important way to our general understanding of eucaryotic gene structure and expression (for a review, see reference. 23). The adenovirus life cycle is divided ...
JOURNAL OF VIROLOGY, Sept. 1988, p. 3258-3264 0022-538X/88/093258-07$02.00/0 Copyright C) 1988, American Society for Microbiology
Vol. 62, No. 9
Gene Product of Region E4 of Adenovirus Type 5 Modulates Accumulation of Certain Viral Polypeptides CATHARINA HEMSTROM,"2 KATARINA NORDQVIST,lt ULF AND ANDERS VIRTANEN1*
PETTERSSON,l
Departments of Medical Genetics' and Microbiology,2 University of Uppsala, Biomedical Center, S-75123 Uppsala, Sweden Received 19 February 1988/Accepted 16 May 1988
An adenovirus type 5 mutant, designated H5ilE4I, was constructed in which region E4 was replaced by a cloned cDNA. The cDNA was a copy of an mRNA which exclusively contains open translational reading frames 6 and 7. The phenotype of the mutant was compared with that of the previously characterized E4 mutant H2dl8O8 and wild-type adenovirus 5. Although the H5ilE4I mutant lacked at least five E4 genes, it was nondefective for growth in HeLa cells. The defects in viral DNA replication, late protein synthesis, and shutoff of host cell protein synthesis associated with the phenotype of the H2dl8O8 mutant were not observed in HeLa cells infected with the H5ilE4I mutant. However, differences were observed regarding the time of onset of viral DNA replication and the accumulation of the hexon polypeptide as well as the 72-kilodalton adenovirus-specific DNA-binding protein. The results thus indicate that open reading frame 6 or 7 or both contain all genetic information required for viral replication in tissue culture cells, whereas another E4 gene modulates the accumulation of certain viral polypeptides. The early onset of viral DNA replication in H5ilE4I-infected cells may be an indirect effect of the enhanced expression of the 72-kilodalton DNA-binding protein.
The adenoviruses provide an excellent model system for studies of eucaryotic genes and have contributed in an important way to our general understanding of eucaryotic gene structure and expression (for a review, see reference 23). The adenovirus life cycle is divided into early and late phases, separated by the onset of viral DNA replication. Each phase is characterized by the expression of specific sets of genes. Generally, the early genes code for proteins that are involved in regulation of gene expression and DNA replication and the late genes code for structural proteins. During the early phase, six transcription units, designated early region 1A (ElA), E1B, E2, E3, E4, and late region 1 (Li), are transcriptionally active. Region E4 is located at the right-hand end of the genome (map units 91.3 to 99.1) (24, 29) and is transcribed leftwards. The nucleotide sequence of the E4 region of adenovirus type 2 (Ad2) (10, 14) reveals seven open translational reading frames (ORFs). A complex set of differentially spliced mRNAs that are expressed from this region have been characterized (6, 9, 16, 32, 33), and several different polypeptides have been identified (13, 20, 31). A few of these have been assigned to individual E4 mRNAs. Three E4 polypeptides have been studied more thoroughly. The product of ORF3 is a 14-kilodalton (kDa) nuclear protein (8, 28). ORF6 is translated into a 34-kDa protein. This protein is associated with the 58-kDa protein encoded by region E1B, forming a complex which is located in the nucleus (27). Finally, a 19.5-kDa product, encoded by a fusion of ORF6 and -7, has been identified (7). Weinberg and Ketner (35) have isolated an Ad2 deletion mutant, H2dl808, which lacks most of the E4 sequences. This mutant was defective in late gene expression, viral DNA replication, and shutoff of host cell macromolecular synthesis. The mutant could only be propagated in cells that expressed the E4
region, i.e., the W162 cell line (34). Moreover, it has been proposed that the E4 products are involved in the transition from the early to late stages of infection (37). Deletions and insertions of DNA sequences affecting the different ORFs of region E4 were introduced by Halbert et al. (12). This analysis indicated that most ORFs could be altered without affecting viral growth in cultured cells. However, mutant H5dl355, which affected ORF6, was modestly defective for growth. H5dl366, a mutant from which most of the E4 region was deleted, showed a phenotype similar to that of H2dl808. In this report we describe an Ad5 mutant, H5ilE4I, in which region E4 was replaced by a cloned cDNA originating from an mRNA which exclusively contained ORF6 and -7. This mutant replicated normally in cultures of HeLa cells and lacked most of the phenotypic properties associated with the H2dl808 and H5dl366 mutants. Minor alterations in the time of onset of viral DNA replication and in the accumulation of certain viral polypeptides were observed, however. MATERIALS AND METHODS Cells. Monolayer cultures of W162, HeLa, and 293 cells were maintained in Dulbecco modified medium containing 10% newborn calf serum. Suspension cultures of HeLa cells were grown by the method of Halbert et al. (12). Construction of mutant viruses. A plasmid containing the EcoRI-B fragment of AdS was reconstructed by replacing the TaqI fragment of region E4 (Fig. 1 shows location of cleavage sites) with the corresponding TaqI fragment of a cDNA representing the E4 mRNA I of Ad2 (clone J6, reference 33). The structure of the reconstructed EcoRI-B clone, named pilE4I (il for intronless), was verified by DNA sequence analysis (21). pilE4I plasmid DNA, 3 ,ug, together with two plasmid DNAs which covered the rest of the Ad5 genome (pEcoRI-A [5 ,ug] and pHindIII-B [3 jig]), was transfected (11) into 293 cells. The cells were overlaid with medium, and infectious virus, generated by in vivo recombination (4), was detected by cytopathic effect and isolated.
