RESISTANCE OF HUMAN CYTOMEGALOVIRUS TO BENZIMIDAZOLE RIBONUCLEOSIDES MAPS TO 2 OPEN READING FRAMES - UL89 AND UL56

2,5,6-Trichloro-1-beta-D-ribofuranosyl benzimidazole (TCRB) is a poten t and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA pro cessing and packaging, Resistance to the 2-bromo analog (BDCRB) has be en mapped to the UL89 open reading frame (ORF), and this gene product was proposed as the viral target of the benzimidazole nucleosides. In this study, we report the independent isolation of virus that is 20- t o 30-fold resistant to TCRB (isolate C4) and the characterization of t he virus. The six ORFs known to be essential for viral DNA cleavage an d packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced f rom wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 w as identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstra ted that each of these mutations individually caused similar to 10-fol d resistance to the benzimidazoles and that the combination of both mu tations caused similar to 30-fold resistance. The rate and extent of r eplication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Ma pping of resistance to UL56 supports and extends recent work showing t hat UL56 codes for a packaging motif binding protein which also has sp ecific nuclease activity (E. Bogner et al., J. Virol. 72:2259-2264, 19 98), Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimida zole ribonucleoside binding site, The results also suggest that the ge ne products of UL89 and UL56 may be antiviral drug targets.