Krueger D K, Kelly S M, Lewicki D N, Ruffolo R, Gallagher T M
Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153, USA.
J Virol. 2001 Mar;75(6):2792-802. doi: 10.1128/JVI.75.6.2792-2802.2001.
The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.
鼠肝炎病毒（MHV）的原型JHM株是一种包膜的、含RNA的冠状病毒，已在体内被选择具有极高的神经毒性。这种病毒编码刺突（S）糖蛋白，它们是细胞间膜融合的极其有效的介质，其独特之处在于即使在没有与主要MHV受体（一种鼠癌胚抗原相关细胞粘附分子（CEACAM））预先相互作用的情况下也能启动融合。在考虑这种过度活跃的膜融合活性在神经毒性中的可能作用时，我们发现JHM在组织培养中的生长选择出了失去鼠CEACAM非依赖性融合活性的变体。在这些变体中，在编码刺突蛋白的受体结合（S1）和融合诱导（S2）亚基的区域中鉴定出了突变。每个突变分别被引入编码原型JHM刺突的cDNA中，并使用痘苗病毒载体表达这组cDNA。这些变体刺突在组装成寡聚体、蛋白水解成S1和S2裂解产物、转运到细胞表面以及对可溶性形式的鼠CEACAM的亲和力方面与JHM相似。然而，这些适应组织培养的刺突作为S1 - S2异二聚体显著稳定，并且它们完全依赖CEACAM的融合活性相对于原型JHM刺突延迟或降低。因此，我们鉴定出的突变指向了S蛋白中特异性调节膜融合反应的区域。我们认为，与某些体内环境不同，培养细胞选择具有延迟的、依赖CEACAM的融合活性的S蛋白，这可能会增加病毒在不可逆脱壳过程之前内化的可能性。
