Attempts to combat HIV have been hampered due to the virus's ability to rapidly mutate and produce genetic variants that can circumvent the immune response and resist drug therapy. Most mutations are generated during genome replication. Recombination serves to reassort them, further amplifying genetic diversity. Two viral proteins, reverse transcriptase (RT) and nucleocapsid (NC) have been clearly implicated in these processes. The goal of this proposal is to answer key questions regarding the mechanism of recombination and to better understand how RT and NC have evolved to interact with replication intermediates. In addition, tight binding nucleic acid molecules (aptamers) that could potentially be used as viral inhibitors will be selected.
Three specific aims are listed: (1) To determine if a recombination hotspot in the gag-pol frameshift region discovered in vitro is also a hotspot in cellular infections and whether occurrence of hotspots is dependent on in vitro test conditions;(2) To determine how HIV nucleocapsid protein inhibits priming of DNA synthesis by random sequence RNAs but not polypurine tract RNA;(3): To isolate primer-template sequences that bind RTs (MuLV, AMV, and TY3) with high affinity and determine if like HIV-RT, other RTs also bind their cognate polypurine tract sequences (ppt) very tightly. A combination of in vitro and cell culture approaches will be used for these experiments. For example, in Aim 1 recombination assays will be conducted under both conditions and compared in order to verify conclusions.
Aim 2 builds on preliminary data from our lab showing that NC inhibits non-ppt RNA priming but does not affect the ppt usage. This could be important to preventing spurious priming 2nd strand synthesis and producing proviruses with multiple discontinuities.
Aim 3 is related to aim 2 and will address the possibility that RTs evolved to recognize and bind tightly to ppt sequences that are used for second strand DNA priming. This tight binding could potentially be used to design inhibitors that may be difficult for the virus to escape from.

Public Health Relevance

This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (i.e. ppt, recombination) for drug intervention. This work is relevant to human health because it will help identify possible leads for nucleic acid-based drug therapies (aptamers). In addition, it will help define the potential of new specific targets (NC, ppt, recombination) for drug intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051140-16
Application #
7848185
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Hagan, Ann A
Project Start
1994-05-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2011-05-31
Support Year
16
Fiscal Year
2010
Total Cost
$297,000
Indirect Cost
Name
University of Maryland College Park
Department
Anatomy/Cell Biology
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Achuthan, Vasudevan; DeStefano, Jeffrey J (2015) Alternative divalent cations (Zn²?, Co²?, and Mn²?) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity. BMC Biochem 16:12
Achuthan, Vasudevan; DeStefano, Jeffrey J (2015) Mismatched Primer Extension Assays. Bio Protoc 5:
Achuthan, Vasudevan; DeStefano, Jeffrey J (2015) Primer Extension Reactions for the PCR- based ?- complementation Assay. Bio Protoc 5:
Achuthan, Vasudevan; Keith, Brian J; Connolly, Bernard A et al. (2014) Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro. J Virol 88:8514-27
Lieberman, Ori J; DeStefano, Jeffrey J; Lee, Vincent T (2013) Detection of cyclic diguanylate G-octaplex assembly and interaction with proteins. PLoS One 8:e53689
Lai, Yi-Tak; DeStefano, Jeffrey J (2012) DNA aptamers to human immunodeficiency virus reverse transcriptase selected by a primer-free SELEX method: characterization and comparison with other aptamers. Nucleic Acid Ther 22:162-76
Nair, Gauri R; Dash, Chandravanu; Le Grice, Stuart F J et al. (2012) Viral reverse transcriptases show selective high affinity binding to DNA-DNA primer-templates that resemble the polypurine tract. PLoS One 7:e41712
Fenstermacher, Katherine J; DeStefano, Jeffrey J (2011) Mechanism of HIV reverse transcriptase inhibition by zinc: formation of a highly stable enzyme-(primer-template) complex with profoundly diminished catalytic activity. J Biol Chem 286:40433-42
Lai, Yi-Tak; DeStefano, Jeffrey J (2011) A primer-free method that selects high-affinity single-stranded DNA aptamers using thermostable RNA ligase. Anal Biochem 414:246-53
Olimpo, Jeffrey T; DeStefano, Jeffrey J (2010) Duplex structural differences and not 2'-hydroxyls explain the more stable binding of HIV-reverse transcriptase to RNA-DNA versus DNA-DNA. Nucleic Acids Res 38:4426-35

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