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Journal of Virology, September 2000, p. 8324-8334, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Sequences in the 5' and 3' R Elements of Human Immunodeficiency Virus Type 1 Critical for Efficient Reverse Transcription

Yuki Ohi and Jared L. Clever*

Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900

Received 31 March 2000/Accepted 18 June 2000

The genome of human immunodeficiency virus type 1 (HIV-1) contains two direct repeats (R) of 97 nucleotides at each end. These elements are of critical importance during the first-strand transfer of reverse transcription, during which the minus-strand strong-stop DNA (-sssDNA) is transferred from the 5' end to the 3' end of the genomic RNA. This transfer is critical for the synthesis of the full-length minus-strand cDNA. These repeats also contain a variety of other functional domains involved in many aspects of the viral life cycle. In this study, we have introduced a series of mutations into the 5', the 3', or both R sequences designed to avoid these other functional domains. Using a single-round infectivity assay, we determined the ability of these mutants to undergo the various steps of reverse transcription utilizing a semiquantitative PCR analysis. We find that mutations within the first 10 nucleotides of either the 5' or the 3' R sequence resulted in virions that were markedly defective for reverse transcription in infected cells. These mutations potentially introduce mismatches between the full-length -sssDNA and 3' acceptor R. Even mutations that would create relatively small mismatches, as little as 3 bp, resulted in inefficient reverse transcription. In contrast, virions containing identically mutated R elements were not defective for reverse transcription or infectivity. Using an endogenous reverse transcription assay with disrupted virus, we show that virions harboring the 5' or the 3' R mutations were not intrinsically defective for DNA synthesis. Similarly sized mismatches slightly further downstream in either the 5', the 3', or both R sequences were not detrimental to continued reverse transcription in infected cells. These data are consistent with the idea that certain mismatches within 10 nucleotides downstream of the U3-R junction in HIV-1 cause defects in the stability of the cDNA before or during the first-strand transfer of reverse transcription leading to the rapid disappearance of the -sssDNA in infected cells. These data also suggest that the great majority of first-strand transfers in HIV-1 occur after the copying of virtually the entire 5' R.

* Corresponding author. Mailing address: Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78229-3900. Phone: (210) 567-3935. Fax: (210) 567-6612. E-mail: cleverj{at}uthscsa.edu.

Journal of Virology, September 2000, p. 8324-8334, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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