The adenovirus type 12 early-region 1B 58,000-Mr gene product is required for viral DNA synthesis and for initiation of cell transformation.

An E1B 58K mutant of adenovirus type 12 (Ad12), dl207, was constructed by the deletion of 852 base pairs in the E1B 58K coding region. The mutant could grow efficiently in 293E1 cells but not in HeLa, KB, or human embryo kidney (HEK) cells. Viral DNA replication of dl207 was not detected in HeLa and KB cells and was seldom detected in HEK cells. Analysis of viral DNA synthesis in vitro showed that the Ad12-DNA-protein complex replicated by using the nuclear extract from Ad12 wild-type (WT)-infected HeLa cells but not by using the nuclear extract from dl207-infected cells. In dl207-infected HeLa and KB cells, early mRNAs were detected, but late mRNAs were not detected. The mutant induced fewer transformed foci than the WT in rat 3Y1 cells. Cells transformed by dl207 could grow efficiently in fluid medium, form colonies in soft agar culture, and induce tumors in rats transplanted with the transformed cells at the same efficiency as WT-transformed cells. Tumors were induced in hamsters injected with WT virions but were not induced in hamsters injected with dl207 virions. The results indicate that the E1B 58K protein is required both for viral DNA replication in productive infection and for initiation of cell transformation, but not for maintenance of the transformed phenotype.     

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.     

Adenovirus E1B 55-kilodalton protein is required for both regulation of mRNA export and efficient entry into the late phase of infection in normal human fibroblasts

The human adenovirus type 5 (Ad5) E1B 55-kDa protein is required for selective nuclear export of viral late mRNAs from the nucleus and concomitant inhibition of export of cellular mRNAs in HeLa cells and some other human cell lines, but its contributions(s) to replication in normal human cells is not well understood. We have therefore examined the phenotypes exhibited by viruses carrying mutations in the E1B 55-kDa protein coding sequence in normal human fibroblast (HFFs). Ad5 replicated significantly more slowly in HFFs than it does in tumor cells, a difference that is the result of delayed entry into the late phase of infection. The A143 mutation, which specifically impaired export of viral late mRNAs from the nucleus in infected HeLa cells (R. A. Gonzalez and S. J. Flint, J. Virol. 76:4507-4519, 2002), induced a more severe defect in viral mRNA export in HFFs. This observation indicates that the E1B 55-kDa protein regulates mRNA export during the late phase of infection of normal human cells. Other mutants exhibited phenotypes not observed in HeLa cells. In HFFs infected by the null mutant Hr6, synthesis of viral late mRNAs and proteins was severely impaired. Such defects in late gene expression were the result of inefficient progression into the late phase of infection, for viral DNA synthesis was 10-fold less efficient in Hr6-infected HFFs than in cells infected by Ad5. Similar, but less severe, defects in viral DNA synthesis were induced by the insertion mutation H224, which has been reported to inhibit binding of the E1B 55-kDa protein to p53 (C. C. Kao, P. R. Yew, and A. J. Berk, Virology 179:806-814, 1990).     

A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture.

The fastidious enteric adenovirus (FEAd) types 40 (Ad40) and 41 (Ad41) are found in stool specimens of infants and young children in association with gastroenteritis. Although they can be isolated routinely from clinical specimens by using 293 cells, they are propagated with variable success in cell lines which support the replication of other adenovirus serotypes. HeLa cells are generally considered to be nonpermissive for the replication of FEAds, but in this study, Ad40 and Ad41 grew to comparable titers in individual 293 and HeLa cells. However, virus was not efficiently released from infected HeLa cells and thus did not undergo multiple cycles of infection in HeLa cell cultures. The block in virus release was not overcome in KB18 cells which, like 293 cells, constitutively express proteins encoded by the E1B region of a subgroup C adenovirus (in this case Ad2). Moreover, it was apparent from these studies that Ad40 and Ad41 have particle-to-infectious unit ratios several orders of magnitude greater than that for Ad5, even in 293 cells which express the E1A and E1B proteins of Ad5 and are considered to be permissive for replication of the FEAds. Neither the block in release of progeny virus nor the high particle-to-infectious unit ratio is explained solely by the defect in expression of the E1B 55K protein identified by Mautner et al. (V. Mautner, N. MacKay, and V. Steinthorsdottir, Virology 171:619-622, 1989; V. Mautner, N. MacKay, and K. Morris, Virology 179:129-138, 1990).     

Dependence of tumor-forming capacities of cells transformed by recombinants between adenovirus types 5 and 12 on expression of early region 1.

