Currently there are no FDA approved drugs targeting viral assembly to combat AIDS. Our long-term goal is to develop a new class of drugs that target Gag-Pol to disrupt integrase (IN)-host protein interactions to inhibit early stages of HIV assembly. During HIV-1 late events, the nascent viral proteins traffic through the cytoplasm to the plasma membrane for assembly and budding. Gag is necessary and sufficient for assembly in vitro. However, genetic studies reveal that mutations in the Integrase (IN) region also leads to defects in particle morphology, virion release, as well as defect in subsequent events of infection. Furthermore, recent work demonstrates that allosteric inhibitors of IN inhibit particle morphogenesis and infectivity. We have shown that mutants of IN defective for binding to a host factor Integrase interactor 1 (INI1)/hSNF5 potently inhibit particle morphogenesis, indicating that disrupting integrase to inhibit assembly is a feasible strategy. INI1/hSNF5 is a component of the SWI/SNF chromatin-remodeling complex that binds to IN. Our studies indicate an essential role for INI1 in HIV-1 assembly. Interfering with INI1 function potently inhibits (>1000 fold) assembly by blocking Gag/Gag-Pol trafficking and stability. INI1 is an adaptor protein and we hypothesize that it bridges interaction of assembly intermediates with essential cellular components to facilitate proper trafficking of the Gag/GagPol to the plasma membrane. New insights reveal that proteins that influence Rev-dependent HIV-1 RNA export are associated with INI1, linking assembly to RNA export. We propose that strategies to target IN-INI1 interactions will lead to inhibition of unique and early steps of assembly namely, Gag trafficking and stability.
In Aim I, we will investigate the mechanism by which INI1 facilitate HIV-1 assembly and how INI1 mutants disrupt this process. We will determine if interfering with INI1 inhibits both RNA export and Gag trafficking.
In Aim II, we will determine how HIV-1 replication influences INI1 nuclear export, as this function of INI1 appears is necessary for mediating assembly.
In Aim III, we will translate our observations into developing a novel class of drugs targeting IN-INI1 interactions to inhibit Gag cytoplasmic trafficking. We will carry out molecular dynamics (MD) homology-based computational modeling to predict the structure of minimal IN binding domain (S6), generate peptidomimetics based on the structure, establish high throughput screening (HTS) assays based on AlphaScreen, and screen for compounds to disrupt IN-INI1 interaction.
Despite advances in the treatment of HIV-1 infection and the widespread use of anti-retrovirals, the AIDS pandemic remains unabated and warrants the continued development of novel antiretroviral therapeutics to combat HIV. The current application is to investigate novel interactions between the viral and cellular proteins to establih a new drug target, namely intracellular trafficking of viral proteins during early stages of HIV-1 virus particle formation. Our studies not only will shed new light on the mechanism of early stages of virus particle formation, but also pave the way to isolate drugs that target this stage t potently inhibit HIV-1 replication.
|La Porte, Annalena; Kalpana, Ganjam V (2016) Quantification of HIV-1 Gag Localization Within Virus Producer Cells. Methods Mol Biol 1354:165-74|
|Bhutoria, Savita; Kalpana, Ganjam V; Acharya, Seetharama A (2016) Computational modeling of Repeat1 region of INI1/hSNF5: An evolutionary link with ubiquitin. Protein Sci 25:1593-604|
|La Porte, Annalena; Cano, Jennifer; Wu, Xuhong et al. (2016) An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability. J Virol 90:9889-9904|