Abstract

BackgroundAn important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.ResultsIn this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA3Lys.ConclusionsThese data support the conclusion that the complex formed between GagPol, mLysRS and tRNA3Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA3Lys into HIV-1 particles.

Highlights

  • An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription

  • Characterization of the interaction between Pol and mitochondrial lysyl-tRNA synthetase (mLysRS) We previously established that the catalytic domain of the mitochondrial species of human lysyl-tRNA synthetase (LysRS) interacts with the Pol domain of the polyprotein GagPol from HIV-1 [17]

  • Human mLysRS was expressed in E. coli with a Cterminal HA-tag, and the Pol polyprotein was expressed in insect cells with a C-terminal His-tag (Fig. 1)

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Summary

Introduction

An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. Reverse transcription of human immunodeficiency virus type-1 (HIV-1) genomic RNA requires annealing of tRNA3Lys to the primer binding site (PBS) [1, 2]. Monospecific antibodies directed to the very N-terminal peptides characteristic of cytoplasmic or mitochondrial LysRS revealed the presence of only the mitochondrial LysRS (mLysRS) species in extracts of HIV-1 virions purified from HIV-1 LAI strain cultured on CEM cells [7]

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