Document 0205
 DOCN  M9440205
 TI    Minimal sequence requirements of a functional human immunodeficiency
       virus type 1 primer binding site.
 DT    9404
 AU    Wakefield JK; Rhim H; Morrow CD; Department of Microbiology, University
       of Alabama at Birmingham; 35294.
 SO    J Virol. 1994 Mar;68(3):1605-14. Unique Identifier : AIDSLINE
       MED/94149851
 AB    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)
 DE    Animal  Base Sequence  Binding Sites  Cells, Cultured  DNA Mutational
       Analysis  Genome, Viral  HIV-1/*GENETICS  Molecular Sequence Data
       Mutation  Proviruses/GENETICS  Reverse Transcriptase/*METABOLISM
       RNA/*METABOLISM  RNA, Double-Stranded/METABOLISM  RNA, Transfer,
       Lys/*METABOLISM  RNA, Viral/*GENETICS/METABOLISM  Support, U.S. Gov't,
       P.H.S.  *Transcription, Genetic  Viral Proteins/BIOSYNTHESIS  Virus
       Integration  Virus Replication  JOURNAL ARTICLE

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       protected by Copyright Law (Title 17, U.S.Code).