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DNA Repair May 2015Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure that resides in the exonuclease domain. Genetic, biochemical and structural...
Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure that resides in the exonuclease domain. Genetic, biochemical and structural studies demonstrate that the phage β hairpin acts as a wedge to separate the primer-end from the template strand in exonuclease complexes. Single amino acid substitutions in the tip of the hairpin or deletion of the hairpin prevent proofreading and create "mutator" DNA polymerases. There is little known, however, about the function of similar hairpin structures in other family B DNA polymerases. We present mutational analysis of the yeast (Saccharomyces cerevisiae) DNA polymerase δ hairpin. Deletion of the DNA polymerase δ hairpin (hpΔ) did not significantly reduce DNA replication fidelity; thus, the β hairpin structure in yeast DNA polymerase δ is not essential for proofreading. However, replication efficiency was reduced as indicated by a slow growth phenotype. In contrast, the G447D amino acid substitution in the tip of the hairpin increased frameshift mutations and sensitivity to hydroxyurea (HU). A chimeric yeast DNA polymerase δ was constructed in which the T4 DNA polymerase hairpin (T4hp) replaced the yeast DNA polymerase δ hairpin; a strong increase in frameshift mutations was observed and the mutant strain was sensitive to HU and to the pyrophosphate analog, phosphonoacetic acid (PAA). But all phenotypes - slow growth, HU-sensitivity, PAA-sensitivity, and reduced fidelity, were observed only in the absence of mismatch repair (MMR), which implicates a role for MMR in mediating DNA polymerase δ replication problems. In comparison, another family B DNA polymerase, DNA polymerase ɛ, has only an atrophied hairpin with no apparent function. Thus, while family B DNA polymerases share conserved motifs and general structural features, the β hairpin has evolved to meet specific needs.
Topics: Amino Acid Motifs; Amino Acid Sequence; DNA Mismatch Repair; DNA Mutational Analysis; DNA Polymerase III; DNA Replication; DNA, Fungal; Exodeoxyribonucleases; Molecular Sequence Data; Mutation; Protein Structure, Secondary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Deletion
PubMed: 25753811
DOI: 10.1016/j.dnarep.2015.02.014 -
Journal of Drugs in Dermatology : JDD Mar 2017
Herpes labialis remains a common worldwide affliction. Recent advances in understanding the basic pathogenesis have led to new therapeutic intervention, both on-label...
Herpes labialis remains a common worldwide affliction. Recent advances in understanding the basic pathogenesis have led to new therapeutic intervention, both on-label and off-label. Aside from reducing the duration and symptomatology of acute outbreaks, another goal of treatment is to decrease the frequency of future episodes. Oral and topical acyclovir and its analogues are the mainstay of both chronic suppressive and episodic therapy. A new muco-adhesive formulation of acyclovir provides a decrease in outbreaks, probably due to a diminution of herpesvirus load in all reservoir sites. Acyclovir-resistant strains are rare in immunocompetent hosts; parenteral foscarnet and cidofovir are administered in this situation. Parenteral acyclovir is the drug of choice for eczema herpeticum, which may begin as herpes labialis in an atopic dermatitis patient. Thermotherapy may be beneficial, and a certified device to deliver heat is available outside the United States.
J Drugs Dermatol. 2017;16(3 Suppl):s49-53.
.Topics: Acyclovir; Administration, Oral; Administration, Topical; Adult; Antiviral Agents; Chronic Disease; Cidofovir; Cytosine; Drug Resistance, Viral; Foscarnet; Herpes Labialis; Herpesvirus 1, Human; Humans; Hyperthermia, Induced; Infusions, Parenteral; Organophosphonates; Recurrence; Stomatitis, Herpetic; Viral Load
PubMed: 28301628
DOI: No ID Found -
Journal of Virology Jun 2019In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we...
