Characterization of RHA:RT interactions in HIV-1 reverse transcription

Project Summary HIV-1 infection is one of the leading causes of death globally. The urgent need for the development of new antiviral therapies arises from the lack of vaccine and emerging of drug- resistant strains. HIV-1 reverse transcriptase (RT) is an error-prone polymerase responsible for the emergence of notorious drug resistant mutants. Due to the low processivity, RT pauses on certain RNA sequences/structures and the pausing is correlated with high mutation and recombination rate. The mechanism that maintains fidelity relatively high in the RNA regulatory regions but error-prone in open reading frames during reverse transcription is poorly understood. Host helicase DHX9/RNA helicase A (RHA) is recruited into virions and promotes processivity of RT. Preliminary data suggest dynamic RT: RHA interactions during reverse transcription and reveal that RHA improves RT’s fidelity on structured regulatory RNA regions. The overall goal of this proposal is to establish the structural basis for RHA-mediated RT fidelity modulation during reverse transcription. Aim 1 will characterize the RHA-dependent fidelity enhanced hotspots in the viral genomic RNA by sequencing the cDNA synthesized in the absence and presence of host RHA. Aim 2 will characterize the RT:RHA interactions by cryo-EM, and validate by cell-based assays. The proposed studies will offer insights into the mechanism of the host helicase mediated modulation of RT pausing and fidelity at various locations of the genomic RNA during reverse transcription, and provide structural basis for the development of novel antiviral therapies.