Processing Membrane of the Rough Endoplasmic Reticulum Glycoproteins of Rotavirus SA11

The synthesis and oligosaccharide processing of the glycoproteins of SA11 rotavirus in infected Mal04 cells was examined. Rotavirus assembles in the rough endoplasmic reticulum (RER) and encodes two glycoproteins: VP7, a component of the outer viral capsid, and NCVP5, a nonstructural protein. A variety of evidence suggests the molecules are limited to the ER, a location consistent with the high mannose N-linked oligosaccharides modifying these proteins. VP7 and NCVP5 were shown to be integral membrane proteins. In an in vitro translation system supplemented with dog pancreas microsomes, they remained membrane associated after high salt treatment and sodium carbonate-mediated release of microsomal contents. In infected cells, the oligosaccharide processing of these molecules proceeded in a timedependent manner. For VP7, ManBGIcNAc2 and Man6GIcNAc2 were the predominant intracellular species after a 5-min pulse with [3H]mannose and a 90 min chase, while in contrast, trimming of NCVP5 halted at Man~GIcNAc2. VP7 on mature virus was processed to MansGIcNAc2. It is suggested that the c~-mannosidase activities responsible for the formation of these structures reside in the ER. In the presence of the energy inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), processing of VP7 and the vesicular stomatitis virus G protein was blocked at Man~GIcNAc2. After a 20-min chase of [3H]mannose-labeled molecules followed by addition of CCCP, trimming of VP7 could continue while processing of G protein remained blocked. Thus, an energy-sensitive translocation step within the ER may mark the divergence of the processing pathways of these glycoproteins. Membrane glycoproteins locate with a high degree of specificity to a number of subcellular compartments. Due to the complex distribution of proteins normally present in membranes, simple model systems have been sought where the behavior of a few or a single molecular species can be readily defined. To this end, studies of the highly abundant glycoproteins of such membrane maturing viruses as vesicular stomatitis (VSV), ~ sindbis, semliki forest, and influenza have greatly expanded our knowledge of the mechanism of transport to the plasma membrane (reviewed in reference 31). The fine structure of the oligosaccharide moiety serves as a useful ~,lhhreviations used in this paper." act D, actinomycin D: CCCP. carbonyl cyanide m-chlorophenylhydrazone; DME, Dulbecco's modified Eagle's medium; endo H, endo-~-N-acetylglucosaminidase H; PMSF, phenylmethylsulfonyl fluoride; RER, rough endoplasmic reticulum: TBS, Tris-buffered saline; VSV, vesicular stomatitis virus. 1270 marker to indicate the subcellular compartments a specific glycoprotein has traversed. In N-linked glycosylation reactions, a glucose3-mannose9-N-acetylglucosamine2 (Glc3Man9GlcNAc2) core is transferred from a dolichol pyrophosphate cartier to an asparagine residue on the nascent polypeptide chain. This carbohydrate core is extensively trimmed before the addition, in some cases, of terminal sugars such as Nacetylglucosamine, galactose, fucose, and sialic acid (reviewed in reference 26). While the removal of the outer glucoses and at least one mannose residue has been shown to occur on nascent chains of VSV G protein (4) and thus in the rough endoplasmic reticulum (RER), the location, linkage specificity, and even the total number of the different a l, 2-mannosidases acting during the remaining processing reactions are not clear. Evidence has been presented for both RER (6, 17, 20) and Golgi apparatus (50, 53) activities. The subsequent steps, which include the addition of an N-acetylglucosamine THE JOURNAL OF CELL BIOLOGY • VOLUME 101 OCTOBER 1985 127