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Antimicrobial Agents and Chemotherapy Jun 2021Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes and ,...
Amino acid substitutions conferring resistance of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) to foscarnet (PFA) are located in the genes and , respectively, encoding the DNA polymerase (pol). In this study, we analyzed the impact of substitutions located in helix K and region II that are involved in the conformational changes of the DNA pol. Theoretical substitutions were identified by sequences alignment of the helix K and region II of human herpesviruses (susceptible to PFA) and bacteriophages (resistant to PFA) and introduced in viral genomes by recombinant phenotyping. We characterized the susceptibility of HSV-1 and HCMV mutants to PFA. In UL30, the substitutions I619K (helix K), V715S, and A719T (both in region II) increased mean PFA 50% effective concentrations (ECs) by 2.5-, 5.6-, and 2.0-fold, respectively, compared to the wild type (WT). In UL54, the substitution Q579I (helix K) conferred hypersusceptibility to PFA (0.17-fold change), whereas the substitutions Q697P, V715S, and A719T (all in region II) increased mean PFA ECs by 3.8-, 2.8- and 2.5-fold, respectively, compared to the WT. These results were confirmed by enzymatic assays using recombinant DNA pol harboring these substitutions. Three-dimensional modeling suggests that substitutions conferring resistance/hypersusceptibility to PFA located in helix K and region II of UL30 and UL54 DNA pol favor an open/closed conformation of these enzymes, resulting in a lower/higher drug affinity for the proteins. Thus, this study shows that both regions of UL30 and UL54 DNA pol are involved in the conformational changes of these proteins and can influence the susceptibility of both viruses to PFA.
Topics: Amino Acid Substitution; Antiviral Agents; Cytomegalovirus; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Herpesvirus 1, Human; Humans; Mutation
PubMed: 33875432
DOI: 10.1128/AAC.00390-21 -
Chemical & Pharmaceutical Bulletin Dec 2022Quantitative H-NMR (H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for...
Quantitative H-NMR (H-qNMR) is useful for determining the absolute purity of organic molecules; however, it is sometimes difficult to identify the target signal(s) for quantitation because of their overlap and complexity. Therefore, we focused on the P nucleus because of the simplicity of its signals and previously reported P-qNMR in DO. Here we report P-qNMR of an organophosphorus compound, sofosbuvir (SOF), which is soluble in organic solvents. Phosphonoacetic acid (PAA) and 1,4-bis(trimethylsilyl)benzene-d (1,4-BTMSB-d) were used as reference standards for P-qNMR and H-qNMR, respectively, in methanol-d. The purity of SOF determined by P-qNMR was 100.63 ± 0.95%, whereas that determined by H-qNMR was 99.07 ± 0.50%. The average half bandwidths of the P signal of PAA and SOF were 3.38 ± 2.39 and 2.22 ± 0.19 Hz, respectively, suggesting that the T relaxation time of the PAA signal was shorter than that of SOF and varied among test laboratories. This difference most likely arose from the instability in the chemical shift due to the deuterium exchange of the acidic protons of PAA, which decreased the integrated intensity of the PAA signal. Next, an aprotic solvent, dimethyl sulfoxide-d (DMSO-d), was used as the dissolving solvent with PAA and sodium 4,4-dimethyl-4-silapentanesulfonate-d (DSS-d) as reference standards for P-qNMR and H-qNMR, respectively. SOF purities determined by P-qNMR and H-qNMR were 99.10 ± 0.30 and 99.44 ± 0.29%, respectively. SOF purities determined by P-qNMR agreed with the established H-qNMR values, suggesting that an aprotic solvent is preferable for P-qNMR because it is unnecessary to consider the effect of deuterium exchange.
Topics: Deuterium; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Reference Standards; Sofosbuvir; Solvents
PubMed: 36223954
DOI: 10.1248/cpb.c22-00639 -
Virologie (Montrouge, France) Oct 2020Herpes simplex virus (HSV) infections remain an important cause of morbidity among immunocompromised patients, such as transplant recipients and human immunodeficiency...
