Molecular analysis of cell entry of SARS coronavirus

SARS冠状病毒（SARS- cov）被认为与其刺突(S)结合在易感靶细胞上表达的受体血管紧张素转换酶2 （ACE2）上。结合后，病毒粒子被运输到一个内体，在那里S蛋白被蛋白酶（最有可能是组织蛋白酶- l）切割，这导致S蛋白的融合性激活。然后，病毒包膜与内体膜融合，病毒遗传物质进入细胞。这是Bates和他的同事从以下两个发现中提出的一种进入机制。首先，SARS-CoV感染被溶酶体增厚剂抑制，表明。SARS-CoV通过内体途径进入，内体内的低pH环境是进入的关键。其次，SARS-CoV感染的细胞在胰蛋白酶诱导S蛋白裂解时发生融合，而在低pH缓冲液处理时，这些细胞不形成融合。如果Bates等人提出的假设是正确的，那么具有裂解S蛋白的病毒显示出更多的融合感染细胞可以从质膜进入细胞。为了验证这种可能性，我们制作了含有sars冠状病毒裂解S蛋白的伪VSV。在SARS-CoV的S蛋白上，有3个氨基酸序列与假定被furin切割的位点相似。我们在这些区域制造了突变，使这些区域被furin切割，并在表达ACE2的细胞上表达突变的S蛋白。在3个突变型S蛋白中，一个在SARS-S蛋白（C3） 795-797氨基酸突变的S蛋白在缺乏诱导SARS-S蛋白裂解的胰蛋白酶的情况下诱导细胞融合，而另外两个突变型S蛋白和野生型S蛋白（wt）则未能做到这一点。其中一个突变的S蛋白以及wt S蛋白在胰蛋白酶存在下诱导融合。在巴菲霉素、溶酶增体剂和组织蛋白酶- l抑制剂的作用下，检测了C3和wt S蛋白假病毒的感染情况，C3 S蛋白假病毒的感染不受这些药物的抑制，而wt S蛋白假病毒的感染则被这些药物阻断。这些结果表明，在795-797位点切割SARS-CoV S蛋白的假型可以直接从细胞表面进入细胞，而wt S假型则通过内体途径进入细胞。这些结果与贝茨和他的同事们提出的假设并没有矛盾。许多包膜病毒通常使用七肽重复肽（HRP）来阻断病毒的进入。据报道，SARS-CoV的HRP对感染也有抑制作用，但抑制效果不如人类免疫缺陷病毒（HIV），甚至明显低于小鼠冠状病毒MHV感染。由于HRP不能通过内体途径阻断SARS-CoV感染，我们在本研究中检测了HRP是否阻断了从细胞表面进入的途径。我们获得的数据表明，虽然HRP不能通过内体途径阻止感染，但它能有效地阻断细胞表面对SARS-CoV的感染。本研究显示了HRP作为抗病毒化合物在SARS治疗中的应用前景。少

SARS coronavirus (SARS-CoV) is believed to bind with its spike (S) to the receptor, angiotensin-converting enzyme2 (ACE2) expressed on susceptible target cells. After binding, virion is transport to an endosome where the S protein is cleaved by proteases, most likely cathepsin-L, which results in the fusogenic activation of the S protein. Then, viral envelope fuses with endosomal membrane and viral genetic material enters into cells. This is an entry mechanism proposed by Bates and his colleagues from the following 2 findings. First, SARS-CoV infection is inhibited by lysosomotropic agent, indicating that. SARS-CoV takes endosomal pathway and low pH environment in the endosome is critical for entry. Secondly, SARS-CoV infected cells undergo fusion when treated by trypsin that induces cleavage of the S protein, however, those cells do not form fusion when treated with low pH buffer. If the hypothesis proposed by Bates et al is correct, then the viruses with cleaved S protein that shows … More the fusion of infected cells can enter into cells from plasma membrane. To verify this possibility, we have made pseudotype VSV that harbors cleaved S protein of SARS-CoV. On the S protein of SARS-CoV, there are 3 regions similar in amino acid sequence to putative sites to be cleaved by furin. We have made mutations in these regions, so that these regions are cleaved by furin and expressed those mutated S proteins on ACE2 expressing cells. Among three mutant S proteins, one with a mutation at amino acid 795-797 of SARS-S protein (C3) induced fusion in cells in the absence of trypsin that induces cleavage of SARS-S protein, though other two mutants and wild type (wt) S failed to do so. One of these mutated S protein as well as wt S protein induced fusion in the presence of trypsin. Pseudotype viruses with C3 or wt S protein were examined for their infection in the presence of bafilomycin, lysosomotropic agent, as well as inhibitors for cathepsin-L The infection of pseudotype with C3 S protein was not inhibited with those agents, but pseudotype with wt S protein was blocked by those reagents. These results suggest that pseudotype with SARS-CoV S protein cleaved at 795-797 could enter into cells directly from cell surface, while wt S pseudotype took an endosomal pathway. These results are not in disagreement with the hypothesis drawn by Bates and his co-workers.Heptad repeat peptide (HRP) of a number of enveloped viruses is generally used to block the virus entry. HRP of SARS-CoV was also reported to exhibit the inhibitory effects upon infection, however, inhibition was not as efficient as that in human immunodeficiency virus (HIV) or even remarkably lower than murine coronavirus MHV infection. Since HRP did not block the SARS-CoV infection via endosomal pathway, we examined in this study whether HRP blocks the entry from cell surface. We have obtained the data that although HRP did not work to prevent the infection via endosomal pathway, it blocked efficiently the infection of SARS-CoV from cell surface. This study showed the possible application of HRP as an anti-viral compound for SARS therapy. Less