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. 2010 Dec 8;85(4):1804–1819. doi: 10.1128/JVI.01347-10

FIG. 7.

FIG. 7.

Nucleocytoplasmic shuttling of betaherpesviral pUL69 homologs as analyzed by interspecies heterokaryon assays. HeLa cells were cotransfected with CFN-βGal (a to d), CFNrev-βGal (e to h), or a combination of CFNrev-βGal and the indicated pUL69 homolog (i to C). CFNrev-βGal served as a shuttling positive control, as it contains β-galactosidase fused to the NLS of the SV40 large T antigen and the NES of HIV-1 Rev. CFN-βGal also codes for β-galactosidase fused to the SV40 NLS but lacks a functional NES and therefore displays the shuttling negative control (41). Interspecies heterokaryons were formed by fusion of transfected HeLa cells with murine NIH 3T3 cells. Four hours later, cells were fixed and the localization of transfected proteins was assessed by indirect immunofluorescence using an antibody specific for β-galactosidase and RAb-FLAG for detection of pUL69 homologs. Upon counterstaining by DAPI, murine nuclei (indicated by arrows) displayed a characteristic punctate pattern and could thereby easily be discriminated from the evenly stained human nuclei.