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Gut Jan 2020Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk...
OBJECTIVE
Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent.
DESIGN
Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection.
RESULTS
Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets.
CONCLUSION
The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.
Topics: Antiviral Agents; Aspartate Carbamoyltransferase; Aspartic Acid; Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); Cell Line; Dihydroorotase; Estrogen Receptor Antagonists; Estrogen Receptor alpha; Fulvestrant; Gene Silencing; Hepatitis D, Chronic; Hepatitis Delta Virus; Hepatocytes; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin Resistance; Life Cycle Stages; Loss of Function Mutation; Phosphonoacetic Acid; Pyrimidines; RNA Interference; RNA, Small Interfering; RNA, Viral; Signal Transduction; Virus Replication
PubMed: 30833451
DOI: 10.1136/gutjnl-2018-317065 -
Transplantation and Cellular Therapy Jul 2021Infections due to herpesviruses resistant to first-line antivirals remains an ever-present and serious complication in recipients of hematopoietic cell transplantation... (Observational Study)
Observational Study
Infections due to herpesviruses resistant to first-line antivirals remains an ever-present and serious complication in recipients of hematopoietic cell transplantation (HCT) and other cellular therapies. Foscarnet is the most common therapy for patients who have resistant herpesvirus infections or intolerable cytopenias due to ganciclovir or valganciclovir; however, the widespread use of foscarnet is limited by its associated nephrotoxicity and challenges in administration. In the earliest published small case series investigating the optimal infusion modality, patients with acquired immunodeficiency syndrome (AIDS) due to the human immunodeficiency virus (HIV) received either continuous infusion or intermittent dosing of foscarnet. Moreover, there was no standardization of hydration strategies to minimize side effects. Eventually, intermittent foscarnet infusions became the standard of care; however, the true impact of hydration and infusion duration on nephrotoxicity has not been adequately studied, and the reports of foscarnet administration in HCT patients has been limited primarily to intermittent infusions. In this report, we characterize the administration of foscarnet as a 24-hour continuous infusion in both the inpatient and outpatient settings compared with intermittent infusion in HCT recipients. This retrospective, single-center, observational study at Stanford University Medical Center assessed HCT recipients who received foscarnet between January 2009 and May 2019. Twenty-eight of 45 patients (62.2%) who received continuous-infusion foscarnet experienced an acute kidney injury (AKI) as defined by the Kidney Disease Improving Global Outcomes classification, compared with 39 of 62 patients (62.9%) who received conventional infusion (P = .94). The average duration of outpatient antiviral days for the continuous infusion group was 9 days (range, 0 to 121 days), compared with 6.3 days (range, 0 to 70 days) in the intermittent infusion group (P = .54). Our findings suggest that foscarnet given as a continuous infusion or as an intermittent infusion have similar rates of adverse reactions, most notably similar rates of AKI. Administering foscarnet as a continuous infusion is a feasible option to facilitate outpatient treatment.
Topics: Cytomegalovirus Infections; Foscarnet; Ganciclovir; Hematopoietic Stem Cell Transplantation; Humans; Retrospective Studies
PubMed: 33891883
DOI: 10.1016/j.jtct.2021.03.018 -
RSC Advances 2020Adenosine 5'-diphosphate ribose (ADPR) is an intracellular signalling molecule generated from nicotinamide adenine dinucleotide (NAD). Synthetic ADPR analogues can shed...
Adenosine 5'-diphosphate ribose (ADPR) is an intracellular signalling molecule generated from nicotinamide adenine dinucleotide (NAD). Synthetic ADPR analogues can shed light on the mechanism of activation of ADPR targets and their downstream effects. Such chemical biology studies, however, are often challenging due to the negatively charged pyrophosphate, also sensitive to cellular pyrophosphatases, and prior work on an initial ADPR target, the transient receptor potential cation channel TRPM2, showed complete pyrophosphate group replacement to be a step too far in maintaining biological activity. Thus, we designed ADPR analogues with just one of the negatively charged phosphate groups removed, by employing a phosphonoacetate linker. Synthesis of two novel phosphonoacetate ADPR analogues is described tandem -dicyclohexylcarbodiimide coupling to phosphonoacetic acid. Neither analogue, however, showed significant agonist or antagonist activity towards TRPM2, underlining the importance of a complete pyrophosphate motif in activation of this particular receptor.
PubMed: 31934327
DOI: 10.1039/C9RA09284F -
Scientific Reports Jul 2018Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of...
Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T ↔ R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.
Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Aspartate Carbamoyltransferase; Aspartic Acid; Catalytic Domain; Cytidine Triphosphate; Enzyme Stability; Models, Molecular; Phosphonoacetic Acid; Protein Multimerization; Protein Subunits; Substrate Specificity
PubMed: 30038211
DOI: 10.1038/s41598-018-27992-z -
Virology Journal Nov 2017Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It's during the latent phase of EBV that all...
