Abstract

RNA helicase A (RHA) promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNALys3 to viral RNA, and elevating the ratio of unspliced to spliced viral RNA. At its amino terminus are two double-stranded RNA binding domains (dsRBDs) that are essential for RHA-viral RNA interaction. Linking the dsRBDs to the core helicase domain is a linker region containing 6 predicted helices. Working in vitro with purified mutant RHAs containing deletions of individual helices reveals that this region may regulate the enzyme's helicase activity, since deletion of helix 2 or 3 reduces the rate of unwinding RNA by RHA. The biological significance of this finding was then examined during HIV-1 production. Deletions in the linker region do not significantly affect either RHA-HIV-1 RNA interaction in vivo or the incorporation of mutant RHAs into progeny virions. While the partial reduction in helicase activity of mutant RHA containing a deletion of helices 2 or 3 does not reduce the ability of RHA to stimulate viral RNA synthesis, the promotion of tRNALys3 annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNALys3 annealing, but reduces its ability to stimulate viral RNA synthesis. Additionally, RHA stimulation of viral RNA synthesis results in an increased ratio of unspliced to spliced viral RNA, and this increase is not inhibited by deletions in the linker region, nor is the pattern of splicing changed within the ∼ 4.0 kb or ∼ 1.8 kb HIV-1 RNA classes, suggesting that RHA's effect on suppressing splicing is confined mainly to the first 5′-splice donor site. Overall, the differential responses to the mutations in the linker region of RHA reveal that RHA participates in HIV-1 RNA metabolism by multiple distinct mechanisms.

Highlights

  • HIV-1, like other retroviruses, does not encode its own RNA helicases, but employs cellular counterparts to facilitate its replication [1]

  • We have examined the ability of mutant RNA helicase A (RHA) to promote some steps of HIV-1 RNA metabolism in vivo and have found that while helices 2 and 3 are indispensable for the promotion of tRNALys3 annealing to viral RNA, helices 4 and 5 are more critical for stimulation of viral RNA synthesis, indicating the distinct mechanisms responsible for RHA to participate in these two viral processes

  • Little rescue of annealing was obtained by expression of the mutant RHADLinker-Helix2 or RHADLinker-Helix3 that contains a deletion of either helix 2 or 3. These results indicate that helices 2 and 3 are required for the promotion of tRNALys3 annealing to viral RNA by RHA, but none of the helices in the linker region analyzed in this report are required for the RHA packaging into HIV-1 particles

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Summary

Introduction

HIV-1, like other retroviruses, does not encode its own RNA helicases, but employs cellular counterparts to facilitate its replication [1]. We have examined the ability of mutant RHAs to promote some steps of HIV-1 RNA metabolism in vivo and have found that while helices 2 and 3 are indispensable for the promotion of tRNALys annealing to viral RNA, helices 4 and 5 are more critical for stimulation of viral RNA synthesis, indicating the distinct mechanisms responsible for RHA to participate in these two viral processes Both wild-type RHA and RHA containing linker region mutations inhibit the splicing events required for the generation of singly (, 4.0 kb) and multiply (, 1.8 kb) spliced HIV-1 RNA species, resulting in a greater production of unspliced (, 9.2 kb) HIV-1 RNA. The splicing patterns within the , 4.0 kb or ,1.8 kb HIV-1 RNA classes remain unchanged, suggesting that RHA’s effect on suppressing splicing is confined mainly to the first 59-splice donor site, SD1

Results
Discussion
Findings
Materials and Methods
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