Efficient transformation of human fibroblasts by adenovirus-simian virus 40 recombinants.

Abstract

The origin-defective simian virus 40 (SV40) mutant 6-1 has been useful in transforming human cells (Small et al., Nature [London] 296:671-672, 1982; Nagata et al., Nature [London] 306:597-599, 1983). However, the low efficiency of transformation achieved by DNA transfection is a major drawback of the system. To increase the efficiency of SV40-induced transformation of human fibroblasts, we used recombinant adenovirus-SV40 virions which contain a complete SV40 early region including either a wild-type or defective (6-1) origin of replication. The SV40 DNA was cloned into the adenovirus vector in place of early region 1. Cell lines transformed by viruses containing a functional origin of replication produced free SV40 DNA. These cell lines were subcloned, and some of the subclones lost the ability to produce free viral DNA. Subclones that failed to produce free viral DNA were found to possess a mutated T antigen. Cell lines transformed by viruses containing origin-defective SV40 mutants did not produce any free DNA. Because of the high efficiency of transformation, we suggest that the origin-defective chimeric virus is a convenient system for establishing SV40-transformed cell lines from any human cell type that is susceptible to infection by adenovirus type 5.Cells range from fully permissive to fully nonpermissive for simian virus 40 (SV40) infection. Permissivity results in full expression of the viral genome, replication of viral DNA, and production of infectious virions. Nonpermissive infection results in expression of the early viral genes, but no viral DNA is synthesized or virions produced. Nonpermissive cells can be transformed, and the viral genome can be integrated into the host cell genome. Human cells are semipermissive for SV40. Virus production occurs to ca. 1% the level produced by permissive monkey kidney cells (12). The majority of viral DNA replication occurs in ca. 1 to 2% of the cells, where it reaches levels equivalent to wild-type infection (18). A portion of the population can become transformed and integrate viral DNA into the host cell genome (18); however, the efficiency is quite low. The frequency of transformation can be enhanced by transfection of SV40 origin-defective mutants (13). Because DNA transfection is an inefficient process, the frequency of transformation is still not very high. We wanted to determine whether we could increase the efficiency of SV40-induced tranformation after infection with recombinant viruses. We were also interested in determining whether having a functional versus a defective SV40 origin of replication would affect the frequency of transformation by recombinant viruses.The chimeric viruses contain the SV40 early region which encodes T antigen (HpaII-BamHI fragment) (16) cloned into the helper independent adenovirus vector, AE1/X (17). The SV40 origin of replication is either wild type or the orimutant 6-1, having six nucleotides deleted at the BglI site (4, 5). The SV40 DNA is cloned into the adenovirus vector in place of early regions la and lb. ...

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