* Corresponding author. t Present address: Department of Microbial Genetics, Medical Nobel Institute, Karolinska Institutet, S-104 01 Stockholm, Sweden.
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VOL. 62, 1988
E4 MUTANT OF ADENOVIRUS TYPE 5
E1AE1B
man et al. (5). A 10-,ug portion of total cytoplasmic RNA was subjected to Si nuclease analysis by the protocol of Berk and Sharp (3). A TaqI-HindIll DNA fragment (92.0 to 97.1 map units) 5'-end labeled at the TaqI site was used as probe, and Si-resistant material was separated on a 6% polyacrylamide gel containing 8.3 M urea (26).
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Protein preparation and analysis. Infected HeLa monolayer cultures were labeled for 30 min in medium lacking methionine supplemented with 2% newborn calf serum, 2% HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and [35S]methionine (20 ,Ci/ml; 1,000 Ci/mmol). Protein extracts were prepared by lysis of the cells in 10 mM Tris hydrochloride (pH 7.9)-0.15 M NaCl-1.5 mM MgCl20.65% Nonidet P-40. The samples were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the method of Laemmli (17). Immunoprecipitation of the E2A-encoded 72-kDa DNA-binding protein (DBP) was performed by the method of Persson et al. (22), using a polyclonal antibody directed against the DBP (18).
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FIG. 1. Physical map of the adenovirus genome and region E4. (Top) Location of the early transcription units. (Bottom) Expansion of the terminal DNA fragment generated by EcoRI digestion of the Ad5 genome. The location of the E4 transcription unit is indicated. ORFs of region E4 are shown by open boxes, numbered 1 through 7. Sequences absent in mutant viruses d1808 and iIE4I are indicated by solid bars. The structures of mRNA species produced by the ilE4I mutant are indicated at the bottom. Numbers to the right correspond to sizes of fragments protected in S1 nuclease mapping experiments from the TaqI site close to the 3' end of the mRNAs. The positions of restriction endonuclease cleavage sites used in the construction and examination of mutant iIE4I are shown. nt, Nucleotides.
