Specific initiation and switch to elongation of human immunodeficiency virus type 1 reverse transcription require the post-transcriptional modifications of primer tRNA3Lys.

Abstract

Initiation of RNA‐dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV‐1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV‐1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo‐ and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV‐1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV‐1 RNA, HIV‐1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer‐template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

“…5), we speculated that NC nucleic acid chaperone activity may be stabilizing extended interactions between the tRNA primer and the RNA template. We considered the possibility that the interaction between residues in the A-rich loop and the anticodon loop of tRNA 3 Lys promote efficient (Ϫ) SSDNA synthesis (1,9). However, although it has been reported that substitution of the six residues in the A-rich loop (nt 162-167 in HIV-1 MAL; GUAAAA) with five residues (CUAUG) can significantly reduce (Ϫ) SSDNA synthesis in vitro (1), others have shown that deletion of the four A residues in HIV-1 HXB2 RNA leads to synthesis of slightly lower or very similar amounts of (Ϫ) SSDNA over time (4,49,79).…”

“…Studies on HIV-1 initiation in vitro indicate that two modes of synthesis are involved in this process: (i) initiation, characterized by a distributive, slow extension of the primer and a high dissociation rate of reverse transcriptase (RT) from the primer-template complex; and (ii) elongation, which follows a transition between incorporation of the sixth and seventh nt and results in a dramatic increase in the processivity and rate of DNA synthesis (1)(2)(3)(4)(5). The inability of HIV-1 RT to initiate (Ϫ) SSDNA synthesis efficiently in vitro may be in part due to the nature of the three-dimensional structure of the initiation complex, which involves extensive intermolecular interactions between the tRNA 3 Lys primer and the RNA template (see below) (6 -11).…”

“…The inability of HIV-1 RT to initiate (Ϫ) SSDNA synthesis efficiently in vitro may be in part due to the nature of the three-dimensional structure of the initiation complex, which involves extensive intermolecular interactions between the tRNA 3 Lys primer and the RNA template (see below) (6 -11). In addition, the efficiency of (Ϫ) SSDNA synthesis appears to be sensitive to the helical conformation of the nucleic acid duplexes that are accommodated by RT; for example, when an 18-nt DNA primer complementary to the PBS (D18) is used, (Ϫ) SSDNA synthesis begins immediately in the elongation mode (1)(2)(3)5).…”

“…In particular, both in vitro and in vivo data pointed to an interaction between the anticodon loop of tRNA 3 Lys and an A-rich loop in HIV-1 RNA, approximately ten bases upstream of the PBS (1, 4, 8 -10, 44, 46 -52). The extended interactions between the primer tRNA and template have been reported to be stabilized by post-transcriptional modification of tRNA 3 Lys (9) and to contribute to efficient initiation of (Ϫ) SSDNA synthesis and transition from the initiation to elongation mode (1). Recently, it was suggested that an interaction between the 5Ј portion of the T⌿C loop of tRNA 3 Lys and a conserved 8-nt sequence in U5, referred to as a primer activation signal, may facilitate initiation of (Ϫ) SSDNA synthesis (53,54).…”

tRNALys3: The Primer tRNA for Reverse Transcription in HIV‐1

“…The interaction of the tRNA Lys3 anticodon loop with the 6-base A-rich loop at the top of the hairpin on the genomic RNA is believed to be important not for tRNA Lys3 annealing to viral RNA, but for an early pausing in reverse transcription, which has been associated with a transition from low processive initiation to high processive elongation (30)(31)(32). However, although all other lentiviruses use tRNA Lys3 as a primer, and have similar stem/loop structures near the PBS, these do not contain an A-rich loop, and therefore, the stem/loop structure rather than the A-rich loop itself may be the important parameter for inducing this pausing (33).…”