The objective of this work is to investigate the role of HIV-1 and other retroviral Gag and nucleocapsid proteins (NCs) in the regulation of processes involving nucleic acid structural transitions and viral self assembly in retroviral systems. The PI has pioneered the development of new single molecule nucleic acid (NA) stretching methods that can be used to quantitatively probe nucleic acid structural rearrangements and protein-nucleic acid interactions. The proposed work continues to develop novel methods to test specific hypotheses concerning nucleic acid-protein interactions that are important for retroviral packaging and replication. The results of these studies are integrated with collaborative in vivo work. The capability of NCs to rearrange nucleic acids to facilitate reverse transcription, referred to as NA chaperone acitivity, is essential for retroviral replication. NA stretching methods are uniquely well suited for probing NA chaperone activity because nucleic acid structural rearrangement and packaging can be directly controlled and the resulting response can be quantified. A new method will be developed to mechanically probe reverse transcription and characterize the effects of proteins important for HIV-1 replication on this process.
The specific aims are: (1) To quantify the interactions of wild type and mutant HIV-1 and other retroviral nucleic acid chaperone proteins with single- and double-stranded DNA and specific RNA structures. This work will test the hypothesis that nucleic acid chaperone activity is comprised of three critical elements: nucleic acid aggregation, nucleic acid destabilization, and rapid protein-nucleic acid interactions kinetics. (2) To characterize the DNA interactions of APOBEC proteins that may interfere with reverse transcription. We hypothesize that APOBEC proteins inhibit reverse transcription by altering specific DNA interactions. To test this hypothesis, will examine the thermodynamics and kinetics of human APOBEC interactions with single- and double-stranded DNA. (3) To quantify the nucleic acid interactions of wild type and mutant HIV-1 Gag. Although HIV-1 NC is the domain of Gag that is primarily responsible for its interactions with NAs, these proteins facilitate significantly different processes in the cell.
This aim seeks to understand how NC and Gag exhibit different NA interactions and how these interactions alter reverse transcription and the NA packaging process. (4) To mechanically measure reverse transcriptase (RT) polymerization in the presence of NC and APOBEC3G and determine how they alter RT's essential processes.

Public Health Relevance

Nucleic acid packaging and reverse transcription are critical components of retroviral replication. This project develops novel methods for characterizing these processes to contribute towards development of a molecular level understanding of retroviral replication, which in turn will help to develop drugs that target these processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM072462-09
Application #
8465239
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Lewis, Catherine D
Project Start
2004-08-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
9
Fiscal Year
2013
Total Cost
$281,029
Indirect Cost
$85,583
Name
Northeastern University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
001423631
City
Boston
State
MA
Country
United States
Zip Code
02115
Chaurasiya, Kathy R; McCauley, Micah J; Wang, Wei et al. (2014) Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein. Nat Chem 6:28-33
Wu, Hao; Wang, Wei; Naiyer, Nada et al. (2014) Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein. Virus Res 193:39-51
Wang, Wei; Naiyer, Nada; Mitra, Mithun et al. (2014) Distinct nucleic acid interaction properties of HIV-1 nucleocapsid protein precursor NCp15 explain reduced viral infectivity. Nucleic Acids Res 42:7145-59
Wu, Hao; Mitra, Mithun; Naufer, M Nabuan et al. (2014) Differential contribution of basic residues to HIV-1 nucleocapsid protein's nucleic acid chaperone function and retroviral replication. Nucleic Acids Res 42:2525-37
Peters, Justin P; Mogil, Lauren S; McCauley, Micah J et al. (2014) Mechanical properties of base-modified DNA are not strictly determined by base stacking or electrostatic interactions. Biophys J 107:448-59
Murugesapillai, Divakaran; McCauley, Micah J; Huo, Ran et al. (2014) DNA bridging and looping by HMO1 provides a mechanism for stabilizing nucleosome-free chromatin. Nucleic Acids Res 42:8996-9004
McCauley, Micah J; Rueter, Emily M; Rouzina, Ioulia et al. (2013) Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility. Nucleic Acids Res 41:167-81
Chaurasiya, Kathy R; Ruslie, Clarissa; Silva, Michelle C et al. (2013) Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III * binding to ssDNA. Nucleic Acids Res 41:8959-68
Wu, Hao; Mitra, Mithun; McCauley, Micah J et al. (2013) Aromatic residue mutations reveal direct correlation between HIV-1 nucleocapsid protein's nucleic acid chaperone activity and retroviral replication. Virus Res 171:263-77
Chaurasiya, Kathy R; Geertsema, Hylkje; Cristofari, Gael et al. (2012) A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3. Nucleic Acids Res 40:751-60

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