Structural Understanding of Remodeling Function of HIV-1 Reverse Transcriptase During Initiation

Abstract

Reverse transcription in HIV-1 is initiated from an intermolecular template:primer duplex formed by annealing of the U5-primer binding site (U5-PBS) region of the HIV genome with a host tRNA. The structure of this RNA:RNA complex is now understood as a complex fold with multiple long-range interactions sequestering the template:primer duplex from RT binding. This complex is engaged by two molecules of reverse transcriptase (RT): the first, or remodeler RT, binds to U5-PBS helix 2 adjacent to the 18-nt primer binding site, releasing long-range interactions to allow the second, processive RT to engage the template:primer. The way in which RT engages helix 2 is unknown. Here we solve the structure of the remodeler RT bound to helix 2 and show that, to accomplish this function as a remodeler, RT binds helix 2 in a unique way. RT binds helix 2 on its surface, outside the canonical nucleic acid binding cleft, and assumes a closed conformation, as opposed to the canonical nucleic acid binding open conformation. In this binding conformation, the thumb subdomain of RT binds helix 2 via specific interactions with three nucleic acid binding motifs within helix 2. Residue Lys 275 engages the A-minor platform composed of A168-A171, Arg 277 engages the arginine sandwich motif composed of the base triple U159-A149-U145 along with C146, and Arg 284 and Lys 287 bind the U-rich apical loop of helix 2. While helix 2 does undergoes little conformation change, the helix alpha I and surrounding loops of RT thumb subdomain shift conformationally to allow these key amino acids to engage helix 2. Thus, our study introduces the remodeler role for the RT enzyme, highlighting its structural specificity in the remodeler role during the process of reverse transcription initiation in HIV-1.