Abstract
Initiation of human immunodeficiency virus-1 (HIV-1) reverse transcription requires formation of a complex containing the viral RNA (vRNA), tRNA(3)(Lys) and reverse transcriptase (RT). The vRNA and the primer tRNA(3)(Lys) form several intermolecular interactions in addition to annealing of the primer 3' end to the primer binding site (PBS). These interactions are crucial for the efficiency and the specificity of the initiation of reverse transcription. However, as they are located upstream of the PBS, they must unwind as DNA synthesis proceeds. Here, the dynamics of the complex during initiation of reverse transcription was followed by enzymatic probing. Our data revealed reciprocal effects of the tertiary structure of the vRNA.tRNA(3)(Lys) complex and reverse transcriptase (RT) at a distance from the polymerization site. The structure of the initiation complex allowed RT to interact with the template strand up to 20 nucleotides upstream from the polymerization site. Conversely, nucleotide addition by RT modified the tertiary structure of the complex at 10-14 nucleotides from the catalytic site. The viral sequences became exposed at the surface of the complex as they dissociated from the tRNA following primer extension. However, the counterpart tRNA sequences became buried inside the complex. Surprisingly, they became exposed when mutations prevented the intermolecular interactions in the initial complex, indicating that the fate of the tRNA depended on the tertiary structure of the initial complex.
Highlights
Reverse transcription is a key step in the retroviral replication cycle [1, 2], during which the virus-encoded reverse transcriptase (RT),1 which possesses RNA- and DNA-dependent DNA polymerase and RNase H activities [3], converts the genomic RNA into double-stranded DNA
The RNase T2 cleavages observed in 123–217 viral RNA (vRNA) either free or bound to tRNA3Lys are consistent with the secondary structure models that we previously proposed for the free human immunodeficiency virus-1 (HIV-1) RNA [34] and for the binary complex (Figs. 1 and 2A) [12, 13]
The efficiency and specificity of HIV-1 reverse transcription relies on intricate interactions between the viral RNA and primer tRNA3Lys [12, 13, 18, 19, 21, 22, 25,26,27,28, 35]
Summary
Chemicals and Enzymes—RNase T2 and Neurospora crassa endonuclease were from Amersham Pharmacia Biotech. RNase H(Ϫ) HIV-1 RT bearing the E478Q mutation was purified essentially as described previously [32]. This mutant polymerase was used to prevent cleavage of the RNA template by the double-stranded RNase activity associated with HIV-1 RNase H [33]. Forty g of heparin were added to half of the reaction mixtures, which were further incubated for 5 min at 37 °C. Enzymatic Probing of tRNA3Lys—3Ј End-labeled tRNA3Lys (ϳ50,000 cpm), either free, in the binary complex formed with 3-fold excess (12 pmol) 1–311 vRNA, or in the ternary complex with 50 pmol of HIV-1 RT, was incubated for 10 min at 37 °C. RNA was ethanol-precipitated and analyzed on a 15% denaturing polyacrylamide gel
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