Resistance of human cytomegalovirus to the benzimidazole L-ribonucleoside maribavir maps to UL27

Abstract

1-(beta-D-Ribofuranosyl)-2,5,6-trichlorobenzimidazole (TCRB) and its 2-bromo analog, BDCRB, are potent and selective inhibitors of human cytomegalovirus (HCMV) DNA processing and packaging. Since they are readily metabolized in vivo, analogs were synthesized to improve biostability. One of these, 1-(beta-L-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (1263W94; maribavir), inhibits viral DNA synthesis and nuclear egress. Resistance to maribavir was mapped to UL97, and this viral kinase was shown to be a direct target of maribavir. In the present study, an HCMV strain resistant to TCRB and BDCRB was passaged in increasing concentrations of maribavir, and resistant virus was isolated. This strain (G2) grew at the same rate as the wild-type virus and was resistant to both BDCRB and maribavir. Resistance to BDCRB was expected, because the parent strain from which G2 was isolated was resistant due to known mutations in UL56 and UL89. However, no mutations were found in UL97 or other relevant open reading frames that could explain resistance to maribavir. Because sequencing of selected HCMV genes did not identify the resistance mutation, a cosmid library was made from G2, and a series of recombinant G2 wild-type viruses were constructed. Testing the recombinants for sensitivity to maribavir narrowed the locus of resistance to genes UL26 to UL32. Sequencing identified a single coding mutation in ORF UL27 (Leu335Pro) as the one responsible for resistance to maribavir. These results establish that UL27 is either directly or indirectly involved in the mechanism of action of maribavir. They also suggest that UL27 could play a role in HCMV DNA synthesis or egress of HCMV particles from the nucleus.

FIG. 1.

Structures of benzimidazole ribonucleosides BDCRB and maribavir.

FIG. 2.

Growth study comparing wild-type HCMV Towne and maribavir-resistant HCMV isolate G2. HFF cells were infected at a 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, titers were determined as described in Materials and Methods.

FIG. 3.

Construction of recombinant viruses. Solid lines represent wild-type HCMV genomic DNA, dotted lines represent G2 HCMV DNA, and bold line segments represent either wild-type or G2 HCMV DNA. (A₁) AD169-BAC with either genes UL13-46 or UL46-68 replaced with the Zeocin resistance cassette. (A₂) G2 HCMV cosmids. (A₃) Recombinant viruses in which the Zeocin resistance cassette was replaced with either UL13-46 or UL46-68 genes from G2 HCMV. (B) Recombinant viruses in which genes UL13 to UL35, UL20 to UL46, UL26 to UL46, or UL32 to UL46 were from G2 HCMV. Details on construction of these viruses are provided in Materials and Methods.