Molecular Analysis Of Retroviral Genes And Their Products

National Institute Of Allergy And Infectious Diseases

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Abstract

HIV-1 encodes genes that are crucial for replication in primate cells exerting functions not provided by the host. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is now strong evidence that these proteins operate in conjunction with specific host factors. In fact, none of the HIV accessory proteins has a known enzymatic activity. Instead, these proteins function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. Over the past few years, our research focus has shifted more and more towards the characterization of host factors and their roles in virus replication. One of the factors we recently identified is human mannose receptor I (hMRC1), a protein expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. HMRC1 contributes to the binding of HIV-1 to monocyte-derived macrophages and is involved in the endocytic uptake of HIV-1 into these cells. In FY18 we continued a project characterizing the function of hMRC1 in the control of HIV-1 replication in macrophages. We identify hMRC1 as a novel macrophage-specific restriction factor that inhibits virus release through a BST-2-like mechanism. Virions produced in the presence of hMRC1 accumulated in clusters at the cell surface but were fully infectious. HIV-1 counteracted the effect by transcriptional silencing of hMRC1. Surprisingly, deletion of the Env protein, which is known to interact with hMRC1, did not relieve the hMRC1-imposed restriction suggesting the involvement of additional cellular factor(s) in the process. Our data reveal a novel host restriction mechanism that is active in primary human macrophages and is counteracted by HIV-1 through downregulation of hMRC1. These data have recently been published in Cell Reports (Sukegawa et al., 2018). Efforts are ongoing to identify and characterize the additional cellular factors involved in hMRC1-mediated restriction of virus release using proteomics approaches. In addition, we identified a second effect of hMRC1 that influences the infectivity of viruses produced in the presence of hMRC1. Unlike the effect on virus release, this effect of hMRC1 is virus-specific and affects primarily R5-tropic virus isolates. We have initiated studies to determine the possible involvement of chemokine receptors in this process. Finally, we continue our effort to fully understand the mechanism of HIV-mediated transcriptional silencing of hMRC1. We have systematically ruled out the contribution of most HIV-encoded accessory proteins, including Vpr, and are now focusing on other viral components. Aside from our work on hMRC1, we continued our characterization of proteins we identified in the process of a proteomic analysis of Vpr-interacting host factors. We have studied the effects of transient over-expression of these factors on HIV replication. We are now in the process of assessing the effects of silencing these genes. Since Vpr effects are typically not seen in immortalized cell lines, we have put extra effort into establishing CRISPR/Cas9-based knockout technology to allow measuring effects of our candidate genes during spreading infection of HIV-1 in primary cells. Our efforts have progressed to the point where we are ready to use primary cells (i.e. PBMC and monocyte-derived macrophages) for our experiments. Aside from knocking out individual host factors identified in our proteomics approach, we have constructed defined mutants of Vpr in the backbone of the R5-tropic AD8 HIV-1 isolate. These mutants will enable us to assess the importance of Vpr-mediated cell cycle arrest or DCAF1-dependent protein degradation for virus replication in primary cell types. Overall, we expect from these experiments important insights into the still poorly understood function of Vpr.