Host-virus interactions in the control of filovirus entry

2014-2015年西非史无前例的埃博拉病毒(EBOV)疫情突显了对疫苗和治疗药物的迫切需求，以预防和治疗EBOV和其他丝状病毒感染。然而，它们的发展受到我们对支撑丝状病毒感染、疾病和传播的病毒-宿主分子相互作用的关键差距的挑战。最近关于丝状病毒进入机制的研究表明，这些制剂是一组广泛利用宿主内切/溶酶体因子和入侵细胞途径的“晚穿透”病毒的原型成员。在之前的资助期间，我们发现宿主内体半胱氨酸蛋白酶(半胱氨酸组织蛋白)编程病毒糖蛋白GP与内体胆固醇转运体Niemann-Pick C1(NPC1)结合，我们发现NPC1是一种重要的细胞内进入受体。然而，尽管有这些和其他的发现，丝状病毒进入的最终步骤-糖蛋白介导的病毒和细胞脂双层之间的融合，以及病毒核衣壳核心的细胞质逃逸-仍然是个谜。这一新应用的首要目标是确定丝状病毒膜融合的分子机制，并鉴定具有抗病毒潜力的新的丝状病毒进入宿主因子。为了实现这些目标，首席研究人员组建了一个多学科团队，其中包括一名蛋白酶化学生物学领域的领先者，一名内切体和溶酶体的生物发生和功能方面的领先专家，以及一名病毒学家，他将把替代病毒的研究结果扩展到真正的丝状病毒。我们将共同：(1)确定丝状病毒膜融合的病毒和细胞要求，剖析融合反应中的步骤，并确定难以捉摸的丝状病毒融合触发因素(S)。(2)确定新发现的宿主因子在病毒转运到膜融合部位中的作用，并利用蛋白质组学方法揭示参与丝状病毒膜融合的新宿主因子。

The unprecedented 2014–2015 Ebola virus (EBOV) outbreak in West Africa has highlighted the urgent, unfulfilled need for vaccines and therapeutics to prevent and treat infections by EBOV and other filoviruses. However, their development is challenged by crucial gaps in our understanding of the virus-host molecular interactions that underpin filovirus infection, disease, and transmission. Recent work on the entry mechanism of filoviruses has revealed that these agents are prototypic members of a group of `late-penetrating' viruses that extensively exploit host endo/lysosomal factors and pathways to invade cells. During the previous grant period, we found that host endosomal cysteine proteases (cysteine cathepsins) program the viral glycoprotein GP to bind the endosomal cholesterol transporter Niemann-Pick C1 (NPC1), which we showed to be an essential intracellular entry receptor. Despite these and other discoveries, however, the culminating steps in filovirus entry—GP-mediated fusion between viral and cellular lipid bilayers, and cytoplasmic escape of the viral nucleocapsid core—remain enigmatic. The overarching goals of this renewal application are to define the molecular mechanism of filovirus membrane fusion, and to identify new filovirus entry host factors with antiviral potential. To fulfill these objectives, the principal investigator has assembled a multidisciplinary team that includes a field leader in the chemical biology of proteases, a leading expert on the biogenesis and functions of endosomes and lysosomes, and a virologist who will extend findings with surrogate viruses to authentic filoviruses. Together we will: (1) Define viral and cellular requirements for filovirus membrane fusion, dissect steps in the fusion reaction, and identify the elusive filovirus fusion trigger factor(s). (2) Determine the roles of newly- identified host factors in viral trafficking to membrane fusion sites, and employ proteomic approaches to uncover novel host factors involved in filovirus membrane fusion.