Herpes simplex virus (HSV) infections remain an important cause of morbidity among immunocompromised patients, such as transplant recipients and human immunodeficiency virus [HIV]-infected individuals. Only few antiviral drugs are available to treat HSV infections: (val)acyclovir, foscarnet, and cidofovir. Prophylactic and curative antiviral treatments administered during prolonged periods among patients with altered T-cell immunity may lead to the emergence of HSV resistance to antivirals, contributing to a challenging therapeutic management of viral infection. The persistence of herpetic lesions after 10 days of well-conducted antiviral therapy is suggestive of viral resistance. Resistance to antivirals can be detected using genotypic methods (identifications of antiviral resistance-associated mutations by sequencing genes encoding viral proteins involved in the mechanism of action of antivirals) or phenotypic methods (measure of antiviral drug concentration inhibiting 50% of viral replication in cell culture). The prevalence of HSV resistance to acyclovir is below 1% in immunocompetent individuals, except those with herpetic keratitis for whom prevalence can reach 7%, and varies from 3.5% to 11% in immunocompromised patients. Adverse effects and the absence of eradication of viral latent infection constitute other limits to the use of antiviral drugs. New antiviral compounds undergoing clinical trials and novel potential viral targets seem very promising to enlarge the panel of efficient compounds to treat HSV infections.
Topics: Acyclovir; Antiviral Agents; Foscarnet; Herpes Simplex; Humans; Simplexvirus
PubMed: 33111706
DOI: 10.1684/vir.2020.0864 -
The Journal of International Medical... Jan 2024This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
OBJECTIVE
This study aimed to examine the mechanism of hyperphosphatemia-induced vascular calcification (HPVC).
METHODS
Primary human aortic smooth muscle cells and rat aortic rings were cultured in Dulbecco's modified Eagle's medium supplemented with 0.9 mM or 2.5 mM phosphorus concentrations. Type III sodium-dependent phosphate cotransporter-1 (Pit-1) small interfering RNA and phosphonoformic acid (PFA), a Pit-1 inhibitor, were used to investigate the effects and mechanisms of Pit-1 on HPVC. Calcium content shown by Alizarin red staining, expression levels of Pit-1, and characteristic molecules for phenotypic transition of vascular smooth muscle cells were examined.
RESULTS
Hyperphosphatemia induced the upregulation of Pit-1 expression, facilitated phenotypic transition of vascular smooth muscle cells, and led to HPVC in cellular and organ models. Treatment with Pit-1 small interfering RNA or PFA significantly inhibited Pit-1 expression, suppressed phenotypic transition, and attenuated HPVC.
CONCLUSIONS
Our findings suggest that Pit-1 plays a pivotal role in the development of HPVC. The use of PFA as a Pit-1 inhibitor has the potential for therapeutic intervention in patients with HPVC. However, further rigorous clinical investigations are required to ensure the safety and efficacy of PFA before it can be considered for widespread implementation in clinical practice.
Topics: Animals; Humans; Rats; Aorta; Foscarnet; Hyperphosphatemia; RNA, Small Interfering; Transcription Factors; Vascular Calcification; Sodium-Phosphate Cotransporter Proteins, Type III
PubMed: 38180904
DOI: 10.1177/03000605231222156 -
Kidney360 Jul 2021
Topics: Allografts; Foscarnet; Kidney; Transplantation, Homologous
PubMed: 35368358
DOI: 10.34067/KID.0001062021 -
Frontiers in Cellular and Infection... 2020Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To...
Human Cytomegalovirus (HCMV) infection may result in severe outcomes in immunocompromised individuals such as AIDS patients, transplant recipients, and neonates. To date, no vaccines are available and there are only few drugs for anti-HCMV therapy. Adverse effects and the continuous emergence of drug-resistance strains require the identification of new drug candidates in the near future. Identification and characterization of such compounds and biological factors requires sensitive and reliable detection techniques of HCMV infection, gene expression and spread. In this work, we present and validate a novel concept for multi-reporter herpesviruses, identified through iterative testing of minimally invasive mutations. We integrated up to three fluorescence reporter genes into replication-competent HCMV strains, generating reporter HCMVs that allow the visualization of replication cycle stages of HCMV, namely the immediate early (IE), early (E), and late (L) phase. Fluorescent proteins with clearly distinguishable emission spectra were linked by 2A peptides to essential viral genes, allowing bicistronic expression of the viral and the fluorescent protein without major effects on viral fitness. By using this triple color reporter HCMV, we monitored gene expression dynamics of the IE, E, and L genes by measuring the fluorescent signal of the viral gene-associated fluorophores within infected cell populations and at high temporal resolution. We demonstrate distinct inhibitory profiles of foscarnet, fomivirsen, phosphonoacetic acid, ganciclovir, and letermovir reflecting their mode-of-action. In conclusion, our data argues that this experimental approach allows the identification and characterization of new drug candidates in a single step.
Topics: Antiviral Agents; Cytomegalovirus; Gene Expression; Herpesviridae; Humans; Infant, Newborn; Pharmaceutical Preparations; Virus Replication
PubMed: 33489928
DOI: 10.3389/fcimb.2020.536150 -
Scientific Reports Feb 2020Paraquat (PQ) is a non-selective herbicide and is exceedingly toxic to humans. The mechanism of PQ toxicity is very complex and has not been clearly defined. There is no...
