Infection of myeloid cells by HIV-2 and SIVsm are restricted at the reverse transcription step unless viral protein X (Vpx) is expressed. Binding of Vpx to the Cul4a-DDB1-DCAF1 ubiquitin ligase was shown to be important to overcome this restriction. It is hypothesized that, in the presence of Vpx, a cellular restriction factor is rapidly ubiquitinated and targeted for proteasome degradation. The goal of this project is to identify cellular restrictive factor(s) targeted by Vpx. This study will utilize candidate restrictive factors identified in previous molecular biology screens, including proteins, identified in the yeast 2-hybrid assay, that interact with Vpx or DCAF1, proteins, found by 2D differential gel electrophoresis, that are over-expressed in myeloid cells infected with HIV-2 Vpx compared to wild type HIV-2, and cDNA clones identified from a functional screen in 293T cells for factors that restrict HIV infection in the absence but not the presence of Vpx. We will also examine members of the Apobec3 family for these activities, based on data that many family members restrict HIV-1 or SIV replication, and Apobec3A binds Vpx.
Aim 1. Analyze candidate restriction factors for the ability to restrict HIV-2 Vpx but not wild type HIV-2. Potential restriction factor cDNAs will be examined for the ability to inhibit infection of HeLa cells by HIV-2 Vpx but not wild type HIV-2. The cDNAs showing this activity will then be examined for their effects on HIV-2 reverse transcription, using real time RT-PCR.
Aim 2. Examine effects of a proteasome inhibitor and a shRNA to DCAF1 on expression of potential Vpx-regulated restriction factors. A 293T cell line in which Vpx expression is induced with insect hormone muristerone will be used to assess effects of Vpx, bortezomib, and shRNA to DCAF1 on the level of expression of potential restriction factors identified in Aim 1.
Aim 3. Examine effects of shRNA to potential Vpx-regulated restriction factor on infection of macrophages. shRNAs to restriction factors identified in Aim 1 will be expressed in macrophages together with a dsRed marker. Levels of infection of HIV-2gfp Vpx compared to HIV-2gfp will be examined in dsRed positive macrophages, to determine if the shRNA relieves the restriction to HIV-2 in the absence of Vpx.
This study will identify cellular factor(s) that limit HIV-2 infection of myeloid cells, but are overcome by viral protein X (Vpx). It is hypothesized that Vpx promotes the degradation of the cellular restriction factor(s). Several potential cellular restriction factors have been identified from molecular biology screens, and their effects on HIV-2 infection of permissive HeLa cells will be examined in the presence or absence of Vpx. Potential restriction factors that are overcome by Vpx will be further examined for the effects of Vpx on their protein stability. In addition, we will determine if small interfering RNA to the potential restriction factor(s) overcomes the block to infection of myeloid cells by HIV-2 lacking Vpx. These experiments are important in identifying a novel mechanism of HIV restriction.
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