Using NMR to Determine the Conformation of the HIV Reverse Transcription Initiation Complex

摘要

Initiation of reverse transcription of genomic RNA is a key early step in replication of the human immunodeficiency virus upon infection of a host cell. Viral reverse transcriptase (RT) initiates from a specific RNA-RNA complex formed between a host transfer RNA (tRNA_3~(Lys)) and region at the 5′end of genomic RNA; the 3′ end of the tRNA acts as a primer for reverse transcription of genomic RNA. We determined the secondary structure of the 50 kDa complex between HIV genomic RNA and human tRNA_3~(Lys) by nuclear magnetic resonance (NMR) spectroscopy. We show that both RNAs undergo large-scale conformational changes upon complex formation. Formation of an 18 base pair primer helix with the 3′ end of tRNA_3~(Lys) drives large conformational rearrangements of the tRNA at the 5′ end, while maintaining the anticodon loop for potential loop-loop interactions. HIV RNA forms an intramolecular helix adjacent to the intermolecular primer helix. This helix, which must be broken by reverse transcription, likely acts as a kinetic block to reverse translation.