Subcellular localization of overexpressed hCoV-NL63 proteins in Huh-7 and HEK-293T cells. Confocal laser scanning microscopy on cells co-expressing GFP-E, GFP-M, GFP-N, respectively, together with FLAG-ORF 3. (A), Huh-7 cells. The green panels on the left show GFP fluorescence from overexpressed E, M, and N proteins. Red pictures in the next column show Cy3 fluorescence from anti-FLAG staining of overexpressed FLAG-ORF 3 fusion protein. Blue pictures show Cy5 fluorescence from staining of the ER-Golgi intermediate compartment (ERGIC) (refer to Materials and Methods section for antibodies and staining technique). Yellow areas in the right hand column represent colocalization of the GFP-proteins with FLAG-ORF 3 whereas white regions in merged pictures show colocalization of GFP proteins with FLAG-ORF 3 within the ERGIC. GFP-E and M show excessive colocalization with FLAG-ORF 3 especially within the ERGIC in both cell lines. GFP-N partially colocalizes with FLAG-ORF 3 mainly within the ERGIC. Analysis was performed with the help of a confocal laser scanning microscope (cLSM 510 Meta, Zeiss). Bars represent 20 μm. (B), to exclude altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated in HEK-293T cells using FLAG-ORF 3 in combination with HA tagged E, M and N proteins. Bars represent 10 μm.