Viral ribonucleotide reductase attenuates the anti-herpes activity of acyclovir in contrast to amenamevir.

Amenamevir is a helicase-primase inhibitor of herpes simplex virus (HSV) and varicella-zoster virus (VZV) and is used for the treatment of herpes zoster in Japan. The half maximal effective concentrations (EC50s) of acyclovir and sorivudine for VZV increased as the time of treatment was delayed from 6 to 18 h after infection, while those of amenamevir and foscarnet were not affected. Susceptibility of infected cells at 0 and 18 h after infection was examined with four anti-herpes drugs, and the fold increases in EC50 for acyclovir, sorivudine, amenamevir, and foscarnet were 13.1, 6.3, 1.3, and 1.0, respectively. The increase in the EC50s for acyclovir in the late phase of infection in VZV and HSV was abolished by hydroxyurea, a ribonucleotide reductase (RR) inhibitor. The common mechanism affecting antiviral activities of acyclovir to HSV and VZV was examined in HSV-infected cells. The amount of HSV DNA in cells treated with amenamevir at 10 x EC50 was similar at 0 and 12 h but less than that in cells treated with acyclovir at 10 x EC50. dGTP, produced through viral RR, peaked at 4 h and decreased thereafter as it was consumed by viral DNA synthesis. Because acyclovir and amenamevir inhibited viral DNA synthesis, thus making dGTP unnecessary, dGTP was significantly more abundant in the presence of acyclovir and amenamevir than in untreated, infected cells. Abundant dGTP supplied by RR may compete with acyclovir triphosphate and attenuate its antiviral activity. In contrast, abundant dGTP did not influence the inhibitory action of amenamevir on viral helicase-primase activity. ATP was significantly decreased at 12 h after infection and significantly more abundant in untreated infected cells compared to cells treated with acyclovir and amenamevir. The anti-herpetic activity of amenamevir was not affected by the replication cycle of VZV and HSV, indicating the suitability of amenamevir for the treatment of herpes zoster and suppressive therapy for genital herpes.

Titration of cell-associated varicella-zoster virus with the MV9G reporter cell line for antiviral studies.

Titration of the cell-associated virus (CAV) of varicella-zoster virus (VZV) is essential for antiviral studies. A VZV reporter cell line, MV9G, generated in our previous study expresses firefly luciferase upon CAV infection in a dose-dependent manner, suggesting that use of the cell line for titration is feasible. In this study, MeWo cells infected with VZV vaccine Oka (vOka) strain or with clinical isolates obtained from patients with varicella or zoster were used as CAV. A co-culture of MV9G cells with the virus-infected MeWo cells were set up and optimized for titration of CAV. Luciferase activities of MV9G cells measured as relative light units (RLUs) of chemiluminescence correlated well (r > 0.9, p < 0.05) both with quantities of viral DNAs measured by TaqMan PCR and with numbers of viral foci detected by immunostaining with a monoclonal antibody against VZV IE62. In addition, the usefulness of MV9G for antiviral studies was exemplified by treatment of the VZV-infected cells with various concentrations of acyclovir. Thus, the reporter cell-based titration of CAV by measuring the induced RLUs may be a reliable way to estimate viral foci and viral DNAs.

Genetic polymorphism of thymidine kinase (TK) and DNA polymerase (pol) of clinical varicella-zoster virus (VZV) isolates collected over three decades.

