title: Template-directed pausing of DNA synthesis by HIV-1 reverse transcriptase during polymerization of HIV-1 sequences in vitro.
abstract: Replication of human immunodeficiency virus type 1 (HIV-1) requires reverse transcriptase (RT) to synthesize double-stranded proviral DNA (9.7 kilobases) through a complex mechanism utilizing both RNA and DNA templates. We have examined DNA synthesis by HIV-1 RT on RNA and DNA templates derived from the HIV-1 genome using a primer extension assay in vitro. Analysis of polymerization products on sequencing gels revealed strong pauses in synthesis, on both RNA and DNA templates, in homopolymeric nucleotide runs, and at regions of predicted secondary structure. Polymerization pauses occurred in runs of template rGs (> or = 4 bases) and rCs (> or = 3 bases) during minus-strand synthesis on RNA templates, and in most runs (> or = 4 bases) of template dTs and dAs during plus-strand synthesis on DNA templates. Pausing also occurred on both templates within the first few nucleotides of the predicted hairpin structures of the Rev response element. The locations of pauses were dependent on template sequence and were unaffected by primer positioning, RT concentration, and ionic strength. Recombinant and virion-derived HIV-1 RTs showed similar pausing patterns. DNA products that accumulated at HIV-1 RT pause sites on RNA templates were extended by continued incubation with excess RT from Moloney murine leukemia virus, showing that the RNA templates were not broken or otherwise unable to support polymerization. Polymerizations conducted in the presence of a poly(rA) oligo(dT) trap showed that pausing results from two mechanisms: 1) RT remaining bound to the primer-template and polymerizing at a greatly reduced rate, or 2) RT dissociating from the primer-template. These results demonstrate that specific HIV-1 RNA and DNA template sequences are capable of interrupting processive DNA synthesis by HIV-1 RT in vitro. Pausing may serve specific functions in HIV-1 replication and mutagenesis. Moreover, these data suggest that one or more accessory factors are required to complete proviral DNA synthesis in vivo and that efficient HIV-1 DNA synthesis may require multiple origins.
authors.affiliation: Laboratory for Cancer Research, College of Pharmacy, Rutgers University, Piscataway, New Jersey 08854.
authors.forename: G J
authors.initials: GJ
authors.lastname: Klarmann
authors.forename: C A
authors.initials: CA
authors.lastname: Schauber
authors.forename: B D
authors.initials: BD
authors.lastname: Preston
mesh_terms: D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015752:Genes, env; D015751:Genes, pol; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D007700:Kinetics; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013698:Templates, Genetic; D014771:Virion / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase