Direct and Indirect Contributions of RNA Secondary Structure Elements to the Initiation of HIV-1 Reverse Transcription

Abstract

Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription requires specific recognition between the viral RNA (vRNA), tRNA 3 Lys , which acts as primer, and reverse transcriptase (RT). The specificity of this ternary complex is mediated by intricate interactions between the HIV-1 RNA and tRNA 3Lys . Here, we compared the relative importance of the secondary structure elements of this complex in the initiation process. To this aim, we used the previously published three-dimensional model of the initiation complex to rationally introduce a series of deletions and substitutions in the vRNA. When necessary, we used chemical probing to check the structure of the tRNA 3 Lys -mutant vRNA complexes. For each of them, we measured the binding affinity of RT and the kinetics of initial extension of tRNA 3 Lys and of synthesis of the (؊) strand strong stop DNA. Our results were overall in keeping with the three-dimensional model of the initiation complex. Surprisingly, we found that disruption of the intermolecular template-primer interactions, which are not directly recognized by RT, more severely affected reverse transcription than deletions or disruption of one of the intramolecular helices to which RT directly binds. Perturbations of the highly constrained junction between the intermolecular helix formed by the primer binding site and the 3 end of tRNA 3 Lys and the helix immediately upstream also had dramatic effects on the initiation of reverse transcription. Taken together, our results demonstrate the overwhelming importance of the overall three-dimensional structure of the initiation complex and identify structural elements that constitute promising targets for anti-initiation-specific drugs.

Efficient Initiation of HIV-1 Reverse Transcriptionin Vitro

“…Apparently, the contribution of nt 201-224 is sufficient for this effect, and therefore, RNA 224 is able to form the secondary structure required for efficient (Ϫ) SSDNA synthesis. This correlates well with the previous observation that an HIV-1 MAL RNA template consisting only of nt 123-217 has a very similar overall secondary structure as a longer HIV-1 MAL template consisting of nt 1-311 (11,44,46).…”

“…These results are generally in accord with earlier in vitro data indicating that extension of tRNA 3 Lys is less efficient if mutant RNA templates contain deletions of sequences within a 54-nt downstream region (4,55,56). However, the present findings and other data (46) appear to be at variance with the observation that the primer activation signal increases (Ϫ) SSDNA synthesis when downstream bases are mutated (53,54).…”

“…Previous work (1, 4, 8 -10, 44, 46 -52) indicated that residues in the A-rich loop interact with the anticodon loop of the tRNA primer. In addition, structure probing suggested an interaction between the 3Ј arm of the anticodon stem and the variable loop of the tRNA (nt 40 -46) and U5 sequences upstream of the A-rich loop, corresponding to nt 140 -143 in helix 3E and nt 158 -160 in helix 5D in HIV-1 MAL RNA (10,44,46). In HIV-1 NL4-3, used in the present work, we find that the corresponding template sequences are contiguous and include nt 143-149 (58).…”

Identification of specific HIV-1 reverse transcriptase contacts to the viral RNA:tRNA complex by mass spectrometry and a primary amine selective reagent

“…We suggest that these contacts may be located on the duplex structure just ahead of the primer [nucleotides 132-139 of HIV-1 RNA paired with nucleotides 168-175 (numbering according to the Mal isolate) and denoted helix 2 in ref. 23]. It is this structure that RT must disrupt for effective elongation to occur.…”

“…Isel et al used RNA-specific nucleases to probe the vRNA-tRNA structure complexed with RT (9). Although this approach was useful in revealing structural reorganizations in several unliganded parts of vRNA and tRNA on RT binding, accurate identification of direct protein-nucleic acid contacts was hampered by high background protection in the vRNA and tRNA footprints derived from the steric conflict between bulky nucleases and RT (9,23). Such limited information on protein contacts to the template-primer prompted us to design a high-resolution protein footprinting methodology allowing comprehensive and fine mapping of direct RT contacts to vRNA:tRNA in the initiation complex.…”