HIV-1 Env gp160 maturation in the Golgi apparatus

HIV-1包膜（Env）糖蛋白（gp）160属于I类融合蛋白，其也由其它蛋白表达。 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)例如SARS-CoV（SARS 1）、MERS和SARS-CoV-2（SARS 2）。它们在病毒上建立尖峰 包膜诱导病毒和细胞膜的融合，以允许病毒进入细胞，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 在内质网（ER）中的前体，并交付给高尔基体成熟。的 高尔基体含有用于糖基化的糖苷酶/糖基转移酶和保守的寡聚高尔基体（COG） 复合物和其他相关蛋白质，如可溶性N-乙基马来酰亚胺敏感因子附着受体 （陷阱）蛋白质贩运。在高尔基体内，高甘露糖型N-聚糖被加工成复杂的- 型和混合型N-聚糖后，广泛的甘露糖修剪，和O-糖基化也发生。这些 除SARS 1-刺突（S）外的前体进一步受到弗林蛋白酶的蛋白水解切割，以完成 成熟过程当这些步骤在高尔基体中被破坏时，就不会产生感染性颗粒， 以完全抑制病毒感染。最近，我们和其他人报道说，3月8日，一个成员的 膜相关RING-CH-型E3泛素连接酶家族，通过靶向广泛抑制病毒复制 各种各样的融合蛋白。重要的是，我们报告了MARC导致I类中的多个缺陷， H8 融合成熟在高尔基体通过一个未知的机制。这些缺陷不仅存在于弗林蛋白酶裂解中， HIV-1 gp 160、IAV-血凝素（HA）、EBOV-糖蛋白（GP）、MERS-S和SARS 2-S，以及N-和 高尔基体中SARS 2-S、MERS-S和EBOV-GP的O-糖基化。虽然3月8日没有触发 尽管这些融合蛋白降解，但其E3连接酶功能仍然是引起这些缺陷所必需的。的目标 本项目旨在阐明HIV-1 gp 160成熟过程中这些多重缺陷的分子机制 通过了解MARCH 8的抗病毒机制。我们假设MARCH 8靶向糖苷酶， 糖基转移酶、弗林蛋白酶、COG复合物和/或SNARE阻断HIV-1 gp 160成熟。我们提出 两个不同但相互关联的目标来检验这一假设。在目标1中，我们将描述3月8日 在HIV-1感染期间阻断gp 160成熟。实验将在原代细胞和人类中进行。 T细胞系与RNA沉默和CRISPR/Cas9敲除的组合以阐明MARCH 8抗- 艾滋病毒活动。在目标2中，我们将确定在HIV-1 gp 160成熟中起关键作用的MARCH 8靶标。 我们将集中在18高尔基体蛋白选择高置信度的生物信息学分析，以确定目标。的 这个项目的意义是非常高的，这不仅将填补空白，在我们的理解我类融合 蛋白质糖基化和高尔基体中的运输，而且还阐明了一种新的抗病毒机制， 广泛应用于几种高致病性人类病毒，包括HIV-1、SARS 2、EBOV和IAV。

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.