PMID: 16474139

 

    Legend: Sugar

Title : Glycosylation of the severe acute respiratory syndrome coronavirus triple-spanning membrane proteins 3a and M

Abstract :
  1. The severe acute respiratory syndrome coronavirus ( SARS-CoV) open reading frame 3a protein has recently been shown to be a structural protein
  2. The protein is encoded by one of the so-called group-specific genes and has no sequence homology with any of the known structural or group-specific proteins of coronaviruses
  3. It does, however, have several similarities to the coronavirus M proteins ; (i) they are triple membrane spanning with the same topology, (ii) they have similar intracellular localizations (predominantly Golgi), (iii) both are viral structural proteins , and (iv) they appear to interact with the E and S proteins , as well as with each other
  4. The M protein plays a crucial role in coronavirus assembly and is glycosylated in all coronaviruses, either by N-linked or by O-linked oligosaccharides
  5. The conserved glycosylation of the coronavirus M proteins and the resemblance of the 3a protein to them led us to investigate the glycosylation of these two SARS-CoV membrane proteins
  6. The proteins were expressed separately using the vaccinia virus T7 expression system, followed by metabolic labeling
  7. Pulse-chase analysis showed that both proteins were modified, although in different ways
  8. While the M protein acquired cotranslationally oligosaccharides that could be removed by PNGaseF, the 3a protein acquired its modifications posttranslationally , and they were not sensitive to the N-glycosidase enzyme
  9. The SARS-CoV 3a protein , however, was demonstrated to contain sialic acids , indicating the presence of oligosaccharides
  10. O-glycosylation of the 3a protein was indeed confirmed using an in situ O-glycosylation assay of endoplasmic reticulum-retained mutants
  11. In addition, we showed that substitution of serine and threonine residues in the ectodomain of the 3a protein abolished the addition of the O-linked sugars
  12. Thus, the SARS-CoV 3a protein is an O-glycosylated glycoprotein , like the group 2 coronavirus M proteins but unlike the SARS-CoV M protein , which is N glycosylated