BACKGROUND: Genotypic resistance testing of varicella-zoster virus (VZV) strains to antivirals is of high relevance in immunocompromised patients with VZV reactivations unresponsive to therapy. However, the knowledge on mutations associated with natural gene polymorphism or resistance is limited. OBJECTIVES: To examine the genotype of the thymidine kinase (TK) and DNA polymerase (pol) of unselected clinical VZV isolates collected between 1984 and 2014 and to verify the phenotype related to novel amino acid (aa) substitutions. STUDY DESIGN: The TK and DNA pol genes of 169 VZV isolates were analyzed by amplification and sequencing. Sequences were compared to that of the reference strain Dumas. The phenotype to acyclovir and other antivirals was examined in isolates with novel aa substitutions using modified plaque reduction assay. RESULTS: In the TK of four strains, four different aa substitutions were detected, apart from the known change S288L that was present in all strains compared to Dumas. All four substitutions have hitherto not been described in the literature and were phenotypically classified as natural gene polymorphisms although two out of them (S51L, K186R) were localized in conserved gene centers. The DNA pol of 34 isolates exhibited 19 different substitutions, 14 out of them were novel, and two (R753K, V777I) were within conserved gene regions. Again, these changes were characterized as natural gene polymorphisms. CONCLUSIONS: Non-synonymous mutations in VZV TK or DNA pol conferring natural gene polymorphism are rare events. Nevertheless, the phenotypic characterization of 18 novel polymorphisms can help to provide a better identification of resistance mutations.

Helicase-primase inhibitor amenamevir for herpesvirus infection: Towards practical application for treating herpes zoster.

Valacyclovir and famciclovir enabled successful systemic therapy for treating herpes simplex virus (HSV) and varicella zoster virus (VZV) infection by their phosphorylation with viral thymidine kinase. Helicase-primase inhibitors (HPIs) inhibit the progression of the replication fork, an initial step in DNA synthesis to separate the double strand into two single strands. The HPIs amenamevir and pritelivir have a novel mechanism of action, once-daily administration with nonrenal excretory characteristics, and clinical efficacy for genital herpes. Amenamevir exhibits anti-VZV and anti-HSV activity while pritelivir only has anti-HSV activity. A clinical trial of amenamevir for herpes zoster has been completed, and amenamevir has been licensed and successfully used in 20,000 patients with herpes zoster so far in Japan. We have characterized the features of the antiviral action of amenamevir and, unlike acyclovir, the drug's antiviral activity is not influenced by the viral replication cycle. Amenamevir is opening a new era of antiherpes therapy.

Profile of anti-herpetic action of ASP2151 (amenamevir) as a helicase-primase inhibitor.

The antiherpetic drugs acyclovir (ACV, valaciclovir) and penciclovir (famciclovir) are phosphorylated by viral thymidine kinase and terminate DNA synthesis. ASP2151 (amenamevir) and foscavir (PFA) directly inhibit viral helicase-primase and DNA polymerase, respectively, and inhibit replication of herpes simplex virus (HSV) and varicella-zoster virus. ACV, ASP2151, and PFA all inhibit HSV with a different mechanism of action and as a consequence, the kinetics of viral DNA accumulation and progeny virus production differ. This study focused on how viral DNA synthesis and its related events in the replication cycle would influence anti-HSV action of ACV, ASP2151, and PFA. ASP2151 suppressed HSV replication more efficiently than ACV at 10 × 50% effective concentration of plaque formation (EC50), when treatments were started 0-24 h after infection. ASP2151 and PFA were more potent than ACV in suppressing viral DNA synthesis and infectious virus production when they were added up to 3 h following infection. The virus replicated in the presence of ACV was compared for the ratios of HSV DNA copy number to infectivity with that without ACV and infectivity of ACV-treated virus was less efficient than that without ACV-treatment. The EC50 of infected cells in the time course after infection was preserved in PFA, limited in ASP2151, and much increased for ACV, indicating that viral DNA synthesis had little effect on antiviral action of PFA and ASP2151 but reduced the susceptibility of ACV. ASP2151 showed a preferable profile as an anti-herpetic agent with a better pharmacokinetic profile than ACV.

Structure of the Varicella Zoster Virus Thymidylate Synthase Establishes Functional and Structural Similarities as the Human Enzyme and Potentiates Itself as a Target of Brivudine.

