Translational control of SV40 T antigen expressed from the adenovirus late promoter

Cell, Vol. 33, 455-464,

June 1983, Copyright

0 1983 by MIT

0092.8674/83/060455-10

$02.00/O

Translational Control of SV40 T Antigen Expressed from the Adenovirus Late Promoter Carl Thummei,*+ Robert Tjian,* Shiu-Lok Hut* and Terri GrodzickerS *Department of Biochemistry University of California Berkeley, California 94720 *Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724

Summary We have constructed four novel adenovirusSV40 hybrid viruses that contain the SV40 A gene at different positions downstream from the adenoviral major late promoter, within the region that encodes the second and third segments of the late tripartite RNA leader. The SV40 insert was precisely positioned in preselected regions of the adenoviral genome by using a combination of in vitro and in vivo recombination. As expected, all four recombinants produce equally high levels of SV40-encoded RNA that initiates at the adenovirus late promoter and contains two or three leader segments at the 5’ end. Yet, in spite of this efficient transcription, only one virus, AdSVR284, directs the synthesis of high levels of SV40 large T antigen in infected cells; the other recombinants all produce approximately 20fold less T antigen. This differential expression is, however, not seen in vitro, where equal amounts of hybrid T mRNA direct the synthesis of equal amounts of SV40 T antigen. Thus, some form of translational regulation is present in adenovirus-infected cells that is missing from the in vitro translation reaction. Introduction The human adenoviruses contain a long (36,000 base pairs) linear double-stranded DNA genome encoding at least 30 proteins. Upon infection of permissive cells, these tumor viruses undergo a temporally regulated pattern of gene expression that can be divided into early and late phases (for a review, see Tooze, 1981). Shortly after infection, a small set of proteins is expressed from several different regions of the adenoviral genome. This early gene expression continues for several hours until, with the onset of viral DNA replication, the late genes are induced. At this time, there is a striking change in both the pattern of adenoviral transcription and host cell protein synthesis. Expression of some early regions is repressed and transcription of the viral genome initiates primarily at the major late promoter (map position 16.55) to form long heterogeneous RNAs (Backenheimer and Darnell, 1975; Chow et al., 1977). These late transcripts contain three short (40+ Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford CA 94305. s Present address: Molecular Genetics, Inc., 10320 Bren Road East, Minnetonka. MN 55343.

90 nucleotides) leader regions located upstream from the structural genes. When the primary transcripts are processed, the leader segments are spliced together and joined to the 5’ end of all late viral mRNAs to form a common 5’untranslated region (Berget et al., 1977; Broker et at., 1977). An additional leader segment, designated the i leader, has been detected in a significant number of transcripts synthesized from the late promoter at early and intermediate times in infection. This leader is located between the second and third late leader regions and, when joined to the third leader, encodes an adenoviral early protein (Virtanen et al., 1982). The function of the late tripartite leader is not known. Concurrent with the induction of late viral transcription, the pattern of proteins synthesized in infected cells is dramatically altered. Host cell protein synthesis is repressed and the adenoviral late mRNAs are preferentially translated (Ginsberg et al., 1967; Russell and Skehel, 1972). This shift in translational specificity, combined with the efficiency of late transcription, results in the selective synthesis of virally encoded proteins at high levels late during infection. In an effort to learn more about the regulation of adenoviral transcription and translation, a series of adenovirusSV40 hybrid viruses have been constructed that contain the SV40 A gene downstream from different adenoviral promoters (Solnick, 1981; Thummel et al., 1981b, 1982). The segment of SV40 DNA that was inserted into the adenoviral genome contains the T antigen-coding region but is missing the SV40 early promoter and the T antigenbinding sites. Thus, the expression of SV40 T antigen is under the control of adenoviral regulatory elements. The advantage of using this particular gene as a marker is the availability of reagents that allow the rapid and accurate quantitation of SV40 T antigen mRNA and protein produced in infected cells. One of the original hybrid viruses that was constructed, Ad-SVRG, which contains the SV40 A gene downstream from the early El b promoter (Grodzicker et al., unpublished data), efficiently expresses T antigen that is structurally and functionally indistinguishable from the authentic SV40 protein (Thummel et al., 1981b). More recently, we have used a combination of in vitro and in vivo recombination to precisely position the T antigen gene close to and downstream from both the major late promoter and the first of the three late leader regions. Cells infected with this virus, designated AdSVR26, produce very high levels of SV40-encoded mRNA; however, the amount of T antigen synthesized is approximately 50-fold less than would be expected from the amount of mRNA present, This inefficient translation of the AdSVR26 T mRNA could be due to either the novel AUG initiation codons used for T antigen synthesis or the absence of the complete tripartite leader region at the 5’ end of the message (Thummel et al., 1982). Our interest in determining what sequences might be necessary for efficient translation of late adenoviral mRNA led to the experiments reported here. Four novel adenovirusSV40 hybrid viruses were constructed that contain the

