MBV-R amino acid substitutions result in exclusion of pUL27 from nucleoli. (A) HFFs were transfected with EGFP-fusion vectors expressing wild-type (WT) UL27, the MBV-S UL27 clinical strain (CS) variant, and the MBV-R variants A269T, R233S, and V353E, and (B) the MBV-R variants W362R, W153R, and L193F and the MBV-S variants D361E and K89N. 48–72 hours later, indirect immunofluorescence for nucleolin was performed, and the localization of each pUL27-EGFP fusion construct and nucleolin was assessed by fluorescence microscopy.