Department of Microbiology, University of Minnesota, Minneapolis, Minnesota, USA.
J Virol. 2012 Dec;86(23):12665-75. doi: 10.1128/JVI.01861-12. Epub 2012 Sep 12.
Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-β-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.
肠道病毒进入细胞的过程已在多种组织培养系统中得到了深入研究。病毒颗粒与连接黏附分子 A（JAM-A）结合后，通过网格蛋白介导的内吞作用进入细胞，随后衣壳蛋白发生蛋白水解，病毒颗粒解体并进入细胞质。然而，在肠道的体内感染过程中，以及在肿瘤微环境中，衣壳蛋白的水解（脱壳）是在细胞外开始的。我们采用多种方法来确定无衣壳肠道病毒颗粒（称为中间亚病毒颗粒，ISVPs）是否通过直接穿透限制膜进入细胞，或者是否利用内吞作用途径来建立有效的感染。我们发现，ISVPs 的进入和感染被dynasore 抑制，dynasore 是一种依赖于动力蛋白的内吞作用抑制剂，以及 genistein 和显性负性 caveolin-1，它们阻断 caveolar 内吞作用。caveolar 内吞作用的抑制也降低了肠道病毒的感染。用甲基-β-环糊精提取膜胆固醇可抑制病毒颗粒的感染，但当用 ISVPs 起始感染时，其无影响。我们发现这条途径既不依赖于网格蛋白也不依赖于 caveolin。综上所述，这些数据表明肠道病毒颗粒在进入细胞过程中可以利用依赖于动力蛋白和不依赖于动力蛋白的内吞作用途径，并且它们揭示了肠道病毒 ISVPs 可以利用 caveolar 内吞作用来建立有效的感染。
