scholarly journals Varicella-Zoster Virus ORF47 Protein Serine Kinase: Characterization of a Cloned, Biologically Active Phosphotransferase and Two Viral Substrates, ORF62 and ORF63

2001 ◽  
Vol 75 (18) ◽  
pp. 8854-8858 ◽  
Author(s):  
T. K. Kenyon ◽  
J. Lynch ◽  
J. Hay ◽  
W. Ruyechan ◽  
C. Grose
Keyword(s):  
Varicella Zoster Virus ◽  
Kinase Activity ◽  
Biologically Active ◽  
Regulatory Proteins ◽  
Kinase Assay ◽  
Serine Kinase ◽  
Varicella Zoster ◽  
Simplex Virus

ABSTRACT Varicella-zoster virus (VZV) codes for a protein serine kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus serine kinase has been biologically active in vitro. We discovered that preservation of the intrinsic kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory proteins: the ORF62 protein (homolog of HSV ICP4) and the ORF63 protein (homolog of HSV ICP22). Of interest, ORF47 kinase also coprecipitated ORF63 protein from the kinase assay supernatant.

scholarly journals Rapid Phenotypic Characterization Method for Herpes Simplex Virus and Varicella-Zoster Virus Thymidine Kinases To Screen for Acyclovir-Resistant Viral Infection

2000 ◽  
Vol 38 (5) ◽  
pp. 1839-1844 ◽  
Author(s):  
Tatsuo Suzutani ◽  
Masayuki Saijo ◽  
Masayoshi Nagamine ◽  
Masahiro Ogasawara ◽  
Masanobu Azuma
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Virus Isolation ◽  
Screening Method ◽  
Phenotypic Characterization ◽  
T7 Promoter ◽  
Varicella Zoster ◽  
Simplex Virus

A rapid phenotypic screening method for herpes simplex virus (HSV) and varicella-zoster virus (VZV) thymidine kinase (TK) genes was developed for monitoring acyclovir-resistant viruses. This method determines the biochemical phenotype of the TK polypeptide, which is synthesized in vitro from viral DNA using a procedure as follows. The TK gene of each sample virus strain is amplified and isolated under the control of a T7 promoter by PCR. The PCR products are transcribed with T7 RNA polymerase and translated in a rabbit reticulocyte lysate. Using this method, enzymatic characteristics and the size of the TK polypeptides encoding HSV and VZV DNA were defined in less than 2 days without virus isolation. The assay should be a powerful tool in monitoring drug-resistant viruses, especially in cases in which virus isolation is difficult.

scholarly journals Histone Deacetylases 1 and 2 Are Phosphorylated at Novel Sites during Varicella-Zoster Virus Infection

2009 ◽  
Vol 83 (22) ◽  
pp. 11502-11513 ◽  
Author(s):  
Matthew S. Walters ◽  
Angela Erazo ◽  
Paul R. Kinchington ◽  
Saul Silverstein
Keyword(s):  
Varicella Zoster Virus ◽  
Histone Deacetylases ◽  
Kinase Domain ◽  
Varicella Zoster Virus Infection ◽  
Varicella Zoster ◽  
Histone Deacetylase 1 ◽  
C Terminus ◽  
Simplex Virus ◽  
In Cells

ABSTRACT ORF66p, a virion-associated varicella-zoster virus (VZV) protein, is a member of a conserved A lphaherpesvirinae kinase family with homology to herpes simplex virus US3 kinase. Expression of ORF66p in cells infected with VZV or an adenovirus expressing only ORF66p results in hyperphosphorylation of histone deacetylase 1 (HDAC1) and HDAC2. Mapping studies reveal that phosphorylation is at a unique conserved Ser residue in the C terminus of both HDACs. This modification requires an active kinase domain in ORF66p, as neither protein is phosphorylated in cells infected with VZV lacking kinase activity. However, hyperphosphorylation appears to occur indirectly, as within the context of in vitro kinase reactions, purified ORF66p phosphorylates a peptide derived from ORF62p, a known substrate, but does not phosphorylate HDAC. These results support a model where ORF66p is necessary but not sufficient to effect hyperphosphorylation of HDAC1 and HDAC2.

