Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA

Abstract

Edited by Karin Musier-Forsyth DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA Lys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of singlenucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (؊)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR

“…Further experimentation is warranted to validate the c-jun 5'-UTR recapitulates activity of JUND PCE and possibly retroviral PCEs, which is necessary for RHA interaction (36,(43)(44)(45) and mTOR-resistant translation.…”

“…Dysregulation of AP-1 is hallmark of viral pathogenesis, neoplastic transformation and tumor progression (35,46). Likewise, dysregulation of dhx9/rha is associated with productive viral infection (31,37,38,45,(47)(48)(49)(50) and tumor survival (51)(52)(53)(54). The recent finding NCBP3 is essential to mount a precise antiviral response (34) posits dysfunction in a RHA-NCBP1-NCBP3 translation pathway contributes to deficient innate response.…”

A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA

“…The metastable nt pairings in U5 and PBS-segment likely contribute to the alternate functionalities of the 5 -UTR in cellulo. Destabilized PBS-segment reduces affinity for DHX9/RHA and diminishes processivity of reverse transcriptase and virus infectivity [28,54,55]. Destabilized A59-U103 pairing significantly increase Gag translation rate in cells [22] and increase biochemical affinity of the HIVMAL 5 -Cap for host eIF4E [6].…”

The importance of virion-incorporated cellular RNA-Binding Proteins in viral particle assembly and infectivity

“…Several studies have revealed that specific cellular ivRBPs either promote (e.g. UPF1 RNA helicase and ATPase [UPF1] [ 130 ], Y-box binding protein 1 [YBX1] [ 131 ], DHX9 [ 132 ], EEF1A [ 133 , 134 ], PPIA [ 135 ] and aminoacyl-tRNA synthetases [ 136 ]) or inhibit (e.g. Mov10 RISC complex RNA helicase [MOV10] [ 137 ], pyruvate kinase M1/2 [PKM] [ 138 ], enolase 1 [ENO1] [ 139 ]) reverse transcription (Table S1E).…”

“…Authors suggested that UPF1 could mediate the remodelling the vRNP to facilitate reverse transcription or, alternatively, promote the annealing of tRNA Lys3 primer to the viral genome, as shown in other helicases such as DHX9 [ 130 , 140 ]. Conversely, recent evidence suggested that DHX9 participates in the elongation phase of reverse transcription but not in the annealing of tRNA Lys3 to the vRNA [ 132 ]. Further research is required to elucidate the exact mechanism of action of UPF1, DHX9 and other cellular RBPs during HIV-1 reverse transcription.…”