BACKGROUND
Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It's during the latent phase of EBV that all EBV-associated cancers, including Burkitt's lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease arise. Interferon-γ-inducible protein 16 (IFI16) is a well-established innate immune sensor and viral transcriptional regulator involved in response to invading DNA viruses. During latency, IFI16 remains in the nucleus, in part bound to the EBV genome; however, neither its role in EBV lytic cycle or latency has been established.
METHODS
Short interfering RNA against IFI16 and IFI16 overexpression were used to identify the role of IFI16 in the maintenance of EBV latency I. We also studied how induction of the lytic cycle affected IFI16 using the EBV positive, latently infected Akata or MUTU-1 cell lines. Akata cells were induced with TPA and MUTU-1 cells with TGF-β up to 96 h and changes in IFI16 protein were analyzed by Western blotting and immunofluorescence microscopy. To assess the mechanism of IFI16 decrease, EBV DNA replication and late lytic transcripts were blocked using the viral DNA polymerase inhibitor phosphonoacetic acid.
RESULTS
Knockdown of IFI16 mRNA by siRNA resulted in enhanced levels of EBV lytic gene expression from all temporal gene classes, as well as an increase in the total EBV genome abundance, whereas overexpression of exogenous IFI16 reversed these effects. Furthermore, 96 h after induction of the lytic cycle with either TPA (Akata) or TGF-β (MUTU-1), IFI16 protein levels decreased up to 80% as compared to the EBV-negative cell line BJAB. Reduction in IFI16 was observed in cells expressing EBV lytic envelope glycoprotein. The decreased levels of IFI16 protein do not appear to be dependent on late lytic transcripts of EBV but suggest involvement of the immediate early, early, or a combination of both gene classes.
CONCLUSIONS
Reduction of IFI16 protein levels following lytic cycle induction, as well as reactivation from latency after IFI16 mRNA knockdown suggests that IFI16 is crucial for the maintenance of EBV latency. More importantly, these results identify IFI16 as a unique host factor protein involved in the EBV lifecycle, making it a potential therapeutic target to combat EBV-related malignancies.
Topics: Burkitt Lymphoma; Cell Line, Tumor; Epstein-Barr Virus Infections; Gene Expression Regulation; Gene Knockdown Techniques; Genome, Viral; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Nuclear Proteins; Phosphonoacetic Acid; Phosphoproteins; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta; Viral Proteins; Virus Activation; Virus Latency
PubMed: 29132393
DOI: 10.1186/s12985-017-0891-5 -
Proceedings of the National Academy of... Aug 2017The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics,...
The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B) and pyridoxal (vitamin B). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. () The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. () Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. () The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.
Topics: Glyceraldehyde 3-Phosphate; Mass Spectrometry; Models, Molecular; Pentosephosphates; Phosphonoacetic Acid; Protein Binding; Protein Conformation; Transferases
PubMed: 28808005
DOI: 10.1073/pnas.1619981114 -
Frontiers in Microbiology 2022The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During...
The appearance of drug-resistant mutations in UL54 DNA polymerase and UL97 kinase genes is problematic for the treatment of human cytomegalovirus (HCMV) diseases. During treatment of HCMV infection in a pediatric hematopoietic cell transplant recipient, H600L and T700A mutations and E576G mutation were independently found in the UL54 gene. Foscarnet (FOS; phosphonoformic acid) resistance by T700A mutation is reported. Here, we investigated the role of novel mutations in drug resistance by producing recombinant viruses and a model polymerase structure. The H600L mutant virus showed an increase in resistance to ganciclovir (GCV) by 11-fold and to FOS and cidofovir (CDV) by 5-fold, compared to the wild type, while the E756G mutant virus showed an increase in resistance to FOS by 9-fold and modestly to CDV by 2-fold. With the FOS-resistant T700A mutation, only H600L produced increased FOS resistance up to 37-fold, indicating an additive effect of these mutations on FOS resistance. To gain insight into drug resistance mechanisms, a model structure for UL54 polymerase was constructed using the yeast DNA polymerase as a template. In this model, HCMV DNA polymerase contains a long palm loop domain of which H600 and T700 are located on each end and T700 interacts with the FOS binding pocket. Our results demonstrate that H600L and E756G mutations in UL54 polymerase are novel drug-resistant mutations and that the acquisition of both H600L and T700A mutations in the DNA-binding loop confers increased resistance to FOS treatment, providing novel insights for the mechanism acquiring foscarnet resistance.
