FIG. 3.

Functions of mutated and rescued IE1 proteins. (A) Dispersion of nuclear domains (ND10). Shown are CLSM images of representative triple-labeled individual cells (MEF) infected for 4 h (corresponding to the E phase) at an MOI of 4 (0.2 PFU/cell × 20; centrifugal enhancement of infectivity for synchronization) with the IE1 deletion mutant mCMV-ΔIE1 (images a to d) or the point mutant mCMV-IE1-Y165C (images e to h). (a and e) Red staining of intranuclear viral proteins E1 (M112-M113) and IE1 (m123), respectively. Note that green Alexa Fluor 488 fluorescence was electronically converted into red for better contrast. (c and g) Green staining of PML protein. Note that red Alexa Fluor 546 fluorescence was electronically converted into green for better contrast. The arrow points to an intranuclear PML body/ND10. (b and f) Visualization of cell nuclei by blue staining of DNA with Hoechst 33342 dye. (d and h) Merge of red E1 or IE1 protein, green PML, and blue Hoechst dye staining. The bar marker represents 10 μm. (B) Quantification of intranuclear PML bodies. MEF were infected (see above) with the viruses indicated. For statistical-significance analysis, intranuclear PML bodies were counted for 30 infected cell nuclei per group. The dots represent the numbers of PML bodies in individual cell nuclei. The median values are marked by horizontal bars. Two-sided P values (Wilcoxon-Mann-Whitney rank sum test; n = 30) are indicated for group comparisons of major interest. (C) Time course of IE- and E-phase protein expression represented by proteins IE1 (pp89/76) and m164 (gp36.5), respectively. Shown are Western blots of MEF total lysate proteins isolated at the indicated time points after infection with the viruses shown below for lanes 1 to 4. n.i., not infected; lysate proteins derived from uninfected MEF.