scholarly journals Role of Helix P of the Human Cytomegalovirus DNA Polymerase in Resistance and Hypersusceptibility to the Antiviral Drug Foscarnet

2006 ◽  
Vol 80 (3) ◽  
pp. 1440-1450 ◽  
Author(s):  
Egor P. Tchesnokov ◽  
Christian Gilbert ◽  
Guy Boivin ◽  
Matthias Götte
Keyword(s):  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Structural Changes ◽  
Antiviral Drug ◽  
Drug Susceptibility ◽  
Structure Model ◽  
Nucleotide Incorporation ◽  
Α Helix ◽  
Conserved Amino Acids

ABSTRACT Mutations in the human cytomegalovirus DNA polymerase (UL54) can not only decrease but also increase susceptibility to the pyrophosphate (PPi) analogue foscarnet. The proximity of L802M, which confers resistance, and K805Q, which confers hypersusceptibility, suggests a possible unifying mechanism that affects drug susceptibility in one direction or the other. We found that the polymerase activities of L802M- and K805Q-containing mutant enzymes were literally indistinguishable from that of wild-type UL54; however, susceptibility to foscarnet was decreased or increased, respectively. A comparison with the crystal structure model of the related RB69 polymerase suggests that L802 and K805 are located in the conserved α-helix P that is implicated in nucleotide binding. Although L802 and K805 do not appear to make direct contacts with the incoming nucleotide, it is conceivable that changes at these residues could exert their effects through the adjacent, highly conserved amino acids Q807 and/or K811. Our data show that a K811A substitution in UL54 causes reductions in rates of nucleotide incorporation. The activity of the Q807A mutant is only marginally affected, while this enzyme shows relatively high levels of resistance to foscarnet. Based on these data, we suggest that L802M exerts its effects through subtle structural changes in α-helix P that affect the precise positioning of Q807 and, in turn, its presumptive involvement in binding of foscarnet. In contrast, the removal of a positive charge associated with the K805Q change may facilitate access or increase affinity to the adjacent Q807.

scholarly journals Differentiation between Polymorphisms and Resistance-Associated Mutations in Human Cytomegalovirus DNA Polymerase

2010 ◽  
Vol 54 (12) ◽  
pp. 5004-5011 ◽  
Author(s):  
Meike Chevillotte ◽  
Ina Ersing ◽  
Thomas Mertens ◽  
Jens von Einem
Keyword(s):  
Drug Resistance ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Antiviral Drug ◽  
Drug Susceptibility ◽  
Resistance Testing ◽  
Resistance Mutations ◽  
Genotypic Resistance ◽  
Drug Resistance Testing ◽  
Genotypic Resistance Testing

ABSTRACT Specific mutations in the human cytomegalovirus (HCMV) DNA polymerase (pUL54) are known to confer resistance against all currently licensed drugs for treatment of HCMV infection and disease. Following the widespread use of antivirals, the occurrence of HCMV drug resistance is constantly increasing. Recently, diagnostic laboratories have started to replace phenotypic drug resistance testing with genotypic resistance testing. However, the reliability and success of genotypic testing highly depend on the availability of high-quality phenotypic resistance data for each individual mutation and for combinations of mutations, with the latter being increasingly found in patients' HCMV isolates. We performed clonal marker transfer experiments to investigate the impacts of 7 different UL54 point mutations and also of combinations of these mutations on drug susceptibility and viral replicative fitness. We show that several mutations—S695T, A972V, K415R, S291P, and A692V—of suspected but uncertain drug susceptibility phenotype, either alone or in combination, were not relevant to antiviral drug resistance. In contrast, the combination of two mutations individually characterized previously—E756K and D413E—conferred high-grade loss of susceptibility to all three antivirals. Our results have been added to the newly available database of all published HCMV resistance mutations (http://www.informatik.uni-ulm.de/ni/mitarbeiter/HKestler/hcmv/index.html ). These data will allow better interpretation of genotypic data and further improve the basis for drug resistance testing.

scholarly journals Structural and Technological Approach to Reveal the Role of the Lipid Phase in the Formation of Soy Emulsion Gels with Chia Oil

Gels ◽  
2021 ◽  
Vol 7 (2) ◽  
pp. 48
Author(s):  
Ana M. Herrero ◽  
Claudia Ruiz-Capillas
Keyword(s):  
Raman Spectroscopy ◽  
Structural Properties ◽  
Structural Changes ◽  
Protein Secondary Structure ◽  
Lipid Phase ◽  
Α Helix ◽  
Β Sheet ◽  
Shed Light ◽  
Chia Oil

