Initiation of HIV-2 reverse transcription: a secondary structure model of the RNA-tRNA(Lys3) duplex

Abstract

Human immunodeficiency virus type 2 (HIV-2) reverse transcription is initiated from cellular tRNA(Lys3) partially annealed to the RNA viral genome at the primer binding site (PBS). This annealing involves interactions between two highly structured RNA molecules. In contrast to HIV-1, in which the reverse transcription initiation complex has been thoroughly studied, there is still little information regarding a possible model to describe the secondary structure of the template-primer complex in HIV-2. To determine whether HIV-2 RNA sequences flanking the PBS may specifically interact with the natural primer tRNA, we performed site-directed mutagenesis and enzymatic footprinting. An RNA fragment corresponding to the HIV-2 U5 RNA domain and tRNA(Lys3) were probed either in their free form or in the binary complex. Important reactivity changes to nucleases were obtained upon complex formation. In addition to the canonical contacts between the viral PBS and the 3' end acceptor stem of tRNA(Lys3), we identified two additional interacting domains: (i) the U-rich region of the anticodon loop with the A-rich sequence of the internal loop within the U5-prePBS region; (ii) nucleotides 48-54 from the TPsiC domain of tRNA(Lys3) and the 240-247 region of viral U5-RNA. In view of these experimental data and sequence comparison between different HIV-2 isolates, we propose a model for the secondary structure of the HIV-2 template-primer initiation complex.

Figure 1

Secondary structures of tRNA^Lys3 and the HIV-2_ROD U5-PBS region [adapted from Berkhout and Schoneveld (13)]. Dark boxes, principal interaction of the reverse transcription initiation complex between the viral PBS region and the primer acceptor stem. White boxes, the primer anticodon loop may interact with one of the two A-rich viral RNA regions.

Figure 2

Digestion patterns of free tRNA^Lys3 or in the binary complex with HIV-2_ROD viral RNA. (A) Enzymatic probing of tRNA^Lys3 with ribonuclease V1. The 3′ end labelled tRNA^Lys3, ([α-³²P]pCptRNA^Lys3), free or complexed with the U5-PBS region of viral RNA (nucleotides 1–545) was incubated for 7 min at 37°C in the presence of 0, 0.001, 0.005 and 0.01 U RNase (lanes 2–5 and 6–9, respectively). Lane 1 corresponds to untreated labelled tRNA^Lys3 and lane M to the alkaline hydrolysis of tRNA^Lys3 (size ladder). (B) Enzymatic probing with the MBN. Free 5′ end labelled tRNA^Lys3 or complexed with viral RNA was incubated for 7 min at 37°C in the presence of 0, 0.01, 0.05 and 0.1 U enzyme (lanes 2–5 and 6–9, respectively). Lane 1 corresponds to untreated labelled tRNA^Lys3. (C) Schematic representation of reactivity to nucleases of free tRNA^Lys3 or hybridised to HIV-2_ROD PBS. The black dots represent MBN cleavages, and the grey triangles represent the sites of cleavage by ribonuclease V1. The preferential cleavage sites of each nuclease are indicated by the increasing number of symbols depicted beside the nucleotides.

Figure 2

Digestion patterns of free tRNA^Lys3 or in the binary complex with HIV-2_ROD viral RNA. (A) Enzymatic probing of tRNA^Lys3 with ribonuclease V1. The 3′ end labelled tRNA^Lys3, ([α-³²P]pCptRNA^Lys3), free or complexed with the U5-PBS region of viral RNA (nucleotides 1–545) was incubated for 7 min at 37°C in the presence of 0, 0.001, 0.005 and 0.01 U RNase (lanes 2–5 and 6–9, respectively). Lane 1 corresponds to untreated labelled tRNA^Lys3 and lane M to the alkaline hydrolysis of tRNA^Lys3 (size ladder). (B) Enzymatic probing with the MBN. Free 5′ end labelled tRNA^Lys3 or complexed with viral RNA was incubated for 7 min at 37°C in the presence of 0, 0.01, 0.05 and 0.1 U enzyme (lanes 2–5 and 6–9, respectively). Lane 1 corresponds to untreated labelled tRNA^Lys3. (C) Schematic representation of reactivity to nucleases of free tRNA^Lys3 or hybridised to HIV-2_ROD PBS. The black dots represent MBN cleavages, and the grey triangles represent the sites of cleavage by ribonuclease V1. The preferential cleavage sites of each nuclease are indicated by the increasing number of symbols depicted beside the nucleotides.