Varicella zoster virus (VZV) is a highly infectious human herpesvirus that is the causative agent for chicken pox and shingles. VZV encodes a functional thymidylate synthase (TS), which is the sole enzyme that produces dTMP from dUMP de novo. To study substrate binding, the complex structure of TSVZV with dUMP was determined to a resolution of 2.9 Å. In the absence of a folate co-substrate, dUMP binds in the conserved TS active site and is coordinated similarly as in the human encoded TS (TSHS) in an open conformation. The interactions between TSVZV with dUMP and a cofactor analog, raltitrexed, were also studied using differential scanning fluorimetry (DSF), suggesting that TSVZV binds dUMP and raltitrexed in a sequential binding mode like other TS. The DSF also revealed interactions between TSVZV and in vitro phosphorylated brivudine (BVDUP), a highly potent anti-herpesvirus drug against VZV infections. The binding of BVDUP to TSVZV was further confirmed by the complex structure of TSVZV and BVDUP solved at a resolution of 2.9 Å. BVDUP binds similarly as dUMP in the TSHS but it induces a closed conformation of the active site. The structure supports that the 5-bromovinyl substituent on BVDUP is likely to inhibit TSVZV by preventing the transfer of a methylene group from its cofactor and the subsequent formation of dTMP. The interactions between TSVZV and BVDUP are consistent with that TSVZV is indeed a target of brivudine in vivo. The work also provided the structural basis for rational design of more specific TSVZV inhibitors.

Pronounced Inhibition Shift from HIV Reverse Transcriptase to Herpetic DNA Polymerases by Increasing the Flexibility of α-Carboxy Nucleoside Phosphonates.

Alpha-carboxynucleoside phosphonates (α-CNPs) are novel viral DNA polymerase inhibitors that do not need metabolic conversion for enzyme inhibition. The prototype contains a cyclopentyl linker between nucleobase and α-carboxyphosphonate and preferentially (50- to 100-fold) inhibits HIV-1 RT compared with herpetic DNA polymerases. A synthesis methodology involving three steps has been developed for the synthesis of a series of novel α-CNPs, including a Rh(II)-catalyzed O-H insertion that connects the carboxyphosphonate group to a linker moiety and an attachment of a nucleobase to the other end of the linker by a Mitsunobu reaction followed by final deprotection. Replacing the cyclopentyl moiety in the prototype α-CNPs by a more flexible entity results in a selectivity shift of ∼ 100-fold in favor of the herpetic DNA polymerases when compared to selectivity for HIV-1 RT. The nature of the kinetic interaction of the acyclic α-CNPs against the herpetic DNA polymerases differs from the nature of the nucleobase-specific kinetic interaction of the cyclopentyl α-CNPs against HIV RT.

Drug resistance of clinical varicella-zoster virus strains confirmed by recombinant thymidine kinase expression and by targeted resistance mutagenesis of a cloned wild-type isolate.

In this study, approaches were developed to examine the phenotypes of nonviable clinical varicella-zoster virus (VZV) strains with amino acid substitutions in the thymidine kinase (TK) (open reading frame 36 [ORF36]) and/or DNA polymerase (Pol) (ORF28) suspected to cause resistance to antivirals. Initially, recombinant TK proteins containing amino acid substitutions described as known or suspected causes of antiviral resistance were analyzed by measuring the TK activity by applying a modified commercial enzyme immunoassay. To examine the effects of these TK and Pol substitutions on the replication of recombinant virus strains, the method of en passant mutagenesis was used. Targeted mutations within ORF36 and/or ORF28 and an autonomously expressed gene of the monomeric red fluorescent protein for plaque identification were introduced into the European wild-type VZV strain HJO. Plaque reduction assays revealed that the amino acid substitutions with unknown functions in TK, Q303stop, N334stop, A163stop, and the deletion of amino acids 7 to 74 aa (Δaa 7 to 74), were associated with resistance against acyclovir (ACV), penciclovir, or brivudine, whereas the L73I substitution and the Pol substitutions T237K and A955T revealed sensitive viral phenotypes. The results were confirmed by quantitative PCR by measuring the viral load under increasing ACV concentrations. In conclusion, analyzing the enzymatic activities of recombinant TK proteins represent a useful tool for evaluating the significance of amino acid substitutions in the antiviral resistance of clinical VZV strains. However, direct testing of replication-competent viruses by the introduction of nonsynonymous mutations in a VZV bacterial artificial chromosome using en passant mutagenesis led to reliable phenotypic characterization results.

