Mutations in the human cytomegalovirus UL27 gene that confer resistance to maribavir

Abstract

Previous drug selection experiments resulted in the isolation of a human cytomegalovirus (CMV) UL97 phosphotransferase mutant resistant to the benzimidazole compound maribavir (1263W94), reflecting the anti-UL97 effect of this drug. Three other CMV strains were plaque purified during these experiments. These strains showed lower-grade resistance to maribavir than the UL97 mutant. Extensive DNA sequence analyses showed no changes from the baseline strain AD169 in UL97, the genes involved in DNA replication, and most structural proteins. However, changes were identified in UL27 where each strain contained a different mutation (R233S, W362R, or a combination of A406V and a stop at codon 415). The mutation at codon 415 is predicted to truncate the expressed UL27 protein by 193 codons (32% of UL27) with a loss of nuclear localization. The expression of full-length UL27 as a green fluorescent fusion protein in uninfected fibroblasts resulted in nuclear and nucleolar fluorescence, whereas cytoplasmic localization was observed when codons 1 to 415 were similarly expressed. Viable UL27 deletion mutants were created by recombination and showed slight growth attenuation and maribavir resistance in cell culture. Marker transfer experiments confirmed that UL27 mutations conferred maribavir resistance. The UL27 sequence was well conserved in a sample of 16 diverse clinical isolates. Mutation in UL27, a betaherpesvirus-specific early gene of unknown biological function, may adapt the virus for growth in the absence of UL97 activity.

FIG. 1.

Isolation of maribavir-resistant virus strains. After nine passages of strain AD169 under a maribavir carbocyclic analog (2916W93), three virus pools (rA, rB, and rC) were further propagated under conditions of drug exposure (rA under 2916W93 and the others under maribavir). At passage 13 (P13) or 14 (P14), the viruses were triply plaque purified (PP×3), resulting in the viruses shown, along with their UL97 genotype. The characterization of strain 2916rA has been published (4). DNA was extracted from 2916rA, cloned in cosmids, cotransfected with strain AD169 DNA, and selected four times under maribavir (4). One by-product of this process (rA5-13) was plaque purified. Sensitivity to maribavir, as determined by yield reduction assay, is shown as IC₅₀ values (± SD, based on at least three determinations) under each strain.

FIG. 2.

Northern blot analysis of UL27 gene expression. (A) Total RNA extracts of infected cells at 1 and 3 days, cultured with and without added ganciclovir, were denatured and run on agarose gels, Northern blotted to nylon membrane, and then hybridized with digoxigenin-labeled UL27 probe. An uninfected-fibroblast total RNA extract was included as a control and showed no signal (right lane). RNA size markers (data not shown) were run in parallel. (B) Ethidium-stained 18S cellular RNA was photographed before blotting as an indicator of RNA content in each extract. (C) At early times postinfection, a 5-kb band predominated, corresponding in length to the distance between polyadenylation (pA) motifs upstream of UL29 and downstream of UL26. At late times this was replaced by various shorter transcripts, a process that is partially inhibited by ganciclovir. GCV, ganciclovir.

FIG. 3.

Fibroblasts expressing GFP fusion proteins. UL97 (full-length) or UL27 (full-length or truncated) coding sequences were fused in frame with GFP coding sequences in vector pEGFP-N1 and transfected into fibroblasts. The UL97 fusion protein shows nuclear localization (A), the full-length UL27 fusion protein shows nuclear and nucleolar localization (B), and the fusion protein with UL27 truncated at codon 415 shows cytoplasmic localization with exclusion from the nucleus (C). Original magnification, ×60 (A and B) and ×40 (C).

FIG. 4.

Southern blot analysis of UL27 deletion mutants. Viral genomic DNA extracts were digested with enzyme PstI, electrophoresed, and blotted to nylon. Multiple replicates of the three extracts (AD169, T2092, and T2182) were run on the same gel and blotted to the same membrane and then cut into strips and hybridized separately with one of five different probes representing the sequences of the UL26 to UL29 genes and GFP. For wild-type AD169, two PstI genomic DNA fragments are detected, one with probes UL26, UL27, and UL28 and the other with probe UL29. T2092 and T2182, which have most of UL27 deleted, show an absence of hybridization with the UL27 (codons 1 to 415) probe. T2092 shows different fragment sizes with UL28 and UL26 probes because of two PstI sites in the GFP sequence inserted in place of UL27, whereas T2182 shows the same fragment with probes UL26 and UL28 because the GFP insert has been removed. The GFP probe hybridizes only to strain T2092 as expected, and neither strain T2092 nor T2182 shows the AD169 fragment size detected by probes UL26 to UL28, indicating absence of contamination with wild-type virus.

FIG. 5.

Single-step growth curves of UL27 and UL97 deletion mutants. Extracellular virus concentrations were sampled daily after inoculation of various strains at an MOI of >1.4. Compared with wild-type strain AD169, the UL97 deletion mutant RCd97-19 (17) was attenuated by ∼1.5 logs at 4 days. The UL27 deletion mutants T2092 and T2182 were slightly attenuated (∼0.5 log at 4 days). The viral quantitations are averages of data from six experiments per virus, and the error bars show ± 1 SD. The day 0 quantitations are of the input virus.

FIG. 6.

Recombinant viruses containing specific UL27 mutations. Marker transfers were accomplished by digesting T2092 genomic DNA with PacI to remove the GFP coding sequence, leaving a gap at UL27. This was repaired by recombination following cotransfection with overlapping plasmids containing the desired UL27 sequence configurations. As a by-product of experiments used to produce T2203, a new strain T2182 was isolated in which no cotransfected UL27 DNA was incorporated, resulting in a nonfluorescent UL27 deletion mutant. Recombinant viruses were found to have the UL27 genotype and maribavir resistance phenotypes shown (IC₅₀ ± SD values from at least three determinations are shown under the strain names).