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Journal of Virology, July 2005, p. 9081-9087, Vol. 79, No. 14
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.14.9081-9087.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Defective Replication in Human Immunodeficiency Virus Type 1 When Non-Primers Are Used for Reverse Transcription

Lady Davis Institute for Medical Research and McGill AIDS Centre, Jewish General Hospital,¹ Departments of Medicine,² Microbiology and Immunology, McGill University, Montreal, Quebec, Canada H3T 1E2³

Received 25 January 2005/ Accepted 26 March 2005

, the primer for reverse transcriptase in human immunodeficiency virus type 1 (HIV-1), anneals to the primer binding site (PBS) in HIV-1 RNA. It has been shown that altering the PBS and U5 regions upstream of the PBS in HIV-1 so as to be complementary to sequences in tRNA^Met or tRNA^His will allow these tRNA species to be stably used as primers for reverse transcription. We have examined the replication of these mutant viruses in Sup-T1 cells. When Sup-T1 cells are infected by cocultivation with HIV-1-transfected 293T cells, viruses using tRNA^His or tRNA^Met are produced at rates that are approximately 1/10 or 1/100, respectively, of rates for wild-type virions that use . When Sup-T1 cells are directly infected with equal amounts of these different viruses isolated from the culture supernatant of transfected 293T cells, virions using tRNA^Met are produced at 1/100 the rate of wild-type viruses, and production of virions using tRNA^His is not detected. Both wild-type and mutant virions selectively package tRNA^Lys only, and examination of the ability of total viral RNA to prime reverse transcription in vitro indicates a >80% reduction in the annealing of tRNA^His or tRNA^Met to the mutant viral RNAs. PCR analysis of which of the three primer tRNAs is used indicates that only is detected as primer in wild-type virions and only tRNA^His is detected as primer in virions containing a PBS complementary to tRNA^His, while the mutant viruses containing a PBS complementary to tRNA^Met use both tRNA^Met and as primer tRNAs.

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