The primer tRNA for reverse transcriptase in HIV-1, human tRNALys3, is selectively packaged into the virion, along with human lysyl-tRNA synthetase (LysRS), the enzyme that aminoacylates tRNALys. Gag alone will package LysRS into Gag particles. Viral LysRS is smaller than the cytoplasmic form, and this truncated form is present even in the absence of viral protease. We hypothesize that LysRS is the signal recognized by viral proteins and targets tRNALys for viral packaging. LysRS truncation may release it from tRNALys, thereby facilitating tRNALys3 annealing to the viral RNA. These processes will be studied in the proposed work, and represent new targets for anti-HIV-1 therapy.
The specific aims of this study are: 1) To investigate the interaction between HIV-1 Gag and human LysRS. Gag/LysRS interactions will be studied in vivo, through the cellular expression of mutant forms of Gag and LysRS, measuring their ability to interact in the cytoplasm (coimmunoprecipitation) and to package LysRS into Gag particles. Interactions between purified components will be studied in vitro, using both qualitative and quantitative methods for measuring protein-protein interactions. 2) To investigate the role of the tRNA aminoacylation state on tRNALys3 packaging into HIV-1 and on priming of reverse transcription. Although all detectable cytoplasmic tRNALys3 is aminoacylated, tRNALys3 must be uncharged to serve as a primer. We shall determine if aminoacylation of tRNALys3 is essential for tRNALys3 packaging by determining if mutant LysRS able to bind tRNALys3, but not aminoacylate it, still facilitates tRNALys3 packaging. The ability of viral proteins to induce tRNALys3 deacylation will also be examined in vitro. 3) To investigate the cellular origin of viral LysRS. Cytoplasmic LysRS is found in a high molecular weight aminoacyl-tRNA synthetase (HMW aaRS) complex composed of 12 proteins, but only LysRS is found in HIV-I. We shall determine if mutant LysRS unable to bind to p38 and therefore unable to participate as a component of the HMW aaRS, is still able to be packaged into HIV-1. Using RNA interference, we shall determine if the source of viral LysRS is newly-synthesized LysRS. 4) To characterize the cellular protease that leads to the truncated form of human LysRS found in HIV-1. Both biochemical purification methods and a genetic strategy for identifying cDNAs coding for LysRS proteases will be used to identify cellular proteases that can cleave LysRS.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI054145-02
Application #
6785379
Study Section
AIDS and Related Research 8 (AARR)
Program Officer
Sharma, Opendra K
Project Start
2003-08-15
Project End
2007-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
2
Fiscal Year
2004
Total Cost
$259,219
Indirect Cost
Name
Sir Mortimer B. Davis Jewish Gen Hosp
Department
Type
DUNS #
208572446
City
Montreal
State
PQ
Country
Canada
Zip Code
H3 1-E2
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Wei, Min; Yang, Yiliang; Niu, Meijuan et al. (2008) Inability of human immunodeficiency virus type 1 produced in murine cells to selectively incorporate primer formula. J Virol 82:12049-59
Guo, Min; Ignatov, Michael; Musier-Forsyth, Karin et al. (2008) Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation. Proc Natl Acad Sci U S A 105:2331-6
Kovaleski, Brandie J; Kennedy, Robert; Khorchid, Ahmad et al. (2007) Critical role of helix 4 of HIV-1 capsid C-terminal domain in interactions with human lysyl-tRNA synthetase. J Biol Chem 282:32274-9
Kovaleski, Brandie J; Kennedy, Robert; Hong, Minh K et al. (2006) In vitro characterization of the interaction between HIV-1 Gag and human lysyl-tRNA synthetase. J Biol Chem 281:19449-56