Probing the Mechanism of HIV-1 Gag Localization to Transcription Sites

Abstract Infection with the human retrovirus HIV-1 is the cause of AIDS, a global pandemic that has claimed the lives of over 44 million people worldwide. Significant progress has been made in the treatment of AIDS, yet the emergence of drug-resistant HIV-1 strains continues, supporting the need for ongoing efforts to develop novel therapies. There are no drugs in clinical use that target the incorporation of the viral genome into nascent virus particles, a process that is directed by the HIV-1 Gag protein. HIV-1 particle assembly is initiated when Gag binds to unspliced viral RNA, which serves as the viral genome. The mechanism by which HIV-1 Gag locates and binds to genomic RNA in infected cells is poorly understood. The traditional model of genomic RNA packaging posits that Gag-viral RNA recognition occurs in the cytoplasm or at the plasma membrane. However, our laboratory and others have observed that the Gag proteins of several retroviruses, including HIV- 1, localize to the nucleus. Because the highest concentration of HIV-1 unspliced RNA is at the viral transcription site, it is reasonable to postulate that Gag could enter the nucleus to bind the genome to initiate packaging. Published and preliminary studies in support of this idea are presented in this proposal, including our finding that HIV-1 Gag binds to unspliced viral RNA at the transcriptional burst site in T cells reactivated from latency. Treatment with transcription inhibitors increased Gag-viral RNA interactions, suggesting that the viral ribonucleoprotein complex forms co-transcriptionally. We and others identified transcription-related host factors in the HIV-1 Gag interactome, including Mediator proteins required for RNA polymerase II gene expression. To gain insight into role of Mediator proteins in the function of nuclear HIV-1 Gag, we propose an innovative set of experiments in this exploratory R21 research proposal to test the hypothesis that Gag co-opts members of the Mediator complex to traffic to the site of nascent viral RNA production. To test this hypothesis, we propose two specific aims. In Aim 1, we will characterize the interaction between HIV-1 Gag and Med26 using advanced confocal microscopy, mapping of interaction sites, biophysical methods, and functional assays. In Aim 2, we will characterize the interactions between HIV-1 Gag and other Mediator proteins that have previously been identified in mass spectrometry experiments as potential Gag binding partners. We will perform knockdown studies to examine the role of Mediator proteins in Gag-viral RNA nuclear colocalization and genomic RNA packaging. Together, these novel, exploratory experiments will advance our understanding of the HIV-1 Gag nuclear interactome and investigate the potential role of Mediator complex members in facilitating Gag-genomic RNA interactions.