Shannon-Lowe, Claire Deidre; (2002) Biochemical and virologic analysis of the human cytomegalovirus encoded UL97. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in immunocompromised patients. It is one of the most important opportunistic agents in the pathology of HFV, where it frequently causes sight-threatening retinitis. Currently approved antiviral drugs include ganciclovir, cidofovir and foscarnet. The HCMV UL97 gene encodes a phosphotransferase that has been shown to catalyse the initial phosphorylation of ganciclovir (GCV) to its monophosphate form, whereafter it is converted to the active triphosphate by cellular kinases. HCMV disease often necessitated long-term antiviral therapy, which in turn led to the emergence of ganciclovir resistant strains of HCMV. Mutations in the UL97 gene have been shown to impair GCV phosphorylation, thus conferring GCV resistance to HCMV and have been associated with disease progression. HCMV has been observed in almost every organ system in the body, but little information is available on the extent of UL97 mutations in the organs of chronically treated AIDS patients. A point mutation assay was employed to determine the prevalence of UL97 mutations in post mortem organs of patients who died with AIDS. The results showed that quantitative differences in resistant genotypes between organs of the same individual and between individuals occurs which has relevance to the management of patients on long term therapy for HCMV. In order to investigate functional effects of UL97 drug resistance mutations, wild type and mutant UL97 proteins were produced using a recombinant baculovirus expression system. Autophosphorylation properties of wild type and mutant UL97 were similar, while the mutations reduced ganciclovir phosphorylation to below 20% of the wild type UL97. The novel antiviral benzimidavir, inhibited autophosphorylation of wild type and mutant UL97 proteins. Enzyme kinetic analysis demonstrated benzimidavir acts by competitive inhibition of ATP binding to UL97. The mutation at codon 397, which confers resistance to benzimidavir, reduced autophosphorylation by 90%. These observations have implications for combination therapy with benzimidavir and ganciclovir.

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