Harris N, Buller R M, Karupiah G
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1995 Feb;69(2):910-5. doi: 10.1128/JVI.69.2.910-915.1995.
Gamma interferon (IFN-gamma)-induced nitric oxide synthase (iNOS) and nitric oxide (NO) production in the murine macrophage-like RAW 264.7 cells were previously shown to inhibit the replication of the poxviruses vaccinia virus (VV) and ectromelia virus and herpes simplex virus type 1. In the current study, we performed biochemical analyses to determine the stage in the viral life cycle blocked by IFN-gamma-induced NO. Antibodies specific for temporally expressed viral proteins, a VV-specific DNA probe, and transmission electron microscopy were used to show that the cytokine-induced NO inhibited late protein synthesis, DNA replication, and virus particle formation but not expression of the early proteins analyzed. Essentially similar results were obtained with hydroxyurea and cytosine arabinoside, inhibitors of DNA replication. Enzymatically active iNOS was detected in the lysates of IFN-gamma-treated but not in untreated RAW 264.7 cells. The IFN-gamma-treated RAW 264.7 cells which express iNOS not only were resistant to productive infection but also efficiently blocked the replication of VV in infected bystander cells of epithelial origin. This inhibition was arginine dependent, correlated with nitric production in cultures, and was reversible by the NOS inhibitor N omega-monomethyl-L-arginine.
γ干扰素（IFN-γ）诱导的小鼠巨噬细胞样RAW 264.7细胞中的一氧化氮合酶（iNOS）和一氧化氮（NO）生成先前已被证明可抑制痘病毒痘苗病毒（VV）、埃可病毒和1型单纯疱疹病毒的复制。在本研究中，我们进行了生化分析，以确定被IFN-γ诱导的NO阻断的病毒生命周期阶段。针对时间表达的病毒蛋白的特异性抗体、VV特异性DNA探针和透射电子显微镜被用于表明细胞因子诱导的NO抑制晚期蛋白合成、DNA复制和病毒颗粒形成，但不抑制所分析的早期蛋白的表达。用DNA复制抑制剂羟基脲和阿糖胞苷获得了基本相似的结果。在IFN-γ处理的RAW 264.7细胞裂解物中检测到具有酶活性的iNOS，但在未处理的RAW 264.7细胞中未检测到。表达iNOS的IFN-γ处理的RAW 264.7细胞不仅对生产性感染具有抗性，而且能有效阻断上皮来源的受感染旁观者细胞中VV的复制。这种抑制是精氨酸依赖性的，与培养物中的一氧化氮产生相关，并且可被NOS抑制剂Nω-单甲基-L-精氨酸逆转。
