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ABSTRACT: CMX001 is an orally available lipid acyclic nucleotide phosphonate that delivers high intracellular levels of cidofovir (CDV)-diphosphate and exhibits enhanced in vitro antiviral activity against a wide range of double-stranded DNA viruses, including cytomegalovirus (CMV). Mutations in the DNA polymerase of CMV that impart resistance to CDV also render the virus resistant to CMX001. Here, we report a novel resistance mutation that arose under the selective pressure of CMX001. The wild-type CMV strain AD169 was propagated in human foreskin fibroblasts under increasing concentrations of CMX001 over 10 months and the resulting strain (CMX001(R)) was less susceptible to CDV and CMX001 in a plaque reduction assay. Genotypic analysis of CMX001(R) via conventional sequencing of the genes encoding the CMV DNA polymerase (UL54) and UL97 kinase (UL97) demonstrated one mutation which changed the wild-type aspartate to glutamate at position 542 in UL54. A recombinant virus with this novel D542E mutation was generated via bacterial artificial chromosome-mediated marker transfer experiments. Subsequent phenotypic resistance analysis of the D542E mutant demonstrated reductions in susceptibility of greater than 10-fold to CMX001 and CDV but no resistance to foscarnet (FOS) or ganciclovir (GCV). Analysis of replicative fitness showed that both CMX001(R) and the D542E mutant viruses demonstrated a smaller plaque phenotype and slower replication kinetics than their respective parent viruses. These data describe the first resistance mutation generated under the selective pressure of CMX001 and suggest that CMX001 may have a unique resistance profile associated with reduced viral replication and maintenance of sensitivity to FOS and GCV.
Antimicrobial Agents and Chemotherapy 05/2013; · 4.84 Impact Factor
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ABSTRACT: The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G₂/M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.
Journal of General Virology 05/2012; 93(Pt 8):1743-55. · 3.36 Impact Factor
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ABSTRACT: Cyclopropavir (CPV) is active against human cytomegalovirus (CMV), as well as both variants of human herpesvirus 6 and human herpesvirus 8. The mechanism of action of CPV against CMV is similar to that of ganciclovir (GCV) in that it is phosphorylated initially by the CMV UL97 kinase, resulting in inhibition of viral DNA synthesis. Resistance to CPV maps to the UL97 kinase but is associated primarily with H520Q mutations and thus retains good antiviral activity against most GCV-resistant isolates. An examination of CMV-infected cultures treated with CPV revealed unusual cell morphology typically associated with the absence of UL97 kinase activity. A surrogate assay for UL97 kinase activity confirmed that CPV inhibited the activity of this enzyme and that its action was similar to the inhibition seen with maribavir (MBV) in this assay. Combination studies using real-time PCR indicated that, like MBV, CPV also antagonized the efficacy of GCV and were consistent with the observed inhibition of the UL97 kinase. Deep sequencing of CPV-resistant laboratory isolates identified a frameshift mutation in UL27, presumably to compensate for a loss of UL97 enzymatic activity. We conclude that the mechanism of action of CPV against CMV is complex and involves both the inhibition of DNA synthesis and the inhibition of the normal activity of the UL97 kinase.
Antimicrobial Agents and Chemotherapy 07/2011; 55(10):4682-91. · 4.84 Impact Factor
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ABSTRACT: A cross-sectional survey of 210 healthcare workers at a pediatric teaching hospital was performed to assess knowledge of published guidelines for proper measurement and documentation of tuberculin skin test results. We conclude that many healthcare workers have inadequate knowledge for optimal measurement and documentation of tuberculin skin test results.
Infection Control and Hospital Epidemiology 12/2009; 30(12):1230-2. · 3.67 Impact Factor
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ABSTRACT: Herpesvirus infections of the central nervous system (CNS) are a significant cause of morbidity and mortality, including long-term neurologic sequelae. Among the family of herpesviruses, the most significant CNS infections are due to herpes simplex virus (HSV) and cytomegalovirus (CMV). The onset of HSV CNS infection can occur in neonates as well as older children and adults. CNS infection associated with CMV occurs predominantly in the perinatal period, but may also be seen rarely in children and adults, especially in immunocompromised individuals. Although advances in antiviral agents have led to improved outcomes, there is still a need for more effective treatments.
Antiviral research 06/2009; 83(3):207-13. · 3.61 Impact Factor
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Pediatrics in Review 05/2009; 30(4):139-45. · 0.55 Impact Factor
