Background: The nucleotide reverse transcriptase inhibitor tenofovir retains potency against NRTI-resistant mutants of HIV-1 RT.  We have employed structural studies to investigate the relationship between the acyclic nature of tenofovir and the mechanism of sensitivity to HIV-1 RT mutants.

Methods:  A modified oligonucleotide template-primer containing a thiol-G was covalently linked to HIV-1 RT (Q258C) and blocked at the 3’-terminus with the acyclic nucleotide chain-terminator PMPA (phosphonylmethoxypropyl[adenine]), also known as tenofovir.  This complex was crystallized together with a monoclonal antibody fragment (Fab28) and the structure was solved to 3.1 Å resolution.

Results:  The overall structure of this RT-DNA-tenofovir binary complex is similar to a complex with a 3’ primer terminal deoxynucleotide.  Refinement of the structure has revealed that there are at least 2 conformations of the terminal tenofovir nucleotide.  As expected, density that would normally be expected for the deoxyribose sugar ring is not seen.  In one conformation the adenine moiety of tenofovir stacks with the penultimate dNMP residue of the primer strand occupying a position similar to that observed for natural dNTPs; however, there appears to be a disruption of Watson-Crick base-pairing.  A second conformation shows tenofovir “flipped out” by approximately 180° from where a base paired dNTP would be expected and makes compensatory contacts with H221 and D110.   The interactions between tenofovir and the amino acid residues at the polymerase active site, notably M184 of the YMDD motif, are minimal in comparison to the protein-DNA contacts that have been observed with the sugar ring of other nucleotides in related crystal structures.

Conclusions:  The acyclic nature of tenofovir may help to explain its favorable resistance profile; the minimal and flexible nature of the acyclic linker allows tenofovir to evade several important mechanisms of NRTI resistance.  M184V discriminates against the modified ribose ring of 3TC at the level of incorporation by steric hindrance.  A valine at 184 would not interact with the relatively small acyclic phosphonate moiety.  AZT resistance involves an excision mechanism whereby ATP is involved in a pyrophosphorolytic cleavage of AZTMP from the primer terminus.  The minimal acyclic structure of tenofovir provides a greater degree of torsional freedom resulting in multiple conformations at the RT active site likely providing an unfavorable environment for excision of tenofovir or for resistance due to steric hindrance