Integrase (IN) is one of three virus-encoded enzymes that are essential for retroviral replication and a validated target for the development of drugs t treat HIV/AIDS. Although there has been success in developing clinically useful drugs that block the final step in the integration reaction, the so-called strand transfer inhibitors, there is a continuing need to augment or replace existing IN therapeutics as viral resistance is encountered. A detailed knowledge of all aspects of the structure, assembly, and catalysis by HIV-1 IN, will reveal unexploited vulnerabilities and novel strategies for inhibiting this critical enzyme. In the current funding period, we applied small angle X-ray scattering (SAXS) and protein-protein cross-linking methods to obtain the first experimentally-derived models of full-length unliganded apo-IN monomers and dimers in solution, using avian sarcoma virus (ASV) IN. The results revealed a dimer architecture (called a reaching dimer) that was previously unsuspected. The configuration of the reaching dimer resembles that of the viral DNA-binding, inner dimer in the crystal structure of the prototype foamy virus (PFV) IN. From these and other data, we have constructed a structural model for an HIV IN reaching dimer, which we hypothesize is pre-positioned to interact with viral DNA ends.
In Aim 1 of this competitive renewal we propose to test this model by determining the solution structures of monomers, dimers, and tetramers of HIV IN, using methods successfully employed with ASV IN. We will identify the interactions that stabilize HIV dimers and determine the effects of substrate binding on their conformation.
In Aim 2 we will identify compounds that alter the stability of HIV apo-IN dimers and inhibit the conformational changes that are required for IN function. The results of our studies will provide critical new information concerning HIV IN structure and function, and contribute to the design of new, allosterically-acting drugs that can complement the active site inhibitors now in clinical use.
HIV-1 integrase is an important target for drugs to treat HIV/AIDS. Although one active-site inhibitor is FDA-approved for this purpose and a second is in advanced clinical trials, the inevitable development of drug resistant HIV mutants drives a continuing need for additional strategies to block the activity of this viral enzyme. Knowledge gained from the proposed studies will lead to the development of a new class of allosteric inhibitors of integrase to complement the present arsenal of AIDS therapies.
|Balasubramanian, Sangeetha; Rajagopalan, Muthukumaran; Bojja, Ravi Shankar et al. (2017) The conformational feasibility for the formation of reaching dimer in ASV and HIV integrase: a molecular dynamics study. J Biomol Struct Dyn 35:3469-3485|
|Andrake, Mark D; Skalka, Anna Marie (2015) Retroviral Integrase: Then and Now. Annu Rev Virol 2:241-64|
|Benleulmi, Mohamed Salah; Matysiak, Julien; Henriquez, Daniel Rodrigo et al. (2015) Intasome architecture and chromatin density modulate retroviral integration into nucleosome. Retrovirology 12:13|
|Skala, Anna Marie (2014) Retroviral DNA Transposition: Themes and Variations. Microbiol Spectr 2:|
|Skalka, Anna Marie (2014) Retroviral DNA Transposition: Themes and Variations. Microbiol Spectr 2:MDNA300052014|
|Bojja, Ravi Shankar; Andrake, Mark D; Merkel, George et al. (2013) Architecture and assembly of HIV integrase multimers in the absence of DNA substrates. J Biol Chem 288:7373-86|
|Shalginskikh, Natalia; Poleshko, Andrey; Skalka, Anna Marie et al. (2013) Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx. J Virol 87:2137-50|
|Bojja, Ravi S; Andrake, Mark D; Weigand, Steven et al. (2011) Architecture of a full-length retroviral integrase monomer and dimer, revealed by small angle X-ray scattering and chemical cross-linking. J Biol Chem 286:17047-59|
|Katz, Richard A; Merkel, George; Andrake, Mark D et al. (2011) Retroviral integrases promote fraying of viral DNA ends. J Biol Chem 286:25710-8|
|Peletskaya, Elena; Andrake, Mark; Gustchina, Alla et al. (2011) Localization of ASV integrase-DNA contacts by site-directed crosslinking and their structural analysis. PLoS One 6:e27751|
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