Recombinant Phenotyping of Cytomegalovirus UL54 Mutations That Emerged during Cell Passages in the Presence of either Ganciclovir or Foscarnet

ABSTRACTSelection of human cytomegalovirus variants in the presence of ganciclovir or foscarnet led to 18 DNA polymerase mutations, 14 of which had not been previously studied. Using bacterial artificial chromosome technology, each of these mutations was individually transferred into the genome of a reference strain. Following reconstitution of infectious viral stocks, each mutant was assessed for its drug susceptibility and growth kinetics in cell culture. Computer-assisted three-dimensional (3D) modeling of the polymerase was also used to position each of the mutations in one of four proposed structural domains and to predict their influence on structural stability of the protein. Among the 10 DNA polymerase mutations selected with ganciclovir, 7 (P488R, C539R, L545S, V787L, V812L, P829S, and L862F) were associated with ganciclovir resistance, whereas 2 (F595I and V946L) conferred only foscarnet resistance. Among the eight mutations selected with foscarnet, only two (T552N and S585A) conferred foscarnet resistance, whereas four (N408D, K500N, L802V, and L957F) had an impact on ganciclovir susceptibility. Surprisingly, the combination of mutations, some of which were not associated with resistance for a specific antiviral, resulted in increasing resistance effects. 3D modeling suggested that none of the mutated residues were directly involved in the polymerase catalytic site but rather had an influence on drug susceptibility by modifying the structural flexibility of the protein. Our study significantly adds to the number of DNA polymerase mutations conferringin vitrodrug resistance and emphasizes the point that evaluation of individual mutations may not accurately reflect the phenotype conferred by multiple mutations.

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DNA Polymerase: Structural Homology, Conformational Dynamics, and the Effects of Carcinogenic DNA Adducts

Journal of Nucleic Acids ◽

10.4061/2010/457176 ◽

2010 ◽

Vol 2010 ◽

pp. 1-15 ◽

Cited By ~ 7

Author(s):

Richard G. Federley ◽

Louis J. Romano

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Dna Adducts ◽

Structural Changes ◽

Dna Polymerases ◽

Conformational Dynamics ◽

Three Dimensional ◽

Structural Homology ◽

Human Right ◽

Selection Of

DNA replication is vital for an organism to proliferate and lying at the heart of this process is the enzyme DNA polymerase. Most DNA polymerases have a similar three dimensional fold, akin to a human right hand, despite differences in sequence homology. This structural homology would predict a relatively unvarying mechanism for DNA synthesis yet various polymerases exhibit markedly different properties on similar substrates, indicative of each type of polymerase being prescribed to a specific role in DNA replication. Several key conformational steps, discrete states, and structural moieties have been identified that contribute to the array of properties the polymerases exhibit. The ability of carcinogenic adducts to interfere with conformational processes by directly interacting with the protein explicates the mutagenic consequences these adducts impose. Recent studies have identified novel states that have been hypothesised to test the fit of the nascent base pair, and have also shown the enzyme to possess a lively quality by continually sampling various conformations. This review focuses on the homologous structural changes that take place in various DNA polymerases, both replicative and those involved in adduct bypass, the role these changes play in selection of a correct substrate, and how the presence of bulky carcinogenic adducts affects these changes.

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In Vitro Three Dimensional Imaging of Human Carotid Atherosclerotic Plaques Using Ultrasonography

ASME 2011 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2011-53463 ◽

2011 ◽

Author(s):

Renate W. Boekhoven ◽

Marcel C. M. Rutten ◽

Marc R. H. M. van Sambeek ◽

Frans N. van de Vosse

Keyword(s):

Mechanical Properties ◽

Carotid Artery ◽

Carotid Endarterectomy ◽

Early Stage ◽

Three Dimensional ◽

Atherosclerotic Plaques ◽

Unstable Plaques ◽

Human Carotid ◽

Selection Of

Ruptured atherosclerotic plaques in the carotid artery are the main cause of stroke (70–80%). To prevent it, carotid endarterectomy is the procedure of choice in patients with a recent symptomatic 70–99% stenosis. Today, the selection of candidates is based on stenosis size only. However, endarterectomy is beneficial for only 1 out of 6 patients [1], the patients with unstable plaques (Fig. 1). Knowledge of mechanical properties of different components in the atherosclerotic arteries is important, because it will allow the identification of plaque stability at an early stage.

