HIV-1 Env gp160 maturation in the Golgi apparatus

HIV-1 Env gp160 在高尔基体中成熟

• 批准号: 10626272
• 负责人: YONG-HUI ZHENG
• 金额: $ 39.98万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-10 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10626272
• 关键词: 2019-nCoV; Ablation; Acetylgalactosamine; Adopted; Bioinformatics; CCL7 gene; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Line; Cell membrane; Cells; Cellular Membrane; Chikungunya virus; Chimeric Proteins; Complex; Coronavirus; Defect; Ebola virus; Endoplasmic Reticulum; Eukaryotic Cell; Family; Glycoproteins; Glycoside Hydrolases; Goals; Golgi Apparatus; HIV; HIV-1; Hemagglutinin; Human; Human immunodeficiency virus test; Hybrids; Infection; Influenza A virus; Investigation; Knock-out; Laboratories; Lymphocytic choriomeningitis virus; Lysosomes; Macrophage; Mannose; Mediating; Membrane; Membrane Fusion; Middle East Respiratory Syndrome; Middle East Respiratory Syndrome Coronavirus; Molecular; Mouse Mammary Tumor Virus; Nucleocapsid; Oligosaccharides; Organelles; Pathogenicity; Play; Polysaccharides; Polyubiquitination; Process; Protein Biosynthesis; Protein Glycosylation; Proteins; Public Health; RNA Interference; Reporting; Rod; Role; SARS coronavirus; SIV; SNAP receptor; Structure; Surface; T-Lymphocyte; Testing; Translating; Translational Research; Vesicular stomatitis Indiana virus; Viral; Viral Fusion Proteins; Viral Physiology; Viral Proteins; Virion; Virus; Virus Diseases; Virus Replication; Zika Virus; alpha helix; experimental study; glycosylation; glycosyltransferase; gp160; influenzavirus; member; monocyte; novel; particle; polypeptide; porcine epidemic diarrhea virus; protein degradation; protein folding; protein oligomer; protein transport; receptor; receptor binding; trans-Golgi Network; ubiquitin-protein ligase; virology

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.

HIV-1 包膜 (Env) 糖蛋白 (gp) 160 属于 I 类融合蛋白，也由其他蛋白表达 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 (SARS-CoV)，例如 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的包膜，使病毒能够进入细胞，这对于病毒进入细胞至关重要 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）中的前体并被输送到高尔基体成熟。这 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，例如可溶性 N-乙基马来酰亚胺敏感因子附着受体 (SNARE) 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- 广泛的甘露糖修剪后形成类型和杂合型 N-聚糖，并且也会发生 O-糖基化。这些 除 SARS1-spike (S) 之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。当高尔基体中的这些步骤被破坏时，不会产生感染性颗粒，从而导致 以彻底抑制病毒感染。最近，我们和其他人报道了 MARCH8 的成员 膜相关的 RING-CH 型 E3 泛素连接酶家族，通过靶向广泛抑制病毒复制 多种融合蛋白。重要的是，我们报告了 MARC 导致 I 类中的多个缺陷 H8 高尔基体中的融合成熟通过未知的机制进行。这些缺陷不仅存在于弗林蛋白酶裂解中 HIV-1 gp160、IAV-血凝素 (HA)、EBOV-糖蛋白 (GP)、MERS-S 和 SARS2-S 以及 N- 和 高尔基体中 SARS2-S、MERS-S 和 EBOV-GP 的 O-糖基化。虽然MARCH8没有触发 这些融合蛋白的降解，其E3连接酶功能仍然是导致这些缺陷所必需的。目标是 该项目旨在阐明HIV-1 gp160成熟过程中这些多重缺陷的分子机制 通过了解 MARCH8 抗病毒机制。我们假设 MARCH8 靶向糖苷酶， 糖基转移酶、弗林蛋白酶、COG 复合物和/或 SNARE 可阻止 HIV-1 gp160 成熟。我们建议 两个不同但相互关联的目的是检验这一假设。在目标 1 中，我们将描述 MARCH8 如何 在 HIV-1 感染期间阻止 gp160 成熟。实验将在原代细胞和人类中进行 T 细胞系结合 RNA 沉默和 CRISPR/Cas9 敲除来阐明 MARCH8 抗 艾滋病毒活动。在目标 2 中，我们将确定在 HIV-1 gp160 成熟中发挥关键作用的 MARCH8 靶标。 我们将重点关注通过高置信度生物信息分析选择的 18 种高尔基体蛋白来识别靶标。这 这个项目的意义非常高，这不仅将填补我们对I类聚变理解的空白 高尔基体中的蛋白质糖基化和运输，而且还阐明了一种新的抗病毒机制 广泛应用于多种高致病性人类病毒，包括 HIV-1、SARS2、EBOV 和 IAV。