Recombinants between an adenovirus type 5 (Ad5) deletion mutant and the Ad12 DNA fragment containing early region 1 (E1) were isolated from cells cotransfected with the EcoRI-C fragment of Ad12 DNA and Ad5 dl312 (deletion in E1A) DNA (rcA) and from cells cotransfected with the SalI-C fragment of Ad12 DNA and Ad5 dl312 DNA (rcB). No recombinant was isolated from cells cotransfected with Ad5 dl313 (deletion in E1B) DNA and restriction fragments of Ad12 DNA. Both rcA and rcB are defective and able to replicate in human embryo kidney (HEK) and KB cells with complementation by dl312. Both rcA and rcB formed Ad12 T antigen g, but not T antigen f, in infected HEK and KB cells. In rcA- and rcB-infected cells, Ad5 E1B and Ad12 E1A genes are transcribed. Heteroduplex and size analyses of rcA-1 or rcB-1 DNA fragments hybridized with Ad12 DNA revealed that rcA-1 DNA has a deletion between 5 and 15 map units with an insertion of a portion of Ad12 DNA (10%) and that rcB-1 DNA has a deletion between 70 and 80 map units with an insertion of a portion of Ad12 DNA (10%). The transformed cell lines, RCAY and RCBY, were established after infection of rat 3Y1 cells with rcA and rcB, respectively. Both Ad5 and Ad12 DNA sequences are contained in these cells. In RCAY cells, Ad12 T antigen g is detected, but Ad12 T antigen f is not. In RCBY cells, both Ad12 T antigen g and f are detected. Only the Ad12 E1A gene is transcribed in RCAY cells, whereas Ad5 E1B, Ad12 E1A, and Ad12 E1B genes are transcribed in RCBY cells. In soft-agar cultures, RCBY cells form large colonies, whereas RCAY cells form only tiny colonies. RCBY cells form tumors as efficiently as 12WY cells in transplanted rats. RCAY cells formed tumors inefficiently. Ad5-transformed 5WY cells do not form tumors. These observations indicate that the efficient tumor formation by RCBY cells is dependent on the expression of the Ad12 E1A and E1B genes, whereas the inefficient tumor formation by RCAY cells is due to the expression of only the Ad12 E1A gene.     

Transforming genes among three different oncogenic subgroups of human adenoviruses have similar replicative functions.

We have examined the functional similarity of the transforming genes for replicative functions among three different subgroups of human adenoviruses (A, B, and C), using mutant complementation as an assay. A host range deletion mutant (dl201.2) of Ad2 (nononcogenic subgroup C) lacking about 5% of the viral DNA covering two early gene blocks (E1a and E1b) involved in cellular transformation was isolated and tested for its ability to replicate in nonpermissive KB cells in the presence of Ad7 (weakly oncogenic group B) or ad12 (highly oncogenic group A). The complementation of the mutant defect was demonstrated by cleaving the viral DNA extracted from mixed infected cells or the DNA extracted from purified virions from mixed infected cells with restriction endonuclease BamHI, which produces a different cleavage pattern with the DNA of each serotype. It was found that the defects in E1a plus E1b of dl201.2 could be complemented by Ad7 and Ad12, indicating that these genes in Ad2, Ad7, and Ad12 have similar functions during productive infection.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Expression of the chloramphenicol acetyl transferase gene in human cells under the control of early adenovirus subgroup C promoters: effect of E1A gene products from other subgroups on gene expression

A hierarchy of dominance has been observed in HeLa cells co-infected with two serotypes of adenovirus belonging to different subgroups. DNA replication and late protein synthesis of one serotype are inhibited by those of the other. The degree of inhibitory effect has the following decreasing order: adenovirus type 3 (Ad3) and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad12 (A), Ad2 and Ad5 (C) [Delsert and D'Halluin, Virus Res. 1 (1984) 365-380]. HeLa cells were first transfected with recombinant plasmids carrying Ad5 E2A or E3 promoters fused to the chloramphenicol acetyl transferase gene (cat), and then infected with human Ad belonging to different subgroups. All the serotypes tested were found to be able to stimulate both E2A and E3 promoters. When HeLa cells were co-transfected with either of the previous plasmids, plus a second plasmid carrying the Ad3 E1A region, the same stimulatory effect was observed. However, an inhibitory effect on Ad5 E2A and E3 promoters seemed to occur when both Ad2 E1A (subgroup C) and Ad3 E1A (subgroup B) genes were present together. To determine which one of the early products was responsible for the observed repression effect, and to assign the target on the genome of subgroup C Ad, a plasmid was constructed in which the sequences at the 5' end of the Ad2 E1A region were fused to the structural sequences of the cat gene. In HeLa cells transfected with this plasmid, CAT activity was significantly increased after co-transfection with a plasmid carrying the Ad2 E1A region, but decreased with a plasmid carrying the Ad3 E1A region.(ABSTRACT TRUNCATED AT 250 WORDS)     