scholarly journals Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase

1994 ◽  
Vol 304 (1) ◽  
pp. 17-21 ◽  
Author(s):  
J F Tanti ◽  
T Grémeaux ◽  
E Van Obberghen ◽  
Y Le Marchand-Brustel
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
Kinase Assay ◽  
Insulin Receptor Substrate ◽  
Phosphatidylinositol 3 Kinase ◽  
Serine Kinase ◽  
Phosphatidylinositol 3

Insulin receptor substrate (IRS) 1, which is tyrosine phosphorylated in response to insulin, presents multiple serine/threonine phosphorylation sites. To search for a serine kinase activity towards IRS 1, immunoprecipitates from basal or stimulated 3T3-L1 adipocytes were used in an in vitro kinase assay. When IRS 1 was isolated from insulin-treated cells, serine phosphorylation of IRS 1 occurred, which we attribute to the kinase activity of the phosphatidylinositol 3-kinase (PI3-kinase). Importantly, in an in vitro reconstitution assay, an excess of the PI3-kinase subunit prevents this phosphorylation. Together, our results suggest that following insulin stimulation, PI3-kinase associates with IRS 1, allowing for its serine phosphorylation. This phosphorylation event could play a role in the modulation of insulin signalling.

Synthesis, Anti-Varicella-Zoster Virus and Anti-Cytomegalovirus Activity of 4,5-Disubstituted 1,2,3-(1H)-Triazoles

2019 ◽  
Vol 15 (7) ◽  
pp. 801-812 ◽  
Author(s):  
Wei-yuan Yuan ◽  
Xue Chen ◽  
Ning-ning Liu ◽  
Yi-ning Wen ◽  
Bei Yang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Varicella Zoster Virus ◽  
Mechanisms Of Action ◽  
High Toxicity ◽  
Varicella Zoster ◽  
Clinical Drugs ◽  
Simplex Virus

Background: Clinical drugs for herpesvirus exhibit high toxicity and suffer from significant drug resistance. The development of new, effective, and safe anti-herpesvirus agents with different mechanisms of action is greatly required. Objective: Novel inhibitors against herpesvirus with different mechanisms of action from that of clinical drugs. Methods: A series of novel 5-(benzylamino)-1H-1,2,3-triazole-4-carboxamides were efficiently synthesized and EC50 values against Human Cytomegalovirus (HCMV), Varicella-Zoster Virus (VZV) and Herpes Simplex Virus (HSV) were evaluated in vitro. Results: Some compounds present antiviral activity. Compounds 5s and 5t are potent against both HCMV and VZV. Compounds 5m, 5n, 5s, and 5t show similar EC50 values against both TK+ and TK− VZV strains. Conclusion: 5-(Benzylamino)-1H-1, 2,3-triazole-4-carboxamides are active against herpesviruses and their activity is remarkably affected by the nature and the position of substituents in the benzene ring. The results indicate that these derivatives are independent of the viral thymidine kinase (TK) for activation, which is indispensable for current drugs. Their mechanisms of action may differ from those of the clinic anti-herpesvirus drugs.

scholarly journals Selection and Characterization of Varicella-Zoster Virus Variants Resistant to (R)-9-[4-Hydroxy-2-(Hydroxymethy)Butyl]Guanine

2001 ◽  
Vol 45 (6) ◽  
pp. 1629-1636 ◽  
Author(s):  
Teresa I. Ng ◽  
Yan Shi ◽  
H. Janette Huffaker ◽  
Warren Kati ◽  
Yaya Liu ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Dna Polymerases ◽  
Resistant Mutant ◽  
Competitive Inhibitor ◽  
Varicella Zoster ◽  
Antiviral Activities ◽  
Predominant Variant ◽  
Infected Cells

