Gallagher T M
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
J Virol. 1997 Apr;71(4):3129-37. doi: 10.1128/JVI.71.4.3129-3137.1997.
Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses with novel pathogenic properties.
鼠肝炎病毒（MHV）是一种冠状病毒，它通过从病毒粒子膜伸出的刺突（S）蛋白与细胞受体（MHVR）结合来引发感染。这些S蛋白的结构在MHV毒株之间有很大差异，这种差异通常被认为在决定MHV感染的毒株特异性病理学方面很重要，可能是通过影响MHV与MHVR之间的相互作用。为了研究S变异与受体结合之间的关系，开发了能够测量MHV与MHVR之间相互作用的检测方法。这些检测方法利用了一种新型的可溶性MHVR形式，即sMHVR-Ig，它由与人类免疫球蛋白G1的Fc部分融合的MHVR的病毒结合免疫球蛋白样结构域组成。sMHVR-Ig在人293 EBNA细胞中稳定表达为二硫键连接的二聚体，并通过Fc结构域固定在琼脂糖蛋白G上。所得的琼脂糖珠用于吸附放射性标记的MHV颗粒。在4℃时，这些珠子特异性吸附了两种原型MHV毒株，MHV JHM（4型毒株）和MHV JHM的组织培养适应突变体JHMX毒株。温度升至37℃会导致JHM洗脱，但不会导致JHMX洗脱。在37℃下从其受体上洗脱JHM（但不是JHMX）的这一体外观察结果与在37℃下从组织培养细胞中洗脱与受体相关的JHM（但不是JHMX）相对应。受体结合维持这种差异的基础与JHMX S蛋白中存在的一个大缺失突变相关，因为sMHVR-Ig与含有该缺失的痘苗病毒表达的S蛋白表现出相对耐热的结合。这些结果表明，冠状病毒S蛋白中的自然发生突变会影响与宿主细胞初始相互作用的稳定性，从而有助于进入的病毒粒子成功感染的可能性。病毒进入特征的这些变化可能导致具有新致病特性的冠状病毒。
