Vpr

描述（由申请人提供）：Vpr和vpx是灵长类慢病毒中表达的两个病毒附属基因，是病毒在体内复制和疾病进展所必需的。Vpr在体外有两个众所周知的活性：在G2期阻滞细胞周期和增强单核细胞源性巨噬细胞中的病毒复制。Vpx与Vpr同源性约25%，仅具有病毒增强活性。虽然Vpr和Vpx都能增强HIV-1的复制，但涉及不同的机制。虽然Vpx可以抵消SAMHD1的抗逆转录病毒活性，但Vpr增强病毒复制的机制尚不清楚。最近，我们报道了人类CD4+ T细胞系CEM中HIV-1的有效限制。NKR (NKR)，从人T淋巴母细胞样细胞系CEM中自然分离得到。尽管NKR细胞同时表达CD4和CXCR4，但从第二轮复制开始，HIV-1的复制受到严重限制。从原始的NKR细胞中，我们分离出三种类型的克隆，显示出不同水平的HIV-1抗性：非允许（NP），半允许（SP）和允许(P)。然后，我们比较了野生型（WT）和Vpr缺陷型（Vpr） HIV-1在这些细胞中的复制。在非受纳细胞中，WT和Vpr病毒都不能复制。值得注意的是，三氧化二砷（As2O3）处理使WT病毒的复制增加了近1000倍，但没有促进Vpr病毒的复制。同样，虽然WT病毒在半允许细胞和允许细胞中都可以复制，但Vpr病毒在半允许细胞中的复制被完全抑制，在允许细胞中的复制被显著延迟。这些结果表明，Vpr对于HIV-1在非允许和半允许细胞中的复制是绝对必需的。因此，我们鉴定出了Vpr特异性HIV-1非允许性人CD4+ T细胞系，这是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细胞可能表达一种对病毒复制敏感的限制因子来阻止病毒复制，或者缺乏一种类似于病毒复制的限制因子

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.