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SV40 A gene at various distances downstream from the adenovirus late promoter, within the region that encodes the second and third segments of the tripartite leader. We have used a variety of techniques to determine the levels of expression and structures of both the hybrid virusencoded T mRNAs and T antigens. Although all four recombinant viruses produce large amounts of SWO-encoded RNA, three of the viruses produce low levels of T antigen, similar to the amount detected in Ad-SVR26infected cells. Only when the SV40 A gene is inserted into the third leader region, in AdSVR284, is the level of translation commensurate with the level of transcription. This differential expression is, however, not reflected in vitro where the amount of T antigen synthesized from each of the hybrid T mRNAs is equal. The amount of SV40 large T antigen synthesized in Ad-SVR284-infected cells is dramatically higher than that produced by any other adenovirus-SV40 hybrid virus.

Results Construction of Adenovirus-SV40 Hybrid Viruses In order to place the SV40 A gene in different positions in the adenoviral genome, we made use of a series of bacterial plasmids in which progressively longer segments of adenovirus DNA containing the major late promoter were fused to the SV40 large T antigen gene (see Experimental Procedures; Figure 1). The SV40 sequences in these plasmids extend from the Hindlll site located 12 base pairs (bp) upstream from the T antigen translation initiation codon, through the coding region, to the Barn site following the poly(A) addition site. This fragment lacks the SV40 early promoter but contains the other sequences necessary for the expression of SV40 T antigen (Gluzman et al., 1980; Rio et al., 1980). The adenovirus DNA segments in the plasmids have one common end at map position 15.4 and extend through the late promoter to four different sites within the region encoding the leader sequences: map position 22.6 for the p6B insert; map position 25.9 for p8B; map position 26.5 for p9B; and map position 29.0 for plOB (see Figure 1). The fused adenovirus-SV40 segments have Barn ends and are inserted into the Barn site of pBR322. The inserts can be excised from the plasmids with Barn for their subsequent insertion into adenovirus vectors. The adenovirus vector 1 X 51i is an adenovirus 2adenovirus 5 hybrid virus that contains two Bell sites at map positions 34 and 41. Adenovirus DNA complexed with the 55-kd terminal protein (Rekosh et al., 1977) was digested with Bell and ligated to an excess of either p68, p8B, p9B, or plOB DNA that had been cleaved with Barn. The ligation products were mixed with full length 1 X 51i DNA-protein complex to provide helper DNA and transfected into human 293 cells by calcium phosphate coprecipitation (Graham and van der Eb, 1973; Frost and Williams, 1978). Wild-type adenovirus grows very poorly in monkey cells because of a block in late gene expression (Rabson et al., 1964). This block can be overcome by a helper function

I 11.4

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Figure 1. Inserts of Viral DNA in Pfasmids

4 29.0

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plOB

p6B, p6B. p9B. and plOB

On top is depicted a segment of the adenoviraf genome. including the late promoter (PI& three late leader regions (small dark boxes), and early i leader segment (hatched box). Below are the four Bam inserts that contain different lengths of adenovirai DNA fused to the SW0 A gene (open box). See Experimental Procedures for details of the plasmid constructions,

provided by the carboxy-terminal portion of SV40 large T antigen (Levine et al., 1973; Grodzicker et al., 1976; Tjian et al., 1978; Fey et al., 1979). Therefore, by passaging the recombinant viruses produced in 293 cells on CV-1 monkey cells, one can amplify selectively those progeny that express the inserted SV40 A gene. After growth and plaque purification on CV-1 cells, individual adenovirusSV40 hybrid virus stocks were grown on CV-1 cells. As expected for a mixture of defective and helper viruses, each recombinant virus population displayed two-hit kinetics in a plaque assay on monkey cells (data not shown). In each stock, the hybrid virus constituted approximately 30% of the viral population. Several independent isolates of each recombinant virus were characterized. DNA was purified from infected CV-1 cells by a modified Hirt (1967) extraction procedure and was analyzed by restriction enzyme digestion and Southern blot hybridization (data not shown). The majority (approximately 80%) of the virus isolates contained the entire SV40 A gene in the predicted position and orientation. All were stable on further passage. The structures of the four hybrid viruses, designated AdSVR274, AdSVR280, AdSVR284, and Ad-SVR289, are depicted in Figure 2. Their genomes most likely resulted from replacing the internal 1 x 51i Bell fragment, from map positions 34 to 41, with the adenovirusSV40 Barn fragment from each bacterial plasmid. These recombinants would contain a duplication of adenovirus DNA with one copy contributed by the vector DNA and one by the inserted adenoviral sequences. Homologous recombination between these duplicated sequences would result in excision of the intervening adenovirus DNA and placement of the SV40 sequences such that the adenovirus/SV40 junction in the final recombinant genome is identical to that present in the original plasmid (see Thummel et al., 1982). Ad-S/R274 and Ad-SVR284 carry the SV40 DNA inserted into a leader region: the early i leader in AdSVR274 and the late third leader in Ad-SVR284. AdSVR280 and AdSVR289, on the other hand, contain the T antigen gene

