Resistance of Human Cytomegalovirus to Benzimidazole Ribonucleosides Maps to Two Open Reading Frames: UL89 and UL56

2,5,6-Trichloro-1-β-d-ribofuranosyl benzimidazole (TCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication. TCRB acts via a novel mechanism involving inhibition of viral DNA processing and packaging. Resistance to the 2-bromo analog (BDCRB) has been 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- to 30-fold resistant to TCRB (isolate C4) and the characterization of the virus. The six ORFs known to be essential for viral DNA cleavage and packaging (UL51, UL52, UL56, UL77, UL89, and UL104) were sequenced from wild-type HCMV, strain Towne, and from isolate C4. Mutations were identified in UL89 (D344E) and in UL56 (Q204R). The mutation in UL89 was identical to that previously reported for virus resistant to BDCRB, but the mutation in UL56 is novel. Marker transfer analysis demonstrated that each of these mutations individually caused ∼10-fold resistance to the benzimidazoles and that the combination of both mutations caused ∼30-fold resistance. The rate and extent of replication of the mutants was the same as for wild-type virus, but the viruses were less sensitive to inhibition of DNA cleavage by TCRB. Mapping of resistance to UL56 supports and extends recent work showing that UL56 codes for a packaging motif binding protein which also has specific nuclease activity (E. Bogner et al., J. Virol. 72:2259–2264, 1998). Resistance which maps to two different genes suggests that their putative proteins interact and/or that either or both have a benzimidazole ribonucleoside binding site. The results also suggest that the gene products of UL89 and UL56 may be antiviral drug targets.Human cytomegalovirus (HCMV) can cause significant morbidity and mortality in immunocompromised populations (3). It is a common opportunistic disease in patients with AIDS and is often a factor in their death (38). HCMV infection has been implicated in increased risk of organ rejection following heart (28) and kidney transplants (8) and in restenosis of diseased arteries following angioplasty (41, 63). It is also a leading cause of birth defects (16).Current therapies for HCMV infection include ganciclovir (GCV) (22), cidofovir (30), and foscarnet (20). Each of these drugs has several limitations to its use: none are orally bioavailable, all have dose-limiting toxicity, and resistance has developed to each (26). Because all three of these drugs inhibit viral replication through an interaction with the virally encoded DNA polymerase (25, 31, 37), the possibility of cross-resistance exists. Thus, additional drugs with unique mechanisms of action are needed for the treatment of HCMV infections.In 1995, we reported that 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB; Fig. ​Fig.1)1) and the 2-chloro analog [2,5,6-trichloro-1-(β-d-ribofuranosyl)benzimidazole TCRB] are potent and selective inhibitors of HCMV replication (55). These compounds have a novel mechanism of action, which unlike the current therapies for HCMV infection, does not involve inhibition of DNA synthesis. The benzimidazole ribonucleosides prevent the cleavage of high-molecular-weight viral DNA concatemers to monomeric genomic lengths (57). Resistance to BDCRB has been mapped to the HCMV UL89 open reading frame (ORF), which, by analogy to gene gp17 from bacteriophage T4, may be a terminase (23, 57). Consequently, we have proposed that the benzimidazole ribonucleosides inhibit the product of this gene and that the UL89 gene product is involved in the viral DNA concatemer cleavage process (57). Open in a separate windowFIG. 1Structure of benzimidazole ribonucleosides. TCRB, R = Cl; BDCRB, R = Br.HCMV replication proceeds in a manner which is conserved among herpesviruses. The virally encoded DNA polymerase produces large, complex head-to-tail concatemers (10, 29, 33) which must be cleaved into genomic-length pieces before insertion into preformed capsids (59). With herpes simplex virus type 1 (HSV-1), temperature-sensitive mutants which are unable to cleave and package the concatemeric DNA have been derived (1, 2, 4, 45, 49, 50, 61). By this process, six HSV-1 genes have been found to be involved in concatemer cleavage and packaging. They are UL6, UL15, UL25, UL28, UL32, and UL33. In addition, recent studies in Homa’s laboratory have established that the product of UL25 is required for viral DNA encapsidation but not cleavage (39). Homologs of these genes exist in HCMV and are UL104, UL89, UL77, UL56, UL52, and UL51, respectively (18).In our continuing investigation of the mode of action of benzimidazole nucleosides, we report herein the independent isolation of HCMV strains resistant to TCRB, characterization of these strains, and identification of the mutations responsible for the development of resistance. The results demonstrate that the mechanism of action of the benzimidazole ribonucleosides is more complex than previously proposed and that a second gene product implicated in DNA cleavage and packaging is involved.