Short communication

Synthesis of DNA, late polypeptides, and infectious virus by host-range mutants of adenovirus 5 in nonpermissive cells

This chapter discusses the organization and expression of viral genes in adenovirus-transformed cells. Not all human adenoviruses are tumorigenic when inoculated into newborn rodents. Although adenoviruses display differences in their ability to induce tumors in animals, they transform the cells in culture. The transformation by adenoviruses is an outcome of a rare interaction between the virus and cells that either fail completely to support replication or are semi-permissive for virus growth, cells that are usually of rodent origin. Adenovirus-transformed cells exhibit an altered morphology and express virus-specific proteins (T-antigens). They also possess many other properties typical of mammalian cells transformed by viruses, including the tumorigenicity in nude mice. Viral products made during productive infection, perhaps in combination with signals present in an intact viral genome, possess the ability to override the signals recognized when segments of the viral genome become integrated.

Various early and late temperature-sensitive mutants of adenovirus (Ad) were able to support adeno-associated virus (AAV) DNA replication in either coinfections or in the rescue of AAV from latently infected human (Detroit 6) cells. Group I Ad host range mutants (hr 3, early region 1a) were unable to help AAV DNA replication in either case. Group II Ad host range mutants (hr 6, early region 1b), however, were able to help AAV DNA replication in coinfection but were unable to rescue AAV DNA replication in latently infected Detroit 6 cells. Thus, an Ad function(s), in addition to those required for AAV DNA replication, is needed for AAV rescue.

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.