Some adenovirus DNA is associated with the DNA of permissive cells during productive or restricted growth

Abstract

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Simiar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency of integration.

Altered cell cycle progression and aberrant mitosis in adenovirus‐infected rodent cells

“…Primary cultures of C57Black6 mouse embryos and PVG rat embryos were prepared as described previously (Bellett and Younghusband, 1979). Ad5 and mutants ts36, ts37, and ts125 were grown in KB cells and titrated in human embryo kidney cells as decribed previously (Tyndall et al, 1978;Younghusband et al, 1979). The method of staining cells for viral early proteins by the indirect fluorescent antibody techniqueusing ' P antiserum has also been published (Hayashi and Russell, 1968;Bellett and Younghusband, 1979).…”

Adenovirus DNA is associated with the nuclear matrix of infected cells

“…Reassociation kinetics. The concentration of viral DNA in the preparations was determined by solution hybridization with 32P-nick-translated AdS DNA by the method of Pettersson and Sambrook (22) as described previously (27).…”

“…The nuclear matrix was digested with DNase I to remove 99% of the cell DNA, and the residual matrix-associated DNA was purified. Equal amounts of total or matrix-associated DNA were used in reassociation experiments (27). The slope of the line is proportional to the amount of Ad5 DNA in the sample.…”

Integration of adenovirus type 2 DNA at a limited number of cellular sites in productively infected cells

“…In particular, the possible ignificance of virus DNA integration in the process of malignant transformation by viruses has been studied extensively (for a review, see reference 6). Adenovirus DNA has been shown to integrate in abortively infected cells (4, 5), in productively infected cells (2, 16,23), and in transformed cells (10)(11)(12)(13)20).…”

“…In human cells permissively infected by adenovirus type 2 (Ad2), a large number of viral DNA molecules recombine with the host DNA (8,23). The host-virus DNA hybrids have been shown to exhibit buoyant densities in alkaline CsCl density gradients intermediate between those of host and viral DNAs (2).…”