HIV-1 Env gp160 maturation in the Golgi apparatus

HIV-1包膜蛋白(Env)糖蛋白(GP)160属于I类融合蛋白，也可由 高致病性人类病毒包括甲型流感病毒(IAV)、埃博拉病毒(EBOV)和冠状病毒 (CoV)，如SARS-CoV(SARS1)、MERS和SARS-CoV-2(SARS2)。他们在病毒上建立尖峰 诱导病毒和细胞膜融合以允许病毒进入细胞的包膜，这是必不可少的 病毒感染。I类融合蛋白被合成为I型跨膜(TM)多肽 内质网(ER)中的前体，并被送到高尔基体成熟。这个 高尔基体含有糖苷酶/糖基转移酶和保守的寡聚体高尔基体(COG) 复合体和其他相关蛋白，如可溶性N-乙基马来酰亚胺敏感因子附着受体 (圈套)用于贩运的蛋白质。在高尔基体内部，高甘露糖型N-聚糖被加工成复杂的- 经过广泛的甘露糖修剪后，还会发生O-糖基化。这些 除SARS1-Spike(S)外的前体进一步受到Furin的蛋白水解性切割，以完成 成熟过程。当这些步骤在高尔基山脉被破坏时，不会产生感染性粒子，导致 彻底抑制病毒感染。最近，我们和其他人报道说，MARCH8，一个 膜相关环-CH型E3泛素连接酶家族通过靶向广泛抑制病毒复制 广泛的融合蛋白。重要的是，我们报告了MARC在I类中导致多个缺陷 H8 高尔基体中的融合成熟机制未知。这些缺陷不仅存在于呋喃裂解中 HIV-1gp160、兔疫病毒血凝素(HA)、EB病毒糖蛋白(GP)、MERS-S和SARS-2-S，以及N-和 高尔基体中SARS2-S、MERS-S和EBOV-GP的O-糖基化。尽管MARCH8不会触发 这些融合蛋白的降解，其E3连接酶功能仍然是造成这些缺陷所必需的。的目标是 本课题旨在阐明HIV-1 gp160成熟过程中这些多重缺陷的分子机制。 通过了解MARCH8的抗病毒机制。我们假设MARCH8针对的是糖苷酶， 糖基转移酶、呋喃、COG复合体和/或SNARE，以阻止HIV-1 gp160成熟。我们建议 检验这一假设的两个截然不同但又相互关联的目标。在目标1中，我们将描述MARCH8如何 阻断HIV-1感染过程中gp160的成熟。实验将在原代细胞和人类 T细胞系结合RNA沉默和CRISPR/Cas9基因敲除来阐明MARCH8抗 艾滋病毒活跃度。在目标2中，我们将确定在HIV-1 gp160成熟过程中起关键作用的MARCH8靶点。 我们将重点关注通过高置信度生物信息学分析选择的18个高尔基蛋白来识别靶标。这个 这个项目的意义是非常高的，它不仅将填补我们对I类聚变的认识空白 蛋白质的糖基化和高尔基体的运输，也阐明了一种新的抗病毒机制，即可以 广泛应用于包括HIV-1、SARS2、EBOV和IAV在内的几种高致病性人类病毒。

HIV-1 envelope (Env) glycoprotein (gp) 160 belongs to class I fusion proteins that are also expressed by other highly pathogenic human viruses including influenza A viruses (IAV), Ebola viruses (EBOV), and coronaviruses (CoV) such as SARS-CoV (SARS1), MERS, and SARS-CoV-2 (SARS2). They build spikes on the viral envelope that induce fusion of viral and cellular membranes to allow viruses to enter cells, which is essential to the viral infection. Class I fusion proteins are synthesized as a type I transmembrane (TM) polypeptide precursor in the endoplasmic reticulum (ER) and delivered to the Golgi apparatus for maturation. The Golgi contains glycosidases/glycosyltransferases for glycosylation and conserved oligomeric Golgi (COG) complex and other associated proteins such as soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins for trafficking. Inside the Golgi, high-mannose-type N-glycans are processed into complex- type and hybrid-type N-glycans after extensive mannose-trimming, and O-glycosylation also occurs. These precursors except for SARS1-spike (S) are further subjected to proteolytic cleavage by furin to complete the maturation process. When these steps are disrupted in the Golgi, no infectious particles are produced, leading to complete inhibition of viral infection. Recently, we and others reported that MARCH8, a member of the membrane-associated RING-CH-type E3 ubiquitin ligase family, broadly inhibits viral replication by targeting a wide range of fusion proteins. Importantly, we reported that MARC causes multiple defects in class I H8 fusion maturation in the Golgi via an unknown mechanism. These defects are found not only in furin-cleavage of HIV-1 gp160, IAV-hemagglutinin (HA), EBOV-glycoprotein (GP), MERS-S, and SARS2-S, but also in N- and O-glycosylation of SARS2-S, MERS-S, and EBOV-GP in the Golgi. Although MARCH8 does not trigger the degradation of these fusion proteins, its E3 ligase function is still required for causing these defects. The goal of this project is to elucidate the molecular mechanism of these multiple defects in HIV-1 gp160 maturation by understanding the MARCH8 antiviral mechanism. We hypothesize that MARCH8 targets glycosidases, glycosyltransferases, furin, COG complex, and/or SNARE to block HIV-1 gp160 maturation. We propose two distinct but inter-related Aims to test this hypothesis. In Aim 1, we will characterize how MARCH8 blocks gp160 maturation during HIV-1 infection. Experiments will be performed in primary cells and human T cell lines in combination with RNA silencing and CRISPR/Cas9 knockout to elucidate the MARCH8 anti- HIV activity. In Aim 2, we will identify the MARCH8 targets that play a critical role in HIV-1 gp160 maturation. We will focus on 18 Golgi proteins selected by high confidence bioinformatic analysis to identify the targets. The significance of this project is very high, which will not only fill in gaps in our understanding of class I fusion protein glycosylation and trafficking in the Golgi, but also elucidate a novel antiviral mechanism that can be broadly applied to several highly pathogenic human viruses including HIV-1, SARS2, EBOV, and IAV.