Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins.

Abstract

Selective binding of lipid to glycoprotein was detected when [3Hjpalmitate-labeled Sindbis virus particles or viral-infected cells were disrupted by heating with sodium dodecyl sulfate, and glycoproteins were isolated by electrophoresis in sodium dodecyl sulfate/lO% polyacrylamide gels. The smaller glycoprotein (E2) retained 2 to 3 times more labeled lipid than did the larger El glycoprotein, and the cell-associated glycoprotein precursor (PE2) bound even less lipid. No lipid was associated with the nonglycosylated glycoproteins that accumulated in infected cells treated with tunicamycin. The labeled lipid remained bound to the glycoproteins after exhaustive extraction with chloroform/methanol of virus particles, infected-cell extracts, or isolated glycoproteins, but it could be extracted by chloroform/methanol after treating glycoproteins with mild alkali. Analysis by gas/liquid chromatography showed that 60% of the label was in palmitate and the balance of label was distributed between oleate and stearate. There were approximately 2 mol of fatty acid bound per mol-of El glycoprotein. Proteolysis of the fatty acid-labeled glycoprotein with pepsin, thermolysin, and Pronase degraded the polypeptide to fragments that retained'the fatty acids in an alkali-labile state. These data suggest that a covalent attachment of fatty acid may occur during maturation of the viral glycoproteins.We have been studying the formation and processing of viralspecific proteins in tissue culture cells infected with Sindbis virus, a small enveloped RNA virus of the family togaviridae. A critical stage in the late events of replication of this virus is a proteolytic cleavage that alters the structure of one of the two viral-specific glycoproteins (1), and this reaction is essential for release of virus particles from the cell (2-5). In the process of studying the glycoprotein cleavage reaction we made the serendipitous observation that glycoproteins isolated by sodium dodecyl sulfate (NaDodSO4)/10% polyacrylamide gel electrophoresis after disruption of virus by NaDodSO4 appeared to retain small amounts of lipid (6). We have investigated this glycoprotein-lipid complex in more detail and describe our initial results in this communication.Two significant observations have emerged from these studies: (i) the lipid remained associated with glycoprotein despite strong denaturing conditions and proteolytic digestion but could be recovered as the methyl ester of fatty acid after mild alkali treatment in methanol, a procedure that also released fatty acid from proteolytic digestion products of the glycoproteins, and (ii) this binding of fatty acid was affected by changes in the glycoprotein structure. These data indicate that fatty acid may be covalently attached to viral glycoprotein and this modification may play an important role in the maturation of viral glycoprotein. Preliminary reports of this work have been presented (7,8).The publication costs of this article were defrayed in part by page charge payment. This article must there...

Structural Protein Relationships Among Eastern Equine Encephalitis Viruses

“…(i) Oligosaccharide side-chain diversity (microheterogeneity), which is characteristically found on glycoproteins, may not be responsible for the differential electrophoretic profiles of EEE virus isolates. Rather, variation in polypeptide structure (too subtle to allow detection by partial proteolysis) or other posttranslational modifications of alphaviruses, such as phosphorylation, acylation or sulphation (Waite et al, 1974;Pinter & Compans, 1975;Schmidt et al, 1979) may have contributed to the glycoprotein banding patterns we observed. (ii) Despite prior dissociation of virus with SDS and 2-ME, cleavage of the carbohydrate moieties from the polypeptide backbone with endoglycosidases may have been incomplete, which could occur if carbohydrate~eptide bonds were not fully accessible to the enzyme (McCarthy & Harrison, 1977;Hsieh et aL, 1983a, b;Chu, 1986;Hirani, 1987).…”

Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA

“…Although there is a high degree of sequence conservation in the first part (10 out of 14 residues), only the hydrophobic character is maintained in the last 10 residues of that section, while the nucleotide sequence is almost totally different. Furthermore, the region contains two conserved serine residues (position 190 and 194), one of which (or both) could be the fatty acid attachment site(s) (Schmidt, Bracha and Schlesinger, 1979). A stretch of 24 amino acids which spans the lipid bilayer would be analogous to the peptide of 22 amino acids proposed as the intramembranous segment of human erythrocyte glycophorin A (Furthmayr et al, 1978) and to a stretch of 25 amino acids forming the portion of the HLA-A and HLA-B antigen heavy chain spanning the membrane (Springer and Strominger, 1976).…”

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

“…A recently discovered modification is the covalent attachment of long-chain fatty acid to eucaryotic cell membrane proteins (9). Although initially detected in the glycoproteins of two enveloped animal viruses, vesicular stomatitis virus and Sindbis virus (21,22), fatty acid-acylated proteins have been found in several other enveloped viruses (20) as well as in cultured cells (19) and intact animals (1). In the study of vesicular stomatitis virus glycoprotein biosynthesis, Schmidt and Schlesinger (23) related the time of fatty acid incorporation to oligosaccharide processing in the glycoprotein and found that acylation occurred shortly before the protein became endoglycosidase H (endo H) resistant.…”