Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells.

Abstract

Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells.

Surface expression of influenza virus neuraminidase, an amino-terminally anchored viral membrane glycoprotein, in polarized epithelial cells.

“…Thus, the HA contains the information needed to direct its transport in the absence of other influenza virus proteins. This directed transport is not dependent on glycosylation (10,11,17,26), indicating that the required information lies in the polypeptide portion of the glycoprotein. We have now investigated the site of surface expression of the NA gene of influenza virus.…”

“…It has been proposed that the cellular transport machinery recognizes a structural feature in the viral polypeptides, thus determining the ultimate insertion sites for these glycoproteins (25,27). Evidence has been obtained that glycosylation is not required for polarized surface expression of viral glycoproteins (3,11,26). Roth et al (25) reported that expression of the cloned hemagglutinin (HA) gene of influenza A virus, in the absence of other influenza components, occurs almost exclusively on the apical surfaces of primary African green monkey kidney (AGMK) epithelial cells, indicating that the cellular machinery involved in directional transport to apical surfaces recognizes structural features of the HA molecule.…”

“…Influenza A, influenza C, and two paramyxoviruses have been found to selectively bud from the apical surface of polarized cells (12,21,25), whereas the basolateral surface is the preferred site of maturation for vesicular stomatitis virus (VSV) (21) and several retroviruses (27). The maturation sites reflect the sites of insertion of the respective viral glycoproteins (11,20). It has been proposed that the cellular transport machinery recognizes a structural feature in the viral polypeptides, thus determining the ultimate insertion sites for these glycoproteins (25,27).…”

Mechanisms for the incorporation of proteins in membranes and organelles.

“…This work demonstrated that the conformational properties ofthe G protein are affected by glycosylation (72) and that the decreased production of viruses in the presence of tunicamycin is likely to result from aggregation of the nonglycosylated protein at higher temperatures. Furthermore, VSV and influenza virions, whichnormally bud from only the basolateral and apical surfaces of polarized cultured epithelial cells (172), respectively, continue to display the polarized budding after glycosylation is inhibited by tunicamycin (78,177), which indicates that even the finer degrees of segregation of membrane proteins can proceed in the absence of glycosylation.…”

Identification of a Novel Apical Sorting Motif and Mechanism of Targeting of the M2 Muscarinic Acetylcholine Receptor

“…Cells were rinsed three times with PBS at the end of the incubation, fixed, labeled, and stained using goat anti-mouse Alexa-568-conjugated secondary antibody (1:500; Molecular Probes) using nonpermeabilized conditions, or stained with ␤-catenin antibody (1:200; Transduction Laboratories) using permeabilized conditions, as described under "Immunocytochemical Analysis." 2 Receptor in MDCK Cells-Many studies have indicated that N-or O-glycosylation is critical not only for the exit of newly synthesized proteins from the endoplasmic reticulum (33,34) and the Golgi (35) but also for targeting of newly synthesized proteins to the correct membrane domain of polarized epithelial cells (35)(36)(37). Despite mounting evidence indicating that apical sorting can occur independently of N-glycosylation (18, 38 -40), a role for N-glycans in apical sorting continues to be supported by recent evidence that N-glycans alone can mediate apical sorting of the membrane dipeptidase, independent of raft association (41).…”