Testing the sensitivities of noncognate inhibitors to varicella zoster virus thymidine kinase: implications for postherpetic neuralgia therapy with existing agents.

Varicella zoster virus (VZV), a member of the human herpesvirus family, affects peripheral or cranial nerves and can reactivate years after the primary infection. Thymidine kinase (TK) is essential for VZV replication, and its active site is highly conserved in the herpesvirus family. A number of small-molecule inhibitors have already been successfully developed that target the TK of herpes simplex virus type 1 (HSV-1), which is one of the most prevalent sexually transmitted infections worldwide. In the present study, we attempted to test the sensitivities of HSV-1 TK inhibitors to their noncognate VZV TK by integrating in silico modeling and an in vitro assay. We tested nine representative HSV-1 TK inhibitors, including three FDA-approved drugs and six compounds that are under clinical development. The structures of the complexes of these inhibitor ligands with HSV-1 TK and noncognate VZV TK had been solved previously by X-ray crystallography or were modeled in the present work using a template-based approach. Subsequently, a rigorous quantum mechanics/molecular mechanics (QM/MM) nonbonded analysis that accounted for the Poisson-Boltzmann/surface area (PB/SA) solvent effect was employed to refine the complex structures and, on this basis, to evaluate the binding potencies of these complexes. As might be expected, the QM/MM-PB/SA-derived free energy was shown to be highly correlated with the HSV-1 TK inhibitory activities of the nine inhibitors. Further, it was found that the HSV-1 TK inhibitors exhibit strong binding affinities for their noncognate VZV TK, although they are still more selective for HSV-1 TK than for VZV TK. In order to test the theoretical results obtained from the computational analysis, we performed an in vitro kinase assay to determine the inhibitory potencies of three commercially available antiviral agents, namely penciclovir, ganciclovir, and aciclovir, against their noncognate target VZV TK, resulting in IC50 values of 86, 127, and 150 µM respectively, which are modestly weaker than the corresponding values obtained for HSV-1 TK. In addition, visual structure examination and virtual mutation/deletion analysis suggested that the residue Arg222 is present at the active site of HSV-1 TK but not at the active site of VZV TK, which is the reason for the difference in inhibitor selectivity between HSV-1 TK and VZV TK.

ProTides of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide as potential anti-tubercular and anti-viral agents.

The flavin-dependent thymidylate synthase X (ThyX), rare in eukaryotes and completely absent in humans, is crucial in the metabolism of thymidine (a DNA precursor) in many microorganisms including several human pathogens. Conserved in mycobacteria, including Mycobacterium leprae, and Mycobacterium tuberculosis, it represents a prospective anti-mycobacterial therapeutic target. In a M. tuberculosis ThyX-enzyme inhibition assay, N-(3-(5-(2'-deoxyuridine-5'-phosphate))prop-2-ynyl)octanamide was reported to be the most potent and selective 5-substituted 2'-deoxyuridine monophosphate analogue. In this study, we masked the two charges at the phosphate moiety of this compound using our ProTide technology in order to increase its lipophilicity and then allow permeation through the complex mycobacterial cell wall. A series of N-(3-(5-(2'-deoxyuridine))prop-2-ynyl)octanamide phosphoroamidates were chemically synthesized and their biological activity as potential anti-tuberculars was evaluated. In addition to mycobacteria, several DNA viruses depend on ThyX for their DNA biosynthesis, thus these prodrugs were also screened for their antiviral properties.

Varicella-zoster virus open reading frame 48 encodes an active nuclease.

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.

Preliminary validation of varicella zoster virus thymidine kinase as a novel reporter gene for PET.

