Extended Data Fig. 2: Apical infection of T84 cells by MRV leads to more de novo virus production compared to basolateral infection.
Polarized T84 cells were infected apically or basolaterally with MRV. (a) Fluorescently labelled MRV was added to polarized T84 cells in a side specific manner. Cells were collected at indicated times points and were washed with a quick acid wash to remove any non-internalized virus particles bound to the cell surface. Following acid wash, cells were lysed, and the relative amount of internalized virus particles was measured by spectrofluorometry. Results show the mean +/- sd. n=3. (b) Infected cells were collected at the indicated time points and viral replication was assayed by q-RT-PCR against the MRV μ2 genome segment. Results are normalized to non-infected controls and show the mean +/- sd. n=3. (c) Same as b except virus replication was addressed by Western blot against the MRV non-structural protein μNS. Actin was used as a loading control (left panel). Representative figure is shown. The relative expression of μNS is normalized to actin is shown (right panel). Results shown the mean +/- sd. n=3. (d) Infected cells were fixed at indicated time points and immunofluorescence was performed against the non-structural protein μNS. The number of infected cells per field of view was counted. 10 fields of view were counted for each time point for each replicate. Results shown the mean +/- sd. n=3. (e) Polarized T84 cells were infected apically or basolaterally with MRV. Infected cells were collected at the indicated time points and viral replication was assayed by q- RT-PCR against the MRV μ2 genome segment. Results are normalized to non-infected controls and show the mean +/- sd. n=3. (a-e) P-values were calculated from a two-tailed unpaired t test using GraphPad Prizm.
