Vpr

电压

基本信息

批准号：
8602711
负责人：
YONG-HUI ZHENG
金额：
$ 21.64万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2015-07-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Vpr and vpx are two viral accessory genes that are expressed in primate lentiviruses, which are required for viral replication and disease progression in vivo. Vpr has two well-known activities in vitro: arrest of cell cycle in G2 phase and enhancement of viral replication in monocyte-derived macrophages. Sharing ~25% homology with Vpr, Vpx only has the viral enhancement activity. Although both Vpr and Vpx enhance HIV-1 replication, different mechanisms are involved. While Vpx counteracts the antiretroviral activity of SAMHD1, the mechanism of Vpr enhancement of viral replication is still unknown. Recently, we reported a potent HIV-1 restriction in the human CD4+ T cell line CEM.NKR (NKR), which was naturally isolated from the human T lymphoblastoid cell line CEM. Although NKR cells express both CD4 and CXCR4, HIV-1 replication is severely restricted from the 2nd round of replication. From the original NKR cells, we isolated three types of clones that show different levels of HIV-1 resistance: non-permissive (NP), semi-permissive (SP), and permissive (P). We then compared wild-type (WT) and Vpr-deficient ( Vpr) HIV-1 replication in these cells. In non-permissive cells, both WT and Vpr viruses were unable to replicate. Notably, a treatment with arsenic trioxide (As2O3) increased the WT virus replication by almost 1000-fold, but did not promote the Vpr virus replication. Similarly, although the WT virus could replicate in the semi-permissive and permissive cells, the Vpr virus replication was completely inhibited in the semi-permissive cells and significantly delayed in the permissive cells. These results suggest that Vpr is absolutely required for HIV-1 replication in the non-permissive and semi-permissive cells. Thus, we have identified Vpr-specific HIV-1 non-permissive human CD4+ T cell line, which represents an important progress in the Vpr field. Our objective is to decipher the mechanism of how Vpr enhances HIV-1 replication in NKR cells. Our hypothesis is that NKR cells may express a Vpr-sensitive restriction factor to block viral replication, or lack a Vpr-like positive factor to support viral replication. Our rationale is that these NKR cells provide a relevant model system for studying Vpr function, which will be useful for further characterization of Vpr activity in vivo. We propose three specific aims: 1) Delineate how Vpr enhances HIV-1 replication in NKR cells; 2) Identify the host factor in NKR cells that is responsible for Vpr- dependent HIV-1 replication; 3) Elucidate the relevance of HIV-1 restriction in NKR cells for HIV-1 biology. We will define the enigmatic role of Vpr in viral life cycle. The discovered new mechanism will be likely translated into innovative tools for antiretroviral therapy.

描述（申请人提供）：Vpr和vpx是在灵长类慢病毒中表达的两种病毒辅助基因，是体内病毒复制和疾病进展所必需的。 Vpr 在体外具有两种众所周知的活性：将细胞周期阻滞在 G2 期和增强单核细胞来源的巨噬细胞中的病毒复制。 Vpx 与 Vpr 具有约 25% 的同源性，仅具有病毒增强活性。尽管 Vpr 和 Vpx 都能增强 HIV-1 复制，但涉及不同的机制。虽然 Vpx 抵消 SAMHD1 的抗逆转录病毒活性，但 Vpr 增强病毒复制的机制仍不清楚。最近，我们报道了人 CD4+ T 细胞系 CEM.NKR (NKR) 中的有效 HIV-1 限制，该细胞是从人 T 类淋巴母细胞系 CEM 中自然分离出来的。尽管NKR细胞同时表达CD4和CXCR4，但HIV-1复制从第二轮复制开始就受到严格限制。从原始的 NKR 细胞中，我们分离出三种类型的克隆，它们表现出不同水平的 HIV-1 抗性：非许可型 (NP)、半许可型 (SP) 和许可型 (P)。然后我们比较了这些细胞中野生型 (WT) 和 Vpr 缺陷型 (Vpr) HIV-1 的复制。在不允许的细胞中，WT 和 Vpr 病毒都无法复制。值得注意的是，三氧化二砷 (As2O3) 处理使 WT 病毒复制增加了近 1000 倍，但并没有促进 Vpr 病毒复制。同样，虽然WT病毒可以在半允许和允许细胞中复制，但Vpr病毒复制在半允许细胞中完全受到抑制，并且在允许细胞中显着延迟。这些结果表明，Vpr 对于 HIV-1 在非许可型和半许可型细胞中的复制是绝对必需的。因此，我们鉴定了Vpr特异性HIV-1非许可型人类CD4+ T细胞系，这代表了Vpr领域的重要进展。我们的目标是破译 Vpr 如何增强 NKR 细胞中 HIV-1 复制的机制。我们的假设是 NKR 细胞可能表达 Vpr 敏感的限制因子来阻止病毒复制，或者缺乏 Vpr 样支持病毒复制的积极因素。我们的理由是这些 NKR 细胞为研究 Vpr 功能提供了一个相关的模型系统，这将有助于进一步表征体内 Vpr 活性。我们提出了三个具体目标：1）描述Vpr如何增强NKR细胞中的HIV-1复制； 2) 鉴定NKR细胞中负责Vpr依赖性HIV-1复制的宿主因子； 3) 阐明NKR细胞中HIV-1限制与HIV-1生物学的相关性。我们将定义 Vpr 在病毒生命周期中的神秘作用。发现的新机制可能会转化为抗逆转录病毒治疗的创新工具。