Considerable attention has been paid to emulsion gels (EGs) in recent years due to their interesting applications in food. The aim of this work is to shed light on the role played by chia oil in the technological and structural properties of EGs made from soy protein isolates (SPI) and alginate. Two systems were studied: oil-free SPI gels (SPI/G) and the corresponding SPI EGs (SPI/EG) that contain chia oil. The proximate composition, technological properties (syneresis, pH, color and texture) and structural properties using Raman spectroscopy were determined for SPI/G and SPI/EG. No noticeable (p > 0.05) syneresis was observed in either sample. The pH values were similar (p > 0.05) for SPI/G and SPI/EG, but their texture and color differed significantly depending on the presence of chia oil. SPI/EG featured significantly lower redness and more lightness and yellowness and exhibited greater puncture and gel strengths than SPI/G. Raman spectroscopy revealed significant changes in the protein secondary structure, i.e., higher (p < 0.05) α-helix and lower (p < 0.05) β-sheet, turn and unordered structures, after the incorporation of chia oil to form the corresponding SPI/EG. Apparently, there is a correlation between these structural changes and the textural modifications observed.

scholarly journals The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 648
Author(s):  
del Prado ◽  
Santos ◽  
Lázaro ◽  
Salas ◽  
de Vega
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Changes ◽  
Binding Capacity ◽  
Dna Polymerases ◽  
Crystallographic Structure ◽  
Genome Replication ◽  
Terminal Protein ◽  
Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

scholarly journals Interstrain Variation in the Human Cytomegalovirus DNA Polymerase Sequence and Its Effect on Genotypic Diagnosis of Antiviral Drug Resistance

1999 ◽  
Vol 43 (6) ◽  
pp. 1500-1502 ◽  
Author(s):  
Sunwen Chou ◽  
Nell S. Lurain ◽  
Adriana Weinberg ◽  
Guang-Yung Cai ◽  
Prem L. Sharma ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Antiviral Drug ◽  
Resistance Mutations ◽  
Sequence Alignments ◽  
Coding Sequence ◽  
Antiviral Drug Resistance ◽  
Almost All

ABSTRACT The polymerase (pol) coding sequence was determined for 40 independent clinical cytomegalovirus isolates sensitive to ganciclovir and foscarnet. Sequence alignments showed >98% interstrain homology and amino acid variation in only 4% of the 1,237 codons. Almost all variation occurred outside of conserved functional domains where resistance mutations have been identified.

scholarly journals Role of Homodimerization of Human Cytomegalovirus DNA Polymerase Accessory Protein UL44 in Origin-Dependent DNA Replication in Cells

2008 ◽  
Vol 82 (24) ◽  
pp. 12574-12579 ◽  
Author(s):  
Elisa Sinigalia ◽  
Gualtiero Alvisi ◽  
Beatrice Mercorelli ◽  
Donald M. Coen ◽  
Gregory S. Pari ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Accessory Protein ◽  
C Terminus ◽  
Cellular Context ◽  
In Cells

ABSTRACT The presumed processivity subunit of human cytomegalovirus (HCMV) DNA polymerase, UL44, forms homodimers. The dimerization of UL44 is important for binding to DNA in vitro; however, whether it is also important for DNA replication in a cellular context is unknown. Here we show that UL44 point mutants that are impaired for dimerization, but not for nuclear localization or interaction with the C terminus of the polymerase catalytic subunit, are not capable of supporting HCMV oriLyt-dependent DNA replication in cells. These data suggest that the disruption of UL44 homodimers could represent a novel anti-HCMV strategy.

scholarly journals Contrasting Effects of W781V and W780V Mutations in Helix N of Herpes Simplex Virus 1 and Human Cytomegalovirus DNA Polymerases on Antiviral Drug Susceptibility

2015 ◽  
Vol 89 (8) ◽  
pp. 4636-4644 ◽  
Author(s):  
Jocelyne Piret ◽  
Nathalie Goyette ◽  
Brian E. Eckenroth ◽  
Emilien Drouot ◽  
Matthias Götte ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Antiviral Drug ◽  
Drug Susceptibility ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Antiviral Drug Susceptibility ◽  
Hsv 1

ABSTRACTDNA polymerases of theHerpesviridaeand bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC50s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC50) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains.IMPORTANCEDNA polymerases of theHerpesviridaeand bacteriophage RB69 belong to the α-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.

scholarly journals Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase

2016 ◽  
Vol 60 (7) ◽  
pp. 4176-4182 ◽  
Author(s):  
Han Chen ◽  
Chengwei Li ◽  
Jiri Zemlicka ◽  
Brian G. Gentry ◽  
Terry L. Bowlin ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Potent Inhibitor ◽  
Antiviral Drug ◽  
Viral Polymerase ◽  
Current Standard ◽  
Hcmv Replication ◽  
Additional Nucleotide ◽  
Chain Terminators

