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To analyze the structure and function of the E1B 19,000-molecular-weight protein (19K protein) (163R) of human adenovirus type 12, mutants were produced at various positions across the 163R-coding sequence. Viruses bearing mutations within the first 100 or so amino acids yielded unstable 163R-related products, induced DNA degradation and enhanced cytopathic effect (cyt/deg phenotype) in KB cells, and transformed primary rodent cells at much lower efficiencies than wild-type (wt) virus. Deletion of the final 16 residues at the carboxy terminus had no phenotypic effect. Alteration of residue 105 reduced transforming efficiency significantly, suggesting that this region of 163R is functionally important. Disruption of the AUG initiation codon at nucleotide 1542 blocked production of 163R completely but resulted in higher levels of E1B 55K-482R protein synthesis and a transforming efficiency similar to that of wt virus. These data suggested that while 163R is of some importance, normal transforming efficiencies can be obtained in its absence if 482R is overexpressed.

The early region E1b of adenovirus type 2 (Ad2) codes for two major tumor antigens of 53 and 19 kilodaltons (kd). The adenovirus lp+ locus maps within the 19-kd tumor antigen-coding region (G. Chinnadurai, Cell 33:759-766, 1983). We have now constructed a large-plaque deletion mutant (dl250) of Ad2 that has a specific lesion in the 19-kd tumor antigen-coding region. In contrast to most other Ad2 lp mutants (G. Chinnadurai, Cell 33:759-766, 1983), mutant dl250 is cytocidal (cyt) on infected KB cells, causing extensive cellular destruction. Cells infected with Ad2 wt or most of these other Ad2 lp mutants are rounded and aggregated without cell lysis (cyt+). The cyt phenotype of dl250 resembles the cyt mutants of highly oncogenic Ad12, isolated by Takemori et al. (Virology 36:575-586, 1968). By intertypic complementation analysis, we showed that the Ad12 cyt mutants indeed map within the 19-kd tumor antigen-coding region. The transforming potential of dl250 was assayed on an established rat embryo fibroblast cell line, CREF, and on primary rat embryo fibroblasts and baby rat kidney cells. On all these cells, dl250 induced transformation at greatly reduced frequency compared with wt. The cells transformed by this mutant are defective in anchorage-independent growth on soft agar. Our results suggest that the 19-kd tumor antigen (in conjunction with E1a tumor antigens) may play an important role in the maintenance of cell transformation. Since we have mapped the low-oncogenic or nononcogenic Ad12 cyt mutants within the 19-kd tumor antigen-coding region, our results further indicate that the 19-kd tumor antigen also directly or indirectly plays an important role in tumorigenesis of Ad12. Our results show that the cyt+ locus is an allele of the lp+ locus and that the cyt phenotype may be the result of mutations in specific domains of the 19-kd tumor antigen.

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.

Several mutants with much reduced oncogenicity (spontaneous mutants H12 cyt 52 and H12 cyt 70 and UV-induced mutants H12 cyt 61, H12 cyt 62, and H12 cyt 68) of the highly oncogenic adenovirus type 12 (Ad12) were studied for their ability to transform primary baby rat kidney cells. Four of the mutants showed much reduced capacity to transform cells in vitro, while H12 cyt 61 transformed cells as efficiently as the wild-type virus. Viral gene expression in several cell lines established from cultures infected by cyt mutants was studied, and it was found that viral sequences belonging to the left 16% of Ad12 were always transcribed. These results suggest that the function of the transformed state is not defective in the cyt mutants studied. Heterotypic complementation studies showed that the defect(s) in a cyt mutant can be corrected by an Ad7 function. Ad5 dl 313, with a deletion between 3.5 and 10.5 map units, transformed rat cells only at high multiplicity. These results suggest that the region E1B of adenoviruses may be required for efficient transformation of rat cells.

Purified human adenovirus type 12 preparations contain defective virions with a lighter density. These defective virions were isolated, and their biological functions and DNA were characterized. They can induce early and late antigens in infected cells and tumors in newborn hamsters with similar efficiency as complete virions. The majority of the DNA molecules from light virions contain deletions mapping near 16% from the left-hand end of the genome. Mechanisms for the generation of these molecules are discussed.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.

Physical and biological properties of highly oncogenic human adenovirus type 12 were compared with a low oncogenic mutant (cyt mutant). Parental and cyt mutant virions had very similar density and DNA size. However, the parental strain virion preparations contained a much higher proportion of defective virions (capable of cell killing, but not able to induce T- or V-antigen) than cyt mutant stock. It was also found that cyt mutant had a reduced virus yield in several human cell lines compared with the parental strain.

A group of mutants (cyt mutants) with much reduced oncogenicity was isolated from the highly oncogenic human adenovirus type 12 (Takemori et al., Virology 36: 575-586, 1968). These mutants induce extensive cellular destruction during lytic infection of human cells and produce low yields of virions. We report here that human KB cells infected with cyt mutants synthesized a reduced amount of viral DNA as compared with cells infected with the parental virus. Furthermore, the newly synthesized viral and cellular DNAs were extensively degraded in mutant-infected cells. Viral DNA was first synthesized as complete genome size, and most of it was degraded to subgenomic size within 6 h after synthesis. This virus-induced DNA degradation function, as well as the low yield of virions, was prevented by co-infection with the parental virus.

