In virio SHAPE analysis of tRNA(Lys3) annealing to HIV-1 genomic RNA in wild type and protease-deficient virus.

Elias Seif,Lawrence Kleiman,Meijuan Niu

doi:10.1186/s12977-015-0171-7

Abstract

BackgroundtRNALys3 annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys3 annealing to viral RNA occurs in two steps. First, Gag promotes annealing of tRNALys3 to the viral RNA during cytoplasmic HIV-1 assembly. Second, mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNALys3, resulting in a more tightly bound and efficient primer for reverse transcription.ResultsIn this report, we have used in virio SHAPE analysis of both the 5´-untranslated region in HIV-1 RNA and the annealed tRNALys3 to determine structural differences of the annealed complex that occur between protease-negative (Pr-) and wild type viruses. Our results indicate that the weaker binding of tRNALys3 annealed by Gag in Pr- virions reflects both missing interactions of tRNALys3 with viral RNA regions in the upper PBS stem, and a weaker interaction with the internal stem-loop found within the unannealed primer binding site in viral RNA.ConclusionsWe propose secondary structure models for the tRNALys3/viral RNA annealed complexes in PR- and wild type viruses that support the two-step annealing model by showing that Gag promotes a partial annealing of tRNALys3 to HIV-1 viral RNA, followed by a more complete annealing by NCp7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0171-7) contains supplementary material, which is available to authorized users.

Highlights

TRNALys3 annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys3 annealing to viral RNA occurs in two steps
Reverse transcription is initiated from a primer tRNALys3 that was previously annealed during viral assembly to an 18 nt sequence in the viral RNA termed the primer binding site (PBS)
In that report, we showed that weaker binding to viral RNA, and the reduced ability of Gag-annealed tRNALys3 to function as a primer for reverse transcription, can be rescued by a transient exposure to purified NCp7 of total viral RNA isolated from Pr(−) virions [17]

Summary

Introduction

TRNALys annealing to the viral RNA of human immunodeficiency virus type-1 (HIV-1) is an essential step in the virus life cycle, because this tRNA serves as the primer for initiating reverse transcription. tRNALys annealing to viral RNA occurs in two steps. Gag promotes annealing of tRNALys to the viral RNA during cytoplasmic HIV-1 assembly. Mature nucleocapsid (NCp7), produced from the processing of Gag by viral protease during viral budding from the cell, remodels the annealed complex to form a more stable interaction between the viral RNA and tRNALys, resulting in a more tightly bound and efficient primer for reverse transcription. Reverse transcription is initiated from a primer tRNALys that was previously annealed during viral assembly to an 18 nt sequence in the viral RNA termed the primer binding site (PBS). The annealing of tRNALys to viral RNA is a multi-step process, promoted by both the viral Gag precursor, and later, after viral protein processing, by mature nucleocapsid (NCp7).

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