Host protein targets of HIV-1 Vpr in gene expression, cell cycle and innate immunity

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The role and mechanism of action of Vpr (Viral Protein R), an accessory protein encoded by HIV- 1, has been enigmatic for decades. Vpr causes cell cycle arrest at G2/M, triggers a DNA damage response, and enhances viral gene expression. It exerts these activities by targeting host protein(s) for degradation, hijacking cullin4-based E3 ubiquitin ligase complex (CRL4) to induce their depletion. We recently identified a host protein CCDC137, also known as cPERP-B, as a key target protein depleted by Vpr in a CRL4 complex dependent manner. Specifically, CCDC137 depletion by RNA interference recapitulates the aforementioned effects of Vpr on host and virus. In this project we seek to study the molecular details of how CCDC137 represses HIV-1 gene expression as well as how it controls cell cycle progression and the DNA damage response. In Aim 1, we will determine whether CCDC137 depletion is a conserved feature of Vpr proteins from diverse HIV and SIV strains, map the CCDC137 determinants required for Vpr-induced depletion, and assess the effect of Vpr from diverse viruses on viral gene expression. In addition, we will define host proteins required for CCDC137 depletion by Vpr. Aim 2 is centered on the mechanisms of CCDC137-mediated repression of HIV-1 gene expression. We will delineate cis- acting sequences required for CCDC137-mediated repression and evaluate the effect of integration and integration site selection on the Vpr/CCDC137-regulated HIV-1 gene expression. We will also combine screening methods (proteomics, yeast 2-hybrid, and CRISPR functional screens) to identify CCDC137 interacting cofactor(s) to illuminate the mechanism of how CCDC137 inhibits HIV-1 gene expression. In Aim 3 we will investigate how CCDC137 prevents DNA damage response and controls cell cycle progression. In particular, we will determine whether CCDC137 protects chromosomal DNA and delineate host factor(s) cooperating with CCDC137 to modulate the DNA damage response.