Previously our lab has shown that the presence of central polypurine tract (cPPT) enhances transduction efficiency of HIV-1 with defective reverse transcriptase (RT) by promoting the proviral DNA synthesis, particularly in non-dividing cells containing low cellular dNTP concentrations. The experiments proposed in this application focus on testing our hypothesis that: 1) if the absence of cPPT delays the viral replication kinetics, then HIV mutant lacking cPPT becomes more sensitive to RT inhibitors such as non-nucleoside RT inhibitors, known to delay the viral replication kinetics. HIV-1 replication kinetics relies on various elements that also affect the biochemical kinetics of DNA synthesis, such as dNTP substrate concentration and catalytic activity of RT protein. Another way to complete the 9.6 kb ds proviral DNA efficiently is to shorten the size of the template. This explains how cPPT, which serves as an additional primer for the (+) DNA synthesis, accelerates the proviral DNA synthesis especially when the DNA replication was kinetically hindered by either limited cellular dNTP pools or enzymatically compromised RT mutants. Since it is well known that NNRTIs binds RT directly and delay the DNA synthesis, we predict that the cPPT removal, which may delay the DNA synthesis, reduce the chance of the virus to complete proviral DNA synthesis, ultimately increasing the sensitivity of HIV to NNRTI. This hypothesis will be tested using in vitro HIV-1 and its vector systems. We also expect that this predicted elevated NNRTI sensitivity can be counteracted by the elevation of cellular dNTP concentration which can accelerate the proviral DNA synthesis and may compensate the cPPT defect. To test this we will employ a dN treatment to elevate cellular dNTP pool in cells with low dNTP concentration, 2) removal of cPPT may further delay the replication of AZT resistant mutant viruses in the presence of AZT, ultimately re- sensitizing the AZT resistant mutants to AZT. The mechanism of HIV resistance to AZT is unique, compare to viral resistance to NNRTI and protease inhibitors, which prevent drugs from binding to the viral enzymes. HIV- 1 renders AZT resistance by removing the incorporated AZTMP from the 3'end of the polymerizing DNA. It is logical to assume that whenever the drug resistant RT molecules remove AZTMP, the DNA synthesis pauses, ultimately delaying the overall replication kinetics. Thus, in this study, we predict that cPPT removal will re- sensitize the AZT resistant HIV mutant to AZT by delaying the completion of proviral DNA synthesis in the presence of AZT. We will test this using HIV vectors harboring AZT resistant mutation. 3) Our structural model predicts that the A114 reside of HIV-1 RT is important for the dNTP binding affinity, and we will test this hypothesis by using biochemical and kinetic approaches. Since these mutants are expected to display delayed DNA synthesis kinetics, especially at low dNTP concentrations, by employing these HIV-1 RT mutants and the cPPT mutations, we will test if there is any mechanistic interplay between cPPT and dNTP binding affinity in the proviral DNA synthesis kinetics in cell types containing high and low cellular dNTP concentration.

Public Health Relevance

Developing effective highly active anti-retroviral therapy (HAART) to combat HIV-1 is a constant challenge due to fast viral evolution and mutagenesis that persistently selects drug resistant strains. The central polypurine tract (cPPT) sequence of the HIV-1 genome, which is encoded near the dimerization region of HIV-1 integrase, is essential for the initiation of the (+) strand proviral synthesis of HIV-1 [54]. Ideally, if the integrase dimerization region is targeted by a drug, the virus will be forced to alter the cPPT sequence in order to become resistant to the drug. We hypothesize that if the cPPT encoding region of integrase is altered, it will disrupt cPPT function making the virus more sensitive to existing HAART. Our studies examine this putative and novel HIV-1 drug target.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-AARR-J (22))
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Gaillard, Shawn R
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University of Rochester
Schools of Dentistry
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Gramberg, Thomas; Kahle, Tanja; Bloch, Nicolin et al. (2013) Restriction of diverse retroviruses by SAMHD1. Retrovirology 10:26
Daddacha, Waaqo; Noble, Erin; Nguyen, Laura A et al. (2013) Effect of ribonucleotides embedded in a DNA template on HIV-1 reverse transcription kinetics and fidelity. J Biol Chem 288:12522-32
Van Cor-Hosmer, Sarah K; Kim, Dong-Hyun; Daly, Michele B et al. (2013) Restricted 5'-end gap repair of HIV-1 integration due to limited cellular dNTP concentrations in human primary macrophages. J Biol Chem 288:33253-62
Kim, Baek; Nguyen, Laura A; Daddacha, Waaqo et al. (2012) Tight interplay among SAMHD1 protein level, cellular dNTP levels, and HIV-1 proviral DNA synthesis kinetics in human primary monocyte-derived macrophages. J Biol Chem 287:21570-4
Baldauf, Hanna-Mari; Pan, Xiaoyu; Erikson, Elina et al. (2012) SAMHD1 restricts HIV-1 infection in resting CD4(+) T cells. Nat Med 18:1682-7
Hofmann, Henning; Logue, Eric C; Bloch, Nicolin et al. (2012) The Vpx lentiviral accessory protein targets SAMHD1 for degradation in the nucleus. J Virol 86:12552-60
Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin et al. (2012) Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase. J Virol 86:10484-93
Mock, David J; Hollenbaugh, Joseph A; Daddacha, Waaqo et al. (2012) Leishmania induces survival, proliferation and elevated cellular dNTP levels in human monocytes promoting acceleration of HIV co-infection. PLoS Pathog 8:e1002635
Lahouassa, Hichem; Daddacha, Waaqo; Hofmann, Henning et al. (2012) SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates. Nat Immunol 13:223-228