Mutational changes in the vesicular stomatitis virus glycoprotein affect the requirement of carbohydrate in morphogenesis

The role of carbohydrate in the morphogenesis of vesicular stomatitis virus was studied, using the antibiotic tunicamycin to inhibit glycosylation. It has been reported previously (Gibson et al., J. Biol. Chem. 254:3600-3607, 1979) that the San Juan strain of vesicular stomatitis virus requires carbohydrate for efficient migration of the glycoprotein (G) to the cell surface and for virion formation, whereas the prototype or Orsay strain of vesicular stomatitis virus is less stringent in its carbohydrate requirement at 30 degrees C. However, there are many differences between the two strains. We found that mutational changes within the G protein of the same strain of virus (prototype or Orsay) alters the requirement for carbohydrate at 30 degrees C. Group V or G protein mutants tsO45 and tsO44, like their prototype parent, did not require carbohydrate for efficient morphogenesis. In contrast, the G protein of another group V mutant, tsO110, was totally dependent upon carbohydrate addition for migration to the cell surface. Furthermore, no tsO110 particles were released in the absence of glycosylation. The wild-type prototype strain did require carbohydrate at 39.5 degrees C for insertion of the G protein into the plasma membrane and virion formation. However, a pseudorevertant of tsO44 (tsO44R), unlike the prototype parent, no longer exhibited this temperature-sensitive requirement for carbohydrate. At 39.5 degrees C in the presence of tunicamycin, tsO44R-infected cells released normal yields of particles and the unglycosylated G reached the cell surface very efficiently. In contrast to tsO110, which absolutely requires carbohydrate, mutational change in the tsO44R G protein has eliminated the requirement for carbohydrate. Thus, simple mutational changes, as opposed to many changes in the molecule, are sufficient to alter the carbohydrate requirement.</jats:p

Vesicular stomatitis virus glycoprotein is anchored to intracellular membranes near its carboxyl end and is proteolytically cleaved at its amino terminus

The intracellular vesicular stomatitis virus glycoprotein (G) is inserted into membranes such that a small portion of one end of the molecule is exposed on the cytoplasmic surface of the endoplasmic reticulum and is susceptible to proteolytic digestion (T.G. Morrison, C.O. McQuain, and D. Simpson, J. Virol. 28:368-374). We have determined that this region of the G protein contains two methionyl tryptic peptides. The methionyl tryptic peptides of the G protein have been ordered by the use of the antibiotic pactamycin, and the two methionyl tryptic peptides removed by proteolytic digestion of intracellular G protein have been shown to be derived from the carboxyl terminal end of the protein. In addition, we have found that the unglycosylated G protein synthesized in a reticulocyte cell-free reaction migrates on polyacrylamide gels slightly slower than the unglycosylated G protein synthesized in tunicamycin-treated infected cells. We have also compared these G proteins derived from different sources by partial proteolysis (D.W. Cleveland, S.G. Fischer, M.W. Kirschner, and V.K. Laemmli, J. Biol. Chem. 252:1102-1106) and by chymotryptic peptide analysis. We have found minor differences between the two proteins consistent with the removal of 10 to 15 amino acids from the amino terminus of the intracellular G protein.</jats:p

Characterization of the soluble glycoprotein released from vesicular stomatitis virus-infected cells

Vesicular stomatitis virus-infected Chinese hamster ovary cells release into the extracellular medium a soluble form of the vesicular stomatitis virus glycoprotein (G protein) termed Gs (Kang and Prevec, Virology 46:678-680, 1971). The properties of this molecule and the cellular site at which it is generated were characterized. By comparing the sizes and the peptide maps of the unglycosylated forms of G and Gs, we found that between 5,000 and 6,000 daltons of the carboxy-terminal end of the G protein is cleaved to generate the Gs molecule. This truncated molecule contains no fatty acid. Gs released from cells grown at 39 degrees C migrated on polyacrylamide gels slightly slower than Gs released at 30 degrees C. The unglycosylated form of Gs also showed this size difference. Furthermore, unglycosylated Gs was resolved into two species upon isoelectric focusing: the relative amounts of the two species depended upon the temperature at which infected cells were incubated. Full-sized unglycosylated virus-associated G also was resolved into two species, but the more basic form predominated at both 30 and 39 degrees C. The appearance of Gs in the extracellular medium depended upon the presence of stable, full-sized G at the cell surface. The amount of Gs released was quantitated in seven different situations in which the migration of G to the cell surface was inhibited. In all cases, the amount of Gs released was also decreased. In addition, incubation of cells surface labeled with 125I resulted in the release of 125I-labeled Gs protein, as well as full-sized G protein. These results suggest that Gs is generated primarily by proteolytic cleavage of plasma membrane-associated G at a site in the molecule just amino terminal to the membrane-spanning region of the molecule.</jats:p

Fatty acid modification of Newcastle disease virus glycoproteins

The fatty acid acylation of Newcastle disease virus hemagglutininin-neuraminidase and fusion glycoproteins was assayed. [3H]palmitate label was associated with cytoplasmic fusion proteins (F0 and F1) and virion-associated F1. In contrast, there was no detectable [3H]palmitate label associated with the hemagglutin-neuraminidase protein in Newcastle disease virus-infected Chinese hamster ovary cells or chicken embryo cells or in virions released from these cells. Thus, fatty acid modification may not be important for the maturation of some glycoproteins.</jats:p