Increased Frequency of Specific Locus Mutation Following Human Cytomegalovirus Infection

Abstract

The effect of human cytomegalovirus (HCMV) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster lung V79 cells. When V79 cells were infected with HCMV (strain AD169) at multiplicities of 0.1 to 50 plaque forming units (PFU) per cell the presumptive mutation frequency, as determined by the number of 6-thioguanine-resistant (TGr) colonies, was increased up to 16.8-fold (P < 0.005), depending on the multiplicity of infection. Increases in the mutation frequency at the hprt locus were also observed for other laboratory-adapted HCMV strains (C-87, Davis) and for low passage clinical isolates (82-1, 84-2). The expression time required for the maximum increase in TGr colonies was 3 days and was consistent among the HCMV strains evaluated in this study. UV-irradiation of HCMV stock up to a dose of 9.6 x 10(4) ergs/mm2 increased the mutation frequency, but further exposure to UV light or to heat (56 degrees for 30 min) significantly decreased the frequency of TGr-resistant colonies, suggesting that expression of HCMV genes was involved in the mutation process. HCMV-induced TGr cells demonstrated substantially reduced (> 96%) incorporation of [3H]hypoxanthine. PCR analysis of the hprt locus demonstrated deletions in 9 of 19 HCMV-induced TGr colonies randomly selected for further study, while 2 of 17 spontaneously developed TGr colonies demonstrated deletions. Although insertions were not detected in spontaneously developed clones, 3 of 19 HCMV-induced TGr clones had insertions in the hprt gene. Neither HCMV-specific DNA sequences nor HCMV-specific proteins were detected in the TGr clones obtained after HCMV infection. Infection of V79 cells with HCMV also increased their sensitivity to mutation with N-methyl-N'-nitro-N-nitrosoguanidine, giving a synergistic enhancement of the mutation frequency. These results indicate that HCMV infection has the capacity to induce mutations in the cellular genome and increase the sensitivity of infected cells to mutation by genotoxic chemicals. Although inactivated HCMV particles are responsible for a modest increase in the mutation frequency, expression of HCMV genes is associated with a substantial enhancement of the mutation frequency.

Infection of Cells with Human Cytomegalovirus during S Phase Results in a Blockade to Immediate-Early Gene Expression That Can Be Overcome by Inhibition of the Proteasome

“…This latter population may be important not so much in permissive cells, which are eventually killed by the virus, but in nonpermissive or semipermissive cells, where IE gene expression alone may lead to mutagenic effects (2,48). The fact that these S-phase-infected cells maintain the ability to divide (at least once) is important in light of our most recent finding that approximately 10% of the mitotic cells in this first round of division sustain specific chromosomal damage at position 1q42 (19).…”

Mutator phenotype of mammalian cells due to deficiency of NEIL1 DNA glycosylase, an oxidized base-specific repair enzyme

“…The ability of 6-thioguanine (6-TG)-resistant cells to replicate DNA and thus allowing cell multiplication in the presence of 6-TG is a reliable indicator of Hprt mutation [32,33]. For mutant frequency analysis, the sense or antisense oligonucleotide-treated V79 cells were subcultured into 100-mm dishes (5×10 5 cells/dish) in the growth medium containing 6-TG (7 µg/ml).…”

“…Experiments were repeated 4 to 5-times to identify presumptive mutants. Plating efficiencies were determined in the absence of the 6-TG [27,32,33]. In parallel experiments, sense or antisense oligonucleotide-treated cells were mock treated or treated with glucose oxidase (GO) to induce oxidative stress.…”

“…Colonies of 6-TGresistant cells grown for 6 to 8 days were fixed in 3.7% formalin, and stained with 0.1% crystal violet. The Hprt mutant frequency was calculated from the number of 6-TG-resistant colonies relative to the total number of colonies in non-selecting medium [32,33]. A549 cells at 60% confluency (~3 × 10 6 ) were treated with NEIL1 antisense or sense oligonucleotide four times in a 30 h time period.…”

“…Because of the ease in scoring its forward mutations [32,33] the single copy, X-linked Hprt locus has been widely used to evaluate the genotoxicity of various chemical and physical agents, as well as the impact of altered levels of DNA repair proteins, [37,38]. Fig 3 shows that NEIL1 downregulation increased the HPRT mutant frequency by about 3-fold in both V79 and A549 cells relative to treatment with the sense (control) oligonucleotide, or to the untreated cells.…”