In order to identify host cellular DNA metabolic enzymes that are involved in the biosynthesis of hepatitis B virus (HBV) covalently closed circular (ccc) DNA, we developed a cell-based assay supporting synchronized and rapid cccDNA synthesis from intracellular progeny nucleocapsid DNA. This was achieved by arresting HBV DNA replication in HepAD38 cells with phosphonoformic acid (PFA), a reversible HBV DNA polymerase inhibitor, at the stage of single-stranded DNA and was followed by removal of PFA to allow the synchronized synthesis of relaxed circular DNA (rcDNA) and subsequent conversion into cccDNA within 12 to 24 h. This cccDNA formation assay allows systematic screening of the effects of small molecular inhibitors of DNA metabolic enzymes on cccDNA synthesis but avoids cytotoxic effects upon long-term treatment. Using this assay, we found that all the tested topoisomerase I and II (TOP1 and TOP2, respectively) poisons as well as topoisomerase II DNA binding and ATPase inhibitors significantly reduced the levels of cccDNA. It was further demonstrated that these inhibitors also disrupted cccDNA synthesis during HBV infection of HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). Mechanistic analyses indicate that whereas TOP1 inhibitor treatment prevented the production of covalently closed negative-strand rcDNA, TOP2 inhibitors reduced the production of this cccDNA synthesis intermediate to a lesser extent. Moreover, small interfering RNA (siRNA) knockdown of topoisomerase II significantly reduced cccDNA amplification. Taking these observations together, our study demonstrates that topoisomerase I and II may catalyze distinct steps of HBV cccDNA synthesis and that pharmacologic targeting of these cellular enzymes may facilitate the cure of chronic hepatitis B. Persistent HBV infection relies on stable maintenance and proper functioning of a nuclear episomal form of the viral genome called cccDNA, the most stable HBV replication intermediate. One of the major reasons for the failure of currently available antiviral therapeutics to cure chronic HBV infection is their inability to eradicate or inactivate cccDNA. We report here a chemical genetics approach to identify host cellular factors essential for the biosynthesis and maintenance of cccDNA and reveal that cellular DNA topoisomerases are required for both synthesis and intracellular amplification of cccDNA. This approach is suitable for systematic screening of compounds targeting cellular DNA metabolic enzymes and chromatin remodelers for their ability to disrupt cccDNA biosynthesis and function. Identification of key host factors required for cccDNA metabolism and function will reveal molecular targets for developing curative therapeutics of chronic HBV infection.
Topics: Antiviral Agents; DNA Topoisomerases; DNA, Circular; DNA, Viral; Foscarnet; Genome, Viral; Hep G2 Cells; Hepatitis B; Hepatitis B virus; Hepatocytes; Humans; RNA, Small Interfering; Virion; Virus Replication
PubMed: 30867306
DOI: 10.1128/JVI.02230-18 -
Antiviral Research Nov 2021Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration,...
Acyclovir (ACV) and penciclovir and their prodrugs are recommended for therapy or prophylaxis of Herpes simplex virus 1 (HSV-1) infections. Their administration, however, can lead to the emergence of resistant strains with altered viral thymidine kinase (TK) function, especially in immunocompromised patients. Furthermore, amino acid (aa) changes of the viral deoxyribonucleic acid polymerase (POL) may contribute to resistance to the aforementioned nucleoside analogues. Given this, treatment with foscarnet (FOS) or cidofovir (CDV) may represent an important alternative. Both drugs directly affect POL activity. Several aa changes of POL, such as L49I, E70K, L359I, E421V, P829S, T1121M, and M1226I, have been observed in ACV-resistant clinical strains which also carried relevant aa changes in their TK. Their contribution to ACV, FOS, and CDV resistance is not fully understood. In this study, these seven aa changes with unknown significance for ACV, FOS and CDV resistance were introduced separately into the POL of a recombinant HSV-1 strain rHSV-1(17+)Lox, equipped with or without information for expression of green fluorescent protein (GFP). The GFP-expressing variants were tested for susceptibility to ACV, FOS and CDV. An rHSV-1(17+)Lox GFP strain with the S724N change conferring resistance to ACV and FOS was generated and included as a control. Only the S724N change was confirmed to induce ACV and FOS resistance, whereas the other changes did not contribute to resistance. The underlying nucleotide substitutions of the POL gene should be therefore considered as natural polymorphism. These data will improve sequence-based prediction of antiviral susceptibility.
