RESUMEN
HCMV-related illness due to infections with antiviral resistant virus was verified by phenotypic and genotypic assays in 17% (8/47) of high-risk immunocompromised Australian patients. Selective PCR-sequencing of UL97 (protein kinase; PK) and UL54 (DNA polymerase; DNApol) regions important for antiviral sensitivity, identified the majority (6/8) of resistant strains through detection of mutations known to confer antiviral resistance. Additionally, eight UL54 (DNApol) mutations (N408K, T691S, A692V, S695T, L737M, A834P, V955I, and A972V) of unknown phenotype were identified in six specimens from patients with clinical evidence of antiviral resistant infections. One isolate was resistant to ganciclovir (GCV) and another resistant to PFA on phenotypic testing where mutations in UL97 (PK) or UL54 (DNApol) were not detected, suggesting a loss of correlation between phenotype and genotype. Selective PCR-sequencing of UL97 (PK) and UL54 (DNApol) provided rapid and comprehensive results, but missed some resistance detected by phenotypic assays. A combination of phenotypic and genotypic assays is recommended for complete analysis of CMV antiviral resistance, as well as further definition of the clinical relationship between novel UL54 (DNApol) mutations and antiviral resistance.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por Citomegalovirus/virología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/genética , ADN Polimerasa Dirigida por ADN/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Virales/genética , Sustitución de Aminoácidos , Antivirales/farmacología , Australia , Aziridinas/farmacología , Citomegalovirus/aislamiento & purificación , Infecciones por Citomegalovirus/tratamiento farmacológico , ADN Viral/química , ADN Viral/aislamiento & purificación , ADN Polimerasa Dirigida por ADN/fisiología , Farmacorresistencia Viral/genética , Ganciclovir/farmacología , Genotipo , Humanos , Huésped Inmunocomprometido , Datos de Secuencia Molecular , Mutación , Fenotipo , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Análisis de Secuencia de ADN , Proteínas Virales/fisiologíaRESUMEN
To study the role of protein phosphorylation in erythropoietin (EPO)-mediated signal transduction, we examined the effects of tyrosine phosphatase and tyrosine and serine-threonine kinase inhibitors as well as activators of serine kinases on DNA synthesis and cell proliferation in the murine EPO-dependent cell line HCD-57. HCD-57 cells were obtained synchronized in G0 by centrifugal elutriation, and DNA synthesis was measured by incorporation of labeled thymidine into DNA. Half-maximal DNA synthesis was stimulated by 0.001 U/ml of EPO. Sodium orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor, at 5 microM potentiated a subsaturating concentration of EPO. Na3VO4 alone stimulated HCD-57 DNA synthesis at concentrations of 0.1-20 microM. Zinc chloride, another tyrosine phosphatase inhibitor, also stimulated HCD-57 DNA synthesis at concentrations of 50-100 microM. Genistein, a tyrosine kinase inhibitor, blocked the effect of EPO at a concentration of 5 micrograms/ml. Bryostatin, a protein kinase C (PKC) activator, stimulated DNA synthesis in HCD-57 cells at concentrations of 10(-9)-10(-10) M, whereas the phorbol ester, phorbol 12,13-dibutyrate (PDBu), was stimulatory only at a concentration of 10(-11) M. Staurosporine, a PKC inhibitor, blocked the effect of EPO at a concentration of 10(-7) M, and H-7, a nonspecific protein kinase inhibitor, was not inhibitory. These agents also had similar effects on the in vitro proliferation of HCD-57 cells. Taken together, the data indicate that the EPO-mediated transition from G0 to S phase in HCD-57 cells involves the activation of both tyrosine and serine-threonine kinases and is modulated by tyrosine phosphatase activity.