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 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, Vpx, and Nef, which are unique to primate lentiviruses. There is now strong evidence that these proteins operate in conjunction with specific host factors. They do not have enzymatic activity but instead 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. We reported that hMRC1 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 not virus isolate specific, its effect on viral infectivity appears to affect primarily R5-tropic viruses. Experiments are ongoing to understand the mechanistic basis. 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 a myeloid-specific transcription factor that regulates the mannose receptor promoter. Interestingly, while Tat activates HIV-1 gene expression through a positive feedback loop, the myeloid-specific cellular transcription factor 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 the myeloid-specific transcription factor. 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. Aside from our work on hMRC1, we continued research on Vif, Vpu, Vpr, and Vpx to understand their importance for HIV-1 and HIV-2 replication. With regard to Vif, we worked on two separate issues. First, we continued to study the mechanism of dominant negative interference by certain Vif mutants. We found that only mutants that retained the ability to bind CBF were capable to interfere with the function o wild type Vif. The significance of this observation is that it opens the possibility of designing drugs that can target the function of virus encoded Vif. Since Vif is critical for HIV-1 replication in vivo, such interference could impose a severe restriction on virus replication. Second, in collaboration with the Barahona lab in Portugal, we investigated the contribution of each member of the APOBEC3 (A3) family for the restriction of HIV-2. We found that A3G is a strong restriction factor of both HIV-1 and HIV-2 while A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against HIV-2 but its effect was negligible against HIV-1. Whereas A3B is packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, both viruses are able to replicate in host cells expressing A3B using different mechanisms of antagonism that likely resulted from distinct adaptation over evolutionary time. 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.