INTRODUCTION: Imaging of gene expression with positron emission tomography (PET) has emerged as a powerful tool for biomedical research during the last decade. The prototypical herpes simplex virus type 1 thymidine kinase (HSV1-TK) PET reporter gene (PRG) is widely used and many other PRGs have also been validated. We investigated varicella zoster virus thymidine kinase (VZV-tk) as new PRG with radiolabeled bicyclic nucleoside analogues (BCNAs) as PET tracers. METHODS: The uptake and washout of four different radiolabeled BCNAs was evaluated in cells expressing VZV-tk after lentiviral vector (LV) transduction and in control cells. Metabolism of the tracers was assayed by high pressure liquid chromatography (HPLC). Mice bearing VZV-TK expressing xenografts were imaged with PET. RESULTS: High uptake in VZV-tk expressing cells was seen for 3 of the 4 tracers tested. The uptake of the tracers could be blocked by the presence of excess thymidine in the incubation solution. Cellular retention was variable, with one tracer showing an acceptable half-life of ~1 hour. The amount of intracellular tracer correlated with the titer of LV used to transduce the cells. VZV-TK dependent conversion into metabolites was shown by HPLC. No specific accumulation was observed in cells expressing a fusion protein containing an HSV1-TK moiety. VZV-tk expression in xenografts resulted in a 60% increase in uptake in vivo as measured with PET. CONCLUSIONS: We have validated the combination of VZV-tk and radiolabeled BCNAs as new PRG/PRP system. Further optimization of the PRPs and the PRG are warranted to increase the signal.

Studying protein complexes by the yeast two-hybrid system.

Protein complexes are typically analyzed by affinity purification and subsequent mass spectrometric analysis. However, in most cases the structure and topology of the complexes remains elusive from such studies. Here we investigate how the yeast two-hybrid system can be used to analyze direct interactions among proteins in a complex. First we tested all pairwise interactions among the seven proteins of Escherichia coli DNA polymerase III as well as an uncharacterized complex that includes MntR and PerR. Four and seven interactions were identified in these two complexes, respectively. In addition, we review Y2H data for three other complexes of known structure which serve as "gold-standards", namely Varicella Zoster Virus (VZV) ribonucleotide reductase (RNR), the yeast proteasome, and bacteriophage lambda. Finally, we review an Y2H analysis of the human spliceosome which may serve as an example for a dynamic mega-complex.

In vitro-selected drug-resistant varicella-zoster virus mutants in the thymidine kinase and DNA polymerase genes yield novel phenotype-genotype associations and highlight differences between antiherpesvirus drugs.

Varicella zoster virus (VZV) is usually associated with mild to moderate illness in immunocompetent patients. However, older age and immune deficiency are the most important risk factors linked with virus reactivation and severe complications. Treatment of VZV infections is based on nucleoside analogues, such as acyclovir (ACV) and its valyl prodrug valacyclovir, penciclovir (PCV) as its prodrug famciclovir, and bromovinyldeoxyuridine (BVDU; brivudin) in some areas. The use of the pyrophosphate analogue foscarnet (PFA) is restricted to ACV-resistant (ACV(r)) VZV infections. Since antiviral drug resistance is an emerging problem, we attempt to describe the contributions of specific mutations in the viral thymidine kinase (TK) gene identified following selection with ACV, BVDU and its derivative BVaraU (sorivudine), and the bicyclic pyrimidine nucleoside analogues (BCNAs), a new class of potent and specific anti-VZV agents. The string of 6 Cs at nucleotides 493 to 498 of the VZV TK gene appeared to function as a hot spot for nucleotide insertions or deletions. Novel amino acid substitutions (G24R and T86A) in VZV TK were also linked to drug resistance. Six mutations were identified in the "palm domain" of VZV DNA polymerase in viruses selected for resistance to PFA, PCV, and the 2-phophonylmethoxyethyl (PME) purine derivatives. The investigation of the contributions of specific mutations in VZV TK or DNA polymerase to antiviral drug resistance and their impacts on the structures of the viral proteins indicated specific patterns of cross-resistance and highlighted important differences, not only between distinct classes of antivirals, but also between ACV and PCV.

