Figure 4.

R18 forms photoadducts with influenza proteins upon illumination. SDS-PAGE of dye-labeled influenza virus with and without illumination yielded fluorescent protein bands. Fluorescence intensities of the bands corresponding to the HA0 monomer and either the HA1 subunit or neuraminidase (∼75 and ∼55 kDa, respectively) were calculated; these were normalized by the Coomassie blue staining intensity of each band, and then the % increase in fluorescence was calculated between unilluminated and illuminated samples. Samples are denoted by the dye (R18 or Texas Red) and concentration (μg/mL) used to label the virus; “dup” denotes duplicate samples. The use of R18 as a lipid marker caused a substantial increase in protein fluorescence in a dye-concentration-dependent manner, whereas Texas Red-DHPE did not when similarly introduced into the viral membrane. A gel shift for the 55 kDA band and also the 25 kDa band where the HA2 and M1 bands comigrate (28) is also evident in the Coomassie-blue-stained bands after illumination of the R18-labeled samples, as shown in the inset. Full gels are shown in Fig. S4. To see this figure in color, go online.