Molecular characterization of interferon-resistant HIV-1 using novel imaging techniques

HIV-1 is a causative pathogen that can cause AIDS in humans. Although type I interferon (IFN) is known to suppress viral replication both in the early and in the late stage of HIV-1’s replication cycle, the molecular mechanism of inhibition remains to be elucidated. Treatment of cells with IFN leads to upregulation of IFN-inducible genes encoding anti HIV-1 host factors such as BST-2 (Tetherin), SAMHD1 and Mx2. We have been interested in the mechanism of suppression imposed by IFN-β treatment in the early stage (steps between entry of viral core into the cytosol and integration into host genome) of HIV-1 infection. Recent studies suggest that the early stage of HIV-1 infection is blocked by Mx2 but also by unidentified mechanisms in IFN-β-treated cells. Consistent with the findings, our preliminary experiments show that HIV-1 infection is blocked by Mx2-indepenent mechanism in IFN-β-treated cells. In this research proposal, we want to clarify the underlying mechanisms that control the IFN-β sensitivity. Our biggest advantage is that we already obtained mutant viruses that are partially/completely resistant to IFN-β treatment. These mutants contain mutations in the capsid protein (CA). CA orchestrates multiple step of HIV-1’s replication cycle including uncoating, reverse transcription, nuclear entry and integration of pre-integration complex into host cell chromatin. We became interested in relationship between uncoating kinetics and IFN-β sensitivity. Our hypothesis is that IFN-β-resistant CA mutants alter the uncoating kinetics in order to change the timing or localization not to be exposed to inhibitory factor. Therefore, the studies proposed here are intended to fill a critical void in our understanding of molecular mechanism of IFN-mediated inhibition of HIV-1. First, we will examine the uncoating kinetics of these CA mutants using newly established, novel live-cell imaging system. Second, we will test the sensitivity of CA to mutants in primary cells such as CD4+ lymphocytes and macrophages. Last, we investigate the mechanism of evasion. The information generated in these studies will substantially alter our view of IFN-β-mediated inhibition of HIV-1 and also lead to a discovery of novel strategy against HIV epidemic.