In five out of six primary hamster tumors induced by adenovirus type 12, less than 55% of the adenovirus type 12 genome is present. Various fragments of the integrated viral DNA were present in non-equimolar amounts.

Earlier, we reported (N. J. Lassam, S. T. Bayley, F. L. Graham, and P. E. Branton, Nature (London) 277:241-243, 1979) detecting protein kinase activity when cytoplasmic extracts of human adenovirus type 5 (Ad5)-infected KB cells immunoprecipitated with 14b antitumor serum directed against the transforming proteins of Ad5, were incubated with [gamma-32P]ATP. Here we show that in the in vitro assay this kinase phosphorylated both the heavy chain of immunoglobulin G and polypeptide than comigrated on sodium dodecyl sulfate gels with the 58,000-dalton Ad5 antigen. It also phosphorylated added histone H3. Evidence is presented that the protein kinase activity found with extracts from Ad5-infected cells is not due to nonspecific trapping of cellular enzymes in immune complexes, but to an enzyme which is distinct from kinases detected at background levels in controls. Serine and threonine were the major phosphorylated amino acids, and essentially no phosphotyrosine was detected. Protein kinase activity detected in Ad12-infected cells immunoprecipitated by an antiserum derived from hamsters bearing Ad12-induced tumors appeared to be immunologically distinct from that immunoprecipitated from Ad5-infected cells by 14b serum.

Several nononcogenic cyt mutants of adenovirus type 12 induced the same E1A (55,000 [55K] and 25K) and E1B polypeptides (55K, 19K, and 17K) as did the wild-type virus, except that cyt 68 did not induce the E1B 19K protein. Tumorigenicity tests showed cells transformed by cyt 68 to be highly oncogenic in vivo. Therefore, it was concluded that the E1B 19K polypeptide is not necessary for tumor induction but may be involved in the efficiency of transformation.

The yield of infectious virus was determined for KB cells infected with both adenovirus types 2 (ad 2) and 12 (ad 12). It was found that the yield of the former was greatly reduced, whereas that of the latter was not affected significantly. The reduction in virus yield was accompanied by an inhibition of ad 2 virus-specific ribonucleic acid (RNA) and viral deoxyribonucleic acid (DNA) synthesis at various times after infection. On the other hand, the rate of synthesis of ad 12 virus-specific RNA and viral DNA was not inhibited, but advanced in time. The total amount of ad 12 viral DNA synthesized was not affected by coinfection with ad 2. These results suggest that ad 2 infection hastens the maturation of ad 12.

The transcription of virus-specific ribonucleic acid (RNA) was studied in KB cells infected with adenovirus type 12 (strain Huie). Viral deoxyribonucleic acid (DNA) synthesis began at 12 to 15 hr after infection, and virus maturation occurred between 20 and 50 hr after infection. The rate of incorporation of (3)H-uridine into RNA per infected cell was stimulated, reaching a maximum of 1.6 times that of uninfected cells at 20 hr after infection. "Early" viral messenger ribonucleic acid (mRNA) constituted 0.1% of RNA synthesized at 11 hr, and "late" viral mRNA constituted 50% of RNA synthesized at 45 hr after infection, as determined by hybridization of viral DNA with labeled RNA from infected cells pulse labeled with (3)H-uridine. The species of virus-specific RNA synthesized at 22 hr after infection (when virus maturation has just begun) and at 45 hr (when virus maturation is nearly complete) were studied further: (i) 22- and 45-hr RNA had the same average guanine plus cytosine content, 47%, (ii) 22- and 45-hr RNA contained mostly the same viral nucleotide sequences, (iii) 45-hr RNA had a five times higher concentration of virus-specific RNA molecules than did 22-hr RNA, and (iv) 22- and 45-hr RNA contained virus-specific nucleotide sequences transcribed from all, or nearly all, of the viral genome.

Purified preparations of human adenovirus type 12 showed two bands when subjected to isopycnic centrifugation in a density gradient of cesium chloride. Their density difference was about 0.003 g/ml, suggesting a small difference in their deoxyribonucleic acid to protein ratio. Virions with a lighter density can kill human KB cells and induce T antigen as efficiently as the heavy virions. However, they appeared incapable to form plaques. Two passages of the heavy infectious virions at low multiplicity of infection did not produce significant amounts of light virions; however, when it was passed at high multiplicity of infection, the light band became visible in a cesium chloride density gradient.

Purified preparations of adenovirus types 2 and 12 were used to infect KB cells at different input multiplicities. The resulting infected cultures were scored for inclusion body formation, production of infectious centers, and cloning efficiency. Both preparations were found to contain some defective particles capable of preventing a cell from cloning but unable to induce inclusion bodies or form plaques. The proportion of such defective particles in adenovirus 12 was about 10 times that in adenovirus 2. At high input multiplicities, the percentage of cells displaying an inclusion body was less than that predicted by the Poisson distribution and reached a maximum of 40 to 60% for adenovirus 2 and 12 to 15% for adenovirus 12. This reduction may be due to interference by large numbers of non-plaque-producing particles infecting each cell. The per cent of cells forming infectious centers was substantially greater for adenovirus 2 than for adenovirus 12 when compared at the same input plaque-forming units, reaching a maximum of 35 to 73% for adenovirus 2 and 5 to 10% for adenovirus 12. The low value for adenovirus 12 may be a result of the same interference phenomenon.