Effect of TAR hairpin stabilization on HIV-1 reverse transcription

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome requires the first strand transfer that relies on a base-pairing interaction between the 3' R sequence of the genomic RNA (gRNA) and the r sequence of the strong-stop DNA (ssDNA). The annealing reaction between the cTAR hairpin of ssDNA and the TAR hairpin of gRNA plays an essential role in r-3' R pairing. Studies suggest that HIV-1 nucleocapsid protein (NC) has only a minor role in ssDNA synthesis but facilitates the annealing reaction by destabilizing the two complementary hairpins. We designed mutations to increase the stability of the cTAR DNA hairpin. These mutations should impair the NC-mediated annealing process, but not other viral processes. We investigated the effects of mutations in the annealing reaction, viral infectivity, and reverse transcription in infected cells. The mutations stabilizing the lower stem of the cTAR hairpin impair the annealing reaction, strongly reduce HIV-1 replication, and the amount of full-length ssDNA in infected cells. The in vitro and ex vivo data support NC as the essential player in the first strand transfer reaction by facilitating the initiation of the cTAR-TAR pairing through the zipper pathway. These results agree with previous studies suggesting a coevolutionary relationship between the stability of the cTAR hairpin and NC activity. Our data are consistent with the hypothesis proposed in an early work, suggesting that ssDNA degradation by cellular nucleases occurs when the first strand transfer cannot take place. This attractive hypothesis warrants further investigation in future studies.IMPORTANCEThe first strand transfer is a crucial step of the reverse transcription process. Because the great majority of first strand transfers in HIV-1 occur with full-length ssDNA, the annealing reaction between the cTAR DNA hairpin and the 3' TAR RNA hairpin plays an essential role in the first strand transfer. To our knowledge, our study is the first to show that mutations stabilizing the lower stem of the cTAR hairpin strongly decrease the annealing reaction, HIV-1 replication, and the amount of full-length ssDNA in infected cells. Our data support the notion that NC is the essential player in the first strand transfer reaction by facilitating the initiation of the cTAR-TAR pairing through the zipper pathway.