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

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.

Gunnerales are sister to other core eudicots: implications for the evolution of pentamery.

Phylogenetic relationships among many lineages of angiosperms have been clarified via the analysis of large molecular data sets. However, with a data set of three genes (18S rDNA, rbcL, and atpB), relationships among lineages of core eudicots (Berberidopsidales, Caryophyllales, Gunnerales, Santalales, Saxifragales, asterids, rosids) remain essentially unresolved. We added 26S rDNA sequences to a three-gene matrix for 201 eudicots (8430 base pair aligned nucleotides per taxon). Parsimony analyses provided moderate (84%) jackknife support for Gunnerales, which comprise the two enigmatic families Gunneraceae and Myrothamnaceae, as sister to all other core eudicots. This position of Gunnerales has important implications for floral evolution. A dimerous or trimerous perianth is frequently encountered in early-diverging eudicots (e.g., Buxaceae, Proteales, Ranunculales, Trochodendraceae), whereas in core eudicots, pentamery predominates. Significantly, dimery is found in Gunneraceae and perhaps Myrothamnaceae (the merosity of the latter has also been interpreted as labile). Parsimony reconstructions of perianth merosity demonstrate lability among early-diverging eudicots and further indicate that a dimerous perianth could be the immediate precursor to the pentamerous condition characteristic of core eudicots. Thus, the developmental canalization that yielded the pentamerous condition of core eudicots occurred after the node leading to Gunnerales.

Cytomegalovirus UL97 phosphotransferase mutations that affect susceptibility to ganciclovir.

Most ganciclovir (GCV)-resistant cytomegalovirus (CMV) isolates contain UL97 gene mutations at codon 460 or 520 or between codons 590 and 607, where an increasing variety of mutations have been detected, including deletions. To determine their phenotypic effect, 9 UL97 mutations not previously studied were transferred to drug-sensitive laboratory CMV strains that contained unique restriction sites developed for this purpose. Deletion of the entire codon range 591-607 conferred a 6-fold increase in GCV resistance, with little effect on viral replication. Some mutations found in clinical isolates, including C592G and A594T, conferred only 2-3-fold decreases in GCV susceptibility. For C592G, this phenotype was confirmed by transfer to different CMV strains and by restoration of full drug susceptibility after removal of the mutation. Low drug levels resulting from oral GCV therapy may predispose the virus to the initial selection of these low-grade UL97 resistance mutations and to later accumulation of other mutations and greater resistance.