Tunicamycin resistant glycosylation of a coronavirus glycoprotein: Demonstration of a novel type of viral glycoprotein

Abstract

Tunicamycin has different effects on the glycosylation of the two envelope glycoproteins of mouse hepatitis virus (MHV), a coronavirus. Unlike envelope glycoproteins of other viruses, the transmembrane glycoprotein El is glycosylated normally in the presence of tunicamycin. This suggests that glycosylation of El does not involve transfer of core oligosaccharides from dolichol pyrophosphate intermediates to asparagine residues, but may occur by O-linked glycosylation of serine or threonine residues. Synthesis of the peplomeric glycoprotein E2 is not readily detectable in the presence of tunicamycin. Inhibition of N-linked glycosylation of E2 by tunicamycin either prevents synthesis or facilitates degradation of the protein moiety of E2. Radiolabeling with carbohydrate precursors and borate gel electrophoresis of glycopeptides show that different oligosaccharide side chains are attached to El and E2. The two coronavirus envelope glycoproteins thus appear to be glycosylated by different mechanisms. In tunicamycin-treated cells, noninfectious virions lacking peplomers are formed at intracytoplasmic membranes and released from the cells. These virions contain normal amounts of nucleocapsid protein and glycosylated El, but lack E2. Thus the transmembrane glycoprotein El is the only viral glycoprotein required for the formation of the viral envelope or for virus maturation and release. The peplomeric glycoprotein E2 appears to be required for attachment to virus receptors on the plasma membrane. The coronavirus envelope envelope glycoprotein El appears to be a novel type of viral glycoprotein which is post-translationally glycosylated by a tunicamycin-resistant process that yields oligosaccharide side chains different from those of N-linked glycoproteins. These findings suggest that El may be particularly useful as a model for studying the biosynthesis, glycosylation, and intracellular transport of O-linked glycoproteins.

Characterization of carbohydrates linked to rubella virus glycoprotein E2

“…No previous reports have demonstrated the presence of O-linked sugars on E2. Olinked oligosaccharides, bound to viral glycoproteins, were first detected in 1981 (Holmes et al, 1981;Niemann & Klenk, 1981 ;Olofsson et al, 1981 ;Shida & Dales, 1981). This study presents three independent lines of evidence indicating that E2 contains carbohydrates of the O-linked type.…”

“…O-linked, or mucin-type carbohydrates, on the other hand, have been demonstrated on a limited number of viral glycoproteins (Holmes et al, 1981;Niemann & Klenk, 1981 ;Olofsson et al, 1981 ;Shida & Dales, 1981;Kari & Gehrz, 1988). N-linked oligosaccharides are synthesized as dolichol-bound precursors, which are common for complex-, hybrid-and high mannose-type carbohydrates.…”

Glycoprotein C of Herpes Simplex Virus Type 1: Characterization of O-linked Oligosaccharides

“…Results obtained in our (Olofsson et al, 1981 a, b) and other (Norrild & Pedersen, 1982;Wenske et al, 1982;Johnson & Spear, 1982 laboratories suggest in addition the occurrence of HSV glycoproteins with oligosaccharides O-glycosidically linked to the polypeptide. This other major class of carbohydrates of membrane glycoproteins has also been observed in vaccinia virus-infected (Shida & Dales, 1981) and mouse hepatitis virus-infected cells (Holmes et aL, 1981 ;Niemann & Klenk, 1981). The O-glycosyl and N-glycosyl classes of carbohydrates differ not only in the type of sugar-protein linkage but also in monosaccharide composition (Kornfeld & Kornfeld, 1976), mode of biosynthesis (Li et al, 1978 ;Strous, 1979) and resistance to glycosylation inhibitors such as tunicamycin (Heifetz et al, 1979;Gahmberg et al, 1980).…”

Coronaviruses: Structure and Genome Expression

“…Addition of antiserum raised against N (but not against S or M) to an in vitro replication system inhibited the synthesis of genome-sized RNA by 90 ~ (Compton et al, 1987). In the presence of tunicamycin, MHV and IBV formed virions which lacked S but contained M and RNP (Holmes et al, 1981 ;Rottier et al, 1981 ;Stern & Sefton, 1982a). In infected cells virion budding occurs at the site of M accumulation (Tooze etaL, 1984).…”

“…The host cell protein probably serves as a virus receptor as cells were protected against infection after incubation with homologous polyclonal and monoclonal antibody (MAb) (K. Holmes, personal communication). Spikeless virions of IBV and MHV are non-infectious (Cavanagh, 1981 ;Stern & Sefton, 1982a;Holmes et al, 1981 ;Rottier et al, 1981). Removal from IBV of $1 (but not of $2) by urea abolished both infectivity and haemagglutinating activity (HA; Cavanagh & Davis, 1986).…”