Plaque-purified stocks of a mutant, designated H5ilE4I (iIE4I), were established. The structure of the mutant DNA was characterized by restriction endonuclease digestion. Purification and titration of viruses. Ad5 and ilE4I viruses were grown in HeLa cell Spinner cultures, and d1808 was grown in W162 cells. Viruses were purified by ultracentrifugation in isopycnic CsCl gradients and stored at -70°C. Viral titers expressed as fluorescent focus units were determined on monolayer cultures of HeLa and W162 cells, using the fluorescent focus assay described by Philipson (25). Viral infections. Monolayer cultures of HeLa cells were infected with wild-type or mutant viruses at a multiplicity of infection of 20 fluorescent focus units per cell based on titers determined on W162 cells. DNA replication. Infected HeLa monolayer cells were labeled with [3H]thymidine (100 ,uCi/ml; 15 Ci/mmol) 1 h before the cells were harvested. Viral DNA was extracted by the protocol of Hirt (15) at various times after infection and analyzed by digestion with restriction endonuclease EcoRI and subsequent electrophoresis on agarose gels (19). RNA preparation and analysis. Cytoplasmic RNA from infected HeLa cells was isolated by the method of Brawer-
RESULTS Construction and propagation of ilE4I virus. A mutant virus was generated by transfection of three DNA fragments which together covered the entire Ad5 genome except for region E4. This region of the genome was replaced by a cDNA representing E4 mRNA I of Ad2 (33). The fragments were transfected into W162 and 293 cells. The 293 cells were used since they are very easy to transfect (1) and since we wanted to test the possibility that ORF6 and ORF6/7 might encode all of the genetic information of region E4 that is required for growth of adenovirus in cultured cells. This assumption was based on the finding of Halbert et al. (12) that mutants with lesions in all ORFs except ORF6 are nondefective for growth. A mutant virus designated H5ilE4I (ilE4I) was recovered from 293 cells. It was plaque purified and propagated in Spinner cultures of HeLa cells. The structure of the mutant genome was characterized by digestion with restriction endonucleases EcoRI (Fig. 2A) and XbaI (data not shown). The results showed that the EcoRI-B and XbaI-C fragments of the mutant displayed an altered mobility compared with the corresponding fragments of AdS and their estimated sizes agreed with those expected. To verify further the structure of the mutant DNA, DNAs purified from Ad2, AdS, ilE4I, and plasmid pilE4I were digested with endonuclease TaqI. The resulting fragments were fractionated by gel electrophoresis. Ethidium bromide staining of the gel showed that the fragment patterns of AdS and ilE4I were identical with the exception of one DNA fragment (data not shown). The fragments were transferred to nitrocellulose as described by Southern (30), and fragments containing the E4 region were revealed by hybridization with a nick-translated E4-specific TaqI fragment (92.0 to 97.1 map units). The resulting autoradiogram showed that the Ad2 and AdS genomes contained TaqI fragments of the same size, while the iIE4I virus and the pilE4I plasmid contained TaqI fragments of identical but considerably smaller size (Fig. 2B). Thus, these results demonstrated that the ilE4I genome had the expected structure in region E4 while the rest of the genome was indistinguishable from Ad5. Mutant ilE4I replicates normally in HeLa cells. The growth properties of iIE4I, d1808, and AdS were studied by infection of monolayer HeLa and W162 cells at a multiplicity of 20 fluorescent focus units per cell. At 4, 24, 48, and 72 h postinfection (p.i.), cells and medium were collected. Virus, recovered by freeze-thawing, was titrated on W162 cells.
3260
HEMSTROM ET AL.
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FIG. 2. Characterization of the mutant genome. (A) DNA was prepared from Ad5-, ilE4I-, d1808-, and Ad2-infected HeLa cells. Purified DNA was digested with EcoRI and subjected to electrophoresis. DNA fragments were visualized by ethidium bromide staining. The arrow marked B indicates the EcoRI B fragment of AdS. The unmarked arrow indicates the corresponding fragment of ilE4I. (B) DNA prepared from Ad2, AdS, iIE4I, and plasmid pilE4I, digested with TaqI. The resulting DNA fragments were fractionated by agarose gel electrophoresis and transferred to nitrocellulose as described by Southern (30). Bands containing E4 sequences were visualized by hybridizing a 32P-labeled TaqI fragment (map units 92.0 to 97.1). The resulting autoradiogram is shown.