Paraquat (PQ) is a non-selective herbicide and is exceedingly toxic to humans. The mechanism of PQ toxicity is very complex and has not been clearly defined. There is no specific antidote for PQ poisoning. 5-hydroxy-1-methylhydantoin (HMH) is an intrinsic antioxidant and can protect against renal damage caused by PQ. The mechanism of PQ toxicology and the possible effects of HMH on PQ-induced lung injury were determined in this study. It was found that PQ decreased superoxide dismutase (SOD) activity and elevated the level of malondialdehyde (MDA), while HMH elevated SOD activity and decreased the level of MDA. Based on metabolomics, the citrate cycle, glutathione metabolism, taurine and hypotaurine metabolism, regulation of lipolysis in adipocytes, inflammatory mediator regulation of TRP channels, purine and pyrimidine metabolism, aldosterone synthesis and secretion, and phenylalanine metabolism were changed in the PQ group. Compared with the PQ group, the levels of N-acetyl-l-aspartic acid, L-glutamic acid, L-aspartic acid, mesaconic acid, adenosine 5' monophosphate, methylmalonic acid, cytidine, phosphonoacetic acid, hypotaurine, glutathione (reduced) and cysteinylglycine increased, while the levels of corticosterone, xanthine, citric acid, prostaglandin G2, 4-pyridoxic acid and succinyl proline decreased in the HMH group. These metabolites revealed that HMH can alleviate inflammation caused by PQ and elevate the activity of intrinsic antioxidants. In conclusion, our results revealed PQ toxicology and the pharmacology underlying the protective effect of HMH on lung injury due to PQ. Toxicity caused by PQ results in lipid peroxidation and an increase in reactive oxygen species (ROS), nitric oxide (NO), damage to the biliary system, gastrointestinal system and nervous system, in addition to lungs, kidneys, and the liver. HMH is a good antioxidant and protects against lung injury caused by PQ. In summary, HMH efficiently reduced PQ-induced lung injury in mice.
Topics: Acute Lung Injury; Glutathione; Herbicides; Humans; Hydantoins; Lipid Peroxidation; Malondialdehyde; Metabolomics; Paraquat; Protective Agents; Superoxide Dismutase; Taurine
PubMed: 32019966
DOI: 10.1038/s41598-020-58599-y -
BioRxiv : the Preprint Server For... Jan 2023The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the...
UNLABELLED
The APOBEC3 family of DNA cytosine deaminases comprises an important arm of the innate antiviral defense system. The gamma-herpesviruses EBV and KSHV and the alpha-herpesviruses HSV-1 and HSV-2 have evolved an efficient mechanism to avoid APOBEC3 restriction by directly binding to APOBEC3B and facilitating its exclusion from the nuclear compartment. The only viral protein required for APOBEC3B relocalization is the large subunit of the ribonucleotide reductase (RNR). Here, we ask whether this APOBEC3B relocalization mechanism is conserved with the beta-herpesvirus human cytomegalovirus (HCMV). Although HCMV infection causes APOBEC3B relocalization from the nucleus to the cytoplasm in multiple cell types, the viral RNR (UL45) is not required. APOBEC3B relocalization occurs rapidly following infection suggesting involvement of an immediate early or early (IE-E) viral protein. In support of this mechanism, cycloheximide treatment of HCMV-infected cells prevents the expression of viral proteins and simultaneously blocks APOBEC3B relocalization. In comparison, the treatment of infected cells with phosphonoacetic acid, which is a viral DNA synthesis inhibitor affecting late protein expression, still permits A3B relocalization. These results combine to show that the beta-herpesvirus HCMV uses a fundamentally different, RNR-independent molecular mechanism to antagonize APOBEC3B.
IMPORTANCE
Human cytomegalovirus (HCMV) infections can range from asymptomatic to severe, particularly in neonates and immunocompromised patients. HCMV has evolved strategies to overcome host-encoded antiviral defenses in order to achieve lytic viral DNA replication and dissemination and, under some conditions, latency and long-term persistence. Here, we show that HCMV infection causes the antiviral factor, APOBEC3B, to relocalize from the nuclear compartment to the cytoplasm. This overall strategy resembles that used by related herpesviruses. However, the HCMV relocalization mechanism utilizes a different viral factor(s) and available evidence suggests the involvement of at least one protein expressed at the early stages of infection. This knowledge is important because a greater understanding of this mechanism could lead to novel antiviral strategies that enable APOBEC3B to naturally restrict HCMV infection.
