Abstract
In human immunodeficiency virus type 1 (HIV-1), the tRNA(Lys.3) primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. Initiation of reverse transcription from this complex has been shown to be distinguished from subsequent elongation by early pausing events, such as at the +1 and +3 nucleotide positions. One major concern regarding the biological relevance of these results is that most kinetic studies of HIV-1 reverse transcription have been performed using tRNA(Lys.3)-viral (v) RNA complexes that were formed by heat annealing. In contrast, tRNA(Lys.3) in viruses is placed onto the primer binding site by nucleocapsid (NC) sequences of the Gag protein. In this study, we have further characterized the initiation features of reverse transcription in the presence of HIV-1 NC protein. In contrast to results obtained with a heat-annealed tRNA(Lys.3).vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the +1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was actually still present during reverse transcription. This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. However, NC was not able to help reverse transcriptase to escape the +3 pausing event. Mutagenesis of a stem structure within the tRNA(Lys.3). vRNA complex led to disappearance of the +3 pausing event as well as to significantly reduced rates of reverse transcription. Thus, this stem structure is essential for optimal reverse transcription, despite its role in promotion of the +3 pausing event.
Highlights
In human immunodeficiency virus type 1 (HIV-1), the tRNALys.3 primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions
In contrast to results obtained with a heat-annealed tRNALys.31⁄7vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the ؉1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was still present during reverse transcription
Transcription to proceed from a NC-derived tRNALys.31⁄7vRNA complex, while avoiding the direct involvement of NC in the reactions at the same time. These results showed that the tRNALys.31⁄7vRNA complex that is formed by NC is already active, and can overcome ϩ1 nt pausing during subsequent polymerization without the further involvement of NC
Summary
In human immunodeficiency virus type 1 (HIV-1), the tRNALys. primer and viral RNA template can form a specific complex that is characterized by extensive inter- and intramolecular interactions. In contrast to results obtained with a heat-annealed tRNALys.31⁄7vRNA complex, we found that polymerization reactions catalyzed by HIV-1 reverse transcriptase did not commonly pause at the ؉1 nucleotide position when a NC-annealed RNA complex was used, and that this was true regardless whether NC was still present during reverse transcription This activity of NC required both zinc finger motifs, as demonstrated by experiments that employed zinc finger-mutated forms of NC protein (H23C NC and ddNC), supporting the involvement of the zinc fingers in the RNA chaperone activity of NC. Using an in vitro reaction system, initiation of reverse transcription can be detected by the formation of short intermediate cDNA products after the tRNA primer is extended by 1, 3, or 5 nt (4 –7) In these studies, reverse transcription had been performed with tRNALys.31⁄7vRNA complexes that were formed by heat annealing.
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