Adenovirus-SW.0 457

Hybrid Viruses

Figure 2. Genome Structures SV40 Hybrid Viruses

Ad-SVR26

The genome structure of Ad-SVR26. one of our previous constructrons (Thummel et al., 1982) IS deprcted above. Below are the genome structures of the four new recombinant viruses: AdSVR274, AdSVR280, AdSVR284, and AdSVR289. as determtned by restriction digestion and Southern (1975) blot hybridization. The late leader regrons (small black boxes), early r leader (small open box), deleted adenovrrus DNA (triangles), and SV40 sequences (large open box) are shown. The numbers are adenoviral map units (1 map unit = 36~3 W

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within introns: between the second and third leaders in AdSVR280 and downstream from the third leader in AdSW289 (see Figure 2). The distal junction of SV40 DNA with adenoviral sequences, at map position 41, was established by restriction enzyme digestion and ligation (Barn/ Bell).

T Antigens Encoded by the Adenovirus-SV40 Hybrid Viruses CV-1 cells infected with the hybrid viruses AdSVR274, R280, R284, and R289 were shown by direct immunofluorescence to display a bright nuclear stain similar to that seen in SV40-infected monkey cells late after infection (data not shown). To determine the size of the T antigens produced by the four hybrid viruses, extracts prepared from infected CV-1 cells were subjected to immunoprecipitation with two different monoclonal antibodies directed against either the amino-terminal (PAb419) or the carboxyterminal (PAb905) regions of the T antigen polypeptide (see Figure 3). A protein that comigrates with authentic SV40 large T antigen was precipitated by PAb905 from each of the hybrid virus-infected cell extracts. As expected, immunoprecipitation using polyclonal hamster anti-T serum yielded an identical pattern (data not shown). By contrast, only the AdSVR284 T antigen was efficiently precipitated by PAb419; neither the AdSVR280nor the Ad-SVR289encoded proteins reacted with this antibody, and only a small proportion of the Ad-SVR274-encoded T antigen was precipitated. This result suggests that the T antigen-sized proteins encoded by Ad-SVR280 and Ad-SVR289, and most of the antigen encoded by AdSVR274, might actually be truncated products lacking some sequences from the amino terminus of T antigen. In addition to the truncated protein, AdSVR274 also appears to produce a small amount of full length T antigen. No small t antigen was detected in extracts prepared from AdSVR274, R280, or R289-infected cells and low levels were seen in AdSVR284-infected cells, similar to the results obtained with other adenovirusSV40 viruses constructed previously (Thummel et al., 1981 b).

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CV-1 cells were Infected with either Ad-SVR26, R274. R28O. R284, or R289 and pulse-labeled for 1 hr with T-methionine 30 hr postinfection. The cells were harvested, and aliquots containing 2 X I@ cpm were subjected to immunoprecipitation as described in Experimental Procedures. As a control, authentrc T antrgen was precrprtated from SV40-infected monkey cells. Two different monoclond antrbodies were used: PAb419 and PAb905 drrected agarnst the amino-terminal and carboxy-termrnal regrons of T antrgen. respectively. Immune complexes were collected on Staphylococcus aureus cells (Kessler, 1975) and fractionated on 7-15% gradient SDS-polyacryamrde gels (Studrer, 1973). Marker proteins are shown on the far left The 96,000.dalton large T and 15,COO-dalton small t polypeptrdes are identified by arrows

Ad-SVR284 Overproduces Authentic SV40 Large T Antigen Based on Coomassie-stained bands of T antigen immunoprecipitated from infected cell extracts (data not shown),

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AdSVR284 produces 20- to 40-fold more T antigen than normal SV40-infected cells. Moreover, Ad-SVR284 appears to produce more full length large T antigen than any of our other constructed recombinant viruses (Figure 3; Thummel et al., 1981 b). Pulse-chase analysis of the different T antigens produced by the hybrid viruses revealed that these proteins are stable in infected cells. Comparison of the proteolytic products produced by partial digestion of AdSVR284 T antigen and SV40 T antigen with S. aureus V8 protease revealed no detectable differences, suggesting that the Ad-SVR284-encoded protein is similar, if not identical, to authentic SV40 large T antigen (Cleveland et al., 1977) (data not shown). We wanted to determine the kinetics of Ad-SVR284 T antigen synthesis and also compare the level of T antigen produced by this virus with that synthesized by our previous high producer virus, Ad-SVRG. As can be seen on the Coomassie-stained gel in Figure 4, cells infected with both recombinant viruses accumulate T antigen at late times after infection; however, by 24 hr postinfection, AdSVR284-infected cells contain approximately 5- to 1O-fold more T antigen than cells infected with Ad-SVRG. Furthermore, analysis of labeled proteins revealed that the synthesis of Ad-SVR6 T antigen peaks at an intermediate time in the infection, whereas Ad-SVR284-encoded T antigen continues to be synthesized at later times after infection. These results are consistent with expression of the Ad-SVR6 T antigen gene from the adenovirus El b early promoter (Grodzicker et al., unpublished data) and expression of the AdSVR284 T antigen gene from the adenovirus late promoter. Thus, the same gene can be expressed under entirely different regulatory control and at different levels depending on where it is positioned in the adenoviral genome.

