Molecular Analysis Of Retroviral Genes And Their Products

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications, trials & patents

Abstract

HIV-1 encodes genes that are crucial for replication in primary 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 to us are the HIV accessory proteins Vif, Vpr, Vpu, and Vpx, which are unique to primate lentiviruses. There is strong evidence that these proteins operate in conjunction with specific host factors. Accessory proteins do not have enzymatic activity but function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. Over the past decade, our research focus has shifted more and more towards the characterization of host factors and their roles in virus replication. HIV-1 targeted host factors we are currently investigating are (i) human mannose receptor I (hMRC1), a protein expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells; (ii) gelsolin, an actin-binding protein known to affect actin dynamics in the cell; and (iii) PU.1, a myeloid-specific transcription factor that which we found to be inhibited by HIV-1 Tat. PU.1 is known to regulate expression of hundreds of cellular genes and may therefore represent a key factor for HIV-dependent host gene regulation. With respect to hMRC1, we reported that it inhibits the detachment of progeny viruses from infected macrophages in a manner that is phenotypically similar to the effect exerted by another host factor, BST-2, but is mechanistically distinct. Our continued investigations revealed that hMRC1 can also affect viral infectivity. Interestingly, while the effect of hMRC1 on virus detachment is virus isolate independent, its effect on viral infectivity appears to affect primarily X4-tropic viruses. We found that even though hMRC1 is packaged into virions in an Env-independent manner, X4 tropic Env can interact with hMRC1 via the V3 loop sequence in Env. We also continued our work on characterizing the mechanism by which HIV-1 infection of macrophages reduces the expression of hMRC1. We have now clear evidence that HIV-1 Tat is a main contributor to this process. We identified a complex interplay between HIV-1 Tat and the myeloid-specific transcription factor PU.1 that regulates the mannose receptor promoter. Interestingly, while Tat activates HIV-1 gene expression through a positive feedback loop, PU.1 is involved in a negative feedback loop that acts on the HIV-1 LTR promoter and inhibits viral gene expression, including Tat. Tat, on the other hand, inhibits the activity of PU.1. Thus, we found that there is a complex equilibrium between viral and host gene expression in HIV-infected macrophages. Indeed, disturbing this equilibrium has opposing effects on HIV-1 and hMRC1 gene expression. Our work has important implications on understanding how HIV-1 gene expression is regulated and we are conducting experiments to see if the regulatory feedback loops identified in our study contribute to the still poorly understood phenomenon of viral latency. In the past year we have continued our work on the detailed characterization of promoter elements responsible for PU.1 responsiveness using electrophoretic gel mobility assays (EMSA). We found that the hMRC1 promoter contains three PU.1 binding sites (PU.1 boxes). Deletion or mutations of these PU.1 boxes severely hampered PU.1 responsiveness. Interestingly we found that one binding site in particular was critical for PU.1-based activation of the hMRC1 promoter while the other two PU.1 binding sites were less important. Finally, transfer of the PU.1 boxes to a heterologous promoter rendered that promoter PU.1 responsive. Binding sites for PU.1 can be found in multiple cellular promoters, incl. the promoter for the MCSF receptor (MCSF-1R) gene. MCSF receptor mediated signaling is crucial for the survival, function, proliferation, and differentiation of myeloid lineage cells, including monocytes/macrophages. Experiments are ongoing to see if and how expression of the MCSF receptor is affected by PU.1 and, consequently, by HIV-1 infection. With regards to gelsolin, we completed and published a study showing that GSN is a macrophage-specific host antiviral factor that reduces expression of HIV-1 Env, which is counteracted by Vpr during infection through proteasome-mediated degradation of GSN. Gelsolin (GSN) is a structural actin-binding protein known to affect actin dynamics in the cell. We identified GSN as a novel Vpr-interacting protein. Endogenous GSN protein was expressed at detectable levels in monocyte-derived macrophages and THP-1 cells but was undetectable at the protein level in multiple other cell lines tested. HIV-1 infection of MDM was associated with a reduction in GSN steady-state levels, presumably caused by Vpr-induced degradation of GSN. Indeed, co-expression of GSN and Vpr in transiently transfected HEK293T cells resulted in the DCAF1-dependent degradation of GSN. This effect was observed for Vprs from multiple virus isolates. Overexpression of GSN in HEK293T cells had no effect on Gag expression and particle release but reduced expression and packaging of the HIV-1 Env glycoprotein resulting in reduced viral infectivity. Analysis of the HIV-1 splicing patterns did not reveal any GSN dependent differences suggesting that the effect of GSN on Env expression was regulated at a post-transcriptional level. Indeed, treatment of transfected cells with lysosomal inhibitors reversed the effect of GSN on Env stability suggesting that GSN reduced Env expression via enhanced lysosomal degradation. With regard to Vpr and Vpx, we have started a collaboration with the Taveira lab in Portugal who have provided us with samples from multiple HIV-2 infected individuals. We have cloned the vpr and vpx genes from these individuals and experiments are ongoing to study their functional properties in comparison to the functional properties of lab-adapted isolates. Importantly, since HIV-2 encodes both Vpr and Vpx, we will compare matched pairs of Vpr and Vpx from primary HIV-2 isolates to determine if Vpr and Vpx can potentially adopt redundant biological activities.