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Electron density maps at 3.0-Å resolution from the crystal structure of the pre-translocated (N) complex of HIV-1 RTDNA. The primer strand has been terminated at the 3-end with AZTMP and cross-linked to HIV-1 RT as described under " Materials and Methods. " Two regions of the structure are shown: A, the region surrounding the tethered CG* base pair in the template-primer and residue Cys-258 in helix H of RT. The coordinates for the tether were not included in the calculation of the composite omit 3F o 2F c map (contoured at 1 ). B, the dNTP-binding site (N site) is occupied by the 3-end of the primer, which has been elongated by AZTMP, but not translocated to the P site because of the covalent cross-link between primer and RT. The coordinates of AZTMP and of residues in a 6-Å radius around AZTMP were not included in the calculation of the simulated annealing F o F c omit map (contoured at 2.5 ). The structural details of the N complex are described elsewhere (PDB code 1N6Q) (24).
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A disulfide cross-linking strategy was used to covalently trap as a stable complex (complex N) a short-lived, kinetic intermediate in DNA polymerization. This intermediate corresponds to the product of polymerization prior to translocation. We also prepared the trapped complex that corresponds to the product of polymerization after translocation (c...
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Citations
... For structural studies of catalytic-competent RT/dsDNA complex, RT is usually cross-linked to a modified DNA base. The predominantly used cross-linking is done between a mutated residue Q258C of the thumb subdomain and a modified guanine base of template or primer, which is the fifth nucleotide from the P site 24,25 . The Q258C cross-linking prevents sliding of RT over DNA and arrests a stable complex for structural studies. ...
HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.
... Steady-state kinetics and in vitro IC 50 s. HIV-1 RT and mutants were expressed and purified as described previously (36,77,(100)(101)(102)(103)(104). RT was expressed in JM109 cells (Invitrogen) and purified by nickel affinity chromatography and Mono-Q anion-exchange chromatography. ...
4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV-1, in Phase III clinical trials. EFdA resistance is not well characterized. To study EFdA-resistance patterns as it may emerge in naïve or tenofovir- (TFV), emtricitabine/lamivudine- (FTC/3TC), or zidovudine- (AZT) treated patients we performed viral passaging experiments starting with wild-type, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless the starting viral sequence, all selected EFdA-resistant variants included the M184V RT mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than WT HIV-1. These mutants also had significantly lower specific infectivity than WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (k cat /K m ) of WT vs. A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data, suggest that EFdA is an excellent therapeutic candidate for naïve, AZT-, FTC/3TC, and especially tenofovir-treated patients.
... This compound was obtained as a yellow oil (679 mg, 75%) according to the procedure used for the synthesis of compound 31a, starting from 6-chloropurine (536 mg, 3.467 mmol) and alcohol derivative 30b (670 mg, 1.725 mmol). 1 (37) A solution of (R)-2-aminopropan-1-ol 36 (500 mg, 6.66 mmol) and diethyl vinylphosphonate 35 (625 mg, 3.81 mmol) in H 2 O (10 mL), was stirred for 2 days at room temperature. The solvents were evaporated in vacuo and the crude residue was purified by silica gel flash chromatography (DCM/MeOH, 10:1), yielding the title compound as a light-yellow oil (855 mg, 94%). 1 (40) This by-product was obtained as a colorless oil (233 mg, 50%) according to the procedure used for the synthesis of compound 39, starting from intermediate 37 (400 mg, 1.672 mmol), K 2 CO 3 (300 mg, 2.173 mmol) and methyl bromoacetate (222 mL, 2.006 mmol). ...
HIV-1 reverse transcriptase (RT) plays a central role in the viral life cycle, and roughly half of the FDA-approved anti-HIV drugs are targeting RT. Nucleoside analogs (NRTIs) require cellular phosphorylation for binding to RT, and to bypass this rate-limiting path, we designed a new series of acyclic nucleoside phosphonate analogs as nucleoside triphosphate mimics, aiming at the chelation of the catalytic Mg²⁺ ions via a phosphonate and/or a carboxylic acid group. Novel synthetic procedures were developed to access these nucleoside phosphonate analogs. X-ray structures in complex with HIV-1 RT/dsDNA demonstrated that their binding modes are distinct from that of our previously reported compound series. The impact of chain length, chirality and linker atom have been discussed. The detailed structural understanding of these new compounds provides opportunities for designing new class of HIV-1 RT inhibitors.
