HIV-1 integrase (IN) has an essential, multifunctional role in virus replication and serves as an important therapeutic target. During the early stage of HIV-1 replication IN catalyzes the covalent insertion of viral cDNA into the host chromosome. This process is mediated by cellular chromatin associated protein LEDGF/p75, which both markedly enhances integration efficiency and preferentially guides HIV-1 integration to actively transcribed genes. Structural interactions between HIV-1 IN and LEDGF/p75 have been well characterized, but until recently little was known about how LEDGF/p75 recognizes select chromatin sites. During the past funding cycle we have elucidated structural and mechanistic basis for preferential and tight binding of the PWWP domain, the key chromatin binding module of LEDGF/p75, to cognate mononucleosomes (MNs). In particular, we have demonstrated that cooperative binding of the hydrophobic cavity and basic surface of LEDGF PWWP to the histone H3 tail containing trimethylated Lys36 (H3K36me3) and DNA wrapped in MNs is essential for high-affinity binding of LEDGF/p75 to chromatin. These studies will now be extended in aim 1 to characterize the interplay between full-length LEDGF/p75, H3K36me3 epigenetic marks, and HIV-1 integration sites in vitro and in infected cells.
Aim 2 of the present application will focus on testing the hypothesis that HIV-1 IN plays an active role during the viru particle maturation. The rationale for this notion was provided by previous findings that certain substitutions in the IN coding region or addition of allosteric IN inhibitors induce eccentric maturation of virus particles. A hallmark of this eccentric phenotype is the mislocalization of the ribonucleoprotein complexes (RNPs) in mature virions. While infectious virions contain RNPs embedded within the capsid core, the eccentric particles have RNPs localized between the empty or translucent capsid core and the particle membrane. These experiments have highlighted a potential role of IN during the late steps of virus maturation. However, little is known as to how IN could contribute to proper particle morphogenesis. Here, we present our preliminary data showing that HIV-1 IN interacts directly and with high affinity with highly structured viral RNA segments in vitro and in virus particles. These preliminary findings suggest previously undescribed roles of HIV-1 IN during virus particle maturation and provide compelling rationale for our structural and virology studies proposed in aim 2. The proposed experiments are expected to fill the critical gap in our knowledge of how multifunctional IN contributes to the late stage of HIV-1 replication.
HIV-1 integrase plays an essential role in viral replication and serves as an important therapeutic target. We propose to investigate structural interactions and multifunctional role of HIV-1 integrase in the virus lifecycle. The findings of our studies will hep us to better understand the mechanism of action of allosteric HIV-1 integrase inhibitors, which are currently in clinical trials, as well as to develop new antiretroviral therapies.
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