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J Virol, July 1998, p. 5464-5471, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Preferential Completion of Human Immunodeficiency Virus Type 1 Proviruses Initiated with tRNA3Lys rather than tRNA1,2Lys

Zhijun Zhang, Qin Yu, Sang-Moo Kang, James Buescher, and Casey D. Morrow*

Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

Received 24 October 1997/Accepted 25 March 1998

All retroviral genomes contain a nucleotide sequence designated as the primer binding site (PBS) which is complementary to the tRNA used for initiation of reverse transcription. For human immunodeficiency virus type 1 (HIV-1), all naturally occurring genomes have a PBS complementary to tRNA3Lys. However, within HIV-1 virions, there are approximately equal amounts of tRNA1Lys, tRNA2Lys, and tRNA3Lys. We have used an endogenous reverse transcription-PCR technique specific for the tRNA species within isolated HIV-1 virions to demonstrate that in addition to tRNA3Lys, tRNA1Lys and tRNA2Lys could be used for initiation of HIV-1 reverse transcription. Using a single-round infection assay which employed an HIV-1 genome with a gpt gene encoding xanthine-guanine phosphoribosyl transferase in place of the env gene, we generated cell lines resistant to mycophenolic acid. Analysis of the U5-PBS from single-cell clones revealed PBS complementary to tRNA3Lys, not tRNA1Lys or tRNA2Lys. A mutant HIV-1 genome was then created which would favor the completion of reverse transcription with tRNA1,2Lys. Using this provirus in the complementation system, we again found only genomes with a PBS complementary to tRNA3Lys from proviral DNA isolated from gpt-resistant single-cell colonies. Finally, infection of cells with a mutant HIV genome with a PBS complementary to tRNA1,2Lys resulted in gpt- resistant cell colonies which contained integrated provirions with a PBS complementary to tRNA1,2Lys. The results of these studies suggest that the selection of tRNA3Lys for initiation of HIV-1 reverse transcription occurs both at the initiation and at a postinitiation step in reverse transcription prior to integration of the proviral DNA.


* Corresponding author. Mailing address: Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294. Phone: (205) 934-5705. Fax: (205) 934-1580. E-mail: casey_morrow{at}micro.microbio.uab.edu.


J Virol, July 1998, p. 5464-5471, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.



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