplasmic RNA were analyzed by the Northern technique (Fig. 2). Growth at high temperature was not accompanied by an increase in the steady state level of V3D mRNAs, in contrast to the transformed lines producing large T-antigen. These results (summarized in Table 1) indicate that the temperature-sensitive A gene function is responsible for controlling the steady state levels of early mRNAs present in R5-1, ts-al, and ts-a3. Autoregulation tion
during
Early
Lytic
Infec-
We wanted to determine whether the 5 to lo-fold increase in early mRNA levels observed at nonpermissive temperature in the single-copy transformants was similar in magnitude to that observed early dur-
REGULATED
BY LARGE
T-ANTIGEN
389
ing lytic infection. Also, previous studies of autoregulation (both in SV40 and polyoma) (Reed et al., 1976; Alwine et al., 1977; Cogen, 1978) have been performed at late times of infection, and it has not been demonstrated that large T-antigen plays a similar role at early times. Therefore, 3T3D fibroblasts were infected at 33” with 10 PFU/cell of either wild-type or ts-a Py virus in the presence of 20 pg/ml of cytosine arabinoside (to prevent viral DNA replication). Ten hours after infection, half of the cultures were shifted to 39.5” for an additional 6 hr, and poly(A)+ RNAs were isolated. Figure 4 shows the Northern blot analysis of cytoplasmic RNAs from wild-type or ts-a-infected cells with or without temperature shift. Equal amounts of poly(A)+ RNA were analyzed and early viral RNAs identified with “Plabeled PstI-1 fragment DNA. The qualitative pattern of transcription is indistinguishable in wild-type and ts-a-infected cultures. However, densitometer analysis shows that while equal amounts of the wild-type mRNAs were present at 33” or 39.5”, 5-10 times more early mRNA was
‘33w.i9
’
'33
ts-a 39’
2900-
2550-
FIG. 4. Relative levels of early mRNAs from cells infected with wild-type (W.T.) lor ts-a virus following shift to the nonpermissive temperature. Infected cells were grown for 16 hr (in the presence of cytosine arabinoside) with or without shift to the nonpermissive temperature (39.5”). F’oly (A)+ cytoplasmic RNAs were isolated and 5 Gg of each sample was analyzed by the Northern blot technique and hybridization to the PstI-1 probe. Quantitative comparison of early mRNA abundance was made by densitometric analysis of the autoradiograms (Table 1).
390
FENTON
AND
detected in ts-a-infected cultures shifted to 39.5” (see Table 1). This increase in the steady state level or viral mRNAs is similar to that observed in the transformed cell lines ts-al and ts-a3. DISCUSSION
The role of the A gene product in regulating early region transcription from integrated viral DNA has been studied under conditions in which no free viral DNA is produced. Three polyoma ts-a transformants were studied that contain an intact early region and synthesize functional large T-antigen, but do not produce detectable free viral DNA, even when grown at permissive temperatures for the A gene function. This reflects the requirement of sequence homology (present in tandem but not single-copy insertions) for the excision of integrated Py DNA from rat cell chromosomes (Colantuoni et ul., 1982). In temperature shift experiments, two of three cell lines (ts-al and ts-a3) showed a 5- to lo-fold increase in the steady state levels of early mRNAs following growth at 39.5”. A polyoma-transformed cell line (V3D) not capable of encoding a functional large T-antigen did not show this temperature-sensitive effect, even though the early viral promoter was functional, and by Sl analysis the 5’ ends of its early mRNAs mapped at a position indistinguishable from early lytic mRNAs. Cultures lytically infected with Py ts-a virus show a similar 5- to lo-fold increase in the steady state mRNA levels following temperature shiftup, whereas wild-typeinfected cells failed to show this increase. It is not known why mRNA levels in R51 were affected less by shiftup than in tsal or ts-a3. This could reflect an undetected mutation in R5-1 large T-antigen or in a regulatory site within the early promoter. Alternatively, the site of viral DNA insertion into host chromosomes may play a role in determining the interaction of large T-antigen with promoter sequences. It was not possible to study the regulation of transcription of integrated DNA in cells transformed by wild-type virus, since those cells invariably con-
BASILIC0
tained tandem insertions (and produce free viral DNA) or were unable to encode a functional large T-antigen (Lania et al., 1981; Dailey et ab, 1982). The evidence presented strongly suggests that transcription of integrated viral DNA in polyoma-transformed rat cells is regulated by the early viral promoter and large T-antigen in a manner indistinguishable from early lytic infection. In transformed cells, regulation of viral DNA expression could occur at an adjacent cellular promoter rather than at the viral promoter. This is unlikely to be the case for the transformed lines we studied. First, Southern blot analysis of the singlecopy cell lines (Colantuoni et al., 1982) shows that each contains viral DNA sequences that extend at least 500 base pairs upstream from the early mRNA cap sites (near position 73.3). This region of polyoma DNA includes the early region TATA box and the upstream control sequences required for in vivo expression of the early region. The presence of a functional viral promoter is further suggested by Sl nuclease analysis of mRNAs isolated from these cell lines which demonstrates that the 5’ ends map at the usual early cap site near 73.3 m.u. Recent evidence (Gaudray et al., 1981) indicates that polyoma large T-antigen binds to a region of polyoma DNA that overlaps with the 5’ ends of the early mRNAs, and supports the hypothesis that autoregulation of polyoma and SV40 early transcription occurs by similar mechanisms. These results may help to explain the selection against large T-antigen that has been shown to occur in Py-transformed cells both in vivo and in vitro (Lania et al., 1981; Dailey et al., 1982). Transformants that have lost the ability to encode a fullsize large T-antigen can be expected to overproduce the mRNAs encoding the viral transforming proteins and may have a selective growth advantage. Also, the presence of a functional large T-antigen causes instability (i.e., excision or amplification) of integrated proviral DNA (Basilico et al., 1979, 1980; Colantuoni et aZ., 1980). Transformed cell lines that have lost the capacity to produce large T-anti-
