In Vitro Studies on tRNA Annealing and Reverse Transcription with Mutant HIV-1 RNA Templates

Nancy Beerens,Ben Berkhout

doi:10.1074/jbc.275.20.15474

Abstract

The human immunodeficiency virus type 1 (HIV-1) RNA genome encodes a semistable stem-loop structure, the U5-PBS hairpin, which occludes part of the tRNA primer binding site (PBS). In previous studies, we demonstrated that mutations that alter the stability of the U5-PBS hairpin inhibit virus replication. A reverse transcription defect was measured in assays with the virion-extracted RNA-tRNA complexes. We now extend these studies with in vitro synthesized wild-type and mutant RNA templates that were tested in primer annealing and reverse transcription assays. The effect of annealing temperature and the presence of the viral nucleocapsid protein on reverse transcription was analyzed for the templates with a stabilized or destabilized U5-PBS hairpin, and in reactions initiated by tRNA or DNA primers. The results of this in vitro assay are consistent with the in vivo findings, in that both tRNA annealing and initiation of reverse transcription are sensitive to stable template RNA structure. Reverse transcription initiated by a DNA primer is less hindered by secondary structure in the RNA template than tRNA primed reactions. The inhibitory effect of template structure on tRNA-primed reverse transcription is more pronounced in this in vitro assay compared with the in vivo material, indicating that the heat-annealed RNA-tRNA complex differs from the virion-extracted viral RNA-tRNA complex.

Highlights

The replication cycle of the human immunodeficiency virus type 1 (HIV-1)1 and other retroviruses is characterized by reverse transcription of the viral RNA genome into a doublestranded DNA, which subsequently becomes integrated into the host cell genome [1]
DNA-primed Reverse Transcription on U5-primer-binding site (PBS) Mutant TemtRNA Annealing and Reverse Transcription plates—The results presented above indicate that structure in the template and primer can influence reverse transcription
Reverse transcription assays with these virion-extracted RNAtRNAs complexes demonstrated that reverse transcription of the mutant Ts template was reduced to 27% of the value measured for the wild-type template

Summary

EXPERIMENTAL PROCEDURES

DNA Constructs—The U5-PBS hairpin of the construct Blue-5Ј-LTR [24] was mutated by oligonucleotide-directed in vitro mutagenesis with a Muta-Gene phagemid in vitro mutagenesis kit (Bio-Rad) as described previously [11]. DNA and tRNA Primer Extension Assays—In the DNA- and tRNAprimed reverse transcription assays, 10 ng of in vitro synthesized RNA template was incubated with 1.5 ␮g of calf liver tRNA (6 pmol total tRNA, of which approximately 1.2 pmol is tRNALys; Roche Molecular Biochemicals) or 20 ng of DNA primer in the presence or absence of 80 ng of NC protein in 12 ␮l of annealing buffer (83 mM Tris-HCl, pH 7.5, 125 mM KCl) at 85 or 60 °C for 10 min, followed by cooling to room temperature over a 1-h period or at 37 °C for 30 min. The antisense primers used were CN1 (positions ϩ123 to ϩ151), Top (positions ϩ165 to ϩ181), and Lys (positions ϩ179 to ϩ199), tRNA Occupancy of the PBS—In the PBS occupancy assay, 10 ng of in vitro synthesized RNA template was incubated with 1.5 ␮g of calf liver tRNA in 12 ␮l of annealing buffer at 85 °C for 10 min, followed by gradual cooling to room temperature over a 1-h period. Reverse transcription and analysis of the cDNA products was performed as described above

RESULTS

NC effect

DISCUSSION