The role of SERINC5 in HIV-1 replication

SERINC5 在 HIV-1 复制中的作用

基本信息

批准号：
9974474
负责人：
YONG-HUI ZHENG
金额：
$ 39.13万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-08 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9974474
关键词：
Adaptor Signaling Protein Affect Alternative Splicing Anabolism Anti-Retroviral Agents Antiviral Agents Binding Binding Proteins Biological Assay Cell membrane Cells Complement Complex Cyclin-Dependent Kinases Degradation Pathway Destinations Development Down-Regulation Drug Targeting Endocytosis Endosomes Equine Infectious Anemia Virus Exhibits Family Fluorescence Gene Expression Genome Stability Goals HIV Envelope Protein gp120 HIV-1 Homo Human Infection Integral Membrane Protein Investigation K-cyclin Laboratories Lead Lysosomes Mediating Membrane Fusion Molecular Molecular Conformation Murine leukemia virus Outcome Pathway interactions Pharmacology Phosphatidylserines Phosphorylation Play Prevalence Primate Lentiviruses Protein Isoforms Proteins Public Health Recycling Reporting Research Resistance Retroviridae Role Sequence Homology Serine Sphingolipids Testing Translating Transmembrane Domain Ubiquitination Viral Viral Proteins Virion Virus Virus Replication Yeasts antiretroviral therapy base dimer env Gene Products env Glycoproteins late endosome member neutralizing antibody novel novel strategies receptor receptor binding receptor mediated endocytosis recruit screening trafficking viral rescue virology yeast two hybrid system

项目摘要

SERINC5 (Ser5) is a novel restriction factor that strongly blocks HIV-1 entry. Ser5 belongs to the serine incorporator (SERINC) family that has five members (1 to 5). They are type III integral membrane proteins with 9-11 transmembrane domains. Ser5 and Ser3 were initially identified as the counteractive target of HIV-1 Nef that increases viral infectivity. Although SERINC proteins share 31-58% sequence homology, only Ser5 and Ser3 have antiviral activities, albeit the Ser3 activity is weak. In addition, although the Ser4 antiviral activity was reported, it is poorly expressed at protein levels. Ser5 is antagonized by HIV-1 Nef as well as MLV glycoGag and EIAV S2. The Nef antagonism of Ser5 plays an important role in the prevalence of primate lentiviruses in their hosts. Thus, Ser5 is an important restriction factor for retroviruses. Our goal is to investigate the molecular mechanisms of how Ser5 inhibits HIV-1 replication and conversely, how Ser5 is counteracted by Nef. First, we will elucidate how Ser5 blocks HIV-1 entry. Ser5 is packaged into virions to block viral entry, but the mechanism is still unclear. It is also unclear why the Ser5 inhibition depends on viral Env glycoproteins. Tier 1 viruses, which are largely laboratory-adapted viruses, are very sensitive, whereas most Tier 2/3 viruses, which are the vast majority of circulating strains, are resistant to Ser5. Notably, native Tier 1 Env trimers predominantly occupy an open conformation, whereas those Tier 2/3 trimers occupy a closed conformation. In addition, Ser5 makes HIV- 1 become sensitive to neutralizing antibodies, suggesting that Ser5 may modify the Env conformation. We hypothesize that Ser5 disrupts Env trimers in an open state, resulting in Env inactivation and blockade of HIV-1 entry. We will use a sensitive bimolecular fluorescence complementation (BiFC) assay to study Env oligomerization and SERINC-Env interactions in live cells and use virologic assays to elucidate how HIV-1 entry is blocked by Ser5 and also likely by Ser4. Second, We will elucidate how Nef antagonizes Ser5. Nef counteracts Ser5 by downregulating Ser5 from plasma membrane and excluding it from virions, but the precise mechanism is still unclear. We reported that Nef rapidly internalizes Ser5 via receptor-mediated endocytosis and re-localizes Ser5 to endosomes. Ser5 is then targeted to lysosomes in a ubiquitination-dependent manner for degradation. Cyclin K (CycK) is a previously identified Nef-binding protein that binds Cyclin-dependent kinase (CDK) 12 and 13 and regulates gene expression and genome stability. We found that CycK and CDK13 are required for Nef-downregulation of Ser5. We hypothesize that Nef recruits CDK13/CycK to plasma membrane to phosphorylate Ser5 and targets Ser5 to endosome/lysosome pathways for degradation. We will study the critical role of the CDK13/CycK complex in Nef downregulation of Ser5 by testing the Ser5 phosphorylation and determine how the phosphorylation affects the Ser5 intracellular trafficking and/or degradation. New antiretroviral mechanisms and/or targets will be discovered from our investigations, which is likely translated into novel antiretroviral therapies.