POLYOMA
EARLY
TRANSCRIPTION
gen (but continue to synthesize middle Tantigen) would have stable proviral insertions and may be selected on this basis. Selection favoring cells that no longer produce a functional large T-antigen could reflect both the loss of transcriptional inhibition and the increased stability of the retained proviral DNA. Our results indicate that autoregulation of polyoma early region transcription occurs to a similar quantitative extent in transformed cell lines and during lytic infection. Autoregulation has also been reported to occur in SV40-transformed hamster cells (Alwine et ah, 19’77), although to a much lesser extent than observed during the SV40 lytic cycle. The steady state levels of the SV40 early mRNAs increased 5to l&fold following shift of SV40-tsa-infected cells to the nonpermissive temperature. Shiftup of SV40 ts-A-transformed hamster cells resulted in a maximal threefold increase and in some cell lines no detectable effect was observed. These experiments probably underestimated the quantitative effect of autoregulation for the following reasons: (1) hamster cells are semipermissive for SV40 DNA replication, and free viral DNA was probably produced at the permissive temperature. This would increase the number of viral DNA templates present at the low temperature, and consequently the amount of viral RNA transcribed; (2) the transformed hamster cell lines studied by Alwine et ah, (1977) lose the phenotypic indicators of transformation when grown at the nonpermissive temperature (Martin and Chou, 1975), and it is likely that the cell populations from which RNA was isolated at high temperature contained a sizable proportion of cells which had stopped cycling. Basilic0 and Zouzias (1976) have shown that Gl arrest of SV40-transformed mouse cells results in the disappearance of detectable large T-Ag and viral mRNA. A similar decrease in SV40 transcription could be occurring in the tsA-transformed hamster cells at high temperature, leading to an underestimation of the effect of large T-antigen. This problem is not encountered in the polyoma transformants we have studied since these
REGULATED
BY LARGE
T-ANTIGEN
391
cells are fully transformed at permissive and nonpermissive temperatures. Most previous studies of autoregulation have been performed at late times of infection with SV40 or polyoma (Reed et al., 1976; Alwine et al., 1977; Khoury and May, 1977; Cogen, 1978). We have shown that autoregulation occurs at early times of infection (and in transformed cells) in the absence of autonomous viral DNA replication. This rules out the possibility that a special class of replicated progeny DNA molecules is specifically derepressed at nonpermissive temperatures. The data presented also demonstrate that autoregulation occurs to a similar extent at early and late times of infection. This seems to be in disagreement with the hypothesis that repression of early transcription by T-antigen plays a major role in the increased production of the late mRNAs (Rio et al., 1980). Polyoma large T-antigen represses viral mRNA production at early times of infection, in the absence of detectable late mRNA. Also, as described earlier (Fenton and Basilica, 1981), late mRNAs are not produced in Py-transformed cells under conditions in which free viral DNA molecules are not produced. This cannot be explained solely by the early promoter repression model, but rather suggests that the most important function of large T-Ag in the production of late mRNAs is its role in the initiation of viral DNA replication. ACKNOWLEDGMENTS This investigation was supported by PHS Grant CA 16239 from the National Cancer Institute. R.G.F. was supported by NIH Training Grant 5T32GM07308. REFERENCES ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels analyzed by diazobenzyloxymethyl-paper and hybridization with DNA probes. froc Nat. Acad. Sci.
USA
74,
5350-5354.
ALWINE, J. C., REED, S. I., Characterization of the virus 40 gene A. J. Viral BASILICO, C., GATTONI, S., VALLE, G. (1979). Loss
and STARK, autoregulation
G. R. (1977). of simian
24, 22-27.
ZO~!ZIAS, D., and DELLA of integrated viral DNA
392
FENTON
AND
sequences in polyoma-transformed cells is associated with an active viral A function. Cell 17, 645659. BASILICO, C., and ZOUZIAS, D. (1976). Regulation of viral transcription and tumor antigen expression in cells transformed by simian virus 40. Proc. Nut. Acad Sci. USA 73, 1931-1935. BASILICO, C., ZOUZIAS, D., DELLA VALLE, G., GATTONI, S., COLANTUONI, V., FENTON, R., AND DAILEY, L. (1980). Integration and excision of polyoma virus genomes. Cold Spring Harbor Symp. @ant. Biol. 44,611-620. BIRG, F., DULBECCO, R., FRIED, (1979). State and organization DNA sequences in transformed ViroL 29, 633-648.
M., and KAMEN, R. of polyoma virus rat cell lines. J.
COGEN, B. (19’78). Virus-specific cells infected by a ts mutant Virology 85, 222-230.
early RNA of polyoma
in 3T6 virus.
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