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Scientific Reports Jun 2021Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and...
Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-β expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus-host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.
Topics: Aedes; Animals; Antiviral Agents; Chikungunya Fever; Chikungunya virus; Chlorocebus aethiops; Culicidae; Humans; Mosquito Vectors; RNA, Viral; RNA-Dependent RNA Polymerase; Vero Cells; Viral Nonstructural Proteins; Virus Replication
PubMed: 34112897
DOI: 10.1038/s41598-021-91830-y -
Journal of the National Cancer Institute Mar 1986In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the...
In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the intracellular pool of UTP and eliciting an increased rate of de novo uridylate synthesis. This rise in de novo synthesis was completely suppressed by addition of 6-azauridine (6-AzaUrd) to the cell suspension in vitro or in the solid TA3 mammary tumor in NMRI mice in vivo. A synergistic depletion of UTP pools to less than 6% of the UTP in controls was observed in TA3 cell suspensions exposed to GlcN and 6-AzaUrd. In solid TA3 tumors in vivo, UTP was reduced by this combination to 19% of the control value. A high sensitivity of the solid tumor to inhibition of pyrimidine synthesis de novo was indicated by the reduction of the UTP content after administration of 6-AzaUrd alone. UTP deficiency in TA3 tumor cells was accompanied by CTP deficiency. In addition, 6-AzaUrd caused a lowering of GTP in the neoplastic tissue. Host liver was resistant to 6-AzaUrd but responded to treatment with GlcN with a decrease in UTP to 67%. Uridine-cytidine kinase was less inhibited in the presence of lowered UTP and CTP, which are potent feedback inhibitors of the enzyme, and enabled an enhanced formation of phosphorylated derivatives of 5-fluorouridine (FUrd). Aside from the formation of 5-fluoro-UTP, we have identified 5-fluoro-UDP-N-acetylhexosamines (FUDPHexNAc), which accumulated when FUrd and GlcN were sequentially administered. Treatment of TA3 cells with FUrd after a pretreatment with 6-AzaUrd and GlcN resulted in a 2.5-fold increase in [14C]FUrd uptake and a duplication of 5-fluorouridylate incorporation into the RNA. The proportion of FUDPHexNAc increased to 58% of the phosphorylated FUrd metabolites, as compared to 6% in TA3 cells exposed exclusively to FUrd. In vivo chemotherapy of mice bearing TA3 ascites tumors was most effective with respect to tumor growth inhibition and animal survival when GlcN and FUrd were combined.
Topics: Adenosine Triphosphate; Animals; Antineoplastic Combined Chemotherapy Protocols; Azauridine; Cytidine Triphosphate; Female; Glucosamine; Guanosine Triphosphate; Mammary Neoplasms, Experimental; Mice; Nucleotides; RNA, Neoplasm; Uridine; Uridine Triphosphate
PubMed: 2419623
DOI: No ID Found -
Nature Mar 1961
Topics: Azauridine; Fertility; Nucleosides; Nucleotides; Research
PubMed: 13746146
DOI: 10.1038/1891015a0 -
Nucleic Acids Research May 1976Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated...
Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).
Topics: Azauridine; Magnetic Resonance Spectroscopy; Methods; Optical Rotation; Uridine
PubMed: 940772
DOI: 10.1093/nar/3.5.1387 -
Murine sarcoma virus-induced cytopathological effects in mouse cells made resistant to 6-azauridine.The Journal of General Virology May 1974
Topics: Animals; Autoimmune Diseases; Azauridine; Cell Fusion; Cell Line; Cytopathogenic Effect, Viral; Fibroblasts; Friend murine leukemia virus; Gammaretrovirus; Inclusion Bodies, Viral; Mice; Mice, Inbred NZB; Mice, Inbred Strains; Moloney murine leukemia virus; Rauscher Virus; Retroviridae; Virus Replication
PubMed: 4364879
DOI: 10.1099/0022-1317-23-2-185 -
Folia Biologica Jun 1970
Topics: Animals; Female; Graft vs Host Reaction; Immunosuppressive Agents; Liver; Male; Mice; Skin Transplantation; Spleen; Testis; Thymus Gland; Transplantation Immunology; Triazines
PubMed: 4393498
DOI: No ID Found -
American Journal of Hematology Mar 19855-Azacytidine (azaC) has previously been shown to raise Hb F levels in the repeatedly phlebotomized baboon (PCV: around 20%). The administration of tetrahydrouridine...
