Gene product of region E4 of adenovirus type 5 modulates accumulation of certain viral polypeptides.

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 H2dl808 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 H2dl808 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.     

Adenovirus early region 4 is required for efficient virus particle assembly.

H2dl807, a defective deletion mutant of human adenovirus type 2 lacking parts of early regions 3 and 4 and all of late region 5, was severely defective for virus particle assembly on HeLa cells, producing about 1% of the normal yield of particles. On Vero cells, H2dl807 produced only 5% as many particles as wild type, while on W162 cells, a Vero cell derivative which supports the growth of early region 4 mutants, H2dl807 produced nearly 40% of the wild-type level of particles. Two other defective deletion mutants, H2dl802 and H5dl1021, which lack parts of early region 3 and which are incapable of making fiber, the product of late region 5, were wild type for virus assembly. These data suggest that the cause of the assembly defect of H2dl807 is the lack of a diffusible early region 4 product. H2dl807-infected Vero cells accumulated nearly wild-type amounts of viral late proteins in the nucleus and cytoplasm. Thus, the defect of the mutant in assembly on Vero cells is not due to a general lack of late proteins. Finally, the fact that H2dl802 and H5dl1021 make wild-type amounts of virus particles suggests that fiber is not essential for adenovirus assembly.     

Deletion of the gene encoding the adenovirus 5 early region 1b 21,000-molecular-weight polypeptide leads to degradation of viral and host cell DNA.

The adenovirus 5 mutant H5dl337 lacks 146 base pairs within early region 1B. The deletion removes a portion of the region encoding the E1B 21,000-molecular-weight (21K) polypeptide, but does not disturb the E1B-55K/17K coding region. The virus is slightly defective for growth in cultured HeLa cells, in which its final yield is reduced ca. 10-fold compared with wild-type virus. The mutant displays a striking phenotype in HeLa cells. The onset of cytopathic effect is dramatically accelerated, and both host cell and viral DNAs are extensively degraded late after infection. This defect has been described previously for a variety of adenovirus mutants and has been termed a cytocidal (cyt) phenotype. H5dl337 serves to map this defect to the loss of E1B-21K polypeptide function. In addition to its defect in the productive growth cycle, H5dl337 is unable to transform rat cells at normal efficiency.     

An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.     

p53-Independent and -Dependent Requirements for E1B-55K in Adenovirus Type 5 Replication

The adenovirus type 5 mutant dl1520 was engineered previously to be completely defective for E1B-55K functions. Recently, this mutant (also known as ONYX-015) has been suggested to replicate preferentially in p53(-) and some p53(+) tumor cell lines but to be attenuated in primary cultured cells (C. Heise, A. Sampson-Johannes, A. Williams, F. McCormick, D. D. F. Hoff, and D. H. Kirn, Nat. Med. 3:639-645, 1997). It has been suggested that dl1520 might be used as a "magic bullet" that could selectively lyse tumor cells without harm to normal tissues. However, we report here that dl1520 replication is independent of p53 genotype and occurs efficiently in some primary cultured human cells, indicating that the mutant virus does not possess a tumor selectivity. Although it was not the sole host range determinant, p53 function did reduce dl1520 replication when analyzed in a cell line expressing temperature-sensitive p53 (H1299-tsp53) (K. L. Fries, W. E. Miller, and N. Raab-Traub, J. Virol. 70:8653-8659, 1996). As found earlier for other E1B-55K mutants in HeLa cells (Y. Ho, R. Galos, and J. Williams, Virology 122:109-124, 1982), dl1520 replication was temperature dependent in H1299 cells. When p53 function was restored at low temperature in H1299-tsp53 cells, it imposed a modest defect in viral DNA replication and accumulation of late viral cytoplasmic mRNA. However, in both H1299 and H1299-tsp53 cells, the defect in late viral protein synthesis appeared to be much greater than could be accounted for by the modest defects in late viral mRNA levels. We therefore propose that in addition to countering p53 function and modulating viral and cellular mRNA nuclear transport, E1B-55K also stimulates late viral mRNA translation.     

Mapping of an adenovirus function involved in the inhibition of DNA degradation.

A function involved in the inhibition of DNA degradation has been assigned through complementation tests to a product of region E1b of the adenovirus genome (between 4.5 and 10.5 map units). DNA degradation induced by the adenovirus type 12 (Ad12) cyt mutant H12cyt70 and the Ad5 early deletion mutant dl313 (with the deletion between 3.5 and 10.7 map units) was inhibited by coinfection with Ad5 region E1a (between 0 and 4.5 map units) mutants dl312 and hr1 and region E1b mutant hr6. The defect of inhibition of DNA degradation in Ad5 dl313 was also complemented in 293 cells. This DNase-inhibitory function does not appear to involve polypeptide IX or the 58,000-dalton polypeptide. Wild-type Ad12 induced DNA degradation in hamster embryo cells, suggesting that the DNase-inhibitory function is not expressed in these nonpermissive cells. Additional evidence suggests the involvement of a second viral product which positively influences the DNase activity and which appears to be an early function.     

An adenovirus mutant unable to express VAI RNA displays different growth responses and sensitivity to interferon in various host cell lines.

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for the efficient translation of host cell and viral mRNAs late after infection. VAI RNA prevented activation of the interferon-induced P1/eIF-2 alpha kinase. In its absence the kinase was activated, eIF-2 alpha was phosphorylated, and translational initiation was inhibited. H5dl331 (dl331), a mutant which cannot express VAI RNA, grew poorly in 293 cells but generated wild-type yields in KB cells. The growth phenotype of the mutant appeared to correlate with the kinetics of kinase induction and activation. Active kinase appeared more rapidly in cell extracts prepared from infected 293 cells, in which dl331 grew poorly, than in extracts of KB cells, in which the mutant grew well. However, when kinase was induced in KB cells by interferon treatment and then activated subsequent to dl331 infection, viral protein synthesis was less severely inhibited than in interferon-treated 293 cells. Thus, activated kinase per se is insufficient to severely inhibit dl331 protein synthesis in KB cells.     

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.     

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.     

Early RNA of adenovirus type 3 in permissive and abortive infections.

Early adenovirus type 3 cytoplasmic polyadenylated RNAs from HeLa and BHK-21 cells were detected and mapped on the viral genome by gel blotting and hybridization techniques. The sizes and locations of the 16 adenovirus type 3 RNAs were identical in the two cell types, although relative molarities of the various RNA species differed. Each of the early adenovirus type 3 RNAs was associated with polysomes in both cell types, suggesting that the abortive infection of hamster cells does not result from a defect in early adenovirus type 3 mRNA biosynthesis. No RNAs from regions transcribed late in infection of permissive cells were detected in BHK-21 cells.