Nitric oxide as an endogenous mutagen for Sendai virus without antiviral activity

Abstract

Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.

FIG. 1.

Effects of NO formed from GS-NO and SNAP on propagation of SeV and influenza virus. (A to D) After MDCK monolayers were inoculated with influenza virus or SeV at a multiplicity of infection of 3.0 PFU per cell, infected cells were incubated in DMEM containing 0.2% BSA and various concentrations of the NO donors GS-NO and SNAP. (E and F) MDCK cells were infected with SeV or influenza virus at a multiplicity of infection (MOI) of 0.1 or 0.01 PFU per cell, followed by culture with 0.1 mM SNAP as just described. At different time points after infection, the yield of virus in culture supernatants was assessed by use of the plaque-forming assay. Data are means ± standard error (n = 3). See text for details.

FIG. 2.

Effect of endogenous NO on SeV replication in cultured cells. A cell line stably expressing iNOS (iNOS-SW480 cells) was established. iNOS protein expression and NO overproduction were confirmed by Western blotting (A) and by assay for nitrite (NO2−) and nitrate (NO3−) formed in the supernatant of the cell culture (B). PEC, mouse peritoneal exudate cells, which served as a positive control for iNOS expression. (C) The effect of an NOS inhibitor (l-NMMA) on SeV replication in iNOS-SW480 cells is shown. iNOS-SW480 cells and parent SW480 cells served as controls for iNOS- and NO-producing and nonproducing cells, respectively. iNOS-SW480 and SW480 cells were infected with SeV at a multiplicity of infection of 3.0 PFU per cell, and the yield of virus in the culture supernatant was quantified by means of the plaque-forming assay. Data are means ± standard error (n = 3). See text for details.

FIG. 3.

Mutation frequency (A and C) and virus yield (B and D) in iNOS-SW480 cells and parent SW480 cells with or without l-NMMA and d-NMMA. The mutagenic potential of NO for SeV was determined by analysis of the mutation of GFP-SeV. (A and B) Monolayers of iNOS-SW480 cells were inoculated with GFP-SeV at a multiplicity of infection of 3.0 PFU per cell, followed by culture with or without l-NMMA or d-NMMA. The culture supernatant obtained 48 h after infection was used for determination of viral mutation by the mutation assay (A). The virus yield in the same samples used for the mutation assay is shown in B. (C and D) Monolayers of iNOS-SW480 cells and SW480 cells were inoculated with GFP-SeV at a multiplicity of infection of 0.1 PFU per cell (multicycle replications). After 72 h of culture of the infected cells with or without l-NMMA, the mutation frequency (C) and virus yield (D) were assessed in the same manner as in A and B. Control, iNOS-SW480 cells without l-NMMA; l-NMMA (1.0 or 10 mM), iNOS-SW480 cells treated with 1.0 or 10 mM l-NMMA; d-NMMA, iNOS-SW480 cells treated with 10 mM d-NMMA. Data are means ± standard error of four independent experiments (>1,000 plaques counted/assay); *, P < 0.05, and **, P < 0.01 versus the control and the d-NMMA-treated cells. See text for details.

FIG. 4.

Increased formation of 8-nitroguanosine in iNOS-SW480 cells. 8-Nitroguanosine formation in iNOS-SW480 cells was identified by immunocytochemical analysis with the monoclonal 8-nitroguanosine antibody. After iNOS-SW480 cells and their parent SW480 cells cultured in DMEM plus 10% fetal bovine serum were fixed and blocked, they were reacted overnight with 10 μg of anti-8-nitroguanosine antibody per ml, followed by reaction with Cy3-labeled secondary antibody. The antibody bound on the cells was visualized by fluorescence microscopy. The reaction for generation of 8-nitroguanosine appears below the fluorescent images.

FIG. 5.

Effect of 8-nitroguanosine on mutation frequency of GFP-SeV replicated in CV-1 cells. GFP-SeV was cultured in CV-1 cells in the same manner as for the culture with iNOS-SW480 cells described for Fig. 3 except that GFP-SeV was allowed to propagate in the cells in the presence of various concentrations of authentic 8-nitroguanosine. Data are means ± standard error of four independent experiments (>1,000 plaques counted/assay); *, P < 0.05 versus the control without 8-nitroguanosine. See text for details.

FIG. 6.

Oxidative stress induced in cells by NO and 8-nitroguanosine. Intensity of fluorescence related to oxidation of dihydrorhodamine 123, as measured by flow cytometry, is shown for iNOS-SW480 cells treated with l-NMMA (A) and for CV-1 cells treated with 8-nitroguanosine (B). (A) iNOS-SW480 cells were incubated with dihydrorhodamine 123 in the presence or absence of 1 mM l-NMMA in KRP (pH 7.4) containing 0.2% BSA and 1 mM l-arginine for 6 h at 37°C. Rhodamine fluorescence was measured as an indication of intracellular peroxidation. (B) Oxidative stress was examined with CV-1 cells after treatment with 500 μM 8-nitroguanosine in KRP containing 0.2% BSA for 12 h. Each panel represents data for three different flow cytometric measurements.

FIG. 7.

Schematic drawing of hypothetical mechanisms for NO-induced viral mutagenesis proposed by the present work. NO may accelerate viral mutation via formation of 8-nitroguanosine (8-nitroGuo), which may be a substantial contributor to erroneous RNA replication of the virus. NO-generated 8-nitroguanosine may cause viral mutation via two different mechanisms: directly, through incorporation into template RNAs for viral replication (pathway shown on the right), and indirectly, by enhanced oxidative stress because of its potent redox-active property (pathway shown on the left).