Novel 1,2,4-triazole and purine acyclic cyclopropane nucleoside analogues: synthesis, antiviral and cytostatic activity evaluations.

BACKGROUND: Several published studies indicate that the acyclic guanine nucleoside analogues possessing bis(1,2-hydroxymethyl) substituted cyclopropane rings mimicking the sugar moiety are potent inhibitors of replication of several herpes viruses. METHODS: Established synthetic methods and antiviral and cytostatic activity assays were used for the evaluation of new 1,2,4-triazole and purine acyclic nucleoside analogues. RESULTS: The synthesis of new types of acyclic nucleoside analogues which incorporate 1,2,4-triazole or purine moiety bound via flexible methylenic spacer to the bis(1,2-hydroxymethyl) cyclopropane ring. None of the new compounds showed pronounced antiviral activities at subtoxic concentrations on a broad panel of DNA and RNA viruses. Evaluation of their affinity for herpes simplex type 1 (HSV-1) and varicella-zoster virus-encoded thymidine kinases (VZV TK) also showed that none of the compounds was able to significantly inhibit 1 µM deoxythymidine phosphorylation by HSV-1 and VZV TK at 500 µM concentrations. The in vitro cytostatic activity evaluation results indicated a weak antiproliferative activity for all tested compounds. Only 6-pyrrolylpurine derivative bearing a carboxylic group substituted cyclopropane ring produced a rather slight inhibitory effect at higher micromolar concentrations on a breast carcinoma cell line (MCF-7) and no cytotoxic effect on human normal fibroblasts (WI 38). CONCLUSIONS: The lack of antiherpetic activity may be due to poor, if any, recognition of the compounds by virus-induced nucleoside kinases as an alternative substrate to become metabolically activated.

Resistance testing of clinical varicella-zoster virus strains.

Acyclovir resistance of varicella-zoster virus (VZV) has been reported in rare cases of immunocompromised patients. In this study, the natural polymorphism of the thymidine kinase (TK) and DNA polymerase (pol) genes was examined in 51 clinical VZV isolates sensitive to acyclovir (ACV). In addition, 16 VZV strains with clinical resistance to ACV were analyzed. None of the ACV-sensitive strains of the clades 1, 3 and 5 showed gene polymorphism of the TK. By contrast, the DNA pol gene exhibited polymorphism-related substitutions as a function of the VZV clade. The novel substitutions M286I, E824Q, R984H and H1089Y were detected in strains of clades 3 and 5. In the TK gene of 7 VZV strains with clinical ACV resistance, the novel substitutions L73I, A163stop, W225R, T256M, N334stop and the deletion of nucleotides 19-223 were found to be associated most likely with resistance. In one strain showing the substitution W225R, ACV resistance could be confirmed by the viral phenotype. In the DNA pol gene, the novel amino acid substitutions T237K and A955T could be detected, but their significance remains unclear. In conclusion, the characterization of resistance using genetic analysis of the TK and DNA pol genes has to be considered the method of choice for the determination of VZV resistance to antiviral drugs. In a considerable number of patients with clinical ACV-resistant VZV infections, resistance cannot be verified by virological methods.

The varicella-zoster virus ORF47 kinase interferes with host innate immune response by inhibiting the activation of IRF3.

The innate immune response constitutes the first line of host defence that limits viral spread and plays an important role in the activation of adaptive immune response. Viral components are recognized by specific host pathogen recognition receptors triggering the activation of IRF3. IRF3, along with NF-κB, is a key regulator of IFN-ß expression. Until now, the role of IRF3 in the activation of the innate immune response during Varicella-Zoster Virus (VZV) infection has been poorly studied. In this work, we demonstrated for the first time that VZV rapidly induces an atypical phosphorylation of IRF3 that is inhibitory since it prevents subsequent IRF3 homodimerization and induction of target genes. Using a mutant virus unable to express the viral kinase ORF47p, we demonstrated that (i) IRF3 slower-migrating form disappears; (ii) IRF3 is phosphorylated on serine 396 again and recovers the ability to form homodimers; (iii) amounts of IRF3 target genes such as IFN-ß and ISG15 mRNA are greater than in cells infected with the wild-type virus; and (iv) IRF3 physically interacts with ORF47p. These data led us to hypothesize that the viral kinase ORF47p is involved in the atypical phosphorylation of IRF3 during VZV infection, which prevents its homodimerization and subsequent induction of target genes such as IFN-ß and ISG15.

The alphaherpesvirus US3/ORF66 protein kinases direct phosphorylation of the nuclear matrix protein matrin 3.

The protein kinase found in the short region of alphaherpesviruses, termed US3 in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) and ORF66 in varicella-zoster virus (VZV), affects several viral and host cell processes, and its specific targets remain an area of active investigation. Reports suggesting that HSV-1 US3 substrates overlap with those of cellular protein kinase A (PKA) prompted the use of an antibody specific for phosphorylated PKA substrates to identify US3/ORF66 targets. HSV-1, VZV, and PRV induced very different substrate profiles that were US3/ORF66 kinase dependent. The predominant VZV-phosphorylated 125-kDa species was identified as matrin 3, one of the major nuclear matrix proteins. Matrin 3 was also phosphorylated by HSV-1 and PRV in a US3 kinase-dependent manner and by VZV ORF66 kinase at a novel residue (KRRRT150EE). Since VZV-directed T150 phosphorylation was not blocked by PKA inhibitors and was not induced by PKA activation, and since PKA predominantly targeted matrin 3 S188, it was concluded that phosphorylation by VZV was PKA independent. However, purified VZV ORF66 kinase did not phosphorylate matrin 3 in vitro, suggesting that additional cellular factors were required. In VZV-infected cells in the absence of the ORF66 kinase, matrin 3 displayed intranuclear changes, while matrin 3 showed a pronounced cytoplasmic distribution in late-stage cells infected with US3-negative HSV-1 or PRV. This work identifies phosphorylation of the nuclear matrix protein matrin 3 as a new conserved target of this kinase group.

Regulation of the expression of the varicella-zoster virus open reading frame 66 gene.

The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a serine/threonine kinase that phosphorylates the major viral transactivator protein, immediate-early (IE) 62, preventing its nuclear importation. Cytoplasmic sequestration of IE62 may alter viral gene transcription and could serve as a mechanism for maintaining VZV latency. We examined the regulation of expression of the ORF66 gene by mapping the promoter region, which was localized to within 150 bases of the start codon. The ORF66 promoter was activated by two viral regulatory proteins, IE62 and IE63. We evaluated the binding of viral regulatory proteins and cellular transcription factors based on recognized cellular transcription factor binding sites identified within the ORF66 promoter. These included Sp1 and TBP binding sites, several of which were essential for optimal promoter activity. Site-directed mutations in Sp1 and TBP binding sites led to varying degrees of impairment of ORF66 gene expression in the context of VZV infection. We also examined the effect of Sp1 and TBP mutations on IE62, Sp1, and TBP binding. These studies reveal that host cell-derived and viral factors contribute to and cooperate in the expression of this important viral kinase gene.

Evaluation of novel phosphoramidate ProTides of the 2'-fluoro derivatives of a potent anti-varicella zoster virus bicyclic nucleoside analogue.

BACKGROUND: Recently, the synthesis and antiviral activity of a series of 2'-fluoro derivatives of the most potent anti-varicella zoster virus (VZV) agent reported to date, the bicyclic nucleoside analogue Cf1743, have been reported. METHODS: Here, we present molecular modelling studies for the interaction of these compounds with VZV-encoded thymidine kinase (TK) and we report the synthesis of a series of phosphoramidate ProTides of these compounds designed to bypass the nucleoside kinase dependence of the parent nucleoside analogues. RESULTS: The phosphoramidate prodrugs were equally effective as their parent compounds against VZV in cell culture, but lost antiviral potency against TK-deficient VZV strains. CONCLUSIONS: ProTide-based kinase bypass is not successful in this case.