Regulation of post-integrational events I HIV-1 replication and implications for novel therapy and eradication

Abstract

In 1996 a breakthrough therapy of combining three antiretroviral drugs, combination antiretroviral treatment (cART) made HIV-1 a manageable illness. Despite this pivotal advance in clinical care, a cure for HIV-1 has remained unattainable. In aviremic patients on CART, HIV-1 persists in latent reservoirs in the form of integrated provirus, which upon reactivation produces infectious virions. Elimination of these latent reservoirs is the main focus of national efforts to eradicate HIV-1.;Our studies focus on the role of a Pol- and Integrase-binding host protein INI1/hSNF5 during HIV-1 late events. Previous studies show that INI1 mutants inhibit assembly and particle production, however the mechanism of inhibition is not understood.;Our hypothesis is that INI1/hSNF5 acts as an adaptor protein that facilitates multiple post-transcriptional and post-translational events by bridging the interaction of viral proteins with cellular host factors required for these events. Therefore interfering with INI1's binding capabilities may lead to defects in HIV-1 replication.;INI1 binds the Sin3a-associated 18kDa (SAP18) protein. Here we tested SAP18Interaction-defective (SID) mutants of INI1 during HIV-1 replication. SID-INI1 mutants dramatically inhibited HIV-1 particle production, due to a cumulative effect on gag RNA levels, intracellular Gag/Gag-Pol levels and trafficking. INI1 knockdown resulted in a decrease of viral Gag/GagPol protein, trafficking and particle production. These results indicated that INI1 influences several post-transcriptional events.;A proteomic screen identified INI1-binding proteins Nucleolin, hnRNPU, ILF3 and Staufen1. These proteins have been shown to influence HIV-1 RNA metabolism and assembly. These novel interactions strengthen our hypothesis that INI1 may be acting as an adaptor molecule.;To visualize transcriptional and post-transcriptional events of HIV-1 replication within the same cell, we developed a novel Single-cell single molecule Multiplex Immunofluorescence and RNA-fluorescence in situ hybridization Assay (SM IRA). Using SM IRA, we quantitated the number of viral transcripts per cell and assessed reactivation kinetics of HIV-1 in a latency cell line model (ACH-2). We believe that SMIRA can be used to study the biology of HIV-1 latency, the kinetics of its reactivation and could be used as an effective drug screening tool, as well as a diagnostic to measure the size of latent reservoirs within patient samples.