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In vitro selection of optimal AbrB-binding sites: comparison to known in vivo sites indicates flexibility in AbrB binding and recognition of three-dimensional DNA structures

Molecular Microbiology ◽

10.1046/j.1365-2958.1996.358882.x ◽

1996 ◽

Vol 19 (1) ◽

pp. 145-158 ◽

Cited By ~ 31

Author(s):

Ke Xu ◽

Mark A. Strauch

Keyword(s):

Binding Sites ◽

In Vitro Selection ◽

Three Dimensional ◽

Dna Structures ◽

Selection Of

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Selection and Recombinant Phenotyping of a Novel CMX001 and Cidofovir Resistance Mutation in Human Cytomegalovirus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00062-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3321-3325 ◽

Cited By ~ 22

Author(s):

Scott H. James ◽

Nathan B. Price ◽

Caroll B. Hartline ◽

E. Randall Lanier ◽

Mark N. Prichard

Keyword(s):

Dna Polymerase ◽

Selective Pressure ◽

Resistance Mutation ◽

Artificial Chromosome ◽

Wild Type ◽

Phenotypic Resistance ◽

Genotypic Analysis ◽

Wide Range ◽

Genes Encoding

ABSTRACTCMX001 is an orally available lipid acyclic nucleotide phosphonate that delivers high intracellular levels of cidofovir (CDV)-diphosphate and exhibits enhancedin vitroantiviral activity against a wide range of double-stranded DNA viruses, including cytomegalovirus (CMV). Mutations in the DNA polymerase of CMV that impart resistance to CDV also render the virus resistant to CMX001. Here, we report a novel resistance mutation that arose under the selective pressure of CMX001. The wild-type CMV strain AD169 was propagated in human foreskin fibroblasts under increasing concentrations of CMX001 over 10 months, and the resulting strain (named CMX001R) was less susceptible to CDV and CMX001 in a plaque reduction assay. Genotypic analysis of virus strain CMX001Rvia conventional sequencing of the genes encoding the CMV DNA polymerase (UL54) and UL97 kinase (UL97) demonstrated one mutation that changed the wild-type aspartate to glutamate at position 542 inUL54. A recombinant virus with this novel D542E mutation was generated via bacterial artificial chromosome-mediated marker transfer experiments. Subsequent phenotypic resistance analysis of the D542E mutant demonstrated reductions in susceptibility of greater than 10-fold to CMX001 and CDV, but no resistance to foscarnet (FOS) or ganciclovir (GCV). Analysis of replicative fitness showed that both strain CMX001Rand the D542E mutant viruses demonstrated a smaller plaque phenotype and slower replication kinetics than their respective parent viruses. These data describe the first resistance mutation generated under the selective pressure of CMX001 and suggest that CMX001 may have a unique resistance profile associated with reduced viral replication and maintenance of sensitivity to FOS and GCV.

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Intestinal Motility: A Computer Simulation of the Effects of Drugs on Colonic Peristalsis, for Teaching Undergraduate Pharmacology Students

Alternatives to Laboratory Animals ◽

10.1177/026119299602400104 ◽

1996 ◽

Vol 24 (1) ◽

pp. 11-19

Author(s):

David Dewhurst ◽

Helen Leathard ◽

Richard Ullyott

Keyword(s):

Undergraduate Students ◽

Intestinal Motility ◽

Teaching And Learning ◽

Rat Colon ◽

Computer Assisted ◽

Chart Recorder ◽

Experimental Protocols ◽

Excitatory Neurotransmission ◽

Selection Of

An interactive computer-assisted learning (CAD program, which simulates experiments performed on segments of rat colon (in vitro) to study the pharmacology of intestinal motility, is described. The program covers: a) the actions of drugs that affect cholinergic excitatory neurotransmission in the colon; b) the effects of sympathomimetic amines; and c) an investigation of the mechanism of action of the laxative, phenolphthalein. The program can be run on any IBM-compatible PC, and makes use of text and high-resolution graphics to provide a background to the experiments and to describe the methodology. The screen display emulates a chart recorder, and simulated results, derived from actual data, are presented, in response to student selection of pre-determined experimental protocols from a menu. The program is aimed at undergraduate students, and is intended to support or replace a conventional laboratory practical class, while achieving the majority of the same teaching and learning objectives.