PubMed: 35185843
DOI: 10.3389/fmicb.2022.771978 -
Antiviral Research Feb 2017Human cytomegalovirus UL54 DNA polymerase gene mutations that confer foscarnet resistance in clinical practice typically cluster in the amino terminal 2, palm and finger...
Human cytomegalovirus UL54 DNA polymerase gene mutations that confer foscarnet resistance in clinical practice typically cluster in the amino terminal 2, palm and finger domains. Exposure to foscarnet in cell culture selected for mutations elsewhere in UL54, including amino acid substitutions S290R in the amino terminal 1 domain and E951D in the palm 2 domain. These are newly confirmed to confer foscarnet resistance and slightly decreased ganciclovir susceptibility. Other emergent substitutions N495K, T552N and T838A are known to confer foscarnet resistance, while additional ones Q783R and V798A only slightly affected susceptibility. An expanded set of domains is involved in foscarnet resistance and its genotypic diagnosis.
Topics: Amino Acid Substitution; Antiviral Agents; Cytomegalovirus; Cytomegalovirus Infections; DNA, Viral; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Ganciclovir; Genotype; Humans; Mutation; Phenotype; Viral Proteins
PubMed: 27940027
DOI: 10.1016/j.antiviral.2016.12.003 -
Bone Marrow Transplantation Dec 2018We studied 97 patients who developed cytomegalovirus (CMV) viremia following an allogeneic hemopoietic stem cell transplant (HSCT) between 2010 and 2015, treated with...
We studied 97 patients who developed cytomegalovirus (CMV) viremia following an allogeneic hemopoietic stem cell transplant (HSCT) between 2010 and 2015, treated with foscarnet, with the aim of assessing efficacy and safety. The donor was unrelated in 30 patients (UD) and a family HLA-haploidentical donor (HAPLO) in 67 patients: the former (UD) received a prophylaxis for graft-versus-host disease (GvHD), based on antithymocyte globulin (ATG); the latter (HAPLO) received GvHD prophylaxis, based on post-transplant cyclophosphamide (PT-CY). Renal and hematological toxicity were defined according to NCI-CTCAE4 criteria. In univariate analysis, CMV response was 84% in HAPLO vs 59% in UD grafts (p = 0.01) and 90 vs 66% (p = 0.02) for patients with a CMV viral load within or over the median value. In multivariate analysis, the CMV viral load was the strongest predictor of response to foscarnet (p = 0.02), followed by donor type (p = 0.06). Renal impairment developed in 14% of the patients. Overall survival was 69%:, advanced phase at transplant (p = 0.01) and ATG-based regimens (p = 0.02), were the only two predicting factor. In conclusion, CMV response to foscarnet treatment is predicted by a lower CMV load and GvHD prophylaxis. Renal toxicity of foscarnet is not a limiting factor.
Topics: Adult; Antiviral Agents; Cytomegalovirus; Female; Foscarnet; Humans; Male; Middle Aged; Unrelated Donors; Young Adult
PubMed: 29795416
DOI: 10.1038/s41409-018-0200-y -
Yakugaku Zasshi : Journal of the... 2024Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for...
Quantitative NMR (qNMR), particularly H-qNMR, is useful for determining the absolute purity of organic molecules. However, identifying the target signal(s) for quantification is difficult, because of the overlap and complexity of organic molecules. Therefore, we focused on the P nucleus, owing to the simplicity of its signals, and investigated the P-qNMR absolute determination method by using organophosphorus drugs, water-soluble cyclophosphamide hydrate (CP), and water-insoluble sofosbuvir (SOF). The optimized and reproducible P-qNMR conditions, such as qNMR sample preparation [i.e., selecting suitable deuterated solvents and a reference standard (RS) for P-qNMR], hygroscopicity and solution stability of the analyte and RS, and qNMR measurements-such as acquisition time, relaxation delay time, and spectral width-were examined. The CP purities determined using P-qNMR agreed well with those for the established H-qNMR method in DO. In contrast, the SOF purity determined using P-qNMR was 1.6% higher than that for H-qNMR in the protic solvent CDOD. Therefore, using a protic solvent, such as CDOD, was not suitable for P-qNMR; the deuterium exchange with the RS for P-qNMR (i.e., phosphonoacetic acid) resulted in a small integrated intensity. Consequently, the aprotic solvent DMSO-d was employed to determine the SOF purity. The data revealed that the SOF purities determined using P-qNMR agreed well with the established H-qNMR values, indicating that the absolute quantification of SOF using both P-qNMR and H-qNMR is possible in DMSO-d. Thus, we established an optimized and reproducible P-qNMR method in validation study across multiple laboratories.
Topics: Organophosphorus Compounds; Dimethyl Sulfoxide; Water; Solvents; Pharmaceutical Preparations
PubMed: 38556308
DOI: 10.1248/yakushi.23-00151-3