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.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

Structural basis of a transcription pre-initiation complex on a divergent promoter

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Identification of a sporulation-specific promoter regulating divergent transcription of two novel sporulation genes in Saccharomyces cerevisiae

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.     

The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase

The human cytomegalovirus UL97 protein is an unusual protein kinase that is able to autophosphorylate and to phosphorylate certain exogenous substrates, including nucleoside analogs such as ganciclovir. However, no natural substrate of UL97 in infected cells has been identified. We report here that recombinant UL44 protein became radiolabeled when incubated with recombinant UL97 and [(32)P]ATP and that both proteins could be coimmunoprecipitated by an antibody that recognizes either protein. Subsequent studies showed that highly purified, recombinant UL97 phosphorylated purified, recombinant UL44. This phosphorylation occurred on serine and threonine residues and was sensitive to inhibition by maribavir and to a mutation that inactivates UL97 catalytic activity. Two-dimensional gel electrophoresis revealed the absence of specific phosphorylated forms of UL44 in immunoprecipitates from lysates of cells infected with a UL97 null mutant virus or with wild-type virus in the presence of maribavir. The results indicate that UL97 is sufficient to phosphorylate UL44 in vitro and is necessary for the normal phosphorylation of UL44 in infected cells. This strongly suggests that UL44 is a natural substrate of UL97.