Yoshitake Jun, Akaike Takaaki, Akuta Teruo, Tamura Fumio, Ogura Tsutomu, Esumi Hiroyasu, Maeda Hiroshi
Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
J Virol. 2004 Aug;78(16):8709-19. doi: 10.1128/JVI.78.16.8709-8719.2004.
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.
一氧化氮（NO）可能会影响各种病原体的基因组，这种诱变作用对于病毒的发病机制和进化尤为重要。在此，我们研究了NO对病毒复制和突变的影响。外源性或内源性NO对甲型流感病毒和仙台病毒均无明显的抗病毒作用。我们用融合了绿色荧光蛋白（GFP）基因的仙台病毒（GFP-SeV）分析了NO的诱变潜力。将GFP-SeV在转染了表达诱导型一氧化氮合酶（iNOS）载体的SW480细胞中培养。通过测量病毒噬斑GFP荧光的丧失来检测GFP-SeV的突变频率。iNOS-SW480细胞中GFP-SeV的突变频率远高于亲本SW480细胞，并且通过用一氧化氮合酶（NOS）抑制剂处理可将其降低至亲本细胞的突变频率水平。免疫细胞化学显示，iNOS-SW480细胞中生成的8-硝基鸟苷比未转染iNOS的SW480细胞更多。将外源性添加的纯8-硝基鸟苷加入感染GFP-SeV的CV-1细胞中可增加病毒突变频率。由8-硝基鸟苷诱导的GFP基因突变谱似乎与在小鼠肺内体内发生的突变谱相似。两种突变体（由8-硝基鸟苷诱导的突变体和体内发生的突变体）共有的一个碱基替换特征是C到U的转变。iNOS-SW480细胞中依赖NO的氧化应激也很明显。总之，结果明确表明，NO对诸如仙台病毒之类的RNA病毒具有诱变潜力，且不影响病毒复制，可能是通过8-硝基鸟苷的形成和细胞氧化应激来实现的。