hours

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R284

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Frgure 4. Trme Course and Relative Quantitation by Ad-SW6 and Ad-S/R284

of T Antigen Expressed

CV-1 cells infected wtth either Ad-SVR6 or Ad-SVR284 were pulse-labeled for 1 hr with Y-methionine at 9, 18, or 26 hr postinfection. Crude extracts containrng 3 x IO’ cpm were subjected to immunoprecipitation with an excess of PAb905 monoclonal antibody and the antigens were analyzed by SDS-polyacrylamide gel electrophoresis. Both the Coomassie bluestarned gel and autoradiogram are shown. SV40 large T antigen (T) and the heavy (IgGH) and light (IgGL) chains of the antibody are marked by arrows.

Structures of the SV40-Encoded RNAs Transcribed from the Adenovirus-SV40 Hybrid Viruses The structures of the SWO-encoded cytoplasmic poly(A)+ RNA produced by Ad-SVR274, R280, R284, and R289 were analyzed by a variety of techniques. Northern blot hybridization revealed that each hybrid virus produces several species of SV40-encoded RNA at levels approximately 50-fold higher than that found in SV40-infected monkey cells (Figure 5). This level of transcription is comparable to normal levels of late adenovirus mRNA synthesis Initial structural characterization of the hybrid RNAs, by sandwich hybridization analysis (Dunn and Hassell, 1977) demonstrated that the adenoviral portions of these messages mapped between position 15.8 (an Xhol site) and the site where SV40 DNA had been inserted into the adenoviral genome. No hybridization to adenoviral sequences was detected downstream from the SV40 A gene (data not shown). These results are consistent with transcription of the T antigen gene initiating at the adenovirus major late promoter and terminating at the SV40 polyadenylation site.

Figure 5. Sizing of Hybrid Northern Blot Hybridizatron

RNAs

by Glyoxd

Gel Electrophoresis

and

Ten rg of poly(A)+ SV40 RNA isolated from COS7 cells (Gluzman, 1961) and 0.5 cg of poly(A)+ RNA extracted from cells infected with each of the adenovirus-SW0 hybrid viruses were denatured by treatment with glyoxai and fractionated on a 1% agarose gel as described (Rio et al., 1960). The RNA was transferred to nitrocellulose (Thomas, 1960) and transcripts contarnrng SV40 sequences were identified by hybridization with radioactive SV40 DNA (Manratis et al., 1975; Rigby et al., 1977). The size markers are DNA fragments corrected for therr relative migratron subsequent to glyoxylatjon.

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Figure 6. Sl Analysrs of Hybrid Transcripts Frve pg of poly(A)+ RNA Isolated from Ad-SVR274, R280, R284, or R289infected cells was hybridized to 50 ng of either the SV40 Elgll-Barn fragment that contarns the T antigen gene or the appropriate plasmid Barn Insert from whrch the vrrus was constructed. The different RNAs and DNA probes are shown above and below the autoradiogram. The single-stranded regions of the RNA-DNA heteroduplexes were drgested with Sl nuclease and the protected DNA fragments were fractionated by glyoxal gel electrophoresis, transferred to nrtrocellulose, and visualized by hybridization with radioactrve SV40 DNA (see Experimental Procedures). Some renatured probe DNA can occasronally be seen. SV40 DNA size markers are present on either srde of the autoradrogram. The RNAs diagrammed below the autoradiogram deprct our interpretation of the Si analyses and primer extension experiments (not shown) for the translated hybrid T mRNAs. The truncated 1650.

