Qu Lin, Vongpunsawad Sompong, Atmar Robert L, Prasad B V Venkataram, Estes Mary K
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA.
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
J Virol. 2014 Sep;88(18):10312-26. doi: 10.1128/JVI.01111-14. Epub 2014 Jul 9.
Norwalk virus (NV) is the prototype strain of human noroviruses (HuNoVs), a group of positive-strand RNA viruses in the Caliciviridae family and the leading cause of epidemic gastroenteritis worldwide. Investigation of HuNoV replication and development of antiviral therapeutics in cell culture remain challenging tasks. Here, we present NoroGLuc, a HuNoV protease reporter system based on a fusion of NV p41 protein with a naturally secreted Gaussia luciferase (GLuc), linked by the p41/p22 cleavage site for NV protease (Pro). trans cleavage of NoroGLuc by NV Pro or Pro precursors results in release and secretion of an active GLuc. Using this system, we observed a cell type-specific activity profile of NV Pro and Pro precursors, suggesting that the activity of NV Pro is modulated by other viral proteins in the precursor forms and strongly influenced by cellular factors. NoroGLuc was also cleaved by Pro and Pro precursors generated from replication of NV stool RNA in transfected cells, resulting in a measurable increase of secreted GLuc. Truncation analysis revealed that the N-terminal membrane association domain of NV p41 is critical for NoroGLuc activity. Although designed for NV, a genogroup GI.1 norovirus, NoroGLuc also efficiently detects Pro activities from GII.3 and GII.4 noroviruses. At noncytotoxic concentrations, protease inhibitors ZnCl2 and Nα-p-tosyl-l-lysine chloromethyl ketone (TLCK) exhibited dose-dependent inhibitory effects on a GII.4 Pro by NoroGLuc assay. These results establish NoroGLuc as a pan-genogroup HuNoV protease reporter system that can be used for the study of HuNoV proteases and precursors, monitoring of viral RNA replication, and evaluation of antiviral agents.
Human noroviruses are the leading cause of epidemic gastroenteritis worldwide. Currently, there are no vaccines or antiviral drugs available to counter these highly contagious viruses. These viruses are currently noncultivatable in cell culture. Here, we report the development of a novel cell-based reporter system called NoroGLuc that can be used for studying norovirus replication and also for screening/evaluation of antiviral agents. This system is based on the fusion between viral protein p41 and a naturally secreted Gaussia luciferase (GLuc) with a cleavage site that can be recognized by the viral protease. Cleavage of this fusion protein by the viral protease results in the release and secretion of an active GLuc. Using NoroGLuc, we demonstrated a cell type-specific activity profile of the viral protease and its precursors and dose-dependent inhibitory effects of two protease inhibitors. This novel reporter system should be useful in probing norovirus replication and evaluating antiviral agents.
诺如病毒（NV）是人类诺如病毒（HuNoVs）的原型毒株，人类诺如病毒是杯状病毒科中的一组正链RNA病毒，是全球流行性肠胃炎的主要病因。在细胞培养中研究HuNoV复制及开发抗病毒疗法仍然是具有挑战性的任务。在此，我们展示了NoroGLuc，这是一种基于NV p41蛋白与天然分泌的高斯荧光素酶（GLuc）融合的HuNoV蛋白酶报告系统，二者通过NV蛋白酶（Pro）的p41/p22切割位点相连。NV Pro或Pro前体对NoroGLuc的反式切割导致活性GLuc的释放和分泌。利用该系统，我们观察到NV Pro和Pro前体具有细胞类型特异性的活性谱，这表明NV Pro的活性在前体形式中受到其他病毒蛋白的调节，并受到细胞因子的强烈影响。NoroGLuc也被转染细胞中NV粪便RNA复制产生的Pro和Pro前体切割，导致分泌的GLuc有可测量的增加。截短分析表明，NV p41的N端膜结合结构域对NoroGLuc活性至关重要。尽管NoroGLuc是为NV（一种基因群GI.１诺如病毒）设计的，但它也能有效检测GII.3和GII.4诺如病毒的Pro活性。在无细胞毒性浓度下，蛋白酶抑制剂ZnCl2和Nα-p-甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）通过NoroGLuc检测对GII.4 Pro表现出剂量依赖性抑制作用。这些结果确立了NoroGLuc作为一种泛基因群HuNoV蛋白酶报告系统，可用于研究HuNoV蛋白酶和前体、监测病毒RNA复制以及评估抗病毒药物。
人类诺如病毒是全球流行性肠胃炎的主要病因。目前，尚无疫苗或抗病毒药物可对抗这些高传染性病毒。这些病毒目前无法在细胞培养中培养。在此，我们报告了一种名为NoroGLuc的新型细胞报告系统的开发，该系统可用于研究诺如病毒复制，也可用于筛选/评估抗病毒药物。该系统基于病毒蛋白p41与天然分泌的高斯荧光素酶（GLuc）的融合，其具有一个可被病毒蛋白酶识别的切割位点。病毒蛋白酶对这种融合蛋白的切割导致活性GLuc的释放和分泌。利用NoroGLuc，我们展示了病毒蛋白酶及其前体的细胞类型特异性活性谱以及两种蛋白酶抑制剂的剂量依赖性抑制作用。这种新型报告系统应有助于探究诺如病毒复制和评估抗病毒药物。