The titers were plotted against length of incubation, and the results are depicted in Fig. 3. From these data it is possible to conclude that the rate of accumulation of newly formed viruses was approximately the same in W162 cells infected with the three viruses and that the final virus yields were similar. In HeLa monolayer cells, mutant d1808 showed the severe growth defect that has been reported before (35); i.e., the final yield was decreased approximately 104-fold. Mutant ilE4I, in contrast, accumulated at the same rate as AdS in HeLa cells and reached the same final yields. Mutant ilE4I is nondefective for viral DNA replication and late protein synthesis. Mutant viruses with large deletions in region E4, like d1808, have a complex phenotype as they are defective in both DNA replication and late protein synthesis. To determine whether mutant ilE4I displayed any of these phenotypic properties, HeLa monolayer cells were infected with ilE4I, d1808, and AdS. The infected cells were labeled at different times after infection with either [3H]thymidine or [35S]methionine. DNA and protein extracts were prepared and analyzed as described in Materials and Methods. The results (Fig. 4 and 5) showed that mutant ilE4I was nondefective with regard to both DNA replication and protein synthesis. A reproducible observation was that mutant ilE4I started to replicate its DNA earlier than the wild-type virus. Small amounts of newly synthesized DNA were observed already after 8 h p.i. (Fig. 4, lane 2). Massive DNA synthesis occurred around 12 h p.i. in cells infected with ilE4I. At this time, AdS had started to replicate its DNA. At 24 h p.i., DNA synthesis was greatly increased for both viruses, while at 48 h p.i. it was decreased, although not terminated. At this time the amount of viral DNA in the cells, as visualized by ethidium bromide staining, was maximal (data not shown). Compared with the ilE4I and wild-type viruses, the DNA replication of d1808 was delayed (Fig. 4), although the amount of newly synthesized DNA had reached wild-type levels at 48 h p.i. It thus appears that the d1808 mutation causes a delayed onset of DNA replication. This delay has also been observed by Yoder and Berget (37).
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VOL. 62, 1988
E4 MUTANT OF ADENOVIRUS TYPE 5
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2
Late proteins appeared with similar kinetics in ilE4I- and AdS-infected cells. At 24 h p.i., late viral proteins were produced at a high rate, whereas the synthesis of host cell proteins was drastically reduced. However, a few minor differences were observed. The E2A-encoded DBP was overproduced in ilE4I-infected cells at 10 and 13 h p.i. (Fig. 5A, cf. lanes 7 with 8 and 10 with 11). The identity of this protein was confirmed by immunoprecipitation, using a polyclonal antibody directed against DBP (data not shown). We also observed that hexon protein appeared earlier in these cells than in AdS-infected cells (Fig. 5A, cf. lanes 10 and 11). Moreover, the hexon protein was slightly overproduced in the ilE4I-infected cells. This overproduction was still observed 48 h p.i. (Fig. SB). Mutant d1808 showed the expected phenotypes with regard to protein synthesis. The DBP was overproduced early after infection (Fig. 5A), the synthesis of late proteins was drastically reduced, and shutoff of host cell protein synthesis was incomplete (Fig. SB), confirming the results reported by Weinberg and Ketner (35). The defects in late protein synthesis and host cell shutoff were seen even 48 h p.i., when the defect in DNA replication was partly overcome. Three different E4 mRNAs are detectable in cells infected with mutant ilE4I. Although the E4 region in the ilE4I mutant was replaced by a cDNA, we suspected that smaller mRNAs might be formed by splicing. Therefore, the structure of cytoplasmic E4 mRNAs, produced in cells infected with iIE4I, was investigated by S1 nuclease analysis. A TaqIHindIIl DNA fragment, 5'-end labeled at the TaqI site located near the polyadenylation site of the E4 mRNAs, was used as probe (Fig. 1). S1-resistant DNA fragments were fractionated in a denaturating polyacrylamide gel, and the
3261
resulting autoradiogram (Fig. 6) revealed protected DNA fragments with estimated sizes of 135, 226, and 1,000 nucleotides. The 1,000-nucleotide-long fragment corresponded to a colinear mRNA designated "I" by Virtanen et al. (33), encoding the ORF6 protein. The 135-nucleotide-long frag-
ment revealed a splice junction at nucleotide 33192, based on the Ad2 sequence of Gingeras et al. (10). This 3' splice site has been identified before by us and others (9, 32, 33). It is likely to be connected to a 5' splice site located at position 33904. The resulting mRNA will encode an ORF6/7 fusion protein. The 226-nucleotide fragment identified a 3' splice site, located at nucleotide 33283. This 3' splice site has also been observed in earlier studies (9, 32). Thus, it appears that mutant ilE4I expressed three spliced mRNAs (Fig. 1).
