Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs.

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.     

Heavy metals enhance the reactivation of nitrous acid-treated adenovirus in HeLa cells

Treatment of HeLa cells with cadmium chloride and zinc chloride increases the survival rate of nitrous acid-treated adenovirus 2 (ade2). This increase is maximal if the time interval between cell treatment and virus infection is delayed by 36 h. The induction process requires protein synthesis only during the 3-h period immediately following treatment; cycloheximide does not prevent the expression of enhanced reactivation if added to the cells after this time.     

An adenovirus early region 1A protein is required for maximal viral DNA replication in growth-arrested human cells.

Two closely related adenovirus early region 1A proteins are expressed in transformed cells. The smaller of these, which is 243 amino acids in length, is required for the transformation of primary rat cells and for the transformation of immortalized rat cells to anchorage-independent growth. This protein is not required for productive infection of exponentially growing HeLa cells but is required for maximal replication in growth (G0)-arrested human lung fibroblasts (WI-38 cells). To determine the function of this protein in viral replication in these G0-arrested cells, we compared viral early mRNA, early protein, and late protein synthesis after infection with wild type or a mutant which does not express the protein. No differences were found. However, viral DNA synthesis by the mutant was delayed and decreased to 20 to 30% that of wild type in these cells. Viral DNA synthesis was much less defective in growing WI-38 cells, and in the transformed human HeLa cell line it occurred at wild-type levels. Furthermore, the mutant which can express only the 243-amino-acid early region 1A protein induced cellular DNA synthesis in G0-arrested rat cells to the same level as wild-type virus. A mutant which can express only the 289-amino-acid early region 1A protein induced less cellular DNA synthesis in G0-arrested rat cells. We propose that the early region 1A 243-amino-acid protein alters the physiology of arrested permissive cells to allow maximal viral DNA replication. In nonpermissive rodent cells, the 243-amino-acid protein drives G0-arrested cells into S phase. This activity is probably important for the immortalization of primary cells.