ABSTRACT (R)-9-[4-Hydroxy-2-(hydroxymethy)butyl]guanine (H2G) is a potent and selective inhibitor of herpesvirus replication. It is a nucleoside analog, and its triphosphate derivative (H2G-TP) is a competitive inhibitor of herpesvirus DNA polymerases. In this study, the antiviral activities of H2G and acyclovir (ACV) and the development of viral resistance to these agents were compared in varicella-zoster virus (VZV)-infected cells. In plaque reduction assays, the 50% effective concentration of H2G for VZV was 60- to 400-fold lower than that of ACV, depending on the virus strain and the cell line tested. The enhanced efficacy of H2G against VZV can be accounted for in part by the fact that the intaracellular H2G-TP level (>170 pmol/106 cells) is higher than the intracellular ACV-TP level (<1 pmol/106 cells). In addition, H2G-TP has extended half-lives of 3.9 and 8.6 h in VZV-infected MRC-5 and MeWo cells, respectively. To assess the emergence of H2G-resistant VZV in vitro, VZV was passaged in the presence of increasing concentrations of H2G. Earlier in the passage, when the concentration of H2G was relatively low, the predominant variant had the (A)76 deletion in the viral thymidine kinase (TK) gene. This mutant was identical to an ACV-resistant mutant generated in parallel experiments. However, higher concentrations of H2G appeared to favor a novel mutant, which had deletions of two consecutive nucleotides at positions 805 and 806 of the TK gene. All of these changes introduced frameshift mutations in the TK gene resulting in the expression of truncated polypeptides. H2G-resistant viruses were cross-resistant to ACV, and vice versa.

scholarly journals Differentiation of Varicella-Zoster Virus ORF47 Protein Kinase and IE62 Protein Binding Domains and Their Contributions to Replication in Human Skin Xenografts in the SCID-hu Mouse

2003 ◽  
Vol 77 (10) ◽  
pp. 5964-5974 ◽  
Author(s):  
Jaya Besser ◽  
Marvin H. Sommer ◽  
Leigh Zerboni ◽  
Christoph P. Bagowski ◽  
Hideki Ito ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Varicella Zoster Virus ◽  
Human Skin ◽  
Kinase Activity ◽  
Cutaneous Lesion ◽  
Protein Kinase Activity ◽  
Varicella Zoster ◽  
Human Skin Xenografts

ABSTRACT To investigate the role of the ORF47 protein kinase of varicella-zoster virus (VZV), we constructed VZV recombinants with targeted mutations in conserved motifs of ORF47 and a truncated ORF47 and characterized these mutants for replication, phosphorylation, and protein-protein interactions in vitro and for infectivity in human skin xenografts in the SCID-hu mouse model in vivo. Previous experiments showed that ROka47S, a null mutant that makes no ORF47 protein, did not replicate in skin in vivo (J. F. Moffat, L. Zerboni, M. H. Sommer, T. C. Heineman, J. I. Cohen, H. Kaneshima, and A. M. Arvin, Proc. Natl. Acad. Sci. USA 95:11969-11974, 1998). The construction of VZV recombinants with targeted ORF47 mutations made it possible to assess the effects on VZV infection of human skin xenografts of selectively abolishing ORF47 protein kinase activity. ORF47 mutations that resulted in a C-terminal truncation or disrupted the DYS kinase motif eliminated ORF47 kinase activity and were associated with extensive nuclear retention of ORF47 and IE62 proteins in vitro. Disrupting ORF47 kinase function also resulted in a marked decrease in VZV replication and cutaneous lesion formation in skin xenografts in vivo. However, infectivity in vivo was not blocked completely as long as the capacity of ORF47 protein to bind IE62 protein was preserved, a function that we identified and mapped to the N-terminal domain of ORF47 protein. These experiments indicate that ORF47 kinase activity is of critical importance for VZV infection and cell-cell spread in human skin in vivo but suggest that it is the formation of complexes between ORF47 and IE62 proteins, both VZV tegument components, that constitutes the essential contribution of ORF47 protein to VZV replication in vivo.

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