ROTAVIRUS VP5 PERMEABILIZES MEMBRANES

描述（改编自申请人摘要）：轮状病毒是二十面体病毒 具有三层蛋白质衣壳的病毒。外壳由以下物质组成： 钙结合糖蛋白VP7和刺突蛋白VP4。轮状病毒结合 通过唾液酸（VP4）或VP4和VP4中的整合素结合结构域与细胞结合。 轮状病毒在中性pH下通过直接膜穿透进入细胞。 需要将VP4刺突蛋白水解切割成VP8和VP5蛋白， 感染性和用于膜的病毒透化。然而， 已知轮状病毒蛋白在进入时与细胞膜的相互作用。 Mackow博士发现，从恒河猴轮状病毒（RRV）中纯化的重组VP5 透化脂质体，膜透化被 VP5特异性中和单克隆抗体。他还表明， 细胞内表达的VP5使细胞透化， 脂质双层内的选择性孔（~10埃）。这些发现表明 轮状病毒进入质膜需要VP5透化。 轮状病毒和其他无包膜病毒穿过血浆的机制 膜和进入细胞是知之甚少。Mackow医生的发现 证明纯化的VP5和表达的VP5 N-末端片段是 能够在没有其他病毒的情况下透化膜和细胞 proteins. VP5在膜中形成孔，其允许 羧基荧光素（CF），而不是4kDa葡聚糖。透化VP5多肽 含有一个长的疏水结构域（HD），其与融合蛋白具有同源性 甲病毒E1蛋白的区域。E1膜融合所需的残留物 由VP5-HD共享，并且在所有轮状病毒株中是保守的。此外，本发明还 VP5诱导的CF释放被中和mAb阻断，表明 阻止VP5膜渗透性是一种可行的中和机制， 轮状病毒Mackow博士假设VP5在早期核内体中诱导孔 其允许Ca流出和从三层颗粒转变为 转录活性双层颗粒。 Mackow博士建议研究轮状病毒VP5蛋白的相互作用 并定义了VP5诱导的孔形成的要求。这些 研究强调了轮状病毒进入过程中的一个重要步骤， 无包膜病毒蛋白透化细胞的机制 膜进入时。

DESCRIPTION (adapted from applicant's abstract): Rotaviruses are icosahedral viruses with a triple-layered protein capsid. The outer capsid is comprised of a calcium binding glycoprotein, VP7, and a spike protein, VP4. Rotaviruses bind to cells by sialic acid (VP4) or integrin binding domains in VP4 and VP4. Rotaviruses enter cells at neutral pH by direct membrane penetration. Proteolytic cleavage of the VP4 spike into VP8 and VP5 proteins is required for infectivity and for virus permeabilization of membranes. However, little is known about the interactions of rotavirus proteins with membranes during entry. Dr. Mackow has found that purified recombinant VP5 from rhesus rotavirus (RRV) permeabilizes liposomes and that membrane permeabilization is inhibited by VP5-specific neutralizing monoclonal antibodies. He has also shown that intracellularly expressed VP5 permeabilizes cells and that VP5 forms size selective pores (~10 angstroms) within lipid bilayers. These findings suggest that VP5 permeabilization of plasma membranes is required for rotavirus entry. The mechanism by which rotaviruses and other non-enveloped viruses cross plasma membranes and enter cells is poorly understood. Dr. Mackow's findings demonstrate that purified VP5 and expressed VP5 N-terminal fragments are capable of permeabilizing membranes and cells in the absence of other viral proteins. VP5 forms pores in membranes which permit the translocation of carboxyfluorescein (CF) but not 4kDa dextrans. Permeabilizing VP5 polypeptides contain one long hydrophobic domain (HD) which shares homology with the fusion region of the alphavirus E1 protein. Residues required for E1 membrane fusion are shared by the VP5-HD and are conserved in all rotavirus strains. Further, VP5-induced CF release is blocked by neutralizing mAbs suggesting that preventing VP5 membrane permeability is a viable mechanism for neutralizing rotavirus. Dr. Mackow hypothesizes that VP5 induces pores in early endosomes which permit Ca efflux and the transition from a triple-layered particle to a transcriptionally active double-layered particle. Dr. Mackow proposes to investigate interactions of the rotavirus VP5 protein with membranes and define requirements for VP5-induced pore formation. These studies address an essential step in the rotavirus entry process and basic mechanisms by which non-enveloped viral proteins permeabilize cellular membranes during entry.