... The cross-linking experiments were conducted as reported in [30][31][32]. NaCl concentrations in the final reactions were, however, changed to 150-200 mM. ...
... The chemistry that was designed to covalently cross-link HIV-1 RT to dsDNA or to RNA/DNA is efficient with a single round of nucleotide incorporation followed by translocation. This allows a thioalkyl-modified guanine base G (at the N2 position) in the primer to be positioned in register with the mutated residue C258, which permits the cross-linking disulfide bond to form [32,45]. The covalently trapped complex can be purified using tandem nickel-heparin columns. ...
In most cases, proteolytic processing of the retroviral Pol portion of the Gag-Pol polyprotein precursor produces protease (PR), reverse transcriptase (RT), and integrase (IN). However, foamy viruses (FVs) express Pol separately from Gag and, when Pol is processed, only the IN domain is released. Here, we report a 2.9 Å resolution crystal structure of the mature PR-RT from prototype FV (PFV) that can carry out both proteolytic processing and reverse transcription but is in a configuration not competent for proteolytic or polymerase activity. PFV PR-RT is monomeric and the architecture of PFV PR is similar to one of the subunits of HIV-1 PR, which is a dimer. There is a C-terminal extension of PFV PR (101-145) that consists of two helices which are adjacent to the base of the RT palm subdomain, and anchors PR to RT. The polymerase domain of PFV RT consists of fingers, palm, thumb, and connection subdomains whose spatial arrangements are similar to the p51 subunit of HIV-1 RT. The RNase H and polymerase domains of PFV RT are connected by flexible linkers. Significant spatial and conformational (sub)domain rearrangements are therefore required for nucleic acid binding. The structure of PFV PR-RT provides insights into the conformational maturation of retroviral Pol polyproteins.
... The predominantly used crosslinking is done between a mutated residue Q258C of the thumb subdomain and a modi ed guanine base of template or primer which is the fth nucleotide from the P site. 23,24 The Q258C crosslinking prevents sliding of RT over DNA and arrests a stable complex for structural studies. The Q258C crosslinking was used to obtain the structures of both N-and P-complexes of RT/DNA when appropriate DNA substrate was crosslinked. ...
HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3’-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structure-based platform provide an opportunity for designing new types of polymerase inhibitors.
... The overall structures of the two crystal structures of RT in complex with the inhibitor d4T are consistent with the previous structures of RT in complex with the dsDNA and the NRTIs [26,27]. For both crystal structures, the F o -F c difference electron density at 3σ was not sufficient to build the amino acids 1-5, 87-95, 212-232, and 430-452 for the p51 subunit and 558-560 for the p66 subunit. ...
... Recombinant RT enzyme containing the mutations C280S and Q258C was expressed in Escherichia coli strain BL21 (DE3) and purified as the following protocols previously described [12,27,32]. For the cross-linking experiments, a 27-mer DNA template (5 -ATGGGCGGCGCCCGAACAGGGACTGTG-3 for the studies with (-)FTC and 5 -ATGGACGGCGCCCGAACAGGGACTGTG-3 for the studies with d4T), standard desalted, was purchase from Integrated DNA Technologies (IDT) (Integrated DNA Technologies, Coralville, IA, USA) and a 20-mer DNA primer (5 -ACAGTCCCTGTTCGGXCGCC-3 ) was purchased from TriLink Biotechnologies (San Diego, CA, USA). ...
... The cross-linking N2-cystamine 2 -deoxyguanosine in the DNA primer is indicated by an X. The cross-linking reaction between RT and 27-mer/20-mer primer/template was carried out as described by Sarafianos S.G. et al. [12,27,32]. Briefly, a solution containing 20 µM of annealed 27-mer/20-mer primer/template DNA and 10 µM of recombinant RT enzyme in 10 mM Tris, 25 mM NaCl, 10 mM MgCl 2 , and 25 µM dGTP set at pH 8 was incubated for 3 h at 37 • C. To obtain the complex with the chain terminated DNA (PDB ID: 6WPJ), a solution containing 20 µM of annealed 27-mer/20-mer primer/template DNA and 10 µM of recombinant RT enzyme in 10 mM Tris, 25 mM NaCl, 10 mM MgCl2, and 25 µM ddGTP (instead of dGTP) was incubated for 3 h at 37 • C. The complexes were then isolated as previously described [12]. ...
Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a “snapshot” for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.
... HIV-1 reverse transcriptase (RT) is a highly dynamic enzyme that carries out multiple reactions during reverse transcription of single-stranded viral RNA to proviral double-stranded DNA (dsDNA). RT undergoes multiple conformational changes during its catalytic cycle upon binding nucleic acids, nucleotide substrates, nucleoside, and non-nucleoside drugs [1][2][3][4][5][6][7][8][9][10]. Previously we showed that small molecule drug-like fragments can be used to detect allosteric changes in RT structure and can be used to identify new binding sites for structure-based drug design efforts [5,11]. ...
The high-resolution crystal structure of HIV-1 reverse transcriptase (RT) bound to a 38-mer DNA hairpin aptamer with low pM affinity was previously described. The high-affinity binding aptamer contained 2’-O-methyl modifications and a seven base-pair GC-rich tract and the structure of the RT-aptamer complex revealed specific contacts between RT and the template strand of the aptamer. Similar to all crystal structures of RT bound to nucleic acid template-primers, the aptamer bound RT with a bend in the duplex DNA. To understand the structural basis for the ultra-high-affinity aptamer binding, an integrative structural biology approach was used. Hydrogen-deuterium exchange coupled to liquid chromatography-mass spectrometry (HDX-MS) was used to examine the structural dynamics of RT alone and in the presence of the DNA aptamer. RT was selectively labeled with ¹⁵N to unambiguously identify peptides from each subunit. HDX of unliganded RT shows a mostly stable core. The p66 fingers and thumb subdomains, and the RNase H domain are relatively dynamic. HDX indicates that both the aptamer and a scrambled version significantly stabilize regions of RT that are dynamic in the absence of DNA. No substantial differences in RT dynamics are observed between aptamer and scrambled aptamer binding, despite a large difference in binding affinity. Small-angle X-ray scattering and circular dichroism spectroscopy were used to investigate the aptamer conformation in solution and revealed a pre-bent DNA that possesses both A- and B-form helical character. Both the 2’-O-methyl modifications and the GC tract appear to contribute to an energetically favorable conformation for binding to RT that contributes to the aptamer’s ultra-high affinity for RT. The X-ray structure of RT with an RNA/DNA version of the aptamer at 2.8 Å resolution revealed a potential role of the hairpin positioning in affinity. Together, the data suggest that both the 2’-O-methyl modifications and the GC tract contribute to an energetically favorable conformation for high-affinity binding to RT.
... The structural perturbations of nucleotide analogues may inhibit translocation or induce conformational change that misaligns the 3'-OH for nucleophilic attack on the α-phosphate of the incoming nucleotide. [51] For this reason, even catalytically efficient single nucleotide insertion is no guarantee that further primer extension can continue. Because RTs perform both RNAand DNA-templated DNA synthesis, read through efficiency may differ in each of these modes. ...
We report on the ability of the reverse transcriptases (RTs) from avian myeloblastosis virus (AMV), Moloney murine leukemia virus (M‐MLV), and human immunodeficiency virus 1 (HIV‐1) to generate labeled DNA using the fluorescent tricyclic cytidine analogues d(tC)TP and d(DEAtC)TP as substrates. Michaelis‐Menten kinetics for the insertion of these analogues show Vmax/KM from 0.09–5 times that of natural dCTP across from G, depending on the polymerase and whether the template is RNA or DNA. The analogues are prone to misinsertion across from adenosine with both RNA and DNA templates. Elongation after analogue insertion is efficient with RNA templates, but the analogues cause stalling after insertion with DNA templates. A model reverse transcription assay using HIV‐1‐RT, including RNA‐dependent DNA synthesis, degradation of the RNA template by the RT's RNase H activity, and synthesis of a second DNA strand to form fluorescently labeled dsDNA, shows that d(tC)TP and d(DEAtC)TP are compatible with a complete reverse transcription cycle in vitro.
