Minimal sequence requirements of a functional human immunodeficiency virus type 1 primer binding site.
The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA(3Lys) primer bound near the 5' end of the genomic RNA at a position termed the primer binding site (PBS). The PBS is an 18-nucleotide sequence of the HIV-1 genome which is complementary to the 3'-terminal 18 nucleotides of the tRNA(3Lys). To investigate the sequence specificity of the interaction between tRNA(3Lys) and the PBS, we have constructed proviral genomes containing mutations in the PBS region. A mutant PBS was constructed in which the 18 nucleotides complementary to tRNA(3Lys) were substituted with 18 nucleotides predicted to be complementary to the 3'-terminal bases of a tRNA(Phe) molecule [pHXB2PBS(phe)]. A second proviral genome was constructed in which the PBS complementary to tRNA(Phe) was changed such that the first six nucleotides correspond to the wild-type PBS [pHXB2PBS(pheC)]. In all models of reverse transcription, the complementarity between the minus- and plus-strand PBS DNA facilitates the template switch and elongation of plus-strand DNA, resulting in a complete proviral genome. To test this model, we have inserted a five-nucleotide sequence 6 bp 3' of the mutant PBSs, which corresponds to the last five nucleotides of the wild-type PBSs [pHXB2PBS(phe+5) and pHXB2PBS(pheC+5)]. Transfection of plasmids containing the wild-type or mutant proviral genomes into COS-1 cells resulted in similar levels of intracellular expression of HIV-1 gag and env gene products as determined by immunoprecipitation with sera from AIDS patients and release of virus as determined by p24 assay. Transfection of pHXB2PBS(phe) or pHXB2PBS(phe+5) did not result in the production of infectious virus, while replication-competent viruses from cells transfected with pHXB2PBS(pheC) were detected very infrequently. Transfection of pHXB2PBS(pheC+5), however, consistently resulted in the production of infectious virus, although the appearance of the virus was delayed compared with those from cells transfected with pHXB2(wild type). Reinfection of SupT1 cells with equal amounts of p24 antigen resulted in similar kinetics of replication. PCR was used to amplify the PBS, and individual DNA products were subcloned into M13mp18. Sequence analysis of the PBS region of integrated proviruses derived from transfection of pHXB2PBS(pheC+5) revealed that the 18-nucleotide PBS complementary to tRNA(3Lys) was regenerated with a deletion of 6 bp 3' to the PBS region in all phage clones examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Wakefield
J K
JK
Department of Microbiology, University of Alabama at Birmingham 35294.
Rhim
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Morrow
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D011533:Proviruses / Q000235:genetics; D012313:RNA / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism; D012357:RNA, Transfer, Lys / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000096:biosynthesis; D016662:Virus Integration; D014779:Virus Replication
C034477:RNA primers; D012330:RNA, Double-Stranded; D012357:RNA, Transfer, Lys; D012367:RNA, Viral; D014764:Viral Proteins; D012313:RNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase