Analysis of phosphorylation pathways of antiherpesvirus nucleosides by varicella-zoster virus-specific enzymes (original) (raw)
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Antimicrobial Agents and Chemotherapy, 1992
The metabolism and mode of action of penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine; BRL 39123] were studied and compared with those of acyclovir. In uninfected MRC-5 cells, low concentrations of the triphosphates of penciclovir and acyclovir were occasionally just detectable, the limit of detection being about 1 pmol/10(6) cells. In contrast, in cells infected with either herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV), penciclovir was phosphorylated quickly to give high concentrations of the triphosphate ester. Following the removal of penciclovir from the culture medium, penciclovir-triphosphate remained trapped within the cells for a long time (half-lives, 20 and 7 h in HSV-2- and VZV-infected cells, respectively). In HSV-2-infected cells, acyclovir was phosphorylated to a lesser extent and the half-life of the triphosphate ester was only 1 h. We were unable to detect any phosphates of acyclovir in VZV-infected cells. (S)-Penciclovir-triphosphate i...
Biochemical Pharmacology, 1985
The antiherpes agent 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) is a much more potent inhibitor of herpes simplex viruses in vivo than acyclovir, yet both are equally active in vitro against these viruses. To explain this difference, studies were conducted to compare the intracellular metabolism and enzymatic phosphorylation of the two compounds. In herpes type 1 and type 2 infected cells, the levels of DHPG triphosphate were only about 2-fold greater than levels of acyclovir triphosphate at virus-inhibitory concentrations (~< 1/~M). At concentrations > 2.5/~M in herpes type 1 but not in type 2 infected cells, acyclovir phosphorylation was inhibited relative to that of DHPG. When drug was removed after 6 hr from infected cells, acyclovir triphosphate rapidly degraded to acyclovir and was excreted into the culture medium. In contrast, DHPG triphosphate persisted at 60-70% of the original level for 18 hr after drug removal, and DHPG excretion from cells was very slow. This finding could be a key factor to the superior potency of DHPG in animals, despite the fact that blood levels of both compounds fall rapidly after dosing. In uninfected cells, low levels of DHPG and acyclovir triphosphates were produced at 100/.tM concentrations. Phosphorylation of DHPG to mono-, di-and triphosphates by purified viral and cell enzymes was more rapid than that of acyclovir. However, acyclovir triphosphate was a much more potent inhibitor of herpes virus and cell DNA polymerases.
Role of the Human Herpesvirus 6 U69-Encoded Kinase in the Phosphorylation of Ganciclovir
Molecular Pharmacology, 2002
The human herpesvirus 6 (HHV-6) U69 gene product (pU69) is the presumed functional homolog of the human cytomegalovirus (HCMV) UL97-encoded kinase (pUL97), which converts ganciclovir to its monophosphate metabolite in HCMV-infected cells. It has been reported that insertion of U69 into baculovirus confers sensitivity to ganciclovir in insect cells (J Virol 73:3284-3291, 1999). Our metabolic studies in HHV-6-infected human T-lymphoblast cells indicated that the efficiency of ganciclovir phosphorylation induced by HHV-6 was relatively poor. Recombinant vaccinia viruses (rVVs), expressing high levels of pU69 from two HHV-6 strains (representing the A and B vari-This work was supported by a grant from the Wetenschappelijk Onderzoek Multiple Sclerose vzw. L.D.B. is a Research Assistant of the Fonds voor Wetenschappelijk Onderzoek Vlaanderen. ABBREVIATIONS: HHV-6, human herpesvirus 6; pU69, human herpesvirus 6 U69 gene product; HCMV, human cytomegalovirus; pUL97, human cytomegalovirus UL97-encoded kinase; rVV, recombinant vaccinia virus; CBLC, cord blood lymphocyte; EGFP, enhanced green fluorescent protein; CDV, cidofovir; GCV, ganciclovir; FCS, fetal calf serum; PCR, polymerase chain reaction; RT, reverse transcriptase; VV, vaccinia virus; CC 50 , compound concentration that causes 50% inhibition of cell growth as determined by cell counting; IC 50 , the compound concentration that produced 50% inhibition of viral DNA replication; PBS, phosphate-buffered saline; HPLC, high-performance liquid chromatography; aa, amino acid; MCMV, murine cytomegalovirus; wtVV, wild-type vaccinia virus.
