Determination of the ex vivo rates of human immunodeficiency virus type 1 reverse transcription by using novel strand-specific amplification analysis

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 FICR 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 (similar to 68 to 70 nucleotides/min). We also determined the rates of minus-strand DNA transfer (similar to 4 min), plus-strand DNA transfer (similar to 26 min), and initiation of plus-strand DNA synthesis (similar to 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.

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