Topics: Acyclovir; Animals; Antiviral Agents; Chlorocebus aethiops; Cidofovir; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Guanine; Herpes Simplex; Herpesvirus 1, Human; Humans; Immunocompromised Host; Microbial Sensitivity Tests; Thymidine Kinase; Vero Cells
PubMed: 34419483
DOI: 10.1016/j.antiviral.2021.105166 -
[Rinsho Ketsueki] the Japanese Journal... 2020Human herpesvirus (HHV)-6B encephalitis has been increasingly recognized as an important central nervous system (CNS) complication after allogeneic hematopoietic stem... (Review)
Review
Human herpesvirus (HHV)-6B encephalitis has been increasingly recognized as an important central nervous system (CNS) complication after allogeneic hematopoietic stem cell transplantation. In this review, the best way to diagnose and treat this devastating complication is described. Diagnostic pitfalls: HHV-6B encephalitis should be diagnosed based on the presence of CNS symptoms, positive results for HHV-6 DNA in the cerebrospinal fluid (CSF), and exclusion of other causes of CNS symptoms. There are potential pitfalls when diagnosing HHV-6B encephalitis. Moreover, false-positive detection of HHV-6 DNA in the CSF can occur. Pleocytosis is observed in few patients, and CSF protein levels are often normal. Limbic encephalitis findings are not commonly detected through a brain MRI at the time of development. Prevention: Neither routine monitoring nor routine prophylactic antiviral therapy is recommended to prevent the development of HHV-6B encephalitis. Treatment: Empiric therapy should be started immediately after HHV-6B encephalitis is suspected. The Guideline Committee of the JSHCT recommends full-dose foscarnet (180 mg/kg/day) for the treatment of HHV-6B encephalitis. Therapeutic effect of anti-HHV6 therapy is assessed based on CNS symptoms and HHV-6 DNA in the CSF, which should be evaluated 1-2 weeks after initiating treatment. Even in patients exhibiting good therapeutic effects, antiviral treatment should be continued for at least 3 weeks.
Topics: Encephalitis, Viral; Foscarnet; Hematopoietic Stem Cell Transplantation; Herpesvirus 6, Human; Humans; Roseolovirus Infections; Transplantation, Homologous
PubMed: 32908059
DOI: 10.11406/rinketsu.61.945 -
Ophthalmic Surgery, Lasers & Imaging... Feb 2015A 60-year-old woman with a history of recurrent headaches and blurred vision presented with bilateral optic disc edema. Optic neuritis was suspected, and intravenous... (Review)
Review
A 60-year-old woman with a history of recurrent headaches and blurred vision presented with bilateral optic disc edema. Optic neuritis was suspected, and intravenous methylprednisonlone was administered. Her vision declined to hand motions in both eyes, and subsequent evaluation revealed bilateral acute retinal necrosis with bilateral central retinal artery occlusions (CRAO). Aqueous humor polymerase chain reaction analysis was positive for herpes simplex virus (HSV), establishing a diagnosis of HSV-associated bilateral acute retinal necrosis (ARN) and meningitis. CRAO has rarely been reported in association with ARN, and a fulminant course with bilateral CRAO in association with ARN has not been previously reported. This case emphasizes the importance of careful peripheral examination in patients with presumptive optic neuritis, judicious use of systemic corticosteroid in this context, and the retinal vaso-obliterative findings that may be observed in the pathogenesis of ARN.
Topics: Antibodies, Viral; Antiviral Agents; Aqueous Humor; DNA, Viral; Drug Therapy, Combination; Eye Infections, Viral; Female; Fluorescein Angiography; Foscarnet; Ganciclovir; Herpes Simplex; Herpesvirus 2, Human; Humans; Magnetic Resonance Imaging; Meningitis, Viral; Middle Aged; Polymerase Chain Reaction; Retinal Artery Occlusion; Retinal Necrosis Syndrome, Acute
PubMed: 25707059
DOI: 10.3928/23258160-20150213-24 -
Transplant Infectious Disease : An... Apr 2018Infection with cytomegalovirus (CMV) is an important cause of morbidity and mortality following solid organ transplantation. Resistance to ganciclovir can rarely develop...
