Role of HCMV genes in benzimidazole ribonucleoside antiviral drug action.

Abstract

Both benzimidazole D- and L-ribonucleosides are potent inhibitors of human cytomegalovirus (HCMV) replication. The D-nucleosides TCRB and BDCRB [2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole and its 2-bromo analog] were previously shown to inhibit HCMV DNA cleavage and processing. Mutations identified in genes UL56 and UL89 which encode a putative HCMV terminase were responsible for this resistance. Two strains with mutations either in UL89 or in both UL89 and UL56, also had a mutation (L21F) in gene UL104. This open reading frame encodes a putative HCMV portal protein that interacts with HCMV terminase. To determine if UL104 was involved in drug action, a recombinant virus with the L21F mutation only was constructed using the genome of HCMV cloned as a bacterial artificial chromosome. This recombinant virus was sensitive to TCRB/BDCRB which established that this mutation alone was not sufficient for resistance and was not involved in drug action unless it compensates for conformational changes in the mutant UL89 and UL56 proteins. The L-nucleoside maribavir [2-isopropylamino-5,6-dichloro-1-(beta- L-ribofuranosyl) benzimidazole) acts by a different mechanism inhibiting DNA synthesis and nuclear egress of HCMV. To investigate this drug's action, a strain of resistant HCMV was isolated. A series of recombinant resistant-wild type viruses were constructed and tested for sensitivity in order to narrow the resistance locus in this large DNA virus. The resistance mutation (L335P) was ultimately mapped to HCMV open reading frame UL27. Since the function of this gene in HCMV replication cycle is unknown, its protein was studied and found to be a nuclear protein with its own nuclear localization signal. Two UL27 deletion viruses also were constructed and their phenotype studied. Results showed that UL27 is not essential for HCMV replication in cell culture because HCMV with UL27 deleted grew at the same rate as the wild-type virus. However, it consistently achieved one-half log10 lower titers. Viral DNA synthesis of the UL27 deletion viruses also was about two fold lower than the wild-type virus. Both UL27 deletion viruses were sensitive to ganciclovir and foscarnet but were resistant to maribavir, confirming the role of this open reading frame in maribavir mechanism of action.Ph.D.Health and Environmental SciencesPharmacologyPharmacy sciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123883/2/3106100.pd