Sequences in the 5' and 3' R elements of human immunodeficiency virus type 1 critical for efficient reverse transcription.
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.
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Department of Microbiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, USA.
Clever
J L
JL
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D004279:DNA, Viral / Q000096:biosynthesis; D016679:Genome, Viral; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014771:Virion / Q000235:genetics; D015215:Zidovudine / Q000494:pharmacology
D004279:DNA, Viral; D018894:Reverse Transcriptase Inhibitors; D015215:Zidovudine; D012194:RNA-Directed DNA Polymerase