Sosnovtsev Stanislav V, Belliot Gaël, Chang Kyeong-Ok, Prikhodko Victor G, Thackray Larissa B, Wobus Christiane E, Karst Stephanie M, Virgin Herbert W, Green Kim Y
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8007, USA.
J Virol. 2006 Aug;80(16):7816-31. doi: 10.1128/JVI.00532-06.
Murine norovirus (MNV) is presently the only member of the genus Norovirus in the Caliciviridae that can be propagated in cell culture. The goal of this study was to elucidate the proteolytic processing strategy of MNV during an authentic replication cycle in cells. A proteolytic cleavage map of the ORF1 polyprotein was generated, and the virus-encoded 3C-like (3CL) proteinase (Pro) mediated cleavage at five dipeptide cleavage sites, 341E/G342, Q705/N706, 870E/G871, 994E/A995, and 1177Q/G1178, that defined the borders of six proteins with the gene order p38.3 (Nterm)-p39.6 (NTPase)-p18.6-p14.3 (VPg)-p19.2 (Pro)-p57.5 (Pol). Bacterially expressed MNV 3CL Pro was sufficient to mediate trans cleavage of the ORF1 polyprotein containing the mutagenized Pro sequence into products identical to those observed during cotranslational processing of the authentic ORF1 polyprotein in vitro and to those observed in MNV-infected cells. Immunoprecipitation and Western blot analysis of proteins produced in virus-infected cells demonstrated efficient cleavage of the proteinase-polymerase precursor. Evidence for additional processing of the Nterm protein in MNV-infected cells by caspase 3 was obtained, and Nterm sequences 118DRPD121 and 128DAMD131 were mapped as caspase 3 cleavage sites by site-directed mutagenesis. The availability of the MNV nonstructural polyprotein cleavage map in concert with a permissive cell culture system should facilitate studies of norovirus replication.
小鼠诺如病毒（MNV）是杯状病毒科诺如病毒属中目前唯一能够在细胞培养中增殖的成员。本研究的目的是阐明MNV在细胞内真实复制周期中的蛋白水解加工策略。生成了ORF1多聚蛋白的蛋白水解切割图谱，病毒编码的3C样（3CL）蛋白酶（Pro）在五个二肽切割位点341E/G342、Q705/N706、870E/G871、994E/A995和1177Q/G1178介导切割，这些位点确定了六种蛋白质的边界，基因顺序为p38.3（N端）-p39.6（NTP酶）-p18.6-p14.3（VPg）-p19.2（Pro）-p57.5（Pol）。细菌表达的MNV 3CL Pro足以介导含有诱变Pro序列的ORF1多聚蛋白的反式切割，产生与体外真实ORF1多聚蛋白共翻译加工过程中观察到的产物以及MNV感染细胞中观察到的产物相同的产物。对病毒感染细胞中产生的蛋白质进行免疫沉淀和蛋白质印迹分析，证明蛋白酶-聚合酶前体的有效切割。获得了在MNV感染细胞中caspase 3对N端蛋白进行额外加工的证据，通过定点诱变将N端序列118DRPD121和128DAMD131定位为caspase 3切割位点。MNV非结构多聚蛋白切割图谱与允许的细胞培养系统相结合，应有助于诺如病毒复制的研究。