We used the Sl nuclease-mapping technique (Berk and Sharp, 1977) (Figure 6) and primer extension analysis of the 5’ ends of the hybrid RNAs (data not shown) to further characterize the structures of the different virus-encoded hybrid transcripts. The RNA structures determined from these experiments are depicted at the bottom of Figure 6. As expected, the sizes of the spliced transcripts correspond to the different RNAs detected by Northern blot hybridization (Figure 5). Furthermore, in agreement with the protein data, no mRNA encoding small t antigen was detected. Each translated hybrid T mRNA contains the normal SV40 1900 nucleotide (n) exon that encodes the carboxyterminal portion of large T antigen (Berk and Sharp, 1978). As observed previously in RNA produced by other adenovirusSV40 hybrid viruses (Khoury et al., 1980; S.-L. Hu, unpublished results), some of the hybrid transcripts contain truncated forms of this exon, 1650 or 1150 n in length. These shortened species are derived from RNAs in which the large T donor splice site was joined to novel splice acceptor sites within the 1900-n exon (Thummel et al., 1982). Transcripts carrying these truncated exons do not encode a detectable T antigen-related protein. In all cases, the adenoviral leader segments are spliced onto the 5’ ends of the hybrid transcripts. However, the sites where the leaders are joined to the T antigen-coding region vary with each recombinant, depending on where the SV40 DNA was positioned in the adenoviral genome (see Figure 6). In AdSVR274 and AdSVR284, where the SV40 insert was positioned within the i leader and the third leader, respectively, the splice acceptor at the 5’ end of the leader segment is used with a high efficiency. In addition, some T mRNAs encoded by the recombinant viruses contain the upstream leaders joined to cryptic splice acceptor sites. Approximately half of the AdSVR274 SV40-encoded RNA and all of the Ad-SVR280 and R289 T mRNA utilize a cryptic splice acceptor located within the 5’coding region of the SV40 A gene (Thummel et al., 1982). The use of this splice acceptor leads to the 220-n exon seen by Sl analysis (Figure 6). Similarly, AdSVR280, R284, and R289 encode hybrid transcripts in which novel adenoviral splice acceptors, located upstream from the T antigen gene, are joined to the 5’ leader segments. It is interesting to note that the cryptic adenoviral splice acceptor site in the 530-n exon encoded by AdSVR280 maps to within 15 n of the splice acceptor site in the 780-n exon encoded by AdSVR284, suggesting that they may be identical. The proposed structures of the recombinant virus-encoded T mRNAs are consistent with the properties of the proteins they encode. Thus, the two different 5’ ends of and 1150-n 3’ exons are not shown. The heavy lanes represent adenovrral sequences and light lanes represent SV40 sequences. The SV40 large T antrgen mRNA IS shown above for comparison. All srzes shown are rn nucleotrdes and the adenoviral leader regions are labeled. The exact structures of the 530-n Ad-SVR280 exon and the 375-n Ad-SVR289 exon are still unclear and reourre further characterization

Cell 460

the mRNAs synthesized by AdSVR274 each encode a different form of T antigen. There are two S-proximal adenovirus AUG codons present in the transcript that contains the i leader sequence. Both codons are out of phase with each other and with the SV40 T antigen-coding region. Yet, in spite of these two upstream AUG codons, low levels of T antigen translation starting from the normal SV40 initiation codon can be detected by immunoprecipitation (Figure 3). The majority of SV40-encoded protein produced by AdSVR274, however, is apparently synthesized from an mRNA in which the cryptic SV40 splice acceptor site has been utilized. In this transcript, the normal T antigen initiation codon has been removed by splicing, leaving the second AUG codon in the SV40 A gene closest to the 5’ end of the message. Translation initiating at this AUG results in the synthesis of a truncated T antigen lacking 13 amino acids from the amino terminus (Thummel et al., 1982). The structure proposed for this RNA is consistent with the observation that the majority of the AdSVR274-encoded T protein will not react with a monoclonal antibody directed against the T antigen amino terminus (Figure 3). Similarly, the cryptic SV40 splice acceptor is used almost exclusively in the AdSVR280 and Ad-SVR289 T mRNAs and the T antigen encoded by these viruses will also not react with the amino terminus-specific monoclonal antibody (Figure 3). AdSVR284 produces one predominant species of SV40-encoded RNA 2300 n in length (Figure 5). This transcript could correspond to either the first and second leader segments and 325-n 5’ exon joined to the 1900-n 3’ exon or the first and second leader segments and 575n 5’ exon joined to the 1650-n 3’ exon. The latter RNA could not, however, encode T antigen due to the presence of four adenoviral AUG codons within the 575-n exon (Gingeras et al., 1982) and the absence of 250 n of 3’ T antigen-coding sequence. Similarly, the 780-n 5’ exon produced by Ad-SVR284 is, most likely, noncoding since it contains six adenoviral AUG codons upstream from the T antigen-coding region (Gingeras et al., 1982). Furthermore, although we can detect an Ad-SVR284 RNA 2700 n in length that could contain the 780-n 5’ exon joined to the full length 1900-n 3’ exon, this transcript represents at most 5% of the total RNA population. Thus, we conclude that the AdSVR284 mRNA that encodes T antigen probably consists of the first and second leader regions and 325-n 5’ exon joined to the 1900-n 3’ exon. The 5’proximal AUG codon in the AdSVR284 T mRNA is the normal SV40 initiation codon, resulting in the synthesis of full length SV40 large T antigen.

