Transformation of rat cells by cyt mutants of adenovirus type 12 and mutants of adenovirus type 5.

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.     

Transcription of the genome of adenovirus type 12. Viral mRNA in productively infected KB cells.

In human KB cells productively infected with adenovirus type 12, viral DNA replication starts between 12 and 14h postinfection. Virus-specific, polysome-associated mRNA was investigated early (6-8h) and late (26-28h) after infection. Most of the viral mRNA was polyadenylated and accounted for 0.46% and 24.1% of the mRNA synthesized early and late postinfection, respectively. The viral-specific mRNA isolated both early and late after infection falls into several distinct size-classes, ranging in molecular weights between 0.3X10(6) and 1.5X10(6) for the early RNA and between 0.6X10(6) and 2.3X10(6) for the RNA synthesized late in the infection.     

Intracellular forms of adenovirus DNA. V. Viral DNA sequences in hamster cells abortively infected and transformed with human adenovirus type 12.

The persistence of viral DNA in BHK-21 cells abortively infected with human adenovirus type 12 has been investigated using reassociation kinetics. No indication of an increase in the amount of viral DNA per cell has been found. On the contrary, the amount of intracellular viral DNA sequences decreases rapidly after infection. Thus, free adenovirus type 12 DNA does not replicate in BHK-21 cells. The influence of the multiplicity of infection on the amount of persisting adenovirus type 12 DNA has also been explored. The viral DNA sequences persisting in four lines of hamster cells transformed in vitro by adenovirus type 12 at various multiplicities of infection have been quantitated and mapped by reassociation kinetics experiments using restriction endonuclease fragments of 3H-labeled adenovirus type 12 DNA. All the EcoRI restriction nuclease fragments of the adenovirus type 12 genome are represented in each of the four cell lines. Individual fragments of the viral genome are represented in multiple copies in non-equimolar amounts.     

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.     

Three new virus-specific low molecular RNAs in adenovirus type 2 infected KB cells.

With the use of alkyl agarose columns which separate proteins according to their lipophilic affinities, a marked correlation has been uncovered between this parameter of proteins and their degradation rates $\text{in}$$\text{vivo}$. The possible relationship between these observations and analogous ones with other properties of proteins and their implications in regard to the mechanism of the turnover process are discussed.     

Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins.

Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.     

Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3.

Virus-specific DNA fragments that are shorter than the full-length viral genomes have been isolated from HeLa cells productively infected with adenovirus type 3. A number of predominant size classes could be detected by gel electrophoresis and hybridization, and the array of sizes was similar or identical to the selection in DNA purified from incomplete particles of this serotype (E. Daniell, J. Virol. 19:685-708, 1976). A large fraction of these short DNA molecules contained long inverted terminal repetitions, as did DNA molecules from incomplete particles. Restriction analysis showed that these subgenomic molecules consist of sequences from the two molecular ends of the normal genome. These results suggest that the predominance of left-hand end fragments seen in packaged incomplete DNAs results from selective packaging, whereas the predominance of certain size classes of intracellular viral DNA is a function of prepackaging events. The incomplete DNAs were generated at all times during viral DNA replication, and the yield relative to complete DNA did not seem to vary significantly with time or multiplicity of infection or when the virus was propagated on different human cell types.     

Cycl AMP in Gl-arrested BHK21 cells infected with adenovirus type 12

Changes in intracellular levels of cyclic AMP and activity of adenyl cyclase and cyclic AMP phosphodiesterase were studied in G1-arrested BHK21 cells infected with adenovirus type 12 (Ad12). The intracellular concentration of cyclic AMP is reduced at 8 hours after infection (h.p.i.) and reaches a minimum at 14 h.p.i. The decrease in activity of adenyl cyclase is detected at 7.5 h.p.i.; at 9 h.p.i. the activity is about 50% of that in mock-infected cells. No significant changes were observed in the activity of cyclic AMP phosphodiesterase until 13 h.p.i., thereafter the activity of cyclic AMP phosphodiesterase decreased. It is suggested that the observed changes in cyclic AMP metabolism are related to expression of the viral genome, as the first changes are detected only after appearance of Ad12 mRNA in the infected cells.     

Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12.

Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.     

Complementation of adenovirus type 5 host range mutants by adenovirus type 12 in coinfected HeLa and BHK-21 cells.

