Article Figures & Data
Figures
- Fig. 1.
Alignment of some conserved motifs in different UL97 homologues. The numbers indicate amino acid positions of the UL97 protein sequence. Published mutations conferring GCV phenotypic resistance on HCMV strains are indicated by boldface, underlined, italic letters. Domain, conserved domains in PK as defined by Hanks et al. (5, 6); S/T-PK, amino acids conserved in serine/threonine PK; Herp, amino acids that are conserved in the UL97 homologues of herpes simplex virus, human herpesvirus 6, varicella-zoster virus, and Epstein-Barr virus according to the work of Chee et al. (2); RhCMV, rhesus cytomegalovirus; MCMV, murine cytomegalovirus; GCMV, guinea pig cytomegalovirus (4, 20); Cons, amino acids that are conserved among the cytomegalovirus proteins; MUTA, mutations introduced by site-directed mutagenesis.
- Fig. 2.
Expression and phosphorylation of newly mutagenized UL97 proteins in cells infected with rVV. Lanes labeled 335 to 617 indicate rVV carrying different point mutations in the UL97 ORF introduced by site-directed mutagenesis; cop, cells infected with the vaccinia virus strain Copenhagen (without the UL97 ORF). CV1 cells were infected at an MOI of 5 with the indicated rVV and were harvested 17 to 20 h postinfection for preparation of nuclear extracts. The extracts were used in parallel for both Western blot analyses and PK assays. (A) For Western blotting nuclear matrix extracts were separated by sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis. Here pUL97 was visualized by chemiluminiscence with the pUL97 antiserum. (B) For determination of pUL97 phosphorylation, nuclear matrix fractions were tested for PK activity. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography.
- Fig. 3.
GCV phosphorylation in rVV-infected 143B (thymidine kinase-deficient) cells. The numbers 335 to 617 indicate the different rVV; cop, vaccinia virus strain Copenhagen. The bars represent mean values and standard errors of the results of 6 to 10 independent experiments. The Roman numerals denote conserved domains in PK (5). Point mutations which were found in phenotypically resistant HCMV at positions 460, 520, and 594 have been previously described (14) and are indicated by asterisks. The invariant lysine (K) at position 355 reported by He et al. (8) is indicated.