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Recording temporal data onto DNA with minutes resolution

10.1101/634790 ◽

2019 ◽

Cited By ~ 1

Author(s):

Namita J Bhan ◽

Jonathan Strutz ◽

Joshua Glaser ◽

Reza Kalhor ◽

Edward Boyden ◽

...

Keyword(s):

Dna Polymerase ◽

Three Dimensional ◽

Terminal Deoxynucleotidyl Transferase ◽

Temporal Data ◽

Environmental Signals ◽

Minute Period ◽

Signal Profile ◽

Over Time ◽

Extract Information

AbstractRecording biological signals can be difficult in three-dimensional matrices, such as tissue. We present a DNA polymerase-based strategy that records temporal biosignals locally onto DNA to be read out later, which could obviate the need to extract information from tissue on the fly. We use a template-independent DNA polymerase, terminal deoxynucleotidyl transferase (TdT) that probabilistically adds dNTPs to single-stranded DNA (ssDNA) substrates without a template. We show that in vitro, the dNTP-incorporation preference of TdT changes with the presence of Co2+, Ca2+, Zn2+ and temperature. Extracting the signal profile over time is possible by examining the dNTP incorporation preference along the length of synthesized ssDNA strands like a molecular ticker tape. We call this TdT-based untemplated recording of temporal local environmental signals (TURTLES). We show that we can determine the time of Co2+ addition to within two minutes over a 60-minute period. Further, TURTLES has the capability to record multiple fluctuations. We can estimate the rise and fall of an input Co2+ pulse to within three minutes. TURTLES has at least 200-fold better temporal resolution than all previous DNA-based recording techniques.

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88 SELECTION OF A CRYOSURVIVAL SPERM POPULATION DOES NOT IMPROVE THE IN VITRO PENETRATING ABILITY OF FROZEN - THAWED BOAR SPERMATOZOA

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab88 ◽

2008 ◽

Vol 20 (1) ◽

pp. 124

Author(s):

M. L. Perals ◽

M. A. Gil ◽

E. M. Garcia ◽

J. Sanchez-Osorio ◽

J. M. Vázquez ◽

...

Keyword(s):