BACKGROUND
Infection with cytomegalovirus (CMV) is an important cause of morbidity and mortality following solid organ transplantation. Resistance to ganciclovir can rarely develop via mutations in UL97 or UL54. There are limited published studies assessing the safety and efficacy of foscarnet for the management of ganciclovir-resistant or refractory cytomegalovirus infection and many centers are reluctant to utilize this important therapy because of concerns about toxicity.
METHODS
Solid organ recipients transplanted between January 1, 2006 and December 31, 2014 who received at least 1 dose of foscarnet were retrospectively reviewed to assess treatment outcomes, tolerability, and safety of foscarnet.
RESULTS
Ten of 31 (32.3%) patients who received foscarnet during the study period died during treatment with foscarnet, whereas all 21 surviving recipients successfully cleared infection. Of these surviving patients, 3 (14.3%) developed significant renal dysfunction, defined as >25% decline in estimated glomerular filtration rate during treatment; one-third had definitive renal biopsy results consistent with foscarnet-induced toxicity.
CONCLUSION
Although mortality was high in this population, foscarnet use, with proper precautions, was generally safe and significant renal dysfunction was lower than previously reported in other sources, even with extended use.
Topics: Adult; Aged; Antiviral Agents; Cytomegalovirus; Cytomegalovirus Infections; Drug Resistance, Viral; Female; Foscarnet; Ganciclovir; Glomerular Filtration Rate; Humans; Male; Middle Aged; Retrospective Studies
PubMed: 29380479
DOI: 10.1111/tid.12852 -
Transplantation Proceedings Dec 2018Cytomegalovirus (CMV) infection is a common complication in solid organ transplant recipients. In patients receiving immunosuppressive treatment, CMV may lead to... (Review)
Review
Cytomegalovirus (CMV) infection is a common complication in solid organ transplant recipients. In patients receiving immunosuppressive treatment, CMV may lead to life-threatening organ complications or graft loss. We describe a case of 31-year-old CMV-seronegative patient who underwent liver transplant from a CMV-seropositive donor with an early acute resistant rejection of the transplanted organ followed by primary CMV infection, despite prophylaxis, and its severe organ complications. Routine treatment of acute allograft rejection through increasing the base immunosuppression and then administering methylprednisolone infusions did not yield significant therapeutic effect. This resulted in anti-thymocyte globulin and ultimately proteasome inhibitor introduction. The cholestasis remitted and liver parameters improved. But 4 weeks later the patient was admitted again due to incorrect liver function tests. Blood tests revealed high CMV viral load, and primary CMV infection was diagnosed. On diagnosis the patient was treated with ganciclovir (GCV) intravenously. As GCV resistance was suspected based on clinical premises, foscarnet (FOS) and leflunomide (LFM) were implemented with concomitant cautious immunosuppression reduction due to the history of recent graft rejection. Despite aggressive treatment introduction, viral clearance was not obtained. Ultimately the patient died due to respiratory distress resulting from lung fibrosis, most probably owing to CMV diseases with Pneumocystis jiroveci coinfection. The presented case proves the importance of strictly following the rules of prophylaxis, especially in patients with a high risk factor of CMV infection development. A quick diagnosis, implementation of appropriate treatment, and fast reaction to the lack of satisfying therapeutic effect can be the key to a successful treatment.
Topics: Adult; Antilymphocyte Serum; Antiviral Agents; Cytomegalovirus Infections; Drug Resistance, Viral; Foscarnet; Ganciclovir; Graft Rejection; Humans; Immunocompromised Host; Immunosuppression Therapy; Immunosuppressive Agents; Liver Transplantation; Male; Methylprednisolone; Risk Factors
PubMed: 30577306
DOI: 10.1016/j.transproceed.2018.05.014 -
Bioorganic Chemistry Nov 2020A series of alkylphosphocholines with foscarnet moiety was synthesized. The structure of these zwitterionic amphiphiles was modified in both polar and non-polar parts of...
