Plasmids expressing partial adenovirus early region 1A (ElA) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. ElA cDNAs expressing the 289-and 243-amino acid proteins expressed both ElA transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289-and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent. These studies suggest that ElA functions which collaborate with ras oncogenes and those which facilitate establishment are linked. Furthermore, ElA transforming functions are not associated with activities of the 289-amino acid ElA proteins required for efficient transcriptional activation of viral early region promoters.

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To distinguish the individual roles of the 13S, 12S, and 9S adenovirus ElA gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected ElA mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA ElA region but not in the presence of a 12S ElA cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type ElA virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the ElA 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.

The adenovirus mutant Ad2tslll has been previously shown to contain a mutation in the early region 2A gene encoding the single-stranded-DNA-binding protein that results in thermolabile replication of virus DNA and a mutation in early region 1 that causes degradation of intracellular DNA. A recombinant virus, Ad2cytlO6, has been constructed which contains the Adltslll early region 1 mutation and the wild-type early region 2A gene from adenovirus 5. This virus, like its parent Ad2tslll, has two temperature-independent phenotypes; first, it has the ability to cause an enhanced and unusual cytopathic effect on the host cell (cytocidal [cyt] phenotype) and second, it induces degradation of cell DNA (DNA degradation [deg] phenotype). The mutation responsible for these phenotypes is a single point mutation in the gene encoding the adenovirus early region 1B (ElB) 19,000-molecular-weight (19K) tumor antigen. This mutation causes a change from a serine to an asparagine in the 20th amino acid from the amino terminus of the protein. Three other mutants that affect the E1B 19K protein function have been examined. The mutants Ad21p5 and Ad5dl337 have both the cytocidal and DNA degradation phenotypes (cyt deg), whereas Ad21p3 has only the cytocidal phenotype and does not induce degradation of cell DNA (cyt deg'). Thus, the DNA degradation is not caused by the altered cell morphology. Furthermore, the mutant Ad5dl337 does not make any detectable E1B 19K protein product, suggesting that the absence of E1B 19K protein function is responsible for the mutant phenotypes. A fully functional E1B 19K protein is not absolutely required for lytic growth of adenovirus 2 in HeLa cells, and its involvement in transformation of nonpermissive cells to morphological variants is discussed.

We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and + 190 by showing that insertion of SV40 sequences within the intron after the first leader segment at + 190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.In a permissive host, the human adenoviruses, which have a double-stranded DNA genome of 36,000 base pairs, undergo a regulated program of gene expression that is divided into early and late phases, demarcated by the initiation of viral DNA replication. During the late phase of infection, viral transcription initiates primarily from the major late promoter, giving rise to primary transcripts that are processed by differential splicing and polyadenylation into more than 30 mRNA species. Each of the late mRNAs carries at its 5' end a common 203-nucleotide untranslated sequence that is spliced to a protein-coding sequence. This 5' region is called the tripartite leader because it is coded by three noncontiguous viral DNA segments that are located between the major start site for late transcription and the late proteincoding regions (for a review, see reference 59). Concomitant with the induction of late transcription, host cell protein synthesis is repressed, and late viral mRNAs are preferentially translated (16,46). The function of the tripartite leader is not fully understood, but there are data which suggest that the leader is a signal that identifies late viral mRNAs for selective translation (4,30,57 indicated that a nearly complete tripartite leader is necessary for optimal levels of hybrid mRNA translation during the late phase of infection. However, it was not possible to use these recombinant viruses to assess systematically the contribution of cis control sequences to late promoter activity because the Ela enhancer is located upstream of the hybrid transcription unit and is likely to affect its activity. Moreover, the hybrid transcription units are missing the intron sequences between the leader segments, and thus pote...