The role of SERINC5 in HIV-1 replication

SERINC5 在 HIV-1 复制中的作用

• 批准号: 10792073
• 负责人: YONG-HUI ZHENG
• 金额: $ 34.12万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-08 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10792073
• 关键词: role; SERINC5; HIV; replication

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）是一个强烈阻止HIV-1进入的新型限制因素。 Ser5属于丝氨酸 有五名成员（1至5）的Incormator（Serinc）家族。它们是III型积分膜蛋白， 9-11跨膜结构域。 Ser5和Ser3最初被确定为HIV-1 NEF的反活性目标 这增加了病毒感染性。尽管序列蛋白具有31-58％的序列同源性，但只有Ser5和 SER3具有抗病毒活性，尽管Ser3活性很弱。另外，尽管Ser4抗病毒活性是 报道说，它在蛋白质水平上表达不佳。 Ser5与HIV-1 NEF以及MLV甘同性 和EIAV S2。 SER5的NEF拮抗作用在灵长类动病毒的流行率中起重要作用 他们的主人。因此，Ser5是逆转录病毒的重要限制因素。我们的目标是研究分子 SER5如何抑制HIV-1复制的机制，相反，Ser5如何由NEF来应对。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，但是机构 仍然不清楚。还不清楚为什么Ser5抑制取决于病毒ENV糖蛋白。第1层病毒，该病毒 主要是实验室适应的病毒，非常敏感，而大多数2/3病毒是广泛的 大多数循环菌株对Ser5具有抗性。值得注意的是，本地层1 env三聚体主要占据 开放的构型，而那些2/3层的三元器占据了封闭的构象。此外，Ser5使HIV- 1对中和抗体敏感，表明Ser5可能会改变ENV构象。我们 假设SER5破坏了处于开放状态的ENV三聚体，从而导致ENV失活和封锁HIV-1 入口。我们将使用敏感的双分子荧光互补（BIFC）测定来研究Env 活细胞中的寡聚化和序列-ENV相互作用，并使用病毒学测定法来阐明HIV-1如何进入 被SER5阻塞，也可能被Ser4阻塞。其次，我们将阐明NEF如何拮抗Ser5。 nef 通过从质膜下下调SER5来抵消Ser5，并将其排除在病毒体中，但精确 机制仍然不清楚。我们报道NEF通过受体介导的内吞作用迅速内化Ser5 并将SER5重新定位到内体。然后以泛素化的方式将Ser5靶向溶酶体 用于退化。 Cyclin K（Cyck）是一种先前鉴定的NEF结合蛋白，该蛋白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-downregulation所必需的。我们假设NEF将CDK13/CYCK招募到质膜 磷酸化Ser5并将Ser5靶向内体/溶酶体途径以降解。我们将研究 CDK13/CYCK复合物通过测试Ser5磷酸化和 确定磷酸化如何影响SER5细胞内运输和/或降解。新的抗逆转录病毒 从我们的调查中发现机制和/或目标，这很可能被转化为新颖 抗逆转录病毒疗法。