HeLa细胞中HCV NS5A的表达对干扰素敏感病毒VSV复制的影响

Effect of HCV NS5A expression in HeLa cells on interferon sensitive virus VSV replication

目的:研究丙型肝炎病毒(HCV)NS5A的表达对水泡性口炎病毒(VSV)复制的影响.方法:将稳定转染的HeLa-NS5A和HeLa-NS5A-ΔISDR细胞在6孔培养板中培养24h后,加入人基因工程α2a型干扰素(rHuIFN-αt2a)至所需浓度.培养24h后加入VSV,继续培养相应时间.取培养上清液,按10-1稀释,50μL稀释液加入含Vero细胞的24孔培养板中.每孔加入含100 mL/L小牛血清的DMEM-7.5g/L羧甲基纤维素钠(CMC)1 mL.继续培养48 h后移走培养液,结晶紫染色,自来水轻柔洗净,记录噬斑形成单位(PFU).结果:干扰素(IFN)浓度为1×105 U/L,VSV对HeLa-NS5A细胞的致病变作用要比对HeLa-NS5A-ΔISDR细胞的致病作用更明显.在整个IFN浓度处理中,HeLa-NS5A细胞培养液中的病毒滴度是HeLa-NS5A-ΔISDR细胞培养液中的病毒滴度的2～5倍(P＜0.05).结论:NS5A能增强IFN敏感病毒的复制,HCVNS5A内的ISDR可能是造成IFN对HCV患者治疗效果的因素.

AIM： To demonstrate the effect of HCV NSSA expression on vesicular stomatitis virus （VSV） replication, METHODS： Stably transfected cells were cultured per well in a 6-well tissue culture plate and cultured for 24 h. Human recombinant IFN-cc2a was then added at various concentrations for 24 h to induce the IFN antiviral state. Infection with VSV was then performed and supernatants were harvested at various time points after infection. Changes in infectious virus yields were measured by a standard virus plaque assay in Vero cells. RESULTS： When the IFN concentration was 1 × 10^5 U/L, HeLa-NSSA ceils infected with VSV had more obvious cytopathic effect compared with that of HeLa- NSSA-AISDR cells. In the various concentration treatments of IFN, the virus titers in the supernatant of HeLa-NSSA cells were about 2 to 5 times those of HeLa-NSSA-AISDR cells （ P 〈 0.05）. CONCLUSION： NSSA expression rescues VSV replication during IFN treatment and this effect is dependent on the putative interferon-sensitivity determining region （ISDR）. This effect will be helpful to demonstrate that HCV can naturally evade the IFN-induced antiviral response in persistently infected patients.