Deciphering the Role of CPSF6 in HIV Infection

Project: Research project

Project Details


Cleavage and polyadenylation specificity factor 6 (CPSF6) is an HIV host factor recruited to incoming viral cores during the early stage of the lifecycle. The interaction between HIV capsid (CA) and CPSF6 is known to dictate the cellular determinants of nuclear translocation and influence integration site preference, but its impact on overall viral infectivity is unclear. While overexpression of cytoplasmic CPSF6 has been shown to restrict viral replication, knock-down of CPSF6 and disruption of the CA-CPSF6 interaction has minimal impact in most ex vivo replication models. Recently, our lab found that knock-out of CPSF6 in primary CD4+ T cells increases HIV replication over an order of magnitude with minimal impact on cell viability. This increase in replication correlates with decreased induction of interferon-stimulated genes. CPSF6, together with its binding partner CPSF5, forms the CFIm cleavage factor complex that acts as an enhancer-dependent activator to direct polyadenylation to distal sites of the 3’ untranslated region (UTR). Inhibition of CFIm activity triggers alternative polyadenylation (APA), which has been previously implicated in the regulation of the innate immune response. Notably, knock-out of CPSF5 in primary CD4+ T cells phenocopies CPSF6 knock-out, dramatically increasing HIV replication. In this proposal, we are testing the hypothesis that disruption of CFIm activity dampens the innate immune response to infection by triggering APA. This hypothesis will be tested in three aims that broadly seek to understand the impact of APA on the viral lifecycle, the impact of HIV infection on CFIm function, and the impact of APA on the innate immune response to HIV infection. In Aim 1, we will use genetic and chemical means to either induce or inhibit APA in primary CD4+ T cells and monocyte-derived macrophages (MDMs). The impact of these manipulations on each stage of the viral lifecycle will be quantified and the breadth of the phenotype assessed across different retroviruses. The impact of the host response will be determined by paired RNA sequencing and in vitro sequencing of APA sites (IVT-SAPAS). In Aim 2, we will characterize the impact of HIV infection on CFIm composition (by affinity purification mass spectrometry), localization (by immunostaining and live cell imaging), and function (by RNA-Seq and IVT-SAPAS). Viruses containing CA mutants that are unable to bind CPSF6 will be used as controls. The impact of infection on CFIm function will likewise be assessed at the single cell level using single-cell RNA-Seq for added rigor. In Aim 3, we will we will explore the impact of APA on the innate immune response to infection. The kinetics of innate immune induction to HIV infection in primary CD4+ T cells and MDMs will be monitored in the presence and absence of CFIm function. The epistatic relationship between CPSF6 and innate immune sensors/signaling modulators will be determine by double knock-out. The role of other early-acting HIV host factors in this phenotype will likewise be assessed. Altogether, this work explores a newly described phenotype for a well-known HIV host factor, CPSF6, and seeks to provide a better understanding of viral manipulation of the innate immune response by post-transcriptional regulation.
Effective start/end date7/13/216/30/26


  • National Institute of Allergy and Infectious Diseases (5R01AI165236-03)


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.