Abstract
Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3. Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription.
Highlights
Reverse transcription is one of the first steps in HIV infection after the viral envelope fuses with the host cell membrane and the viral capsid enters the cell cytoplasm
Within the capsid are two copies of the single-stranded positive-sense viral RNA genome that must be reverse transcribed into double-stranded linear DNA, which is subsequently transported into the nucleus and integrated into the host cell genome
To understand the interactions of the viral RNA genome (vRNA) reverse transcription initiation site with the primer tRNALys3, we focused on a region of 100 nt between residues 123 and 217 in the HIV-1 Mal isolate previously shown to form a complex with tRNALys3, recapitulating the biochemical properties of complexes formed with full-length viral RNAs (Isel et al 1998)
Summary
Reverse transcription is one of the first steps in HIV infection after the viral envelope fuses with the host cell membrane and the viral capsid enters the cell cytoplasm. The initiation of reverse transcription, during which the first approximately 15 dNTPs are incorporated onto the primer tRNA, is slow and nonprocessive relative to the subsequent elongation phase that proceeds toward the vRNA 5′ end (Pop and Biebricher 1996; Suo and Johnson 1997a,b; Liang et al 1998; Goldschmidt et al 2002). Such a slow initiation phase likely plays an important role in regulating the timing of reverse transcription initiation. The roles of these RNA-pairing interactions in regulating initiation kinetics are supported by mutagenesis and biochemical experiments (Aiyar et al 1992; Essink et al 1995; Puglisi and Puglisi 1998; Beerens and Berkhout 2002a,b; Rigourd et al 2003; Abbink et al 2004; Ooms et al 2007; Beerens et al 2013; Jones et al 2013; Sleiman et al 2013; Seif et al 2015)
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