Examining Interactions of HIV-1 Reverse Transcriptase with Single-stranded Template Nucleotides by Nucleoside Analog Interference

Abstract

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.The structures of unliganded, inhibitor-bound, and nucleic acid-containing human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) 4 have been instrumental in dissecting the multifunctional properties of this key retroviral enzyme and exploiting it as a target for antiviral therapy (1-6). In particular, co-crystals of RT with duplex DNA (2, 7) and an RNA/DNA hybrid (6) have highlighted critical contacts mediated by protein motifs of its N-terminal DNA polymerase and C-terminal ribonuclease H domains. Examples of such motifs include the "translocation track" (8, 9) and primer grip of the p66 thumb subdomain (2), the template grip (10), and a second primer grip in the vicinity of the ribonuclease H catalytic center (6). Roles for these motifs have been investigated in vitro by site-directed mutagenesis (8,9,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and complemented by studying the effects of strategic placement of nucleoside analogs into either the template or primer (21-28). In parallel, replication of viruses harboring mutations in these motifs have been extensively examined in vivo (17, 29 -31).Although a number of studies have investigated the geometry of duplex nucleic acid positioned between the catalytic centers of HIV-1 RT, the manner in which the single-stranded template traverses the p66 fingers subdomain before accessing the DNA polymerase catalytic center is less well understood. Stated differently, when is the relatively disordered, singlestranded nucleic acid oriented to serve as the template base for accurate and efficient incorporation of the incoming dNTP? Although less precise in the resolution they provide, enzymatic footprinting and photocross-linking studies (32, 33) sugg...

p66 Trp24 and Phe61 Are Essential for Accurate Association of HIV-1 Reverse Transcriptase with Primer/Template

“…By combining stopped-flow and quench-flow approaches, we demonstrated that Phe61 on p66 plays key roles in the binding of p/t by controlling the interaction between finger-thumb domains and stabilizing the 5′ overhang of the template strand, and in RT processing by stabilizing the conformation of the polymerase catalytic site. These results are along the same lines as those of previous work showing that Phe61 influences the orientation of the templating base and the geometry of the duplex, 34 and controls RT fitness and incorporation after mismatching. 32,33 Fisher et al have demonstrated that the implication of p66 Phe61 in strand displacement, DNA synthesis and RT fidelity is independent.…”

“…This result confirms the requirement of Phe61 for stabilization of the p/t in the catalytic site as previously reported. 32,34 Interestingly, similar movement of the thumb domain was observed on the structures of RT-NNRTI complexes, where the NNRTIs maintain RT in an open conformation 11,30 and improve binding of p/t in a noncatalytic conformation. 36,37 In contrast, mutation of Trp24 does not specifically affect the collisional step of the p/t binding mechanism, but alters overall kinetics, suggesting that this residue is involved in stabilization of the complex, rather than the initial RT-p/t interaction, which is not surprising, as the implication of Trp24 in the fingers-thumb domains contact in the closed conformation of RT is limited (Fig.…”

“…Previous work has clearly demonstrated that contacts between the fingers domain of p66 and template 5′ overhang are essential for polymerase activity and control of the geometry of the dNTP binding pocket. 26,34,[42][43][44] The structure of the covalently trapped HIV RT-p/t catalytic complexes pointed out direct contacts Table 5. Kinetic parameters of p/t Mis binding onto variants of RT between RT and p/t and major conformational changes on the p66 subunit induced by the binding of p/t and dNTP.…”

“…32,33 In general, residues in the β3-β4 loop hairpin of the fingers subdomain of p66 have been reported to control several aspects of RT activity, including fidelity, processivity, pyrophosphorolysis and strand displacement synthesis and harbor hotspot residues for nucleoside analog resistance mutations. 26,[32][33][34][35] In contrast, the role of Trp24 in p66 on RT polymerase activity and on the stability of the RT-p/t complex is poorly documented. This residue contacts nucleosides + 3 and + 2 of the template strand as shown in the structure of RT-trapped p/t.…”

Mechanistic insights into the roles of three linked single-stranded template binding residues of MMLV reverse transcriptase in misincorporation and mispair extension fidelity of DNA synthesis

“…They are found to play critical roles in viral DNA replication, in fidelity, and in drug resistance (Agopian et al, 2007;Dash et al, 2006;Depollier et al, 2005;Kim et al, 1998Kim et al, , 1999.…”