Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein

The gene encoding the VP7 protein was inserted into the vector pJC119 under the control of the SV40 late promoter to generate the plasmid pJC9 (as described in the Materials and Methods). The expression of the VP7 protein from this gene was examined in transfected COS 7 cells to confirm the ER location of this protein. An indirect immunofluorescent procedure using a secondary fluorescein-coupled goat antirabbit Ig and a primary monospecific polyclonal rabbit anti. serum was used to localize VP7. A concomitant display of WGA, conjugated to rbodamine (R-WGA), and known to specifically bind to sialic acid and terminal glucosamines in the Golgi apparatus (40), allowed us to determine if VP7 was present in this organelle. The immunolocalizafion of VP7 expressed from pJC9 shows a distinct, arborizing, reticular pattern of fluorescein staining radiating from the nucleus, and a perinuclear concentration of stained, reticular material (Fig. These are composed of several viral proteins and nucleic acid which assemble into viral cores (arrows) that bud from the periphery of the viroplasm into the rough endoplasmic reticulum (RER). Immature viral particles with surrounding ER membrane appear in the lumen of the ER (large black arrowheads), as do numerous, nonenveloped, mature forms of the virion which are devoid of membrane (small black arrowheads). Several viral particles are indicated which apparently are in the process of losing the surrounding membrane (white arrowheads); possible membrane remnants can also be seen (m). M, mitochondrion, x 41,500. 5). This probably corresponds to the RER and to transitional elements of ER, spatially related to, but exclusive of, the Golgi apparatus (see Fig. 4b and inset). Nuclear staining is also evident. Fig. 5 b shows the same cell stained with R-WGA. A staining pattern consisting of punctate material and a perinuclear localization, probably coincident with part of the Golgi apparatus and distinct from that of the VP7 localization, is seen. Thus, VP7 protein expressed from the wild-type gene in pJC9 appears to localize to the ER and does not appear to reach the Golgi apparatus.