8th Conference on Retroviruses and Opportunistic Infections Chicago, IL - February 4 - 8, 2001

Conf Retroviruses Opportunistic Infect 2001 Feb 4-8; 8:78 (abstract no. 122)

Cen S, Khorchid A, Javanbakht H, Shiba K, Musier-Forsyth K, Kleiman L; Lady Davis Inst for Med Res, Montreal, Quebec, Canada.

BACKGROUND: tRNA(Lys)3 is the primer for reverse transcription in human immunodeficiency virus. During viral assembly, the tRNA(Lys)3 is selected for incorporation into virions. In the cell tRNA is found in complexes with proteins, such as aminoacyl-tRNA synthetase and eEF1alpha, associated with translation. These cellular proteins could play a role in facilitating an interaction between the primer tRNA(Lys)3 and the viral proteins.

METHODS: Sucrose and OptiPrep gradient, velocity sedimentation ultracentrifugation, subtilisin digestion assay and Western blot.

RESULTS: Aminoacyl- tRNA(Lys) synthetase(LysRS) is present in HIV-1 virions and is resistant to digestion with the protease subtilisin. While the major cytoplasmic species of LysRS in infected cells has an M(r)= 70,000 (large species), viral incorporation of primer tRNA(Lys)3 is correlated with the packaging of an intermediate-size LysRS species, M(r)= 63,000. This intermediate species is the major form of LysRS found in virions produced from chronically infected cell lines (H9, U937, PLB, CEMss), while in wild-type or protease-negative HIV-1 produced from transfected COS7 cells, both the large and intermediate LysRS species are found. The presence of the intermediate-size LysRS in protease- negative viruses indicates that a cellular protease is involved in LysRS processing. The intermediate LysRS species becomes the major LysRS species when viral tRNA(Lys)3 packaging is increased as a result of cotransfecting COS7 cells with HIV-1 proviral DNA and a tRNA(Lys)3 gene. Two mutations (P31L and Dr2) and Gag virus-like particles, which inhibited tRNA(Lys) specific incorporation, prevent the incorporation of intermediate-size LysRS species into virions. Rescue of tRNA(Lys)3 packaging in the P31L mutant with wild-type Gag-Pol also results in an increase within the virus of the intermediate form of LysRS.

CONCLUSIONS: LysRS could play an important role in incorporation of tRNA(Lys)3 into HIV-1 particles.

Keywords: AEGIS, RNA, Transfer, Amino Acyl, HIV-1, Virion, Lysine-tRNA Ligase, RNA, Transfer, HIV-1 Reverse Transcriptase, Ligases, Blotting, Western, Lysine, Transcription, Genetic, Human, genetics, AIDS

2001-02-04
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Copyright © 2001 - Foundation for Retrovirology and Human Health. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health. Licensed (AIDSLINE) from National Library of Medicine.