... The X in the primer sequence represents N 2 -cystamine-deoxyguanosine which covalently cross-links to Q258C in the p66 subunit. The method of covalently tethering HIV-1 RT dsDNA to generate the RT-DNA complex has been formally described 27,29,40 . To form the covalent complex, RT and dsDNA substrate were incubated together at 25 µM and 50 µM, respectively at room temperature for approximately 18 h. ...
Emtricitabine (FTC) and lamivudine (3TC), containing an oxathiolane ring with unnatural (-)-stereochemistry, are widely used nucleoside reverse transcriptase inhibitors (NRTIs) in anti-HIV therapy. Treatment with FTC or 3TC primarily selects for the HIV-1 RT M184V/I resistance mutations. Here we provide a comprehensive kinetic and structural basis for inhibiting HIV-1 RT by (-)-FTC-TP and (-)-3TC-TP and drug resistance by M184V. (-)-FTC-TP and (-)-3TC-TP have higher binding affinities (1/Kd) for wild-type RT but slower incorporation rates than dCTP. HIV-1 RT ternary crystal structures with (-)-FTC-TP and (-)-3TC-TP corroborate kinetic results demonstrating that their oxathiolane sulfur orients toward the DNA primer 3'-terminus and their triphosphate exists in two different binding conformations. M184V RT displays greater (>200-fold) Kd for the L-nucleotides and moderately higher (>9-fold) K
d
for the D-isomers compared to dCTP. The M184V RT structure illustrates how the mutation repositions the oxathiolane of (-)-FTC-TP and shifts its triphosphate into a non-productive conformation.
... 28 Overall, the structure of the RT protein in complex with the dsDNA and the NRTIs are similar to previously reported protein-DNA structures. 29,30 The electron density for all six F I G U R E 2 Crystal structures of RT in complex dsDNA primer/template and NRTIs. Fo-Fc difference electron densities are shown as gray mesh at a contour level of 3σ. ...
... Recombinant RT enzyme containing the mutations C280S and Q258C was expressed in Escherichia coli and purified as following protocols previously described. 28,30,41,42 For the cross-linking experiments, a 27-mer DNA template (5 0 -ATGGGCGGCGCCCGAACAGGGACTGTG-3 0 ), standard desalted, was purchase from IDT (Integrated DNA Technologies, Coralville, Iowa), and a 20-mer DNA primer (5 0 -ACAGTCCCTGTTCGGXCGCC-3 0 ) was purchased from TriLink BioTechnologies (San Diego, California). The cross-linking N 2 -cystamine 2 0 -deoxyguanosine in the DNA primer is indicated by an X. ...
... The cross-linking N 2 -cystamine 2 0 -deoxyguanosine in the DNA primer is indicated by an X. The cross-linking reaction between RT and 27-mer/20-mer primer template and the purification of the protein-dsDNA complex were carried out as described by Sarafianos et al. 30,41 SDS PAGE was used to confirm the purity of the dsDNA-protein complex (results not shown). dCTP and ddCTP were purchased from Sigma. ...
The retrovirus HIV‐1 has been a major health issue since its discovery in the early 80s. In 2017, over 37 million people were infected with HIV‐1, of which 1.8 million were new infections that year. Currently, the most successful treatment regimen is the highly active antiretroviral therapy (HAART), which consists of a combination of three to four of the current 26 FDA‐approved HIV‐1 drugs. Half of these drugs target the reverse transcriptase (RT) enzyme that is essential for viral replication. One class of RT inhibitors is nucleoside reverse transcriptase inhibitors (NRTIs), a crucial component of the HAART. Once incorporated into DNA, NRTIs function as a chain terminator to stop viral DNA replication. Unfortunately, treatment with NRTIs is sometimes linked to toxicity caused by off‐target side effects. NRTIs may also target the replicative human mitochondrial DNA polymerase (Pol γ), causing long‐term severe drug toxicity. The goal of this work is to understand the discrimination mechanism of different NRTI analogues by RT. Crystal structures and kinetic experiments are essential for the rational design of new molecules that are able to bind selectively to RT and not Pol γ. Structural comparison of NRTI‐binding modes with both RT and Pol γ enzymes highlights key amino acids that are responsible for the difference in affinity of these drugs to their targets. Therefore, the long‐term goal of this research is to develop safer, next generation therapeutics that can overcome off‐target toxicity.