ABSTRACTCyclopropavir (CPV) is a promising antiviral drug against human cytomegalovirus (HCMV). As with ganciclovir (GCV), the current standard for HCMV treatment, activation of CPV requires multiple steps of phosphorylation and is enantioselective. We hypothesized that the resulting CPV triphosphate (CPV-TP) would stereoselectively target HCMV DNA polymerase and terminate DNA synthesis. To test this hypothesis, we synthesized both enantiomers of CPV-TP [(+) and (−)] and investigated their action on HCMV polymerase. Both enantiomers inhibited HCMV polymerase competitively with dGTP, with (+)-CPV-TP exhibiting a more than 20-fold lower apparentKithan (−)-CPV-TP. Moreover, (+)-CPV-TP was a more potent inhibitor than GCV-TP. (+)-CPV-TP also exhibited substantially lower apparentKmand somewhat higher apparentkcatvalues than (−)-CPV-TP and GCV-TP for incorporation into DNA by the viral polymerase. As is the case for GCV-TP, both CPV-TP enantiomers behaved as nonobligate chain terminators, with the polymerase terminating DNA synthesis after incorporation of one additional nucleotide. These results elucidate how CPV-TP acts on HCMV DNA polymerase and help explain why CPV is more potent against HCMV replication than GCV.

scholarly journals Antiviral Drug Resistance of Human Cytomegalovirus

2010 ◽  
Vol 23 (4) ◽  
pp. 689-712 ◽  
Author(s):  
Nell S. Lurain ◽  
Sunwen Chou
Keyword(s):  
Drug Resistance ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Treatment Options ◽  
Antiviral Drug ◽  
Resistance Mutations ◽  
Phenotypic Analysis ◽  
Antiviral Drug Resistance ◽  
Phenotypic Methods ◽  
Approved Drugs

SUMMARY The study of human cytomegalovirus (HCMV) antiviral drug resistance has enhanced knowledge of the virological targets and the mechanisms of antiviral activity. The currently approved drugs, ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV), target the viral DNA polymerase. GCV anabolism also requires phosphorylation by the virus-encoded UL97 kinase. GCV resistance mutations have been identified in both genes, while FOS and CDV mutations occur only in the DNA polymerase gene. Confirmation of resistance mutations requires phenotypic analysis; however, phenotypic assays are too time-consuming for diagnostic purposes. Genotypic assays based on sequencing provide more rapid results but are dependent on prior validation by phenotypic methods. Reports from many laboratories have produced an evolving list of confirmed resistance mutations, although differences in interpretation have led to some confusion. Recombinant phenotyping methods performed in a few research laboratories have resolved some of the conflicting results. Treatment options for drug-resistant HCMV infections are complex and have not been subjected to controlled clinical trials, although consensus guidelines have been proposed. This review summarizes the virological and clinical data pertaining to HCMV antiviral drug resistance.

scholarly journals Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods

2021 ◽  
Vol 9 (5) ◽  
pp. 928
Author(s):  
Veronica Di Antonio ◽  
Giorgio Palù ◽  
Gualtiero Alvisi
Keyword(s):  
Energy Transfer ◽  
Dna Binding ◽  
Viral Replication ◽  
Dna Polymerase ◽  
Human Cytomegalovirus ◽  
Conformational Changes ◽  
Antiviral Drug ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Amino Acid

Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

scholarly journals Role of Hoogsteen Edge Hydrogen Bonding at Template Purines in Nucleotide Incorporation by Human DNA Polymerase ι

2006 ◽  
Vol 26 (17) ◽  
pp. 6435-6441 ◽  
Author(s):  
Robert E. Johnson ◽  
Lajos Haracska ◽  
Louise Prakash ◽  
Satya Prakash
Keyword(s):  
Hydrogen Bonding ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Human Dna ◽  
Nucleotide Incorporation ◽  
Template Specificity ◽  
Hydrogen Bonding Potential ◽  
Dna Polymerase Ι

ABSTRACT Human DNA polymerase ι (Pol ι) differs from other DNA polymerases in that it exhibits a marked template specificity, being more efficient and accurate opposite template purines than opposite pyrimidines. The crystal structures of Pol ι with template A and incoming dTTP and with template G and incoming dCTP have revealed that in the Pol ι active site, the templating purine adopts a syn conformation and forms a Hoogsteen base pair with the incoming pyrimidine which remains in the anti conformation. By using 2-aminopurine and purine as the templating residues, which retain the normal N7 position but lack the N6 of an A or the O6 of a G, here we provide evidence that whereas hydrogen bonding at N6 is dispensable for the proficient incorporation of a T opposite template A, hydrogen bonding at O6 is a prerequisite for C incorporation opposite template G. To further analyze the contributions of O6 and N7 hydrogen bonding to DNA synthesis by Pol ι, we have examined its proficiency for replicating through the 6 O-methyl guanine and 8-oxoguanine lesions, which affect the O6 and N7 positions of template G, respectively. We conclude from these studies that for proficient T incorporation opposite template A, only the N7 hydrogen bonding is required, but for proficient C incorporation opposite template G, hydrogen bonding at both the N7 and O6 is an imperative. The dispensability of N6 hydrogen bonding for proficient T incorporation opposite template A has important biological implications, as that would endow Pol ι with the ability to replicate through lesions which impair the Watson-Crick hydrogen bonding potential at both the N1 and N6 positions of templating A.

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