Journal of Virological Methods, 1992
Varicella zoster virus (VZV) is responsible for a primary infection (varicella) and, upon reactivation, zoster, which in immunocompromised patients, may both lead to life-threatening disseminated disease. There is a great need for antiviral compounds that are effective inhibitors of VZV replication and for rapid and accurate methods for evaluating viral sensitivity to candidate anti-VZV drugs. With the monoclonal antibody (mAb) (VL8), which is directed against the gp1 of VZV, and using the fluorescence-activated cell sorter (FACS) we could readily demonstrate expression of the VZV gp1 antigen at 3-4 days after VZV infection. (E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU), (a-9-(3hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMC) were shown to be potent inhibitors of VZV replication by this assay. HPMPA and HPMPC were also active against thymidine kinase-deficient (TK-) VZV whereas BVDU was not. The flow cytometric method based on the use of mAb VL8 may be of considerable help for the early diagnosis of VZV infection and evaluation of viral sensitivity to antiviral drugs.
Journal of Virology, 2008
including T lymphocytes, keratinocytes, and neurons, and spreads rapidly in confluent cultured dermal fibroblasts (HFFs). In VZV-infected HFFs, atypical expression of cyclins D3 and B1 occurs along with the induction of cyclin-dependent kinase (CDK) activity. A specific CDK1 inhibitor blocked VZV spread, indicating an important function for this cellular kinase in VZV replication. CDK activity assays of infected cells revealed a large viral phosphoprotein that was identified as being the major immediate-early transactivator, IE62. Since IE62 colocalized with CDK1/cyclin B1 by confocal microscopy, we investigated whether this cellular kinase complex interacts with IE62. Using recombinant fragments of IE62 spanning the entire amino acid sequence, we found that purified CDK1/cyclin B1 phosphorylated IE62 at residues T10, S245, and T680 in vitro.
1999
Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2004
Herpesviruses utilize viral and cellular kinases for replication, and these mediate essential functions that are important for viral pathogenesis. Elucidating the roles of kinases in herpesvirus infections may highlight virus -host interactions that are possible targets for kinase inhibitors with antiviral activity. Varicella zoster virus (VZV) encodes two kinases that phosphorylate viral proteins involved in regulation, assembly, and virulence. VZV infection also induces the activity of host cell cyclin-dependent kinases (cdk4 and cdk2) in nondividing cells, causing a disregulation of the cell cycle. Roscovitine and Purvalanol, kinase inhibitors that target cdks, prevent VZV replication at concentrations with few cytotoxic effects. Cdk inhibitors therefore have potential as antivirals that may extend to a broad range of viruses and have the added advantage that resistance does not arise easily. D
Characterization of regulatory functions of the varicella-zoster virus gene 63-encoded protein
Journal of virology, 1992
Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression expe...
Medical Microbiology and Immunology, 2007
The cAMP-dependent protein kinase A (PKA) is a key enzyme for many cellular mechanisms. In this study, we investigated the importance of this kinase for the replication of the alphaherpesvirus Varicella-zoster virus (VZV). We report that the expression of the catalytic subunit of PKA was strongly increased at the beginning of the viral cycle. The presence of a peptide inhibitor of PKA had no consequence on viral replication in a melanoma cell line whereas in Wbroblasts, it resulted in a drastic decrease of replication. An overall analysis of PKA substrates phosphorylation patterns during VZV replication showed that the phosphorylation of PKA substrates was modulated. These results were completed by investigating the accumulation and phosphorylation patterns of the PKA target cAMP response element binding protein (CREB). This transcription factor remained available throughout the VZV replication, but its phosphorylation decreased in the early phase of infection before it rose later on. These results indicate that the PKA signalling plays a celltype dependent role for VZV replication and that the infection resulted in a regulated CREB-dependent gene expression.