Role of a mutation in human cytomegalovirus gene UL104 in resistance to benzimidazole ribonucleosides

Abstract

The benzimidazole D-ribonucleosides TCRB and BDCRB are potent and selective inhibitors of human cytomegalovirus (HCMV) replication. Two HCMV strains resistant to these compounds were selected and had resistance mutations in genes UL89 and UL56. Proteins encoded by these two genes are the two subunits of the HCMV "terminase" and are necessary for cleavage and packaging of viral genomic DNA, a process inhibited by TCRB and BDCRB. We now report that both strains also have a previously unidentified mutation in UL104, the HCMV portal protein. This mutation, which results in L21F substitution, was introduced into the genome of wild-type HCMV by utilizing a recently cloned genome of HCMV as a bacterial artificial chromosome. The virus with this mutation alone was not resistant to BDCRB, suggesting that this site is not involved in binding benzimidazole nucleosides. As in previous proposals for mutations in UL104 of murine cytomegalovirus and HCMV strains resistant to BAY 38-4766, we hypothesize that this mutation could compensate for conformational changes in mutant UL89 and UL56 proteins, since the HCMV terminase is likely to interact with the portal protein during cleavage and packaging of genomic DNA.

FIG. 1.

Structure of the benzimidazole ribonucleoside BDCRB.

FIG. 2.

Construction of HCMV with the L21F mutation in UL104. (A) Genome of HCMV cloned as a bacterial artificial chromosome (AD169-BAC); (B₁) expanded region surrounding the UL104 ORF; (B₂) AD169-BAC in which a portion of UL104 was replaced with the zeocin cassette; (B₃) AD169-BAC in which the zeocin cassette is replaced with the UL104 fragment containing the L21F mutation; (C) EcoRI digest of AD169-BAC and UL104ΔBAC.

FIG. 3.

Growth study comparing wild-type HCMV AD169-RV and HCMV with L21F mutation in UL104 (UL104L21F rec). HFF cells were infected at an MOI of 0.01 PFU/cell, incubated at 37°C, and harvested at the times indicated over the course of 10 days. After all samples were collected, viral titers were determined as described in Materials and Methods.