Initiation of HIV Reverse Transcription

Abstract

Reverse transcription of retroviral genomes into double stranded DNA is a key event for viral replication. The very first stage of HIV reverse transcription, the initiation step, involves viral and cellular partners that are selectively packaged into the viral particle, leading to an RNA/protein complex with very specific structural and functional features, some of which being, in the case of HIV-1, linked to particular isolates. Recent understanding of the tight spatio-temporal regulation of reverse transcription and its importance for viral infectivity further points toward reverse transcription and potentially its initiation step as an important drug target.

Keywords: retrovirus; reverse transcriptase; tRNA.

Figure 1.

Secondary (a) and tertiary (b) structures of tRNA₃^Lys.

Figure 2.

Partners and model for the tRNA₃^Lys packaging complex (a) Organisation of the Gag-Pol and Gag precursors. MA: matrix ; CA: capsid ; NC: nucleocapsid ; p6: HIV-1 p6 protein ; PR: protease; RT: reverse transcriptase, with its Fingers, Palm, Thumb, Connection (Conn.) and RNase H domains; IN: integrase. The dark blue rectangle in the capsid domain of the precursors corresponds to the C-terminal helix 4 that was shown to interact with lysyl-tRNA synthetase (LysRS) (b) Organisation of LysRS: the anticodon (AC) binding domain is located between positions 125 and 207 and the LysRS dimerisation (Dz) domain between positions 238 and 266. The two boxes toward the C-terminus of the protein, between positions 314 and 343 and 544 and 559 are important for amino acid recognition. The area highlighted in red (208–259), overlapping the dimerisation domain, is involved in binding to the capsid. (c) Model for the packaging complex. The purple patch corresponds to the anticodon binding domain and the red patch to amino acids 208–259 that interact with the CA domain of Gag.

Figure 3.

Schematic representation of HIV-1 specific initiation (a) versus unspecific elongation (b) of reverse transcription. The vRNA template is represented by a thin grey line. The natural tRNA₃^Lys primer or an 18 mer DNA primer are in black and the newly synthesized DNA is represented by thick blue and red lines representative of the initiation and elongation steps of reverse transcription, respectively. In the presence of the natural tRNA primer, transition between initiation and elongation occurs after the addition of the first 6 nucleotides to the 3′ end of the primer. In the case of the HIV-1 MAL isolate, transition is facilitated by the anticodon/A-rich loop interaction upstream of the PBS, represented by the close contact between tRNA₃^Lys and the vRNA.

Figure 4.

Secondary structure of the RNA partners of the HIV-1 initiation complex of reverse transcription and of the binary primer/template complexes, in the case of the HIV-1 MAL (representative of a subtype A PBS domain) and subtype B isolates. The regions undergoing intra- or intermolecular rearrangements upon formation of the primer/template complex are highlighted in various colours. Boxes or sequences of the same color represent areas that are base-paired in the binary complex. (a) The human tRNA₃^Lys. (b) The PBS sub-domain in the free form of the HIV-1 MAL vRNA. (c) The HIV-1 MAL vRNA/tRNA₃^Lys complex. (d) The PBS sub-domain in the free form of the HIV-1 NL-4.3 (subtype B) isolate. The PAS and mutations 2L and 2R are indicated. (e) Localization of the anti-PAS region of tRNA₃^Lys.

Figure 5.

Secondary structure models of the HIV-2 vRNA (a) and of the vRNA/tRNA₃^Lys complex (b). The tRNA is in red and the vRNA in black.

Figure 6.

Schematic representation of the temporal regulation of reverse transcription in producer and target cells.