In Vitro Translation of T mFtNAs Encoded by the Adenovirus-SV40 Hybrid Viruses Each of the four adenovirus-SV40 recombinant viruses produces approximately equal amounts of SV40-encoded mRNA, present at about 50-fold higher levels than found in SWO-infected monkey cells. However, only Ad-SVR284 overproduces T antigen to the level expected from the amount of stable mRNA present. It thus seems likely that

the different T mRNAs are not translated with equal efficiency. In order to investigate the translational efficiency of the hybrid virus-encoded T mRNAs, rabbit reticulocyte lysates were programmed with equal amounts of poly(A)+ RNA isolated from either Ad-SVR26-, R274-, R280-, R284-, or R289-infected cells. As can be seen in the Northern hybridization analysis (Figure 5) equal amounts of the different virus-encoded poly(A)+ RNAs contain approximately equal amounts of SV40-encoded T mRNA. The in vitro-synthesized T antigen was detected by immunoprecipitation with the monoclonal antibodies PAb419 and PAb905 (Figure 7). The amounts of T antigen synthesized in vitro from the different hybrid virus-encoded RNAs are approximately equal, as shown by immunoprecipitation with PAb905, directed against the carboxy-terminal region of T antigen. Furthermore, the initiation codons used in vivo were apparently also used in vitro, as demonstrated by the pattern of proteins immunoprecipitated with the amino terminus-specific monoclonal antibody (Figure 7). In all cases, the addition of more poly(A)+ RNA resulted in the synthesis of proportionally more T antigen (data not shown). This suggests that the reactions were performed within the linear range of the in vitro translation reaction, In addition, equal amounts of total protein were synthesized in the translation reactions programmed with the different virus-encoded RNAs, as expected, and the pattern of proteins displayed by gel electrophoresis resembled those seen late during an adenovirus infection (data not shown). It is striking that the preferential translation of AdSVR284 T mRNA seen in vivo is not seen in vitro. These results suggest that the translation of T mRNAs is regulated in adenovirus-infected cells and that the in vitro rabbit reticulocyte lysate is lacking this regulatory function.

Discussion We have constructed four novel adenovirusSV40 hybrid viruses by using site-directed homologous recombination to position the SV40 insert within the adenoviral genome. These viruses, along with one of our previous hybrid viruses, AdSVR26 (Thummel et al., 1982) constitute a series of five recombinants that contain the SV40 A gene at various positions downstream from the adenovirus late promoter, within the region that encodes the 5’ RNA leader segments. AdSVR26, R280, and R289 contain the T antigen gene close to and downstream from the first, second, and third segments of the tripartite leader region, respectively. AdSVR274 contains the T antigen gene inserted into the early i leader and AdSVR284 contains the T antigen gene inserted into the third leader (Figure 2). Although all five viruses produce equally high levels of SV40-encoded RNA, only Ad-SVR284 produces high levels of T antigen in infected cells (approximately 20-fold higher than the the other recombinants). In vitro translation of the hybrid virus-encoded T mRNAs in a reticulocyte lysate system, however, results in the synthesis of approximately equal levels of T antigen, suggesting that some form of translational regulation present in adenovirus-infected cells

AdenovirusSV40 461

Hybrid Viruses

Ftgure 7. In Vitro Translation Encoded T mRNAs

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Aliquots containrng the rabbit reticulocyte translation extract were supplemented with hybrrd virusencoded poly(A)+ RNA as described rn Experrmental Procedures. Once protein synthesis was complete, the afiquots were divided rn half and subJected to immunoprecrpitatron with an excess of etther PAb419 or PAb905 monoclonal antibodies, directed agatnst the amino-tentnal and carboxy-termrnal regions, respectively, of 3’40 large T antigen. The preciprtated antigen was then analyzed by SDS-polyacrylamrde gel electrophoresis and autoradiography. Protein markers (III kd) and authentrc SV40 large T antigen are also present.

27-

NH,- terminal monoclonal

COOH- terminal monoclonal

is lost in vitro. The translational control present in adenovirus-infected cells discriminates among the different hybrid T mRNAs and directs the efficient translation of only the Ad-S/R284 message. It should also be noted that the same T antigen species are synthesized in vivo and in vitro, suggesting that the same AUG codons are recognized in both cases. It is possible that some factor(s), present in vivo, is missing from the reticulocyte lysate or was inactivated upon making the extract. The mechanisms whereby adenovirus regulates its gene expression at the translational level are just now being elucidated (Klessig et al., unpublished results; Thimmappaya et al., 1982). Adenovirus VA, RNA appears to be essential for efficient initiation of translation in infected cells. Thus, mutants that produce no VA, RNA synthesize normal levels of late mRNA but produce very little late viral protein (Thimmappaya et al., 1982). There is also evidence that VA, RNA can bind to the tripartite leader region of late viral mRNA (Mathews, 1980). Furthermore, nine nucleotides near the 5’ end of the first leader region are complementary to the 3’ end of 18s rRNA (Ziff and Evans, 1978). Thus, the tripartite leader may play an essential role in the initiation of translation by bringing the ribosome, VA, RNA, and the viral mRNA into close proximity. Ad-SVR284 produces a T mRNA that contains a nearly complete tripartite leader linked closely to the initiating AUG of the adenovirus A gene, whereas the other viruses do not. We suggest that it is this mRNA which is efficiently translated in infected cells. There are two sequences upstream from the third leader segment found on some T mRNAs; however, it seems unlikely that these RNAs are efficiently translated to yield wild-type T antigen because the adenoviral sequences contain multiple AUG codons

