Initiation of RNA‐dependent DNA synthesis by retroviral reverse transcriptases is generally considered as unspecific. In the case of human immunodeficiency virus type 1 (HIV‐1), the natural primer is tRNA3Lys. We recently found evidence of complex interactions between tRNA3Lys and HIV‐1 RNA that may be involved in the priming process. In this study, we compare the ability of natural and unmodified synthetic tRNA3Lys and 18mer oligoribo‐ and oligodeoxyribonucleotides complementary to the viral primer binding site to initiate replication of HIV‐1 RNA using either homologous or heterologous reverse transcriptases. We show that HIV‐1 RNA, HIV‐1 reverse transcriptase and primer tRNA3Lys form a specific initiation complex that differs from the unspecific elongation complex formed when an oligodeoxyribonucleotide is used as primer. Modified nucleosides of tRNA3Lys are required for efficient initiation and transition to elongation. Transition from initiation to elongation, but not initiation of reverse transcription itself, is facilitated by extended primer‐template interactions. Elongation, but not initiation of reverse transcription, is inhibited by Mn2+, which further differentiates these two different functional states of reverse transcriptase. These results define initiation of reverse transcription as a target to block viral replication.

We recently showed that primer tRNA3Lys, human immunodeficiency virus type 1 (HIV‐1) RNA and HIV‐1 reverse transcriptase (RT) form a specific complex of initiation of reverse transcription that can be functionally distinguished from the elongation complex, which can be obtained by substituting an 18mer oligodeoxyribonucleotide (ODN) for the natural primer (Isel et al., 1996). Here, we compared the binding properties and the single and multiple turnover kinetics of HIV‐1 RT in the initiation and elongation complexes. Even though the equilibrium dissociation constants of HIV‐1 RT are not very different for the two complexes, RT dissociates approximately 200‐fold faster from the initiation complex. Furthermore, nucleotide incorporation by the pre‐formed primer‐template‐RT complexes is reduced by a approximately 50‐fold factor during initiation of reverse transcription, compared with elongation. As a consequence, processivity of HIV‐1 RT in the initiation complex is close to unity, while it increases by four orders of magnitude during elongation, as expected for a replication enzyme. This processivity change is reminiscent of the transition from initiation to elongation of transcription. Furthermore, our results indicate that the post‐transcriptional modifications of tRNA3Lys play a role similar to that of the sigma factor in transcription by the Escherichia coli RNA polymerase: they favour the formation of the specific initiation complex but do not affect the polymerization rate of the bound enzyme.

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Expression of the human immunodeficiency virus type I pol open reading frame in Escherichia coli led to several protease-mediated processing steps of the pol precursor polyprotein. Accumulation of two polypeptides with molecular sizes of 64 and 52 kilodaltons, with which reverse transcriptase activity is associated, was observed. The protease moiety of the precursor polyprotein accumulated as a 10-kilodalton species as a result of two specific cleavages. Furthermore, a single-amino-acid substitution in the putative active site of protease totally abolished processing of the precursor polyprotein.

A series of 29 madurahydroxylactone derivatives was evaluated for dual inhibition of human immunodeficiency virus type 1 (HIV-1) integrase and RNase H. While most of the compounds exhibited similar potencies for both enzymes, two of the derivatives showed 10-to 100-fold-higher selectivity for each enzyme, suggesting that distinct pharmacophore models could be generated. This study exemplifies the common and divergent structural requirements for the inhibition of two structurally related HIV-1 enzymes and demonstrates the importance of systematically screening for both integrase and RNase H when developing novel inhibitors.