PubMed: 36778493
DOI: 10.1101/2023.01.30.526383 -
Journal of Virology Jul 2022Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the family. We showed earlier that infection of porcine epithelial cells with PRV triggers...
Pseudorabies virus (PRV) is a porcine alphaherpesvirus that belongs to the family. We showed earlier that infection of porcine epithelial cells with PRV triggers activation of the nuclear factor κB (NF-κB) pathway, a pivotal signaling axis in the early immune response. However, PRV-induced NF-κB activation does not lead to NF-κB-dependent gene expression. Here, using electrophoretic mobility shift assays (EMSAs), we show that PRV does not disrupt the ability of NF-κB to interact with its κB target sites. Assessing basal cellular transcriptional activity in PRV-infected cells by quantitation of prespliced transcripts of constitutively expressed genes uncovered a broad suppression of cellular transcription by PRV, which also affects the inducible expression of NF-κB target genes. Host cell transcription inhibition was rescued when viral genome replication was blocked using phosphonoacetic acid (PAA). Remarkably, we found that host gene expression shutoff in PRV-infected cells correlated with a substantial retention of the NF-κB subunit p65, the TATA box binding protein, and RNA polymerase II-essential factors required for (NF-κB-dependent) gene transcription-in expanding PRV replication centers in the nucleus and thereby away from the host chromatin. This study reveals a potent mechanism used by the alphaherpesvirus PRV to steer the protein production capacity of infected cells to viral proteins by preventing expression of host genes, including inducible genes involved in mounting antiviral responses. Herpesviruses are highly successful pathogens that cause lifelong persistent infections of their host. Modulation of the intracellular environment of infected cells is imperative for the success of virus infections. We reported earlier that a DNA damage response in epithelial cells infected with the alphaherpesvirus pseudorabies virus (PRV) results in activation of the hallmark proinflammatory NF-κB signaling axis but, remarkably, that this activation does not lead to NF-κB-induced (proinflammatory) gene expression. Here, we report that PRV-mediated inhibition of host gene expression stretches beyond NF-κB-dependent gene expression and in fact reflects a broad inhibition of host gene transcription, which correlates with a substantial recruitment of essential host transcription factors in viral replication compartments in the nucleus, away from the host chromatin. These data uncover a potent alphaherpesvirus mechanism to interfere with production of host proteins, including proteins involved in antiviral responses.
Topics: Animals; Herpesvirus 1, Suid; Host Microbial Interactions; NF-kappa B; Pseudorabies; Swine; Swine Diseases; Transcription, Genetic
PubMed: 35730976
DOI: 10.1128/jvi.00714-22 -
Molecules (Basel, Switzerland) Sep 2022The chemical reactivity of 3-[()-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1)-quinolinone () towards some phosphorus reagents was studied. The enaminone was...
The chemical reactivity of 3-[()-3-(dimethylamino)-2-propenoyl]-4-hydroxy-1-methy-2(1)-quinolinone () towards some phosphorus reagents was studied. The enaminone was cyclized into pyranoquinolinylphosphonate via treatment with diethyl phosphite in basic medium. However, its reaction with triethoxy phosphonoacetate gave the substituted oxopyranylphosphonate . Using the same reaction conditions, both thioxopyridinylphosphonate and oxopyranylphosphonate were produced via a reaction of enaminone with both diethyl 2-amino-2-thioxoethylphosphonate and diethyl vinylphosphonate, respectively, in low yields. In addition, the two novel oxopyridinylphosphonates and were obtained by treatment of enaminone with a diethyl cyanomethylphosphonate reagent. Two oaxathiaphosphininyl derivatives, and were obtained by treatment of the enaminone with -diethyl dithiophosphoric acid under different reaction conditions. Diazaphosphininyl and oxazaphosphininyl derivatives were obtained in excellent yields using a -phenylphosphonic diamide reagent under different reaction conditions. The treatment of the enaminone with phosphorus pentasulfide produced the non-phosphorylated product thioxothiopyranoquinolinone . Finally, the enaminone was turned into oxathiaphosphininyl using Lawesson's reagent. The possible reaction mechanisms of the formation of these products were discussed. The structures of newly isolated products were established by elemental analysis and spectral tools. The compounds were evaluated for their antioxidant activities.
Topics: Antioxidants; Diamide; Indicators and Reagents; Organophosphonates; Phosphites; Phosphonoacetic Acid; Phosphorus; Quinolones
PubMed: 36144697
DOI: 10.3390/molecules27185960