Molecular Analysis Of Retroviral Genes And Their Products

National Institute Of Allergy And Infectious Diseases

Investigators

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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. In FY17 we continued projects related to Vif and its interaction with the host proteins APOBEC3G (A3G) and CBF. With respect to A3G, we initiated a project to study the functional relevance of the A3G N-terminal domain. One of the proposed functions of this domain is to target A3G to HIV-1 virions where it exerts its antiviral effects. We bypassed the need for packaging signals by tagging A3G with the HIV-1 Vpr, which allows for packaging into virions through an interaction with HIV-1 Gag. Preliminary data indicate that aside from targeting A3G into virions, the N-terminal domain plays additional functions critical to the antiviral activity of A3G. We are in the process of studying biochemical and biophysical properties of virus-associated A3G to identify functionally important motifs in A3G. With regard to CBF, we previously reported that it acts as a molecular chaperone to enhance Vif biosynthesis, to stabilize mature Vif protein, and to facilitate the assembly of an A3G-Vif-Cul5 E3 ligase complex that overall results in more efficient degradation of A3G. Our studies to address the role of CBF in the dominant-negative properties of specific Vif mutants point to a positive correlation between the ability of Vif variants to bind CBFb and their dominant-negative properties. We are in the process of preparing a manuscript reporting our findings. In FY17, we also continued a project involving the characterization of HIV and SIV Vpr proteins and their contributions to the control of host restriction factors. We performed mass spectrometry to identify Vpr-interacting host factors using conditions that avoided the loss of potential targets of Vpr by protein degradation. We identified several proteins and confirmed specific binding to Vpr by co-IP analysis for several of them. When transiently expressed in mammalian cells, wild type Vpr but not a DCAF1-binding mutant (Q65R) induced a reduction of steady-state expression of some of these proteins. We have produced antibodies to three candidate proteins and will use them to perform functional assays. We further initiated a project characterizing the function of mannose receptor I (hMRC1) in the control of HIV-1 replication in macrophages. hMRC1 is 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. 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. The effect of hMRC1 was not virus-specific. 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 been submitted for publication.