Zhang Rong, Li Yize, Cowley Timothy J, Steinbrenner Adam D, Phillips Judith M, Yount Boyd L, Baric Ralph S, Weiss Susan R
Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2015 Apr;89(7):3598-609. doi: 10.1128/JVI.03535-14. Epub 2015 Jan 14.
Mouse hepatitis virus (MHV) isolates JHM.WU and JHM.SD promote severe central nervous system disease. However, while JHM.WU replicates robustly and induces hepatitis, JHM.SD fails to replicate or induce pathology in the liver. These two JHM variants encode homologous proteins with few polymorphisms, and little is known about which viral proteins(s) is responsible for the liver tropism of JHM.WU. We constructed reverse genetic systems for JHM.SD and JHM.WU and, utilizing these full-length cDNA clones, constructed chimeric viruses and mapped the virulence factors involved in liver tropism. Exchanging the spike proteins of the two viruses neither increased replication of JHM.SD in the liver nor attenuated JHM.WU. By further mapping, we found that polymorphisms in JHM.WU structural protein M and nonstructural replicase proteins nsp1 and nsp13 are essential for liver pathogenesis. M protein and nsp13, the helicase, of JHM.WU are required for efficient replication in vitro and in the liver in vivo. The JHM.SD nsp1 protein contains a K194R substitution of Lys194, a residue conserved among all other MHV strains. The K194R polymorphism has no effect on in vitro replication but influences hepatotropism, and introduction of R194K into JHM.SD promotes replication in the liver. Conversely, a K194R substitution in nsp1 of JHM.WU or A59, another hepatotropic strain, significantly attenuates replication of each strain in the liver and increases IFN-β expression in macrophages in culture. Our data indicate that both structural and nonstructural proteins contribute to MHV liver pathogenesis and support previous reports that nsp1 is a Betacoronavirus virulence factor.
The Betacoronavirus genus includes human pathogens, some of which cause severe respiratory disease. The spread of severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) into human populations demonstrates the zoonotic potential of emerging coronaviruses, and there are currently no vaccines or effective antivirals for human coronaviruses. Thus, it is important to understand the virus-host interaction that regulates coronavirus pathogenesis. Murine coronavirus infection of mice provides a useful model for the study of coronavirus-host interactions, including the determinants of tropism and virulence. We found that very small changes in coronavirus proteins can profoundly affect tropism and virulence. Furthermore, the hepatotropism of MHV-JHM depends not on the spike protein and viral entry but rather on a combination of the structural protein M and nonstructural replicase-associated proteins nsp1 and nsp13, which are conserved among betacoronaviruses. Understanding virulence determinants will aid in the design of vaccines and antiviral strategies.
小鼠肝炎病毒（MHV）毒株JHM.WU和JHM.SD会引发严重的中枢神经系统疾病。然而，JHM.WU能强劲复制并引发肝炎，而JHM.SD在肝脏中无法复制或引发病变。这两种JHM变体编码的同源蛋白仅有少量多态性，对于JHM.WU的肝脏嗜性是由哪些病毒蛋白导致的，人们了解甚少。我们构建了JHM.SD和JHM.WU的反向遗传系统，并利用这些全长cDNA克隆构建了嵌合病毒，从而确定了与肝脏嗜性相关的毒力因子。交换这两种病毒的刺突蛋白，既没有增加JHM.SD在肝脏中的复制，也没有减弱JHM.WU的致病性。通过进一步定位，我们发现JHM.WU结构蛋白M以及非结构复制酶蛋白nsp1和nsp13中的多态性对于肝脏发病机制至关重要。JHM.WU的M蛋白和螺旋酶nsp13是其在体外和体内肝脏中高效复制所必需的。JHM.SD的nsp1蛋白在第194位赖氨酸处发生了K194R替换，这是所有其他MHV毒株中保守的一个残基。K194R多态性对体外复制没有影响，但会影响肝脏嗜性，将R194K引入JHM.SD可促进其在肝脏中的复制。相反，JHM.WU或另一种嗜肝毒株A59的nsp1中发生K194R替换，会显著减弱各毒株在肝脏中的复制，并增加培养的巨噬细胞中IFN-β的表达。我们的数据表明，结构蛋白和非结构蛋白都对MHV肝脏发病机制有影响，并支持了之前关于nsp1是β冠状病毒毒力因子的报道。
β冠状病毒属包括人类病原体，其中一些会引发严重的呼吸道疾病。严重急性呼吸综合征冠状病毒（SARS-CoV）和中东呼吸综合征冠状病毒（MERS-CoV）向人类群体的传播证明了新兴冠状病毒的人畜共患病潜力，目前尚无针对人类冠状病毒的疫苗或有效抗病毒药物。因此，了解调节冠状病毒发病机制的病毒-宿主相互作用非常重要。小鼠感染鼠冠状病毒为研究冠状病毒-宿主相互作用提供了一个有用的模型，包括嗜性和毒力的决定因素。我们发现冠状病毒蛋白中非常小的变化就能深刻影响嗜性和毒力。此外，MHV-JHM的肝脏嗜性不取决于刺突蛋白和病毒进入，而是取决于结构蛋白M以及与非结构复制酶相关的蛋白nsp1和nsp13的组合，这些蛋白在β冠状病毒中是保守的。了解毒力决定因素将有助于疫苗设计和抗病毒策略的制定。