DISCUSSION Region E4 of human adenoviruses is extremely complex. The nucleotide sequence reveals six ORFs, all capable of encoding proteins which consists of at least 100 amino acids. Studies of the E4 mRNAs by electron microscopy, Northern (RNA) blot analysis, S1 nuclease analysis, and cDNA cloning have likewise revealed a very complex pattern of differentially spliced mRNAs (6, 9, 16, 32, 33). Still, our current knowledge of the function of E4 gene products is very limited. Several investigators have introduced deletions and insertions into different parts of the E4 region, and the results have shown that most of the E4 genes are dispensible when the virus is propagated in tissue-cultured cells. However, mutants such as d1808 and d1366, in which almost the entire E4 region has been deleted, display a complex phenotype including defects in viral DNA replication, late protein synthesis, and shutoff of host cell protein synthesis. Mutant d1355, affecting only ORF6, shows a similar but less severe phenotype. From these studies it was concluded that the 3' portion of the E4 region encodes one or more gene products essential for viral growth in tissue-cultured cells. To study the properties of region E4 in more detail, we have constructed mutant adenoviruses in which region E4 was replaced by cloned cDNAs. In one mutant, designated ilE4I, region E4 was replaced by a cDNA which included the ORF6 and ORF7. Thus mutant was able to replicate in both 293 and HeLa cells. Its phenotype was studied to determine whether the mutant displayed any of the phenotypic properties associated with mutants d1808, d1355, and d1366. The results showed, surprisingly, that the DNA of the mutant replicated even faster than the DNA of wild-type Ad5 (Fig. 4), that the late proteins were efficiently produced, and that host cell protein synthesis was shut off (Fig. 5). Thus, the E4 protein(s), expressed from the mutant, must alleviate these defects displayed by mutants d1808, d1366, and d1355. It could be argued that second site mutations located outside region E4 account for the growth properties of mutant iIE4I. It should be noted that plaques appeared on 293 cells with comparable frequencies when wild-type fragments and fragments from the pilE4I plasmid were used for reconstruction of infectious viruses. Moreover, it has been possible to transiently rescue the d1808 virus with plasmid pilE4I in several independent experiments which also speaks against the generation of second-site mutations (G. Ketner, E. Bridge, U. Pettersson, and A. Virtanen, manuscript in preparation). An interesting finding was that mutant idE4I showed a slightly different phenotype from that of AdS. Newly synthesized DNA occurred already 8 h p.i., when no viral DNA replication was observed in AdS-infected cells (Fig. 4). A
3262
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Protein synthesis in HeLa cells infected with Ad5, ilE4I, and d1808. Proteins were labeled for 30 min with [35S]methionine before harvesting of the cells. Protein extracts were prepared and subjected to electrophoresis in a 10% polyacrylamide gel containing sodium dodecyl sulfate. Labeled proteins were visualized by fluorography. (A) Infected cells were harvested at 4, 7, 10, and 13 h p.i. An extract prepared from cells infected with Ad5 and harvested 24 h p.i. is shown to indicate the position of late viral proteins. Polypeptides II (hexon) and IV (fiber) and DBP are indicated. (B) Infected cells harvested 4, 12, 24, 48, and 72 h p.i. The locations of polypeptides II (hexon) and IV (fiber) and DBP are indicated by an open triangle, a filled triangle, and a diamond, respectively. DBP is positioned to the left of the diamond. An extract prepared from mock-infected cells is also shown.
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slight overproduction of hexon protein and of the DBP, encoded by region E2A, was observed, while production of the fiber protein was decreased at 24 h p.i. (Fig. 5). Underproduction of the fiber protein was also observed with mutants d1355 and d1366 (12) and d1808 (35). It is possible, however, that this defect is not a direct consequence of the genes in region E4 but rather is caused by the deletion of sequences required for proper transcriptional termination of
the fiber pre-mRNA (35). Mutants d1808 and d1366, like ilE4I, showed an overproduction of the DBP. Thus, we propose that one or more gene products of region E4 modulate the accumulation of selected viral proteins. It is possible that the early onset of viral DNA replication is an indirect effect, caused by an enhanced expression of the DBP. The cDNA introduced into the ilE4I mutant was expected
E4 MUTANT OF ADENOVIRUS TYPE 5
VOL. 62, 1988
-
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3263
was replaced by a cDNA corresponding to E4 mRNA L (33). This mutant can only express the ORF6/7 fusion polypeptide. Preliminary data indicate that this mutant produces low amounts of late proteins, thus supporting the idea that the ORF6 product is of key importance for viral growth (C. Hemstrom, E. Bridge, G. Ketner, U. Pettersson, and A. Virtanen, manuscript in preparation). Mutants with defects in region E1B or E4 have very similar phenotypes regarding the levels of late viral proteins and incomplete shutoff of host protein synthesis (2, 36). The gene product of ORF6 is associated with the 58-kDa polypeptide of region E1B in infected cells (27). It has been suggested that this complex, located in the nucleus, is of importance for the selective transport of viral mRNAs late during infection (36). This defect is apparently overcome in the ilE4I virus.