We have studied the ability of adenovirus type 12 (Ad12) to complement the Ad5 transformation-defective host rang (hr) mutants during infection of human cells (HeLa) or hamster cells (BHK-21). The group I mutant hr3 (mapped within 1.3 to 3.7 map units), which is incapable of synthesizing viral DNA, was complemented for both DNA synthesis and infectious virus production in nonpermissive HeLa cells during coinfection with Ad12. Similarly, the group II mutant hr6 (6.1 to 9.4 map units), which does synthesize DNA, was also shown to be complemented for virus production. When the host cells were BHK-21, an established hamster cell line that is permissive for Ad5 but nonpermissive for Ad12 DNA synthesis and virus production, coinfection with Ad5 and Ad12 did not overcome the block to Ad12 DNA synthesis. Coinfection of BHK-21 cells with Ad12 and either hr3 or hr6 leads to the complementation of only the group I mutant (hr3). The inability of Ad12 to complement hr6 in BHK-21 cells may be due to the failure of Ad12 to express an early gene product from the region corresponding to early region 1B (4.5 to 11 map units) Ad5 where hr6 and the other group II mutations are located.     

Expression of adenovirus type 2 DNA polymerase in insect cells infected with a recombinant baculovirus.

Sequences encoding adenovirus type 2 DNA polymerase were placed under control of the polyhedrin promoter and inserted into the baculovirus Autographa californica nuclear polyhedrosis virus by homologous recombination. Insect cells infected with the recombinant virus produced substantial amounts of the adenovirus type 2 DNA polymerase protein which was functional in both DNA polymerase and replication initiation reactions. Thus, the baculovirus expression system can provide active adenovirus type 2 DNA polymerase that is produced in quantities suitable for biochemical and structural analysis.     

Expression of adenovirus type 12 early region 1 in KB cells transformed by recombinants containing the gene.

The adenovirus type 12 (Ad12) early region 1 (E1) gene was introduced into KB cells by using a dominant selection vector, pSV2-gpt, and over 80 Gpt+ KB cell clones were established. Three types of recombinant DNAs (gAE1A, gARC, and gABA) were constructed. They contained the AccI-H, EcoRI-C, and BamHI-A fragments, respectively, of Ad12 DNA in pSV2-gpt. Five of 50 (10%) gABA-transformed cell clones, 12 of 18 (67%) gAE1A-transformed cell clones, and 10 of 18 (56%) gARC-transformed cell clones complemented the growth of Ad5 dl312 (deletion in E1A) and were designated as Gpt+ Ad+ cell clones. In these cell clones at their early passages, recombinant genome sequences were detected in cellular DNA and were expressed. T antigen g (the E1A gene product) was detected by immunofluorescence. The Gpt+ Ad+ cell clones supported the growth of Ad5 deletion mutants in parallel with the expression of Ad12 E1A or E1A plus E1B genes. After infection of Gpt+ Ad+ cell clones with Ad5 dl312, the early genes of dl312 were efficiently transcribed, indicating the expression of the pre-early function of the Ad12 E1A gene. Two clones each from gAE1A-,gARC-, and gABA-transformed cells were subcultured for a long period to determine the stability of the transfecting DNAs. Subculture in a nonselective medium resulted in cells which lost the transfecting DNAs. Subculture in a selective medium resulted in the selection of cells which maintained the gpt gene expression but lost the Ad12 gene expression. These results indicate that the transfecting DNA is present in an unstable state in KB cells.     

Viral DNA synthesis in cells infected with temperature-sensitive mutants of herpes simplex virus type 1.

Temperature-sensitive mutants of herpes simplex virus type 1 representing eight DNA-negative complementation groups were grouped into the following three categories based on the viral DNA synthesis patterns after shift-up from the permissive to the nonpermissive temperature and after shift-down from the nonpermissive to the permissive temperature in the presence and absence of inhibitors of RNA and protein synthesis. (i) Viral DNA synthesis was inhibited after shift-up in cells infected with tsB, tsH, and tsJ. After shift-down, tsB- and tsH-infected cells synthesized viral DNA in the absence of de novo RNA and protein synthesis whereas tsJ-infected cells synthesized no viral DNA in the absence of protein synthesis. The B, H, and J proteins appear to be continuously required for the synthesis of viral DNA. (ii) Viral DNA synthesis continued after shift-up in cells infected with tsD and tsK whereas no viral DNA was synthesized after shift-down in the absence of RNA and protein synthesis. Mutants tsD and tsK appear to be defective in early regulatory functions. (iii) Cells infected with tsL, tsS, and tsU synthesized viral DNA after shift-up and after shift-down in the absence of RNA and protein synthesis. The functions of the L, S, and U proteins cannot yet be determined.