Chromosomal damage induced by human adenovirus type 12 requires expression of the E1B 55-kilodalton viral protein.

Infection of human embryonic kidney cells with adenovirus type 12 results in the induction of damage at specific (17q21-22, 1p36, 1q21, and 1q42-43) and random sites in the cellular chromosomes. A previous study by Durnam et al. (D. M. Durnam, P. P. Smith, J. C. Menninger, and J. K. McDougall, Cancer Cells 4:349-354, 1986) indicated that the expression of viral early region 1 (E1) is sufficient for the induction of damage at band 17q21-22. In the present report we used an adenovirus type 12-adenovirus type 5 recombinant with E1A hybrid sequences as well as viruses with mutations in the adenovirus type 12 E1B genes to map adenovirus type 12 E1 functions involved in the induction of genetic damage. Our results show that the expression of the E1A proteins is not sufficient for this effect. On the other hand, mutations within the E1B 55-kilodalton protein but not the E1B 19-kilodalton protein affect the ability of the virus to induce both specific and random chromosomal damage.     

Complementation of human adenovirus type 5 ts mutants by human adenovirus type 12.

Temperature-sensitive mutants of type 5 adenovirus belonging to eight complementation groups were complemented in mixed infection by type 12 adenovirus, whereas mutants of 7 other groups were not enhanced. In some crosses, phenotypic mixing took place. No evidence of recombination between type 5 ts mutants and type 12 was found.     

Hyperproduction of adenovirus type 12 E1B gene product in monkey cells, using a simian virus 40 vector.

Simian virus 40 recombinant DNAs carrying the adenovirus type 12 E1B gene were constructed, propagated, and packaged in monkey cells. Monkey cells infected with the resulting virus stocks hyperproduced the E1B gene products in more than 80% of the cells as revealed by immunofluorescence. The products were distributed in both the nuclei and the cytoplasm, and a condensed form of fleck structure was observed in the cytoplasm. Polyacrylamide gel electrophoresis of the cell extracts and their immunoprecipitates detected the E1B-coded 19,000-molecular-weight protein but not the 50,000-molecular-weight protein. The 19,000-molecular-weight protein and the simian virus 40 VP1 protein were synthesized in nearly equal amounts.     

Cycloheximide induces expression of the human interferon beta 1 gene in mouse cells transformed by bovine papillomavirus-interferon beta 1 recombinants.

Mouse cells transformed by a bovine papillomavirus recombinant vector containing the human interferon (IFN) beta 1 (IFN-beta 1) gene could be induced to produce human as well as mouse IFNs. The optimal conditions for induction of human IFN and of its mRNA in these transformants resembled those needed for mouse IFN: high concentrations of DEAE-dextran and low concentrations of polyriboinosinic acid-polyribocytidylic acid. Superinduction by inhibitors of protein synthesis which strongly stimulate IFN-beta 1 induction in human cells had only a small effect on human IFN induction in bovine papillomavirus IFN-beta 1-transformed mouse cells. In contrast, cycloheximide without double-stranded RNA could induce significant levels of human IFN in the bovine papillomavirus IFN-beta 1 mouse transformants. After cycloheximide treatment, these cells contained IFN-beta 1 mRNA whose 5' ends originated in the authentic start site of the human IFN-beta 1 gene, as shown by S1 nuclease mapping. The transferred human gene, propagated extrachromosomally in the mouse cells, was, therefore, inducible under conditions different from those in human cells. The results also confirmed that the inhibitor of protein synthesis, cycloheximide, can induce expression of a human IFN gene.     

Constructing chimeric type 12/type 5 adenovirus E1A genes and using them to identify an oncogenic determinant of adenovirus type 12.

The E1A gene of highly oncogenic type 12 adenovirus (Ad12) possesses a segment unique to this serotype and comprising 60 base pairs contiguous with and separating conserved regions 2 and 3 in the gene. A similar but slightly longer segment is also present in the E1A gene of highly oncogenic simian adenovirus type 7 (D. Kimelman, J. S. Miller, D. Porter, and B. E. Roberts, J. Virol. 53:399-409, 1985). This segment is missing entirely from the E1A gene of type 5 adenovirus, which is nononcogenic. To test the hypothesis that this unique separating or "spacer" region influences the oncogenicity of Ad12, we constructed ClaI and SmaI restriction sites on either side of it, which allowed reciprocal exchange between this and the equivalent cassette from type 5 adenovirus E1A, bounded by the same restriction sites intrinsic to that gene. The resultant Ad12-based chimeric viruses, ch702 and ch704, in which the spacer region is replaced with (in-frame) type 5 sequence, grow normally on human A549 cells and display wild-type transformation frequencies on baby rat and mouse kidney cells. In contrast, the oncogenic capacity of these chimeric viruses, as measured by tumor induction following virus inoculation in Hooded Lister rats, is greatly reduced. Likewise, cells transformed by ch702 and ch704 display reduced tumorigenicity compared with wild-type transformants in syngeneic rats. These results, coupled with recent preliminary tests using a mutant with a point mutation in this region, support the view that the unique spacer region of type 12 is an oncogenic determinant of this virus.     

Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein.

The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.     

Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)