Analysis of primer extension and the first template switch during human immunodeficiency virus reverse transcription

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Abstract
Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNAlys3 priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNAlys3 had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNAlys3. We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h.