FIG. 5.

FHV protein A is a transmembrane protein. (A) Protein A expression and viral RNA replication in yeast transformed with pF1_fs alone or pF1_fs plus pFA, pFA-C/HA, or pFA-N/HA. Total protein samples from an equivalent number of yeast per sample were separated by SDS-PAGE and immunoblotted with rabbit anti-protein A (upper blot) or mouse anti-HA (middle blot). Total protein staining showed equivalent protein loading in all lanes (data not shown). The full-length protein A band (arrow) was quantitated by densitometry, and the values shown are averages of two independent experiments and represent the relative protein A accumulation compared to that in yeast transformed with pF1_fs plus pFA. We separated 2 μg of total RNA per sample on denaturing formaldehyde-agarose gels and blotted this with a ³²P-labeled complementary riboprobe that detected positive-strand RNA1 and RNA3 (lower blot). The positions of RNA1 and RNA3 are indicated on the left. Positive-strand RNA3 was quantitiated by densitometry, and the values shown are averages of two independent experiments and represent the relative positive-strand RNA3 accumulation compared to that in yeast transformed with pF1_fs plus pFA. The positive-strand RNA1 band in the far left control lane represents DNA-directed transcription from pF1_fs and not viral RdRp-directed replication (43). (B) Selective membrane permeabilization of yeast expressing HA-tagged protein A derivatives. Yeast transformed with pF1_fs plus pFA-C/HA or pFA-N/HA were permeabilized with the indicated detergent and immunostained with mouse anti-CoxIII and rabbit anti-HA as described in the Fig. 1 legend. Representative confocal immunofluorescence images for HA (left images, red), CoxIII (middle images, green), and merged signals (right images) are shown.