Analysis of anti-interferon mechanism by HCV NS5A protein using HCV replicon system

据报道，丙型肝炎病毒（HCV）亚基因组复制子在人肝癌Huh-7细胞中有效且持续地复制。我们已将这些结果扩展到其他分离的HCV克隆，并从黑猩猩传染性克隆之一HC-J4构建了另一个HCV复制子。以黑猩猩感染性克隆HC-J4为基础构建了HCV复制体（RpJ4），该复制体由HCV-5′-UTR、新霉素磷酸转移酶基因、脑心肌炎病毒IRES、HCV-非结构区、NS3 ~ NS5B和HCV-3′-UTR组成。已知HCV-Con1复制子所需的适应性突变在RpJ4复制子aa中引入。（氨基酸编号根据HC-J4） 2197丝氨酸到脯氨酸，aa。2201丝氨酸缺失，aa。2204丝氨酸到异亮氨酸（RpJ4-S2197P， RpJ4-S22001del和RpJ4-S2204I）。RpJ4/ISDRmutant和RpJ4- s2201del /ISDRmutant也通过将6个氨基酸突变引入干扰素敏感性决定区（ISDR）构建。将复制子RNA转染到Huh-7细胞中，通过G418选择建立了稳定的复制子表达细胞系。转染初代Huh-7细胞后，RpJ4和RpJ4/ISDRmutants未产生任何g418抗性菌落。相比之下，通过引入RpJ4-S2197P、RpJ4-S2204I、RpJ4-S2201del和RpJ4-S2201del/ISDR突变体，g418耐药细胞的转导效率最高，其中RpJ4-S2201del/ISDR突变体的转导效率最高。在ISDR上游区域引入适应性突变后，HC-J4衍生的HCV复制体可以有效地复制，并且在ISDR中引入额外的突变进一步增强了其复制。这些发现表明NS5A结构域的遗传结构在HCV-1b复制中至关重要，对于HCV-Con1和HC-J4复制子都是如此。细胞抗病毒反应部分由干扰素刺激基因的表达介导，由病毒基因组、转录本和复制中间体触发。丙型肝炎病毒（HCV）复制子的持续复制表明复制子不会引起细胞先天抗病毒反应。在本研究中，我们研究了表达HCV复制的细胞中ifn介导的抗病毒系统的调节因子。荧光素酶报告基因分析显示，在含有复制子的细胞中，干扰素刺激反应元件（ISRE）的基线活性明显低于未处理细胞。在参与IFN/Jak/STAT通路和ISRE活性的蛋白中，干扰素调节因子-1 （IRF-1）的表达水平在含有该复制子的细胞中显著降低。将IRF-1表达构建体转染到含有该复制子的细胞中，会导致ISRE活性增加，同时抑制HCV复制子的表达。此外，在HCV复制子被消除的治愈的Huh7细胞中，IRF-1的表达水平和ISRE活性也受到抑制，这表明IRF-1的降低不是由于病毒蛋白的主动抑制，而是由于细胞的适应性使HCV亚基因组能够复制。通过转基因补充IRF-1表达，消除了固化细胞对复制子的高度许可性。综上所述，IRF-1是通过调节isg介导的抗病毒反应来调节细胞内HCV复制的关键宿主因子之一。少

Hepatitis C virus (HCV) subgenomic replicon has been reported to replicate efficiently and continuously in human hepatoma Huh-7 cells. We have extended these results to other isolated HCV clones, and we have constructed another HCV replicon from HC-J4, one of the chimpanzee-infectious clones. An HCV replieon (RpJ4) was constructed from a chimpanzee-infectious clone, HC-J4, which consists of HCV-5'-UTR, neomycin phosphotransferase gene, the encephalomyocarditis virus IRES, HCV-non-structural region, NS3 to NS5B, and HCV-3'-UTR. The adaptive mutations known to be required for HCV-Con1 replicon were introduced in RpJ4 replicon, aa.(amino acids number according to HC-J4) 2197 serine to praline, aa.2201 serine to deletion, and aa.2204 serine to isoleucine (RpJ4-S2197P, RpJ4-S22001del, and RpJ4-S2204I). RpJ4/ISDRmutant and RpJ4-S2201del/ISDRmutant were also constructed by introducing six amino acid mutations into the interferon sensitivity determining region (ISDR). Replieon RNA was transfec … More ted into Huh-7 cells, and stable replicon-expressing cell lines were established by G418 selection. After transfection to naive Huh-7 cells, RpJ4 and RpJ4/ISDRmutants did not produce any G418-resistant colonies. In contrast, G418-resistant cells were transduced efficiently by the introduction of RpJ4-S2197P, RpJ4-S2204I, RpJ4-S2201del and RpJ4-S2201del/ISDRmutants, with the RpJ4-S2201del/ISDR mutant being most efficient. The HCV replioon derived from HC-J4 can replicate efficiently following the introduction of adaptive mutations into the upstream region of ISDR Moreover, additional introduction of mutations into ISDR further enhances its replication. These findings demonstrate that the genetic structure of the NS5A domain is critical in HCV-1b replications, for both the HCV-Con1 and the HC-J4 replicons.Cellular antiviral responses are mediated partly by the expression of interferon-stimulated genes, triggered by viral genomes, their transcripts and replicative intermediates. Persistent replication of a hepatitis C virus (HCV) replicon suggests that the replicon does not elicit cellular innate antiviral responses. In the present study, we investigated regulatory factors of the IFN-mediated antiviral system in cells expressing an HCV replieon. Luciferase reporter assays revealed that the baseline activity of the interferon-stimulated response element (ISRE) was significantly lower in cells harboring the replicon than in naive cells. Among the proteins involved in the IFN/Jak/STAT pathway and in ISRE activity, the expression level of interferon regulatory factor-1 (IRF-1) was found to be significantly lower in cells harboring the replicon. Transfection of an IRF-1 expression construct into cells harboring the replieon caused an increase of ISRE activity, accompanied by suppression of expression of the HCV replieon. Moreover in cured Huh7 cells, from which the HCV replicon had been eliminated, expression levels of IRF-1 and ISRE activity also were suppressed, demonstrating that the decrease of IRF-1 is attributable, not to active suppression by the viral proteins, but to adaptation of cells that enables replication of the HCV subgenome. The high permissiveness of the cured cells for the replicon was abolished by transgenic supplementation of IRF-1 expression. Taken together, IRF-1 is one of the key host factors that regulate intracellular HCV replication through modulation of ISG-mediated antiviral responses. Less