Topology of recombinant FLAG-tagged ORF 3 protein. Recombinant N-terminal tagged FLAG-ORF 3 protein was transiently expressed in HEK-293T cells and localization was analyzed by confocal laser scanning microscopy (cLSM 510 Meta, Zeiss). FLAG-ORF 3 protein was stained with rabbit-anti-ORF 3 recognizing the C-terminus and mouse-anti-FLAG for detection of the FLAG-tagged N-terminus (upper panel). Permeabilized cells (+TritonX100) show colocalized signals mainly in perinuclear regions for protein ORF 3 C-terminus and N-terminus whereas without permeabilization (-TritonX100) only FLAG-tagged N-terminus of protein ORF 3 could be detected at the plasma membrane (lower panel). Bars represent 10 μm.