Abstract
BackgroundRNA helicase A (RHA), a DExH box protein, promotes annealing of tRNALys3, a primer for reverse transcription, to HIV-1 RNA and assembles into virus particles. A-kinase anchoring protein 95-like protein (HAP95) is a binding partner of RHA. The role of HAP95 in the annealing of tRNALys3 was examined in this study.ResultsHAP95 associates with the reverse transcriptase region of Pol protein of HIV-1. Decreasing endogenous HAP95 in HIV-1-producing 293T cells by siRNA reduces the amount of tRNALys3 annealed on viral RNA. This defect was further deteriorated by knockdown of RHA in the same cells, suggesting a cooperative effect between these two proteins. Biochemical assay in vitro using purified GST-tagged HAP95 shows that HAP95 may inhibit the activity of RHA.ConclusionThe results support a hypothesis that HAP95 may transiently block RHA’s activity to protect the annealed tRNALys3 on viral RNA in the cells from removing by RHA during the packaging of RHA into virus particles, thus facilitating the annealing of tRNALys3 to HIV-1 RNA.
Highlights
RNA helicase A (RHA), a DExH box protein, promotes annealing of tRNALys3, a primer for reverse transcription, to Human immunodeficiency virus 1 (HIV-1) RNA and assembles into virus particles
We found that knockdown of A-kinase anchoring protein 95-like protein (HAP95) in virus-producing 293T cells does not affect the production of HIV-1 particle nor does the viral RNA packaging, but reduces the annealing of tRNALys3 to viral RNA
Roy et al used this method to study the cellular proteins that associate with HIV-1 Gag [15] and identified RHA that is incorporated into HIV-1 particles and promotes the synthesis of viral cDNA upon viral infection of the new cells
Summary
RNA helicase A (RHA), a DExH box protein, promotes annealing of tRNALys, a primer for reverse transcription, to HIV-1 RNA and assembles into virus particles. HIV-1, a member of lentivirus family of retroviridae, selectively packages tRNALys into virus particle and anneals tRNALys to viral genomic RNA as a primer in reverse transcription [2]. The annealing of tRNALys to viral genomic RNA was initially mediated by Gag [10,11] and fine-tuned by nucleocapsid (NC) protein, a proteolytic derivative of Gag during budding of virus particles, to render the annealed tRNALys an increased capability as a reverse transcription primer [12,13]
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