Control of Adenovirus Gene Expression: Cellular Gene Products Restrict Expression of Adenovirus Host Range Mutants in Nonpermissive Cells

Adenovirus type 5 (Ad5) host range mutants dl312 and hr-1, with lesions in region E1A (0 to 4.5 map units) of the viral genome, fail to accumulate virus-specific early RNA during infection in HeLa cells. In a recent report, we showed that the addition of anisomycin, a stringent inhibitor of protein synthesis, at 1 h after infection of HeLa cells with hr-1 virus resulted in the accumulation of properly spliced and translatable mRNA from all early regions (M. G. Katze, H. Persson, and L. Philipson, Mol. Cell. Biol. 1:807-813, 1981). Based on these results we proposed a model in which expression of early mutant RNA was achieved through inactivation of a cellular protein normally causing a reduction in the amount of viral RNA. These studies have been extended in the present report, which shows that early viral proteins can be detected in Ad5 dl312- and Ad5 hr-1-infected HeLa cells which have been treated for several hours with anisomycin either shortly after infection or before infection. A pulse of drug treatment also resulted in expression of substantial amounts of adenovirus structural proteins after infection with both Ad5 hr-1 and Ad5 dl312, whereas in drug-free controls no late proteins were detected. The Ad5 hr-1 virus previously reported to be DNA replication negative in nonpermissive HeLa cells was found to replicate its DNA, albeit at low levels, when anisomycin was present either from 1 to 5 h postinfection or for 5 h before infection. When infectious virus production was examined in mutant-infected cells the titer of Ad5 dl312 virus was found to increase at least 500-fold in anisomycin-treated HeLa cells. Taken together, these and our previous results suggest that the block in gene expression characteristic for complementation group I Ad5 host range mutants in HeLa cells can be overcome by inactivating cellular gene products serving as negative regulators of viral gene expression.     

Integration and transcription of group C human adenovirus sequences in the DNA of five lines of transformed rat cells

We have used the Southern blotting technique to analyze the integration patterns of human adenovirus sequences in the DNA of four rat cell lines, F17, 8617, T2C4 and F4, which were transformed by Ad-2 virus, and 5RK clone 6, which was transformed by Ad-5 HindIII-G fragment. We have also analyzed the Ad-specific messenger RNAs synthesized in these cell lines, in 293 cells (an Ad-5 transformed human cell line), and in Ad-2 early infected human KB cells, using the RNA geltransfer hybridization technique. We were interested in whether the Ad sequences are integrated, what the integration patterns are, whether the transforming region is present in an intact form, and whether the transforming region and other early regions are expressed at the mRNA level.Our results show that the integration patterns of Ad sequences range from simple to quite complex. Cells from line 8617 contain a single copy of right-end sequences flanked by left-end sequences. T2C4 cells have four different left-end sequences and two different right-end sequences. 5RK cells contain multiple different pieces of left-end sequences. In agreement with the results of Sambrook et al. (1979a,b), F17 cells contain a single copy of the left 17% of the genome, and F4 cells contain multiple copies of the right 5% of the genome fused to the left ~ 68% of the genome. The complete Ad genome is not present in any of the cell lines, and different regions may not be equimolar. There are no specific sites on the cellular or viral genome at which integration occurs. In 8617, F17 and F4 cells the Ad-2 sequences appear to be located close together on a single chromosome, suggesting that the Ad sequences in these cells arose from a single integration event. F17, 8617, T2C4, F4, and probably 5RK, cells all have an intact early region E1a (map position 1·3–4·6); F17, 8617, T2C4 and F4 cells also have E1b (m.p. 4·6–11·2) intact. E1a and E1b are the regions responsible for transformation. 8617 cells also have an intact early region E4 (m.p. 99-91·5) and T2C4 cells have an intact early region E3 (m.p. 76–86).Ad-2 early infected KB cells were shown to synthesize major E1a-specific mRNAs of 13 S, 12 S and 9 S, and major E1b-specific mRNAs of 22 S and 13 S. All the transformed cells synthesize the E1a 13 S and E1a 12 S mRNAs, and all cells except 5RK synthesize the E1b 22 S and E1b 13 S mRNAs. Early infected KB cells synthesize E3-specific mRNAs of 26 S, 24 S, 22 S, 19 S, 12 S and 9 S: T2C4 cells synthesize the major 22 S and 19 S RNA species, and possibly the less pronounced E3 mRNAs. Early infected cells and 8617 cells synthesize E4-specific mRNAs of 19 S, 17 S, 14 S, 12 S, 11 S, 9 S and 8 S. 8617 cells also synthesize E4 mRNAs of about 23 to 24 S and 21 S. F4 cells synthesize 24 S and 19 S hybrid mRNAs that contain both E4 and E1a sequences: these RNAs arise because F4 cells contain a portion of the E4 region fused to the left end (m.p. 0) of the genome.Our results, as well as those from other laboratories, are consistent with the idea that the transformed phenotype of Ad transformed cells is maintained by expression of Ad genes in E1a and E1b.     

Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells.

High-titer monospecific antiserum against highly purified adenovirus 2 (Ad2) single-stranded DNA binding protein (DBP) was used to study, by indirect immunofluorescence (IF), the synthesis of DBP in Ad2-infected human cells and adenovirus-transformed rat, hamster, and human cell lines. In infected cells the synthesis of DBP was first detected in the cytoplasm at 2 to 4 h postinfection and reached a maximum intensity at 6 h postinfection. At this time DBP began to accumulate in the nucleus, where it reached maximum intensity at about 14 h postinfection. The cytoplasmic IF was diffuse, whereas nuclear IF appeared as dots that coalesced into large globules as infection progressed. In cells treated with 1-beta-d-arabinofuranosylcytosine to inhibit viral DNA synthesis, strong nuclear IF was observed in the form of dots, but the large fluorescent globules were not observed. The Ad2 (oncogenic group C) anti-DBP serum reacted very strongly by IF with Ad5 (group C)-infected, to a lesser extent with Ad7 and Ad11 (group B)-infected, and weakly with Ad12 and Ad18 (group A)-infected KB cells (treated with 1-beta-d-arabinofuranosylcytosine). These results may indicate that Ad2 DBP is closely related immunologically to DBPs induced early after infection by adenovirus serotypes in oncogenic group C, moderately related to DBPs of serotypes in oncogenic group B, and perhaps distantly related to DBPs of serotypes in oncogenic group A. The following adenovirus-transformed cell lines were examined for DBP synthesis by IF with the Ad2 anti-DBP serum: six rat cell lines (T2C4, F17, 8662, 8638, 8617, and F161) transformed by Ad2 virus, three hamster cell lines transformed by Ad2 virus (Ad2HT1) and Ad2-simian virus 40 hybrid virus (ND1HK1 and ND4HK4), and one rat (5RK) and one human (293-31) cell line transformed by transfection with Ad5 DNA. T2C4 and 8662 appeared weakly positive, whereas Ad2HT1 and ND4HK1 were strongly positive. The other transformed cell lines did not produce DBP detectable by IF. Thus, some but not all transformed cell lines produce DBP, which indicates that DBP is not required for maintenance of cell transformation and that transformed cells can express "nontransforming" viral genes as protein.     

Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein.

A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5d1801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr404, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous study suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions 1A, 1B, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5- and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.     

Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection.     

Efficient expression of small RNA polymerase III genes from a novel simian virus 40 vector and their effect on viral gene expression.

In the past, simian virus 40 (SV40) has been used as a cloning vehicle to clone foreign genes by substituting portions of the viral genome vital for viral replication. Propagation of these defective viruses required a helper virus and the recombinant viruses obtained could be grown only as a mixture. In this study, we describe a novel nondefective SV40 vector to clone small RNA polymerase III genes. Two small RNA polymerase III genes, an amber suppressor human serine tRNA gene and the adenovirus (Ad) VAI RNA gene, were cloned in the intron region of the large-T antigen gene of SV40 after deleting DNA sequences coding for the small-t polypeptide. The recombinant viruses grew to wild type levels and showed no growth defects. When CV-1p cells were infected with these viruses, the cloned RNA polymerase III genes were expressed at high levels at late times. Interestingly, large amounts VAI RNA in CV-1p cells infected with SV40-VA recombinant virus, did not enhance translation of viral mRNAs significantly but did lead to a 3 to 4 fold increase in the steady state levels of large-T mRNA suggesting a novel function for VAI RNA in SV40 infected monkey cells. Furthermore, VAI mutants which fail to function in Ad infected human cells also failed to enhance the levels of large-T mRNAs in monkey cells infected with SV40. The simple SV40 vector described here may be useful to study the structure and function of small RNA polymerase III genes in the context of a eucaryotic chromosome. In addition, the nondefective recombinant SV40 which expresses the suppressor tRNA gene at high levels may provide a useful helper system to propagate animal viruses with amber mutations in essential genes.     

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses

We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K³ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA.Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.