ROTAVIRUS VP5 PERMEABILIZES MEMBRANES

描述(摘自申请人的摘要)：轮状病毒是二十面体 具有三层蛋白质衣壳的病毒。外层衣壳由 钙结合糖蛋白VP7和尖峰蛋白VP4。轮状病毒结合 通过唾液酸(VP4)或VP4和VP4中的整合素结合域与细胞结合。 轮状病毒在中性pH条件下通过直接穿膜进入细胞。 需要将VP4蛋白裂解为VP8和VP5蛋白 感染性和病毒对膜的渗透性。然而，几乎没有什么是 已知轮状病毒蛋白在进入过程中与膜的相互作用。 Mackow博士发现，从恒河猴轮状病毒(RRV)纯化的重组VP5 使脂质体通透性和膜通透性受到抑制 VP5特异性中和单抗。他还表明， 细胞内表达的VP5使细胞通透性和VP5形成尺寸 脂双层内有选择性的孔(~10埃)。这些发现表明 轮状病毒的侵入需要质膜的VP5通透性。 轮状病毒和其他非包膜病毒跨越血浆的机制 对细胞膜和进入细胞的了解很少。Mackow博士的发现 证明纯化的VP5和表达的VP5 N-末端片段是 在没有其他病毒的情况下能够透过膜和细胞 蛋白质。VP5在膜上形成孔，允许移位 羧基荧光素(CF)，而不是4 kDa右旋糖苷。通透性VP5多肽 含有一个与融合有同源性的长疏水结构域(HD) 甲型病毒E1蛋白的区域。E1膜融合所需的残基 都是VP5-HD共有的，并在所有轮状病毒株中保守。此外， VP5诱导的CF释放可通过中和单抗来阻断，这表明 防止VP5膜通透性是一种可行的中和机制 轮形病毒。Mackow博士假设VP5在早期的内小体中诱导毛孔 它允许钙外流，并从三层粒子转变为 转录活性的双层颗粒。 Mackow博士建议研究轮状病毒VP5蛋白之间的相互作用 并定义了对VP5诱导的孔形成的要求。这些 研究涉及轮状病毒进入过程中的一个基本步骤和基本 非囊膜病毒蛋白通透细胞的机制 进入过程中的膜。

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.