Temporal Coordination between Initiation of HIV (+)-Strand DNA Synthesis and Primer Removal

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146

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.

Section: Methodsmentioning

confidence: 99%

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

Marchand

Götte

2003

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146

“…The fact that this was observed only beyond the addition of 18 nucleotides, which corresponds to the distance between the polymerase and RNase H active sites of RT, suggests that both active sites need to be in contact with the DNA part of the primer/template hybrid substrate to favor the polymerase binding orientation. On the other hand, we cannot exclude the possibility that the RNA PPT sequence gets cleaved only after addition of the 18th DNA residue, instead of the 12th as previously demonstrated (32). If this were indeed the case, following the cleavage of the RNA PPT at the RNA⅐DNA junction by the RNase H activity of RT, the primer would serve as a de facto DNA primer.…”

Section: Discussionmentioning

confidence: 93%

“…As observed here and previously, the RNA PPT primer extended by 12 deoxyribonucleotides is still acting like the pure RNA PPT in terms of sensitivity to NNRTIs and NNTIs suggesting that the RNA PPT sequence is still part of the primer/template substrate. In previous work it was shown that RT pauses after the addition of the 12th DNA residue during the initiation of the (ϩ)-strand synthesis from the RNA PPT primer causing the cleavage of the PPT sequence at the RNA PPT-DNA junction (32). One hypothesis that could explain this discrepancy might be that the pausing of RT after the addition of 12 nt, which leads to the cleavage at the RNA PPT-DNA junction, occurs only during ongoing polymerization and not when using a pre-synthesized chimeric primer.…”

Section: Discussionmentioning

confidence: 99%

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

Auger

Beilhartz

Zhu

et al. 2011

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The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre-and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA⅐DNA primer/template substrates versus DNA⅐DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA⅐DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.HIV RT is a multifunctional enzyme that catalyzes the conversion of the single-stranded RNA HIV genome to doublestranded DNA that is then incorporated into the host genome by the HIV integrase. In the process, RT must support RNAdirected and DNA-directed DNA synthesis along with ribonuclease H (RNase H) 2 activities to create an integration-competent product. A key step during reverse transcription is the incomplete hydrolysis of the (ϩ)-stranded RNA that leaves behind a purine-rich RNA sequence referred to as the polypurine tract (PPT). The remnant RNA PPT sequence serves as a primer to initiate synthesis of the (ϩ)-strand DNA (second strand) (1-3). The unique structure of the RNA PPT sequence has been shown to be a key determinant of the RNase H cleavage specificity at the PPT/U3 junction (4). A recent report described the mechanism by which RT discriminates between polymerase and RNase H activities thereby enabling more efficient initiation of polymerization from the RNA PPT primer versus other remnant RNA primers (5). Using single-molecule spectroscopy experiments, it was shown that RT binds nucleic acid substrates in two distinct orientations in a manner that is governed by the sugar backbone composition of the four or five nucleotides at each end of the primer. Depending on the binding orientation, RT either initiates polymerization at the 3Ј-end of the primer (polymerase binding mode on a DNA primer), or alternatively, RNA hydrolysis through the RNase H domain (RNase H bindi...

“…We have recently studied the interplay between both active sites under saturating dNTP concentrations and demonstrated that a specific pausing site at position ϩ12 correlated precisely with the emergence of RNase H cleavage at the RNA-DNA junction that removes the RNA primer from newly synthesized DNA (23). In the present study, we have investigated the impact of diminished and biased dNTP pools on efficiency and accuracy of the polymerization process and the consequences regarding the interdependence between the two active sites of RT.…”

mentioning

confidence: 99%

“…During this process, a purinerich fragment near the 3Ј-end of the genomic RNA, i.e. polypurine tract (PPT), remains resistant to RNase H degradation and serves as a primer for (ϩ)-strand DNA synthesis (21)(22)(23)(24). Following its selection, the PPT primer is initially extended, and specific RNase H cleavage at the RNA-DNA junction removes the RNA fragment from newly synthesized DNA.…”

mentioning

confidence: 99%

Analysis of Efficiency and Fidelity of HIV-1 (+)-Strand DNA Synthesis Reveals a Novel Rate-limiting Step during Retroviral Reverse Transcription

Götte

Kameoka

McLellan

et al. 2001

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We have analyzed the efficiency and accuracy of polymerization at several different stages during the initiation of human immunodeficiency virus type 1 (HIV-1) (؉)-strand DNA synthesis. This reaction is of particular interest, as it involves the recruitment by reverse transcriptase of an RNA primer that serves as substrate for both the polymerase and RNase H activities of the enzyme. We found that the correct incorporation of the first two nucleotides was severely compromised and that formation of mismatches was completely absent at this stage of initiation. Although the fidelity of incorporations decreased concomitantly with ensuing polymerization, the elongation of mispaired primers was literally blocked. Instead, mispaired primer strands initiated a switch from active synthesis of DNA to premature RNase H-mediated primer removal. These findings suggest the existence of a fragile equilibrium between these two enzymatic activities that is shifted toward RNase H cleavage once the polymerization process is aggravated. Our data show that the initiation of HIV-1 (؉)-strand DNA synthesis differs significantly from reactions involving other primer/template combinations, including tRNAprimed (؊)-strand DNA synthesis.