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Molecular analysis of cell entry of SARS coronavirus

SARS冠状病毒进入细胞的分子分析

基本信息

批准号：
17390138
负责人：
TAGUCHI Fumihiro
金额：
$ 9.28万
依托单位：
National Institute of Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

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

SARS冠状病毒（SARS-CoV）以其刺突（S）与易感靶细胞上表达的受体血管紧张素转换酶2（ACE 2）结合。结合后，病毒体被转运至内体，在那里S蛋白被蛋白酶（最可能是组织蛋白酶-L）切割，这导致S蛋白的融合激活。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和他的同事根据以下2个发现提出的进入机制。首先，SARS-CoV感染被亲溶酶体剂抑制，表明。SARS-CoV通过内体途径进入，内体中的低pH环境是其进入的关键。其次，当用诱导S蛋白裂解的胰蛋白酶处理时，SARS-CoV感染的细胞发生融合，然而，当用低pH缓冲液处理时，这些细胞不形成融合。如果贝茨等人提出的假设是正确的，那么具有切割的S蛋白的病毒， ...更多信息融合的感染细胞可从质膜进入细胞。为了验证这种可能性，我们已经制造了假型VSV，其具有SARS-CoV的切割的S蛋白。在SARS冠状病毒S蛋白上，有3个氨基酸序列与推测的弗林蛋白酶切割位点相似的区域。我们已经在这些区域中进行了突变，使得这些区域被弗林蛋白酶切割，并在表达ACE 2的细胞上表达那些突变的S蛋白。在三个突变体S蛋白中，一个在SARS-S蛋白的氨基酸795-797处突变的突变体（C3）在不存在诱导SARS-S蛋白裂解的胰蛋白酶的情况下诱导细胞融合，而另外两个突变体和野生型（wt）S不能这样做。这些突变的S蛋白之一以及野生型S蛋白在胰蛋白酶的存在下诱导融合。在巴弗洛霉素、溶酶体促动剂以及组织蛋白酶-L抑制剂存在下，检查了具有C3或wt S蛋白的假型病毒的感染。这些试剂不抑制具有C3 S蛋白的假型病毒的感染，但这些试剂阻断具有wt S蛋白的假型病毒。这些结果表明，SARS-CoV S蛋白在795-797位裂解的假型可直接从细胞表面进入细胞，而野生型S假型通过内体途径进入细胞。这些结果与Bates等人提出的假说相一致。许多包膜病毒的七肽重复肽（Heptad repeat peptide，HRP）通常被用来阻断病毒的侵入。SARS-CoV的HRP也被报道对感染表现出抑制作用，然而，抑制效果不如人免疫缺陷病毒（HIV），甚至显著低于鼠冠状病毒MHV感染。由于HRP不能阻断SARS冠状病毒通过内体途径的感染，我们在这项研究中检查HRP是否阻断从细胞表面的进入。我们已经获得的数据表明，虽然HRP不能阻止通过内体途径的感染，但它可以有效地阻断SARS-CoV从细胞表面的感染。本研究为HRP作为抗SARS病毒药物的应用提供了可能。少