The long-term goals of our research are to delineate biochemical steps in key early events during HIV replication. To this end, we will continue our efforts on dissecting the structural determinants of critical functions performed by viral reverse transcriptase (RT) and we will expand our original observation of RT interaction with the viral integrase (IN) to understand its virological significance.
Three specific aims are proposed.
Aim (1) RT determinants of dNTP selection: We have made a new observation that the fingers beta3-Deta4 hairpin loop, specifically the contact between K65 residue and the gamma phosphate of incoming dNTP, is responsible for RT's low fidelity. We will use pre-steady state kinetics to generate evidence that the insertion fidelity is governed at the level of initial recognition of correct vs. incorrect dNTPs. Forward mutation rates and mispair extension efficiencies will be measured to determine if the increased fidelity extends beyond dNTP misinsertion to other forms of errors. We will also employ halogenated analogs of dNTPs to determine the role of active site tightness in dNTP selection. These studies should enhance our knowledge of the structural determinants of the RT's active site.
Aim (2) Determinants of strand displacement DNA synthesis: We hypothesize that F61 and W24 residues control strand displacement synthesis by 'gating'the duplex ahead of the active site. Mutant RTs with natural and unnatural amino acid substitutions (at F61 or W24) with variable predicted abilities to stabilize the terminal base pair will be used to examine their strand displacement synthesis in vitro. In virological experiments, effect of mutations that affect strand displacement during viral replication will be studied.
Aim (3) Functional role of RT-IN Interaction in HIV replication: Using reverse yeast 2-hybrid screen, we will isolate interaction-defective mutants of RT or IN. Mutations will be built into viral clones to examine the importance of RT-IN interaction in HIV replication.
|Kuniholm, Mark H; Liang, Hua; Anastos, Kathryn et al. (2017) Association of a 3' untranslated region polymorphism in proprotein convertase subtilisin/kexin type 9 with HIV viral load and CD4+ levels in HIV/hepatitis C virus coinfected women. AIDS 31:2483-2492|
|Kuniholm, Mark H; Liang, Hua; Anastos, Kathryn et al. (2017) Association of a 3' Untranslated Region Polymorphism in PCSK9 with HIV Viral Load and CD4+ Levels in HIV/Hepatitis C Virus Co-Infected Women. AIDS :|
|Ruiz, Arthur P; Prasad, Vinayaka R (2016) Measuring the Uptake and Transactivation Function of HIV-1 Tat Protein in a Trans-cellular Cocultivation Setup. Methods Mol Biol 1354:353-66|
|Duclair, Sonald; Gautam, Archana; Ellington, Andrew et al. (2015) High-affinity RNA Aptamers Against the HIV-1 Protease Inhibit Both In Vitro Protease Activity and Late Events of Viral Replication. Mol Ther Nucleic Acids 4:e228|
|Neogi, Ujjwal; Rao, Shwetha D; Bontell, Irene et al. (2014) Novel tetra-peptide insertion in Gag-p6 ALIX-binding motif in HIV-1 subtype C associated with protease inhibitor failure in Indian patients. AIDS 28:2319-22|
|Hanna, Luke Elizabeth; Neogi, Ujjwal; Ranga, Udaykumar et al. (2014) Phylogenetic characterization of six full-length HIV-1 subtype C molecular clones from three patients: identification of rare subtype C strains containing two NF-?B motifs in the long terminal repeat. AIDS Res Hum Retroviruses 30:586-91|
|Garforth, Scott J; Lwatula, Chisanga; Prasad, Vinayaka R (2014) The lysine 65 residue in HIV-1 reverse transcriptase function and in nucleoside analog drug resistance. Viruses 6:4080-94|
|Rao, Vasudev R; Neogi, Ujjwal; Eugenin, Eliseo et al. (2014) The gp120 protein is a second determinant of decreased neurovirulence of Indian HIV-1C isolates compared to southern African HIV-1C isolates. PLoS One 9:e107074|
|Prasad, Vinayaka R; Bukrinsky, Michael I (2014) New clues to understanding HIV nonprogressors: low cholesterol blocks HIV trans infection. MBio 5:e01396-14|
|Mathew, Sheeba; Nguyen, Minh; Wu, Xuhong et al. (2013) INI1/hSNF5-interaction defective HIV-1 IN mutants exhibit impaired particle morphology, reverse transcription and integration in vivo. Retrovirology 10:66|
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