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. 2000 Jan 15;28(2):634-40.
doi: 10.1093/nar/28.2.634.

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription

F Brulé  1 , G Bec, G Keith, S F Le Grice, B P Roques, B Ehresmann, C Ehresmann, R Marquet
Affiliations

In vitro evidence for the interaction of tRNA(3)(Lys) with U3 during the first strand transfer of HIV-1 reverse transcription

F Brulé et al. Nucleic Acids Res. .

Abstract

Over the course of its evolution, HIV-1 has taken maximum advantage of its tRNA(3)(Lys)primer by utilizing it in several steps of reverse transcription. Here, we have identified a conserved nonanucleotide sequence in the U3 region of HIV-1 RNA that is complementary to the anticodon stem of tRNA(3)(Lys). In order to test its possible role in the first strand transfer reaction, we applied an assay using a donor RNA corresponding to the 5'-part and an acceptor RNA spanning the 3'-part of HIV-1 RNA. In addition, we constructed two acceptor RNAs in which the nonanucleotide sequence complementary to tRNA(3)(Lys)was either substituted (S) or deleted (Delta). We used either natural tRNA(3)(Lys)or an 18 nt DNA as primer and measured the efficiency of (-) strand strong stop DNA transfer in the presence of wild-type, S or Delta acceptor RNA. Mutations in U3 did not decrease the transfer efficiency when reverse transcription was primed with the 18mer DNA. However, they significantly reduced the strand transfer efficiency in the tRNA(3)(Lys)-primed reactions. This reduction was also observed in the presence of nucleocapsid protein. These results suggest that tRNA(3)(Lys)increases (-) strand strong stop transfer by interacting with the U3 region of the genomic RNA. Sequence comparisons suggest that such long range interactions also exist in other lentiviruses.

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Figures

Figure 1
Figure 1
(A) Schematic representation of synthesis of the (–) strand strong stop DNA and of the first strand transfer. The redundant (R) sequence is present at both ends of the genomic RNA, while U5 (unique to the 5′-end) and U3 (unique to the 3′-end) are present at the 5′- and 3′-ends, respectively. DNA synthesis is initiated from the 3′-end of tRNA3Lys annealed to the primer binding site (PBS). The polypurine tract (PPT) generates the primer for (+) strand DNA synthesis. The poly(A) tail of the genomic RNA is also shown. (B) The experimental system used in this study. The complementarity between U3 and the anticodon stem is schematized. In addition to the wild-type acceptor RNA, two RNAs were used in which the sequence complementary to the primer was either substituted or deleted.
Figure 2
Figure 2
DNA-primed strand transfer. (A) Gel fractionation of the transfer products. Labeled ODN (40 nM) was heat annealed to donor RNA (100 nM) and incubated in the absence (lanes 1–3) or presence of 200 nM WT (lanes 4–14), S (lanes 5–15) or Δ (lanes 16–26) acceptor RNAs and 200 nM HIV-1 RT. Reaction was for 30, 36, 42, 48, 54, 60, 66, 72, 78, 84 or 90 min. (B) Quantification of the full-length transfer product obtained under the same conditions, except that the RT concentration was reduced to 100 nM.
Figure 2
Figure 2
DNA-primed strand transfer. (A) Gel fractionation of the transfer products. Labeled ODN (40 nM) was heat annealed to donor RNA (100 nM) and incubated in the absence (lanes 1–3) or presence of 200 nM WT (lanes 4–14), S (lanes 5–15) or Δ (lanes 16–26) acceptor RNAs and 200 nM HIV-1 RT. Reaction was for 30, 36, 42, 48, 54, 60, 66, 72, 78, 84 or 90 min. (B) Quantification of the full-length transfer product obtained under the same conditions, except that the RT concentration was reduced to 100 nM.
Figure 3
Figure 3
tRNA3Lys-primed strand transfer. (A) Gel fractionation of the transfer products. Labeled tRNA3Lys (80 nM) was heat-annealed to donor RNA (200 nM) and incubated in the absence or presence of 400 nM WT, S or Δ acceptor RNA and 100 nM HIV-1 RT. Reaction was for 1, 2 or 4 h. (B) Quantification of the full-length product obtained after 2 (gray) and 4 h (black) incubation.
Figure 4
Figure 4
PCR amplification of the full-length transfer product generated in the tRNA3Lys-primed reaction. Transfer products generated after 6–90 min in the presence of WT, S or Δ acceptor RNAs were amplified and quantified as described in Materials and Methods. When using the WT acceptor, amplification rapidly became limited by the primer concentration.
Figure 5
Figure 5
tRNA3Lys-primed strand transfer in the absence and presence of NCp. (A) Gel fractionation of the transfer products. tRNA3Lys (40 nM) was heat-annealed to 100 nM donor RNA and DNA synthesis was initiated by addition of 50 µM dNTP, 200 nM acceptor RNA, 100 nM RT and saturating amounts of NCp (1 NCp:7–8 nt). (B) Quantification of the gel shown in (A). Reaction time was 1 (gray) or 2 h (black).
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