The Role of Nucleotide Excision by Reverse Transcriptase in HIV Drug Resistance

Abstract

Nucleoside reverse transcriptase (RT) inhibitors of HIV block viral replication through the ability of HIV RT to incorporate chain-terminating nucleotide analogs during viral DNA synthesis. Once incorporated, the chain-terminating residue must be removed before DNA synthesis can continue. Removal can be accomplished by the excision activity of HIV RT, which catalyzes the transfer of the 3'-terminal residue on the blocked DNA chain to an acceptor substrate, probably ATP in most infected cells. Mutations of RT that enhance excision activity are the most common cause of resistance to 3'-azido-3'-deoxythymidine (AZT) and exhibit low-level cross-resistance to most other nucleoside RT inhibitors. The resistance to AZT is suppressed by a number of additional mutations in RT, most of which were identified because they conferred resistance to other RT inhibitors. Here we review current understanding of the biochemical mechanisms responsible for increased or decreased excision activity due to these mutations.

Figure 1.

ATP-mediated excision of AZTMP and the use of Ap₄AZT as substrate for AZTMP incorporation. (A) ATP attacks the phosphodiester bond removing the drug-MP from the primer terminus, forming a dinucleoside polyphosphate and an unblocked primer [32]. (B) Ap₄AZT is recognized as an analog of AZTTP leading to incorporation of AZTMP and release of ATP [34,35]. The template and primer are shown in blue and green, respectively; AZT is in red.

Figure 2.

Locations of residues altered by PFA-resistance mutations in the structures of binary and ternary complexes of HIV-1 RT. (A) Binary complex of HIV-1 RT with AZTMP-terminated primer-template occupying the dNTP-binding site (N site) [PDB structure 1N6Q, Ref. 45]. (B) Ternary complex of HIV-1 RT with ddAMP-terminated primer-template and dTTP occupying the N site [PDB structure 1RTD, Ref. 85]. The template (T) is shown in blue, and the primer strand (P) in green. The structure occupying the N site in each complex is shown as a space-filling model (atoms indicated by CPK color scheme). Residues that are substituted in the indicated PFA-resistant mutants are also shown as space-filling models. The fingers-domain residue K65 is shown in gray. Palm-domain residues are shown in yellow or cyan using contrasting colors to distinguish overlapping structures [37].

Figure 3.

Positioning of RT with respect to the 3′-end of the primer. The pre-translocation complex places the RT active site in position to carry out excision with ATP or PPi as acceptor substrate. The primer terminus occupies the dNTP-binding site (N site) on RT. RT is stabilized in this position by binding PFA. The post-translocated complex places the RT in a position to add the next deoxynucleotide. The primer terminus occupies the primer-binding site (P site) on RT. RT is stabilized in this position by binding the next complementary dNTP [,–47,87]. The template and primer are shown in blue and green, respectively. The three orange dots correspond to active site catalytic residues, which are located near the boundary between the N and P sites.