5-Azacytidine (azaC) has previously been shown to raise Hb F levels in the repeatedly phlebotomized baboon (PCV: around 20%). The administration of tetrahydrouridine (THU), an inhibitor of the enzymatic conversion of azaC to 5-azauridine, made it possible to reduce the amount of azaC and also of 2-deoxy-5-azacytidine (d-azaC) by more than 90% and still achieve maximal Hb F elevations. However, the granulocytopenia, usually occurring after 5-azaC, was not altered by the lowering of the dosages in the presence of THU. Thus, the granulocytopenia is not due to 5-azauridine or other catabolic products resulting from deamination. It is also unlikely that it is caused by a direct influence of azaC on RNA since d-azaC also causes granulocytopenia. The persistence of reticulocytosis throughout the treatment with azaC or d-azaC makes it appear likely that the observed increase in Hb F levels to more than 60% of total hemoglobin is not due to a cytotoxic effect on erythropoiesis resulting in a shift of cell populations toward greater immaturity, but to a direct influence of the drug on the regulation of gamma globin chain production.
Topics: Agranulocytosis; Animals; Azacitidine; Cytidine Deaminase; Decitabine; Fetal Hemoglobin; Gene Expression Regulation; Globins; Granulocytes; Papio; Tetrahydrouridine; Uridine
PubMed: 2579548
DOI: 10.1002/ajh.2830180310 -
Clinica Chimica Acta; International... Aug 1969
Topics: Alanine; Amino Acid Metabolism, Inborn Errors; Amino Acids; Autoanalysis; Cellulose; Chromatography; Chromatography, Thin Layer; Homocystinuria; Humans; Male; Psoriasis; Triazines
PubMed: 5801395
DOI: 10.1016/0009-8981(69)90268-x -
Pharmacology & Therapeutics 1980
Review
Topics: Animals; Antiviral Agents; Aza Compounds; Azacitidine; Azauridine; Chemical Phenomena; Chemistry; Disease Models, Animal; Embryo, Mammalian; Humans; Immunosuppressive Agents; Kinetics; Pyrimidine Nucleosides; Structure-Activity Relationship; Virus Replication
PubMed: 6157166
DOI: 10.1016/s0163-7258(80)80018-0 -
Infection and Immunity May 1975The replication of mumps virus in susceptible chicken embryonic heart cells was escalated by daily treatment of cultures with 6-azauridine (6-AU). On the other hand,...
The replication of mumps virus in susceptible chicken embryonic heart cells was escalated by daily treatment of cultures with 6-azauridine (6-AU). On the other hand, virus production by less susceptible liver cells was depressed by 6-AU. The population of infected susceptible cells was not increased, but the release of virus by infected susceptible cells was enhanced 20-fold by 10 mug of 6-AU per ml. Less susceptible cells, which incorporated less ribonucleic acid and protein precursor than susceptible cells, sustained a constant level of viral release during 6-AU treatment; however, the number of infected less susceptible cells underwent substantial decline.
Topics: Animals; Azauridine; Carbon Radioisotopes; Chick Embryo; Culture Techniques; Liver; Mumps virus; Myocardium; Paramyxoviridae; RNA, Viral; Tritium; Trypsin; Uridine; Viral Proteins; Virus Cultivation; Virus Replication
PubMed: 1123255
DOI: 10.1128/iai.11.5.915-918.1975