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

Abstract

Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre-and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

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

“…However, the presence of dNTP substrate can stabilize the complex, which prevents dissociation in the presence of trap. These findings are consistent with site-specific footprinting experiments showing that the nucleotide traps the complex in the posttranslocated state (27,31). Here we used complexes PPTϩ3, as an example for a pre-translocated complex, and PPTϩ6, as an example for a post-translocated complex, to look at the stability of ternary complexes with PFA and the next nucleotide, respectively (Fig.…”

“…A related, more complex version of this model has also been proposed for multisubunit RNA polymerases (34,35). 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).…”

“…Treatment of these RT-DNA complexes with the chemical nuclease potassium peroxynitrite (KOONO), which is a metal-free source of hydroxyl radicals, was shown to cleave the template site specifically at positions Ϫ7 and/or Ϫ8. Position Ϫ1 corresponds to the template nucleotide that is base paired with the 3Ј-terminal nucleotide of the primer (27). Cleavage at position Ϫ7 is indicative for the post-translocated state, whereas cleavage at position Ϫ8 is indicative for the pretranslocation state.…”

“…We have recently developed site-specific footprinting techniques that allow the distinction between pre-and post-translocated states (27). Here we demonstrate that the sequence of the nucleic acid substrate affects the ratio of pre-and posttranslocational states at a given template position.…”

“…The latter configuration is referred to as the post-translocational state. Cross-linking experiments revealed that pyrophosphorolysis or the ATP-dependent excision can only occur in the former pre-translocational state (26,27). To account for the non-competitive mode of inhibition it has been suggested that PFA may likewise bind to the pre-translocated complex (8).…”