Nucleotide-Induced Stable Complex Formation by HIV-1 Reverse Transcriptase

Abstract

Nondenaturing gel electrophoresis was used to study the nucleotide substrate-induced conformational change in reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). Dead-end complex was formed between HIV-1 RT, dideoxynucleotide chain-terminated primer, and DNA template in the presence of deoxynucleotide triphosphate (dNTP) complementary to the next position on the template. Complexes which form in the absence of the next complementary dNTP were disrupted by adding excess poly(rA)/oligo(dT) or heparin just prior to electrophoresis. Dead-end complex formation by noncomplementary dNTP's or ribonucleotides was at least 2000-fold less efficient than with the complementary nucleotide. When dA was the next nucleotide on the template, analogues of dTTP supported dead-end complex formation with increased apparent Kd (dTTP < dideoxy-TTP approximately alpha-thio-dTTP < dUTP < 3'-azidothymidine triphosphate). A similar relationship was observed for dGTP analogues across from dC on the template (dGTP < dideoxy-GTP < alpha-thio-dGTP << dITP < dideoxy-ITP). The optimal length of the primer/template duplex region for dead-end complex formation was between 20 and 32 base pairs. Primer-template with a mismatched primer terminus did not support dead-end complex formation, and primer terminated with 3'-azidothymidine formed dead-end complex with 25-fold elevated apparent Kd. By contrast, dead-end complex formation on primer terminated with dideoxy-IMP base paired with dC on the template was more efficient than on primer terminated with dideoxy-GMP. Implications for the mechanisms of discrimination between nucleotide analogues by HIV-1 RT are discussed.

The Pyrophosphate Analogue Foscarnet Traps the Pre-translocational State of HIV-1 Reverse Transcriptase in a Brownian Ratchet Model of Polymerase Translocation

“…Our previous site-specific footprinting experiments with stalled HIV-1 RT complexes revealed that the presence of the templated nucleotide can stabilize and trap the post-translocated complex (27). The next complementary nucleotide facilitates the formation of a stable ternary complex that blocks pyrophosphorolysis and the ATP-dependent excision of chain terminating nucleotides (15,29). Together, these observations are consistent with a Brownian ratchet model for polymerase translocation (36).…”

“…Band shift experiments have revealed that binary complexes of HIV-1 RT and primer/template are unstable when challenged with a trap, e.g. heparin (29). However, the presence of dNTP substrate can stabilize the complex, which prevents dissociation in the presence of trap.…”

“…Crystallographic data have shown that dNTP binding is associated with a closure of the fingers subdomain of HIV-1 RT (20). It is probably this conformational change that stabilizes the ternary complex as compared with its binary form (29). Binding of PFA to the pre-translocated complex may cause a similar structural change.…”

“…The results were quantified as the fraction of shifted primer/template for each concentration of substrate used. Apparent K d was estimated by fitting the quantified results to a hyperbola for one site binding (Y ϭ B max ϫ X(K d ϩ X)) as previously described (29).…”

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

“…Phosphorolytic cleavage of incorporated 2Ј,3Ј-dideoxynucleotides, including ddAMP and ddTMP, is compromised at nucleotide concentrations as low as 1 M, whereas significantly higher concentrations are required to inhibit the excision of AZT-MP. The formation of a stable ternary dead end complex is diminished with AZT-terminated primer strands, which provides an explanation for the efficient removal of AZT-MP (16).…”