Figure 3

Digestion patterns of the HIV-2_ROD viral RNA complexed or not with tRNA^Lys3. The cleavage sites were detected by reverse transcription of the digested fragments by using an anti-PBS ODN primer. (A) Enzymatic digestion of the prePBS RNA (nucleotides 244–305) with MBN. The viral RNA complexed or not with tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.05 and 0.1 U enzyme (lanes 1–3 and 4–6, respectively). Lane C corresponds to the reverse transcription products on the viral RNA fragment before any nuclease treatment. (B) Viral RNA reactivity to ribonuclease V1. Free viral RNA or complexed to tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.001, 0.005 and 0.01 U enzyme (lanes 7–10 and 11–14, respectively). The prePBS sequence was used as a size marker. (C) Schematic representation of the prePBS-PBS HIV-2_ROD RNA region hybridised or not to the tRNA^Lys3. Reactivities to nucleases. The black dots correspond to sites of cleavage by MBN, and the grey triangles to those by ribonuclease V1. The preferential cleavage sites of each nuclease are indicated by the increasing number of symbols depicted beside the nucleotides.

Figure 3

Digestion patterns of the HIV-2_ROD viral RNA complexed or not with tRNA^Lys3. The cleavage sites were detected by reverse transcription of the digested fragments by using an anti-PBS ODN primer. (A) Enzymatic digestion of the prePBS RNA (nucleotides 244–305) with MBN. The viral RNA complexed or not with tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.05 and 0.1 U enzyme (lanes 1–3 and 4–6, respectively). Lane C corresponds to the reverse transcription products on the viral RNA fragment before any nuclease treatment. (B) Viral RNA reactivity to ribonuclease V1. Free viral RNA or complexed to tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.001, 0.005 and 0.01 U enzyme (lanes 7–10 and 11–14, respectively). The prePBS sequence was used as a size marker. (C) Schematic representation of the prePBS-PBS HIV-2_ROD RNA region hybridised or not to the tRNA^Lys3. Reactivities to nucleases. The black dots correspond to sites of cleavage by MBN, and the grey triangles to those by ribonuclease V1. The preferential cleavage sites of each nuclease are indicated by the increasing number of symbols depicted beside the nucleotides.

Figure 4

Stem–loop structures of the HIV-2_ROD RNA prePBS region. The prePBS region corresponding either to wild-type RNA, mut_(274–276) RNA or mut_(287–290) RNA is represented. The stability values corresponding to the three structures presented in this figure were determined by using the Zuker program.

Figure 5

MBN digestion patterns of the mutated HIV-2^ROD viral RNAs complexed or not with tRNA^Lys3. Conditions were the same as those described in Figure 3. The mut_(274–276) and mut_(287–290) RNA fragments were incubated for 7 min at 37°C in the presence of 0, 0.001, 0.05 and 0.1 U enzyme, in the absence of tRNA^Lys3 (lanes 1–4 and 9–12, respectively) or hybridised to the tRNA^Lys3 (lanes 5–8 and 13–16, respectively).

Figure 6

(A) MBN digestion patterns of the HIV-2_ROD RNA (321–371) region complexed or not with the natural tRNA^Lys3 primer. The cleavage sites were evidenced by reverse transcription of the digested products from wild-type RNA using an ODN primer complementary to nucleotides 372–390. The viral RNA complexed or not with the tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.05 and 0.1 U enzyme (lanes 1–3 and 4–6, respectively). The sequence of the RNA fragment was used as size marker. (B) Schematic representation of reactivity to MBN of the HIV-2_ROD RNA region (nucleotides 321–371) complexed or not to tRNA^Lys3. The MBN cleavage sites are indicated by black dots, and the level of viral RNA sensitivity by the number of symbols.

Figure 6

(A) MBN digestion patterns of the HIV-2_ROD RNA (321–371) region complexed or not with the natural tRNA^Lys3 primer. The cleavage sites were evidenced by reverse transcription of the digested products from wild-type RNA using an ODN primer complementary to nucleotides 372–390. The viral RNA complexed or not with the tRNA^Lys3 was incubated for 7 min at 37°C in the presence of 0, 0.05 and 0.1 U enzyme (lanes 1–3 and 4–6, respectively). The sequence of the RNA fragment was used as size marker. (B) Schematic representation of reactivity to MBN of the HIV-2_ROD RNA region (nucleotides 321–371) complexed or not to tRNA^Lys3. The MBN cleavage sites are indicated by black dots, and the level of viral RNA sensitivity by the number of symbols.

Figure 7

Structural model of the HIV-2_ROD–tRNA^Lys3 binary complex. The shaded box corresponds to the principal interaction PBS–acceptor stem. The white boxes correspond to the additional interactions: A-rich sequence of the internal loop with the U-rich loop of the anticodon; nucleotides 48–54 from the TΨC domain of tRNA with the region 240–247 of viral RNA.