Single unpaired nucleotides facilitate HIV-1 reverse.  Transcriptase displacement synthesis through duplex RNA

doi:10.1074/jbc.M404117200

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Papers In Press, published online ahead of print May 28, 2004
J. Biol. Chem, 10.1074/jbc.M404117200
Submitted on April 13, 2004
Revised on May 27, 2004
Accepted on May 27, 2004

Single unpaired nucleotides facilitate HIV-1 reverse. Transcriptase displacement synthesis through duplex RNA

Christian Lanciault and James J. Champoux

Microbiology, University of Washington, Seattle, WA 98195

Corresponding Author: champoux{at}u.washington.edu

During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or “bulge”, along RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of non-displacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the non-template strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension.

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