A series of alkylphosphocholines with foscarnet moiety was synthesized. The structure of these zwitterionic amphiphiles was modified in both polar and non-polar parts of surfactant molecule. Investigations of physicochemical properties are represented by the determination of critical micelle concentration, the surface tension value at the cmc and the surface area per surfactant head group utilising surface tension measurements. Hydrodynamic diameter of surfactant micelles was determined using the dynamic light scattering technique. Alkylphosphocholines exhibit significant cytotoxic, anticandidal (Candida albicans) and antiamoebal (Acanthamoeba spp. T4 genotype) activity. The relationship between the structure, physicochemical properties and biological activity of the tested compounds revealed that lipophilicity has a significant influence on biological activity of the investigated surfactants. More lipophilic alkylphosphocholines with octadecyl chains show cytotoxic activity against cancer cells which is higher than that of the compounds with shorter alkyl chains. The opposite situation was observed in case of anticandidal and antiamoebal activity of these surfactants. The most active compounds were found to have pentadecyl chains. The foscarnet analogue of miltefosine C15-PFA-C showed the highest anticandidal activity. The minimum value of anticandidal activity of this compound is 1,4 μM thus representing the highest anticandidal activity found within the group of alkylphosphocholines.
Topics: 3T3 Cells; Acanthamoeba; Amebicides; Animals; Antifungal Agents; Antineoplastic Agents; Candida albicans; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Foscarnet; Humans; Hydrodynamics; Mice; Micelles; Microbial Sensitivity Tests; Molecular Structure; Parasitic Sensitivity Tests; Phosphorylcholine; Structure-Activity Relationship; Surface Tension
PubMed: 32892068
DOI: 10.1016/j.bioorg.2020.104224 -
Medicine Dec 2021Intraocular infection of Epstein-Barr virus (EBV) may cause severe visual loss. However, it is relatively rare, and there is no consensus on its treatment.
RATIONALE
Intraocular infection of Epstein-Barr virus (EBV) may cause severe visual loss. However, it is relatively rare, and there is no consensus on its treatment.
PATIENT CONCERNS
A 44-year-old woman complained of a right-eye floater and exhibited a unilateral exudative change along the retinal veins at the Department of Ophthalmology, St. Luke's International Hospital.
DIAGNOSIS
EBV retinitis was diagnosed based on EBV-positive (9.09 × 103 copies/μl) and cytomegalovirus-negative results in the aqueous humor.
INTERVENTIONS
Oral prescription of valaciclovir hydrochloride, and an intravitreal injection of foscarnet sodium hydrate was administered. However, the retinal infiltration progressed, and vitreous opacity with cellular infiltration appeared. Intravitreal methotrexate (MTX) injection effectively suppressed retinal and vitreous infiltration. However, she developed optic-nerve papillitis, and central retinal vein occlusion related to the severe swelling of the optic-nerve, and began steroid pulse therapy. Considering the increase in intraocular EBV levels to 6.4 × 104 copies/ml, we restarted intravitreal foscarnet injections replacing MTX. This in turn rapidly reduced the EBV levels to 3.27 × 104 copies/ml, followed by papillitis alleviation.
OUTCOMES
The intraocular MTX administration reduced the inflammatory vitreous and retinal infiltration, but not the EBV load, while foscarnet reduced the EBV load and papillitis, but not vitreous infiltration.
LESSONS
The retinal infiltration may have involved EBV infection to the retinal neurons but also EBV-free reactive inflammatory cells. EBV infection to the neurons may have been, at least partially, treated by intravitreal foscarnet treatment, and the reactive inflammatory cells by intravitreal MTX. Further observations are warranted to reach a consensus on treating intraocular EBV infection.
Topics: Adult; Epstein-Barr Virus Infections; Female; Foscarnet; Herpesvirus 4, Human; Humans; Methotrexate; Papilledema; Pulse Therapy, Drug; Retinitis; Steroids; Treatment Outcome
PubMed: 35049237
DOI: 10.1097/MD.0000000000028101