Abstract
RNA helicase A (RHA) promotes multiple steps in HIV-1 production including transcription and translation of viral RNA, annealing of primer tRNA(Lys3) 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 tRNA(Lys3) annealing to viral RNA is blocked. In contrast, deletion of helices 4 or 5 does not affect the ability of RHA to promote tRNA(Lys3) 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.