both in and out of frame with respect to the downstream T antigen coding sequences (see for example, Kozak, 1978; Mulligan and Berg, 1981; Subramani et al., 1981). It is tempting to speculate that the presence of a nearly complete tripartite leader contributes to the high efficiency of AdSVR284 T mRNA translation in vivo. None of the other viral T mRNAs that contain less of the tripartite leader are translated as well. Furthermore, there are adenovirusSV40 hybrid viruses that produce T mRNA that is initiated at the major late promoter and contain, at their 5’ end, the proximal portion of the first segment of the tripartite leader linked to the same SV40 sequences present in AdSVR284 T mRNA. This mRNA is translated in vivo at approximately 10% the efficiency of AdSVR284 T mRNA (Solnick, 1981; D. Solnick and Y. Gluzman, unpublished data). The presence of the three leaders is not, however, sufficient to confer upon any mRNA the extremely high levels of in vivo translation seen with the AdSVR284 T mRNA. The AdSVR26, R280, and R289 T mRNAs form a matched set of transcripts that contain either one, two, or all three leader segments, respectively, joined to the cryptic SV40 splice acceptor. Yet these recombinants all produce low levels of T antigen in infected cells. A difference between the AdSVR289 T mRNA and the translated AdSVR284 T mRNA is that the R284 transcript contains 48 nucleotides of SV40 sequences that are not present in the R289 message (from nucleotide -12 to +36 relative to the R284 T antigen initiation codon, with the A residue being position +l). It is therefore likely that this short region contains some sequences that play a role in the translation of T mRNA in vivo. It should be noted that the sequences around the cryptic SV40 AUG codon (C - AUGG) represent a relatively rare class of eucaryotic initiation sites while

the sequences surrounding the SV40 AUG codon (A - AUGG) in AdSVR284 T mRNA represent a more common class (Kozak, 1981). We are currently constructing two new adenovirusSV40 hybrid viruses that, together with AdSVR284, should produce a matched set of transcripts that contain either one, two, or all three leader segments joined to this critical 48nucleotide segment. These recombinants should all produce full length T antigen and will allow us to determine what effect the different leader segments may have on the efficiency of translation. The recombinant virus Ad-SVR284 produces the highest levels of wild-type SV40 large T antigen reported to date. The level of T antigen expression directed by this virus is similar to the amount of D2 protein produced by Ad2+D2 (Hassel et al., 1978). Approximately 2 mg of T antigen can be purified from 1 liter of HeLa spinner cells infected with AdSVR284 (Clark, Jones, and Tjian, unpublished results); this level may represent the highest level of expression possible in adenovirus vectors. Having thus identified a region of the adenoviral genome in which the SV40 A gene can be efficiently expressed, it will now be interesting to see whether overproduction of other eucaryotic proteins can be achieved by inserting different coding regions into this position in the viral genome. Experimental

Procedures

Cells and Viruses Eschenchia coli MM294 was grown rn L broth (Miller, 1972) and bacteriacarrying plasmids were propagated in the presence of 30 pg/ml amprcillin (Srgma). Monkey CV-1 cells and human HeLa and 293 cells were maintained on plastic dishes wrth Dulbecco’s modified Eagle’s medium (DME) supplemented wrth 50 pg/ml streptomycin and penicillin (Irvine) and 10% calf serum (Irvine). After viral rnfectron. the cells were marntained on DME supplemented with antibiotics and 2% fetal calf serum (Irvrne). Plaque-purified SV40 strain 776 was propagated on CV-I cells as described (Todaro and Takemoto. 1969). Plaque-purified adenovirus 1 x 511 was maintained on HeLa cells and grown preparatively as described (Pettersson and Sambrook, 1973). Thus interserotyprc vrrus was foned as an in viva wild-type recombinant from crosses between temperaturesensitive mutants of Ad5 and Ad2+NDl (Sambrook et al., 1975; Grodzicker et al., 1977). Methods for the construction and amplification of AdSVR274, AdSVR280. Ad-SVR284, and Ad-SVR289 are as described previously for our other hybrid virus constructtons (Thummel et al., 1981 b). All experiments were performed in a P2 containment facility rn accordance with the NIH guidelines. Construction of Bacterial Plasmids All enzymes and technrques have been described previously (Hu and Manley, 1981). Our source of adenoviral DNA was a pBR322 derivative that contained a fragment of adenoviral DNA, extending from the Alul site at map position 15.4 (convened to an EcoRl end with linkers) to the Hindlll sate at map position 31, inserted between the pBR322 EcoRl and Hrndlll sites. Thus plasmrd was cut either partially wfth Xhol (for p6B and p9B), partrally with Sall (for pEB), or completely wrth Xbal (for plOB). The singlestranded ends were filled in by treatment with the Klenow fragment of E. coli DNA polymerase I and the plasmid was cut completely with Barn. DNA fragments of the appropriate srze. extending from the proper blunt adenovrrus end (Xhol site at map position 22.6 for p&I, Sall site at map position 25.9 for pEB, Xhol site at map position 26.5 for p9B. and Xbal site at map posrtion 29.0 for plOB) to the pBR322 Barn site were gel-purified. These vectors were then lrgated to a Hindlll-Barn fragment of SV40 DNA, extending from nucleotide 2537 to 5175 (see Appendrx in Tooze, 1981) which had been treated wrth Klenow DNA polymerase I to fill rn the SV40 Hindlll end. The EcoRl sate in the resultant plasmrds (located at the Alul end of the adenovirus DNA) was converted to a Barn site with linkers, and the

