Comparison of ORF 3 protein in viral infection and overexpression by Western blot. (A), LLC-MK2 cells were inoculated with hCoV-NL63 (MOI 0.01) and analyzed by Western blot after 4 days using antibodies against the ORF 3 protein C-terminus (top) and against M (bottom). The bands named ORF 30 and M0 are corresponding to the predicted molecular weights of both proteins (26 kDa). Larger bands ORF 3 g and Mg were assumed to be the result of posttranslational modification. (B, left panel): HEK-293T cells transfected with N-terminally FLAG-tagged ORF 3 do not show signs of posttranslational modification as observed in (A). (B, right panel): overexpression of ORF 3 protein in the same system without an N-terminal fusion tag reconstitutes the additional band of higher molecular weight observed in infected cells. The "mock" lane represents a control transfected with an empty vector.