Human immunodeficiency virus type 1 (HIV-1) uniquely infects both dividing/activated CD4+ T cell and nondividing cells such as macrophages. These dividing and nondividing HIV-1 target cell types have very different cellular landscapes in terms of the availability of cellular DNA building blocks, dNTPs (macrophages, 20~50nM, and CD4+ T cells, 2-5?M). Recently, our LC-MS/MS study revealed that, unlike dNTPs, macrophages have high rNTP concentrations similar to those of activated T cells. This nondividing cell type thus contains low levels of dNTPs but high levels of rNTPs, unlike activated T cells which contain high levels of both. As a consequence, there is a much larger disparity between the levels of dNTPs vs. rNTPs in macrophages, compared to CD4+ T cells, leading us to hypothesize that HIV-1 may incorporate rNTPs during proviral DNA synthesis in macrophages, but not in activated T cells. Indeed, our recent biochemical simulations revealed that HIV-1 RT uses rNTPs as substrates for DNA synthesis under assay conditions that mimic the dNTP/rNTP pool levels present in macrophages, but not in CD4+ T cell dNTP pool. Furthermore, a ribose-adenosine analog lacking 3'OH inhibited HIV-1 proviral DNA synthesis in macrophages but not in CD4+ T cells, supporting the rNTP incorporation by HIV-1 RT in macrophages. Here, first, we will investigate mechanistic aspects of rNTP and 3'deoxy rNTP incorporation by HIV-1 RT and viral escape from 3'deoxy rNs which will be validated as a new class of macrophage-specific RT chain terminators. Since incorporated rNMPs (ribonucleoside mono-phosphate) in templates are mutagenic, dividing cells harbor a specific repair system to circumvent the mutagenic impact of rNTP incorporation in chromosomes, and RNase H2 and FEN1 are two key enzymes that execute the removal of the singly incorporated rNMPs in ds DNAs. Thus, second, we will test if RNase H2 and FEN1 are expressed in macrophages, and then if the knockdowns of RNase H2 and FEN1 affect HIV-1 infectivity and mutagenesis in macrophages. Lastly, HIV-1 RT has been postulated to undergo a series of conformational changes during dNTP incorporation before chemical catalysis. Recently time-resolved stopped flow florescence technology was employed to monitor the kinetics of this conformational change, and we also recently adopted this technology in our laboratory. In this proposal, third, we will investigate the conformational changes of HIV-1 RT during mutation synthesis events: 1) incorporation of incorrect dNTPs, 2) mismatch extension, 3) rNTP incorporation, and 4) use of mutagenic rNMP containing template by HIV-1 RT and other RT variants. In this renewal application, we will systematically investigate our recent new observations, and continue exploring the mechanistic features of HIV-1 genomic hyper-mutability, and this proposed work is essential for our long-term research goals to develop anti-HIV strategies that can specifically limit the unique viral capability to efficiently mutate and escape during the course of viral pathogenesis and anti-viral treatments.

Public Health Relevance

Unique genomic hypermutability of human immunodeficiency virus (HIV) is a powerful evolutionary tool that promotes effective viral escapes from both host anti-viral immune selection and pharmacological anti-viral treatments, leading to the loss of host immune capability and ultimately to lethal opportunistic infections. However, anti-HIV agents, which completely eliminate viral production in patients, currently lack. This proposal aims at not only identifying mechanistic elements involved in highly error-prone viral replication and viral hypermutability but also developing novel concepts and drugs, which can specifically restrict HIV evolution and escape capability, as a potential anti-HIV strategy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI049781-10A1
Application #
8329759
Study Section
Special Emphasis Panel (ZRG1-AARR-D (02))
Program Officer
Ussery, Michael A
Project Start
2000-12-01
Project End
2017-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
10
Fiscal Year
2012
Total Cost
$386,250
Indirect Cost
$136,250
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Antonucci, Jenna M; St Gelais, Corine; de Silva, Suresh et al. (2016) SAMHD1-mediated HIV-1 restriction in cells does not involve ribonuclease activity. Nat Med 22:1072-1074
Hollenbaugh, Joseph A; Kim, Baek (2016) HIV-1 Reverse Transcriptase-Based Assay to Determine Cellular dNTP Concentrations. Methods Mol Biol 1354:61-70
Sommer, Andreas F R; Rivière, Lise; Qu, Bingqian et al. (2016) Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle. Sci Rep 6:26616
Anacker, Daniel C; Aloor, Heather L; Shepard, Caitlin N et al. (2016) HPV31 utilizes the ATR-Chk1 pathway to maintain elevated RRM2 levels and a replication-competent environment in differentiating Keratinocytes. Virology 499:383-396
Hollenbaugh, Joseph A; Montero, Catherine; Schinazi, Raymond F et al. (2016) Metabolic profiling during HIV-1 and HIV-2 infection of primary human monocyte-derived macrophages. Virology 491:106-14
Bonifati, Serena; Daly, Michele B; St Gelais, Corine et al. (2016) SAMHD1 controls cell cycle status, apoptosis and HIV-1 infection in monocytic THP-1 cells. Virology 495:92-100
Bhattacharya, Akash; Wang, Zhonghua; White, Tommy et al. (2016) Effects of T592 phosphomimetic mutations on tetramer stability and dNTPase activity of SAMHD1 can not explain the retroviral restriction defect. Sci Rep 6:31353
Juvekar, Ashish; Hu, Hai; Yadegarynia, Sina et al. (2016) Phosphoinositide 3-kinase inhibitors induce DNA damage through nucleoside depletion. Proc Natl Acad Sci U S A 113:E4338-47
Wang, Feifei; St Gelais, Corine; de Silva, Suresh et al. (2016) Phosphorylation of mouse SAMHD1 regulates its restriction of human immunodeficiency virus type 1 infection, but not murine leukemia virus infection. Virology 487:273-84
Daly, Michele B; Roth, Megan E; Bonnac, Laurent et al. (2016) Dual anti-HIV mechanism of clofarabine. Retrovirology 13:20

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