Watanabe Rie, Matsuyama Shutoku, Shirato Kazuya, Maejima Masami, Fukushi Shuetsu, Morikawa Shigeru, Taguchi Fumihiro
Department of Virology III, National Institute of Infectious Diseases, Murayama Branch, 4-7-1 Gakuen, Mushai-Murayama, Tokyo 208-0011, Japan.
J Virol. 2008 Dec;82(23):11985-91. doi: 10.1128/JVI.01412-08. Epub 2008 Sep 10.
Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is known to take an endosomal pathway for cell entry; however, it is thought to enter directly from the cell surface when a receptor-bound virion spike (S) protein is affected by trypsin, which induces cleavage of the S protein and activates its fusion potential. This suggests that SARS-CoV bearing a cleaved form of the S protein can enter cells directly from the cell surface without trypsin treatment. To explore this possibility, we introduced a furin-like cleavage sequence in the S protein at amino acids 798 to 801 and found that the mutated S protein was cleaved and induced cell fusion without trypsin treatment when expressed on the cell surface. Furthermore, a pseudotype virus bearing a cleaved S protein was revealed to infect cells in the presence of a lysosomotropic agent as well as a protease inhibitor, both of which are known to block SARS-CoV infection via an endosome, whereas the infection of pseudotypes with an uncleaved, wild-type S protein was blocked by these agents. A heptad repeat peptide, derived from a SARS-CoV S protein that is known to efficiently block infections from the cell surface, blocked the infection by a pseudotype with a cleaved S protein but not that with an uncleaved S protein. Those results indicate that SARS-CoV with a cleaved S protein is able to enter cells directly from the cell surface and agree with the previous observation of the protease-mediated cell surface entry of SARS-CoV.
已知严重急性呼吸综合征（SARS）冠状病毒（SARS-CoV）通过内体途径进入细胞；然而，当受体结合的病毒粒子刺突（S）蛋白受到胰蛋白酶影响时，人们认为它会直接从细胞表面进入，胰蛋白酶会诱导S蛋白的裂解并激活其融合潜能。这表明携带裂解形式S蛋白的SARS-CoV可以在不经过胰蛋白酶处理的情况下直接从细胞表面进入细胞。为了探究这种可能性，我们在S蛋白的798至801位氨基酸处引入了一个类弗林蛋白酶裂解序列，发现当在细胞表面表达时，突变的S蛋白在不经过胰蛋白酶处理的情况下被裂解并诱导细胞融合。此外，携带裂解S蛋白的假型病毒在存在溶酶体促渗剂以及蛋白酶抑制剂的情况下能够感染细胞，这两种物质都已知会通过内体阻断SARS-CoV感染，而未裂解的野生型S蛋白的假型病毒感染则被这些试剂阻断。一种源自SARS-CoV S蛋白的七肽重复序列，已知能有效阻断细胞表面的感染，它能阻断携带裂解S蛋白的假型病毒的感染，但不能阻断携带未裂解S蛋白的假型病毒的感染。这些结果表明，具有裂解S蛋白的SARS-CoV能够直接从细胞表面进入细胞，这与之前关于蛋白酶介导的SARS-CoV细胞表面进入的观察结果一致。
