This application is a revision to the awarded R01 1R01GM112520-01, Integrase Binding Proteins as Drug Targets to Inhibit HIV-1 Assembly. The reason for this revision is the availability of new NMR based solution structure for INI1. Availability of NMR structure enhances our ability to perform the goals of funded R01, whose Aims are as follows.
The Aim I is to understand the mechanism by which INI1 influences assembly;
Aim II is to understand the nuclear export properties of INI1; and the Aim III is to: (i) define the interaction of minimal IN binding domain of INI1 with IN using computational modeling and identify the interface residues involved in IN-INI1 interactions; and (ii) to screen for drugs/peptidomimetics that disrupt IN-INI1 interaction using Alpha Screen. However, NMR based structural determination of INI1 or IN-INI1 interactions are not part of the goals of the R01. The newly proposed supplemental studies will enhance our ability to perform the goals of Aims I and III, as described below. INI1 is an integrase binding protein that has been shown to influence multiple stages of HIV-1 replication including LTR-mediated transcription, assembly and particle production and integration. In addition, INI1 is an epigenetic regulator and a component of SWI/SNF complex involved in chromatin remodeling. It is a tumor suppressor mutated in large number of human cancers. Despite its wide spread importance in cancer, epigenetic and transcriptional regulation and HIV-1 replication, the structure of INI1 is unknown, largely due to inability to obtain large quantities of pure protein or inability to crystallize. W have been recently successful in purifying and obtaining NMR structure of the transdominant negative mutant of INI1, also termed, S6(Rpt1) or INI1183-265. Having solution structure of INI1183-265 rapidly enhances our ability to understand the structural basis of IN-INI1 interactions and enriches our ability to attain the goals proposed in the aims of R01, especially aim III. With INI1183-265 structure in hand, the goals for the supplemental application are as follows. In Supplemental Aim I we will use NMR-based chemical shift perturbation (CSP) mapping to identify the interface residues within IN-INI1 complexes and use standard NMR methods to determine the complete structure of the INI1183-265 - IN complex. In Supplemental Aim II, we will carry out structure-based functional studies by generating mutations of interface residues of IN and INI1 and test the effect of these mutants on HIV-1 replication, especially assembly and particle production. These studies will allow, for the first time, to gain understanding of the structural basis of IN-INI1 interactions, their effect on HIV-1 replication, and pave the way for drug development to disrupt these interactions.
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 goal of the parent R01 is to investigate novel interactions between the viral and cellular proteins to establish a new drug target, namely IN-INI1 interactions. In the parent R01, due to lack of structural information, we proposed to use computational methods of modeling IN-INI1 interactions. The revision to R01 is due to the fact that we recently obtained three- dimensional structure of IN binding domain of INI1, which provides new opportunities and invaluable tools to gain insights into the structural basis of IN-INI1 interactions. The proposed revision enhances our ability to perform goals of R01 and pave the way to develop novel class of drugs to inhibit HIV-1 replication.
| 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 |
| 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 |