SERINC5 (Ser5) 是一种新型限制因子，可强力阻止 HIV-1 进入。 Ser5 属于丝氨酸有五个成员（1 至 5）的公司发起人 (SERINC) 家族。它们是 III 型整合膜蛋白 9-11跨膜结构域。 Ser5和Ser3最初被确定为HIV-1 Nef的反作用靶点从而增加病毒的传染性。尽管 SERINC 蛋白具有 31-58% 的序列同源性，但只有 Ser5 和 Ser3具有抗病毒活性，但活性较弱。此外，虽然 Ser4 的抗病毒活性据报道，它在蛋白质水平上表达较差。 Ser5 被 HIV-1 Nef 以及 MLV gluGag 拮抗和 EIAV S2。 Ser5 的 Nef 拮抗作用在灵长类慢病毒的流行中起着重要作用他们的主人。因此，Ser5是逆转录病毒的重要限制因子。我们的目标是研究分子 Ser5 如何抑制 HIV-1 复制的机制，以及 Ser5 如何被 Nef 抵消的机制。首先，我们将阐明 Ser5 如何阻止 HIV-1 进入。 Ser5被包装到病毒体中以阻止病毒进入，但其机制目前还不清楚。目前还不清楚为什么 Ser5 抑制依赖于病毒 Env 糖蛋白。 1 级病毒，大部分是实验室适应的病毒，非常敏感，而大多数 2/3 级病毒是巨大的大多数流行菌株对 Ser5 具有抗性。值得注意的是，天然 Tier 1 Env 三聚体主要占据开放构象，而那些 2/3 层三聚体则占据封闭构象。此外，Ser5 使 HIV- 1 对中和抗体变得敏感，表明 Ser5 可能会改变 Env 构象。我们假设 Ser5 破坏处于开放状态的 Env 三聚体，导致 Env 失活并阻断 HIV-1 入口。我们将使用灵敏的双分子荧光互补 (BiFC) 测定来研究 Env 活细胞中的寡聚化和 SERINC-Env 相互作用，并使用病毒学检测来阐明 HIV-1 进入的方式被 Ser5 阻断，也可能被 Ser4 阻断。其次，我们将阐明 Nef 如何拮抗 Ser5。内夫通过下调质膜上的 Ser5 并将其排除在病毒粒子之外来抵消 Ser5，但精确的机制尚不清楚。我们报道 Nef 通过受体介导的内吞作用快速内化 Ser5 并将 Ser5 重新定位到核内体。然后 Ser5 以泛素化依赖性方式靶向溶酶体用于降解。细胞周期蛋白 K (CycK) 是一种先前鉴定的 Nef 结合蛋白，可结合细胞周期蛋白依赖性激酶 (CDK) 12 和 13 并调节基因表达和基因组稳定性。我们发现 CycK 和 CDK13 是 Ser5 的 Nef 下调所需。我们假设 Nef 将 CDK13/CycK 招募到质膜磷酸化 Ser5 并将 Ser5 靶向内体/溶酶体途径进行降解。我们将研究通过测试 Ser5 磷酸化和 CDK13/CycK 复合物在 Nef 下调 Ser5 中的关键作用确定磷酸化如何影响 Ser5 细胞内运输和/或降解。新型抗逆转录病毒药物我们的调查将发现机制和/或目标，这很可能转化为新颖的抗逆转录病毒疗法。