Sperm Quality ◽

Membrane Integrity ◽

Molecular Probes ◽

Computer Assisted ◽

Immature Oocytes ◽

Sperm Analysis ◽

Boar Spermatozoa ◽

Casa System ◽

Selection Of

Boars can be classified as good or bad sperm freezers according to their sperm cryosurvival. Different sperm selection techniques, such as PureSperm� (PS; MidAtlantic Diagnostics, Inc., Mount Laurel, NJ, USA), have been developed to improve functional competence of spermatozoa. The aim of this experimental study was to assess the ability of PS for improving the in vitro penetrating ability of frozen–thawed boar spermatozoa from good and bad sperm freezers. The sperm-rich fractions from two boars, good (Boar A) and bad (Boar B) freezers, were extended in a lactose/eggyolk/ glycerol/Equex Stem (Noba Chemical Sales, Inc., Scituate, ME, USA) mixture (1 � 109 sperm mL–1), dispensed into 0.5-mL straws, and frozen using a programmable cell freezer. After thawing (1.200�C min–1), semen from each boar was split into two aliquots of 500 µL. One aliquot was used as the control. The second was placed into a tube of PS gradient (90%/45%) and centrifuged at 425g for 20 min; the pellet re-suspended in 1 mL of BTS and re-centrifuged at 320g for 10 min (PS sample). Control and PS samples were diluted in supplemented TCM-199 (TCMm; Roca et al. 1998 Reprod. Fertil. Dev. 10, 479–485) at 200 � 106 sperm mL–1. Sperm survival (SV) was assessed afterTCMm dilution according to progressive sperm motility (PSM, %) using a computer-assisted sperm analysis (CASA) system (ISAS�), and plasma and acrosome membrane integrity (PMI; %) by flow cytometry (SYBR�-14/PE-PNA/PI; Molecular Probes, Leiden, The Netherlands). A homologous in vitro penetration (hIVP) assay, using immature oocytes (20 oocytes/2 mL TCMm supplemented with caffeine and calcium lactate), was used to assess sperm penetrating ability (Martinez et al. 1993 Theriogenology 40, 547–557). A total of 960 immature oocytes were inseminated (200 � 103 sperm/oocyte) in 3 batches. After 18 h of co-incubation at 39�C under 5% CO2 in air, the oocytes were washed, mounted on slides, fixed with ethanol:acetic acid (3:1, v/v) for 48 h, stained with 1% lacmoid, and examined under a phase contrast microscope (�400). Oocytes with swollen or unswollen heads of sperm found in the vitellus were considered as penetrated. Sperm penetrability ability (SPA) was assessed according to penetration rate (PR) and the mean number of sperm per oocyte (S/O). Data were analyzed using a PROMIXED model and expressed as mean � SEM. Boar A showed better (P ≤ 0.01) results for both SV and SPA parameters than boar B, independent of sperm treatment. PureSperm improved (P ≤ 0.05) PSM and PMI in both boar A (control v. PS: 48.0 � 5.8 v. 66.5 � 3.6 and 63.1 � 7.7 v. 88.4 � 1.3, respectively) and boar B (12.3 � 1.2 v. 22.2 � 3.7 and 44.3 � 3.5 v. 58.7 � 7.0, respectively). However, no differences (P ≥ 0.05) were observed in PR and S/O in either boar A (71.2 � 3.4 v. 78.3 � 3.1 and 5.0 � 0.4 v. 5.2 � 0.4, respectively) or boar B (34.3 � 3.6 v. 37.3 � 3.9 and 1.5 � 0.1 v. 1.5 � 0.1, respectively). In conclusion, under our laboratory conditions, PureSperm selection improves sperm quality but not in vitro penetrating ability of frozen–thawed spermatozoa of both good and bad sperm freezers. This work was supported by CICYT (AGF2005-00760), Madrid, Spain.

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A computer-assisted analysis of conserved residues in the three-dimensional structures of the polymerase domains of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)42408-2 ◽

1994 ◽

Vol 269 (1) ◽

pp. 716-720

Author(s):

P.N. Yadav ◽

J.S. Yadav ◽

E. Arnold ◽

M.J. Modak

Keyword(s):

Escherichia Coli ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Three Dimensional ◽

Computer Assisted ◽

Dna Polymerase I ◽

Conserved Residues ◽

Polymerase I ◽

Assisted Analysis ◽

Hiv 1

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Growth and Drug Resistance Phenotypes Resulting from Cytomegalovirus DNA Polymerase Region III Mutations Observed in Clinical Specimens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00736-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4160-4162 ◽

Cited By ~ 45

Author(s):

Sunwen Chou ◽

Gail I. Marousek ◽

Laura C. Van Wechel ◽

Shaobing Li ◽

Adriana Weinberg

Keyword(s):

Drug Resistance ◽

Bacterial Artificial Chromosome ◽

Dna Polymerase ◽

Artificial Chromosome ◽

Wild Type ◽

Clinical Specimens ◽

Wild Type Sequence ◽

Ganciclovir Resistance ◽

Type Sequence

ABSTRACT Recombinant phenotyping of cytomegalovirus (CMV) pol region III mutations from clinical specimens showed that T813S and G841A each conferred foscarnet resistance and approximately threefold increased ganciclovir resistance; adding the UL97 mutation C592G increased ganciclovir resistance to approximately sixfold. Bacterial artificial chromosome CMV clones containing pol mutation L845P were nonviable unless repaired with the wild-type sequence.

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