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.
The proposed research is relevant to public health, because it will discover novel antiretroviral mechanisms and/or drug targets, which is the first step in a continuum of research expected to lead to the development of new pharmacologic strategies for blocking HIV-1 infection.
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