title: Determination of the ex vivo rates of human immunodeficiency virus type 1 reverse transcription by using novel strand-specific amplification analysis.
abstract: Replication of human immunodeficiency virus type 1 (HIV-1), like all organisms, involves synthesis of a minus-strand and a plus-strand of nucleic acid. Currently available PCR methods cannot distinguish between the two strands of nucleic acids. To carry out detailed analysis of HIV-1 reverse transcription from infected cells, we have developed a novel strand-specific amplification (SSA) assay using single-stranded padlock probes that are specifically hybridized to a target strand, ligated, and quantified for sensitive analysis of the kinetics of HIV-1 reverse transcription in cells. Using SSA, we have determined for the first time the ex vivo rates of HIV-1 minus-strand DNA synthesis in 293T and human primary CD4(+) T cells ( approximately 68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer ( approximately 4 min), plus-strand DNA transfer ( approximately 26 min), and initiation of plus-strand DNA synthesis ( approximately 9 min) in 293T cells. Additionally, our results indicate that plus-strand DNA synthesis is initiated at multiple sites and that several reverse transcriptase inhibitors influence the kinetics of minus-strand DNA synthesis differently, providing insights into their mechanism of inhibition. The SSA technology provides a novel approach to analyzing DNA replication processes and should facilitate the development of new antiretroviral drugs that target specific steps in HIV-1 reverse transcription.
authors.affiliation: Viral Mutation Section, HIV Drug Resistance Program, Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Thomas
authors.forename: Yegor A
authors.initials: YA
authors.lastname: Voronin
authors.forename: Galina N
authors.initials: GN
authors.lastname: Nikolenko
authors.forename: Jianbo
authors.initials: J
authors.lastname: Chen
authors.forename: Wei-Shau
authors.initials: WS
authors.lastname: Hu
authors.forename: Vinay K
authors.initials: VK
authors.lastname: Pathak
mesh_terms: D001483:Base Sequence; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology; D002460:Cell Line; D015342:DNA Probes / Q000235:genetics; D004277:DNA, Single-Stranded / Q000502:physiology*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides; D048348:Reverse Transcription / Q000502:physiology*; D014779:Virus Replication
chemical_list: D015342:DNA Probes; D004277:DNA, Single-Stranded; D009841:Oligonucleotides