Reverse transcription of HIV-1 viral RNA uses human tRNA 3 Lys as a primer. Some of the modified nucleotides carried by this tRNA must play a key role in the initiation of this process, because unmodified tRNA produced in vitro is only marginally active as primer. To provide a better understanding of the contribution of base modifications in the initiation complex, we have designed a recombinant system that allows tRNA 3 Lys expression in Escherichia coli. Because of their high level of overexpression, some modifications are incorporated at substoichiometric levels. We have purified the two major recombinant tRNA 3Lys subspecies, and their modified nucleotide contents have been characterized by a combination of NMR and biochemical techniques. Both species carry Cs, Ds, T, t 6 A, and m 7 G. Differences are observed at position 34, within the anticodon. One fraction lacks the 5-methylaminomethyl group, whereas the other lacks the 2-thio group. Although the s 2 U 34 -containing recombinant tRNA is a less efficient primer, it presents most of the characteristics of the mammalian tRNA. On the other hand, the mnm 5 U 34 -containing tRNA has a strongly reduced activity. Our results demonstrate that the modifications that are absent in E. coli (m 2 G 10 , C 27 , m 5 C 48 , m 5 C 49 , and m 1 A 58 ) as well as the mnm 5 group at position 34 are dispensable for initiation of reverse transcription. In contrast, the 2-thio group at position 34 seems to play an important part in this process.

HIV-1 reverse transcription is initiated from a tRNA 3Lys molecule annealed to the viral RNA at the primer binding site (PBS), but the structure of the initiation complex of reverse transcription remains controversial. Here, we performed in situ structural probing, as well as in vitro structural and functional studies, of the initiation complexes formed by highly divergent isolates (MAL and NL4.3/HXB2). Our results show that the structure of the initiation complex is not conserved. In MAL, and according to sequence analysis in 14% of HIV-1 isolates, formation of the initiation complex is accompanied by complex rearrangements of the viral RNA, and extensive interactions with tRNA 3Lys are required for efficient initiation of reverse transcription. In NL4.3, HXB2, and most isolates, tRNA 3 Lys annealing minimally affects the viral RNA structure and no interaction outside the PBS is required for optimal initiation of reverse transcription. We suggest that in MAL, extensive interactions with tRNA 3Lys are required to drive the structural rearrangements generating the structural elements ultimately recognized by reverse transcriptase. In NL4.3 and HXB2, these elements are already present in the viral RNA prior to tRNA 3 Lys annealing, thus explaining that extensive interactions with the primer are not required. Interestingly, such interactions are required in HXB2 mutants designed to use a non-cognate tRNA as primer (tRNA His ). In the latter case, the extended interactions are required to counteract a negative contribution associate with the alternate primer.

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.