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ACKNOWLEDGMENTS We thank G. Ketner for valuable discussions and for providing the d1808 virus and W162 cells. We are also grateful to K. L. Berkner and P. A. Sharp for giving us plasmids pEcoRI-A and pEcoRI-B and Tommy Linne for antisera against the DBP. We thank Inga Hallin for skillful technical assistance. This investigation was supported by grants from the Swedish Medical Research Council and the Swedish National Board for Technical Development and by Marcus Borgstrom Foundation grants from Uppsala University. LITERATURE CITED 1. Alwine, J. C. 1985. Transient gene expression control: effects of transfected DNA stability and trans-activation by early viral proteins. Mol. Cell. Biol. 5:1034-1042. 2. Babiss, L. E., and H. S. Ginsberg. 1984. Adenovirus type 5 early region lb gene product is required for efficient shutoff of host protein synthesis. J. Virol. 50:202-212. 3. Berk, A. J., and P. A. Sharp. 1978. Structure of the adenovirus 2 early mRNAs. Cell 14:695-711. 4. Berkner, K. L., and P. A. Sharp. 1983. Generation of adenovirus by transfection of plasmids. Nucleic Acids Res. 11:6003-6020. 5. Brawerman, G., J. Mendecki, and S. Y. Lee. 1972. A procedure for isolation of mammalian messenger ribonucleic acid. Bio-
chemistry 11:637-641. 1 35 -----
FIG. 6. S1 nuclease analysis of E4 mRNAs produced by ilE4I. HeLa cells were infected with ilE4I at a multiplicity of infection of 20 fluorescent focus units per cell. Total cytoplasmic RNA was prepared (5) from infected (ilE4I) and mock-infected (mock) cells and subjected to S1 nuclease analysis (3). A TaqI-HindlIl fragment (92.0 to 97.1 map units) 5'-end labeled at the TaqI site was used as probe. DNA size markers were fractionated in Lane M. Sizes are given in nucleotides.
to give rise to an mRNA which encodes the ORF6 polypeptide. An mRNA with this structure was indeed identified by S1 nuclease analysis (Fig. 6). In addition to this mRNA, two smaller mRNAs were observed (Fig. 6). We predict that these mRNAs correspond to previously identified E4 mRNAs (9, 32, 33) encoding an ORF6/7 fusion polypeptide (7, 13, 20, 31) and a polypeptide related to the ORF6 protein. It is not possible to conclude from the present study that all of these polypeptides are required for viral growth. However, we have recently isolated a mutant in which region E4
6. Chow, L. T., J. M. Roberts, J. B. Lewis, and T. R. Broker. 1977. A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids. Cell 11:819-836. 7. Cutt, J. R., T. Shenk, and P. Hearing. 1987. Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells. J. Virol. 61:543-552. 8. Downey, J. F., D. T. Rowe, S. Bacchetti, F. L. Graham, and S. T. Bayley. 1983. Mapping of a 14,000-dalton antigen to early region 4 of the human adenovirus 5 genome. J. Virol. 45:514523. 9. Freyer, G. A., Y. Katoh, and R. J. Roberts. 1984. Characterization of the major mRNAs from adenovirus 2 early region 4 by cDNA cloning and sequencing. Nucleic Acids Res. 12:35033519. 10. Gingeras, T. R., D. Sciaky, R. E. Gelinas, J. Bing-Dong, C. E. Yen, M. M. Kelly, P. A. Bullock, B. L. Parsons, K. E. O'Neill, and R. J. Roberts. 1982. Nucleotide sequences from the adenovirus-2 genome. J. Biol. Chem. 257:13475-13491. 11. Graham, F. L., and A. J. van der Eb. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456-467. 12. Halbert, D. N., J. R. Cutt, and T. Shenk. 1985. Adenovirus early region 4 encodes functions required for efficient DNA replication, late gene expression, and host cell shutoff. J. Virol. 56:
250-257.
13. Harter, M. L., and J. B. Lewis. 1978. Adenovirus type 2 early proteins synthesized in vitro and in vivo: identification in
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15.
16. 17.
18.
19.
20.
21.
22. 23.
24.
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