Point mutation of the globular-globular interface residues impairs ORF57 dimerization.

(A) Representative diagrams of the constructs encoding C-terminal FLAG- or GFP-tagged ORF57 and its mutants. (B) Effect of interface point mutations on ORF57 expression. HEK293 cells transfected with indicated FLAG-tagged ORF57 expression vectors were harvested at 24 h after transfection for extraction of total proteins and RNA. The protein levels of ORF57 or its mutants were determined by Western blotting using anti-FLAG antibody (top). Cellular β-tubulin was used as a loading control. The RNA levels were determined by Northern blotting (bottom). The level of 18S rRNA was used to ensure equal RNA loading. (C) ORF57 interface mutants are partially degraded through proteasome pathway. HEK293 cells were transfected with indicated ORF57 expression vectors and treated at 20 h after transfection with MG132 for additional 4 h or with DMSO (vehicle) as a control. The cell lysates were examined for ORF57 protein by Western blotting using anti-FLAG antibody. Cellular β-tubulin was used as loading control. (D) ORF57 mutants with point mutation of the globular-globular interface residues are incapable of protein dimerization. HEK293 cells were transfected with equal amount (2 μg) of the vectors expressing either wild type (WT) or globular domain mutant ORF57-FLAG. The cells at 24 h after transfection were treated with proteasome inhibitor MG132 for 6 h (lanes 1–12). Alternatively, the expression of wild type and mutant protein was adjusted by transfection of variable amount of the plasmid DNA with 0.5 μg for WT and 2 μg for mutant (lanes 13–16). The cells were harvested in PBS and exposed to chemical crosslinker DSS (50 μM in final concentration) or DMSO (vehicle) for 30 min. The dimer formation was monitored by Western blotting using an anti-FLAG antibody. M, monomer; D, dimer. (E) COS-1 cells seeded on glass cover slips were cotransfected with a GFP-tagged ORF57 mt1+2+3 (a three nuclear localization signal-deficient mutant, mt1+2+3) and a FLAG-tagged full-length of ORF57 interface mutant. The empty vector was used as a negative control and ORF57 WT as a positive control. The previously reported dimerization-incompetent ORF57-W292P and dimerization-competent ORF57-K345A were also used as controls. The localization of GFP-tagged proteins (green) was detected using direct fluorescent microscopy. The FLAG-tagged proteins (red) were visualized by indirect immunofluorescent staining using an anti-FLAG M2 antibody. Cell nuclei (blue) were counterstained with Hoechst 33342 dye. Indicated in the merged panel are % cells with nuclear translocation from 50 double-color positive cells. N/A, not applicable.