adenovirus/SV40 inserts (shown in Figure 1A) were excised by treatment with Bam and cloned into the Bam site of pBR322 to form the plasmids p6B. ~86, p9B, and plOB. lmmunoprecipitation of 1 Antigens CV-1 cells were infected with approximately 10 pfu/cell of virus and, after 24-30 hr, labeled with ?S-methionine (Amersham) as described (Thummel et al., 1981a). Extracts were prepared by lysis with NP40 and T antigen was precipitated with either PAb419 (gift from E. Harfow) or PAb905 (gift from A. Lewis and S. Tevethia) monoclonal antibodies as described (Thummel et al., 1981a). PAb419 is directed against a deteninant formed by the 25 ammo-terminal amino acids of T antigen (Harlow et al., 1981; R. Clark, unpublished data). PAb905 antibody is directed against, at most, 50 amino acids at the carboxy terminus of T antigen (Tevethia, unpublished data). The immunoprecipitated proterns were displayed on SDS-polyacrylamide gels with a 7-15% gradient of acrylamide (Studier, 1973). RNA Analysis CV-1 cells were infected with the adenovirusSV40 hybrid viruses at a multiplicity of approximately IO pfu/cell. After 24 hr, the cells were harvested and lysed with NP40, and the RNA was extracted by treatment with urea and SDS as described by Berk and Sharp (1977). SV40 RNA was extracted from COS7 cells (Gluzman. 1981). We can obtain approxrmately 5-fold more SV40 early RNA from COS7 cells than from normal SV40-infected monkey cells (C. Thummel, unpublished results). Poly(A)+ RNA was isolated by chromatography on oligo(dT)-cellulose (Collaborative Research) (Aviv and Leder, 1972) and stored at -20°C as an ethanol peciprtate. Northern blot hybridization analysis was performed as described previously (Thummel et al., 1981 b). Si analysis was performed essentially as described by Berk and Sharp (1977) with the following modification (D. Rio and S. Mansour. personal communication). After digestion with Sl nuclease (Boehringer Mannheim) and ethanol precipitation, the pellet was resuspended in 50 mM NaOH and left at room temperature for 1 hr. The pH was then adjusted to neutral and the DNA was ethanol precipitated and resuspended in glyoxal denaturation buffer (Rio et al., 1980). After electrophoresis on a 1.9% agarose gel rn 10 mM phosphate buffer (pti 6.8) the DNA was transferred to nitrocellulose (Thomas, 1980) and the SV40 sequences were identified by hybridization to nick-translated SV40 DNA (Rigby et al., 1977; Maniatis et al., 1975). In Vitro Translation Reticulocyte lysates were prepared from anemic rabbits and the endogenous RNA was removed by treatment with micrococcal nuclease as described by Pelham and Jackson (1976). For each reaction, 23 ~1 of lysate was mixed with 4.5 pl of translation cocktail, 4.5 pl of 1 M potassium acetate, 1.5 pl of 25 mM magnesium acetate, 11 pl of water, 5 pl of %Smethronine (50 &I, Amersham). and 2 pg of poly(A)+ RNA (or water for the no RNA control) (Pettersson and Mathews, 1977). Afler 1 hr at 30°C. 200 pl of buffer B (Lane and Robbins, 1978) was added and each reaction was divided into two aliquots. One set of aliquots was treated with PAb419 antibody and one set was treated with PAb905 antibody at O°C as described above. The immunoprecipitated T antigen was displayed by SDS gel electrophoresis and autoradiography. Acknowledgments We thank Taffy Mullenbach, Carmella Stephens, and Mary Merle for excelent technrcal assrstance and Karen Erdfey for preparation of the manuscript. This research was supported by grants from the National Cancer Institute (CA13106-11 and CA25417) and the American Cancer Society (MV94). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

December

22, 1982; revised April 4. 1982

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