Differential maturation and subcellular localization of severe acute respiratory syndrome coronavirus surface proteins S, M and E.
Post-translational modifications and correct subcellular localization of viral structural proteins are prerequisites for assembly and budding of enveloped viruses. Coronaviruses, like the severe acute respiratory syndrome-associated virus (SARS-CoV), bud from the endoplasmic reticulum-Golgi intermediate compartment. In this study, the subcellular distribution and maturation of SARS-CoV surface proteins S, M and E were analysed by using C-terminally tagged proteins. As early as 30 min post-entry into the endoplasmic reticulum, high-mannosylated S assembles into trimers prior to acquisition of complex N-glycans in the Golgi. Like S, M acquires high-mannose N-glycans that are subsequently modified into complex N-glycans in the Golgi. The N-glycosylation profile and the absence of O-glycosylation on M protein relate SARS-CoV to the previously described group 1 and 3 coronaviruses. Immunofluorescence analysis shows that S is detected in several compartments along the secretory pathway from the endoplasmic reticulum to the plasma membrane while M predominantly localizes in the Golgi, where it accumulates, and in trafficking vesicles. The E protein is not glycosylated. Pulse-chase labelling and confocal microscopy in the presence of protein translation inhibitor cycloheximide revealed that the E protein has a short half-life of 30 min. E protein is found in bright perinuclear patches colocalizing with endoplasmic reticulum markers. In conclusion, SARS-CoV surface proteins S, M and E show differential subcellular localizations when expressed alone suggesting that additional cellular or viral factors might be required for coordinated trafficking to the virus assembly site in the endoplasmic reticulum-Golgi intermediate compartment.
Nal
Béatrice
B
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Chan
Cheman
C
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Kien
Francois
F
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Siu
Lewis
L
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Tse
Jane
J
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Chu
Kid
K
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Kam
Jason
J
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
Staropoli
Isabelle
I
Unité d'Immunologie Virale, Institut Pasteur, 25 rue du Dr Roux, Paris, France.
Crescenzo-Chaigne
Bernadette
B
Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, 25 rue du Dr Roux, Paris, France.
Escriou
Nicolas
N
Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, 25 rue du Dr Roux, Paris, France.
van der Werf
Sylvie
S
Unité de Génétique Moléculaire des Virus Respiratoires, Institut Pasteur, 25 rue du Dr Roux, Paris, France.
Yuen
Kwok-Yung
KY
Department of Microbiology, The University of Hong Kong, Hong Kong, China.
Altmeyer
Ralf
R
HKU-Pasteur Research Centre, 8 Sassoon Road, Hong Kong, China.
D000818:Animals; D000086862:Coronavirus M Proteins; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D008358:Mannose / Q000032:analysis; D008562:Membrane Glycoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D011134:Polysaccharides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D045473:Severe acute respiratory syndrome-related coronavirus / Q000254:growth & development*; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014763:Viral Matrix Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*
D000086862:Coronavirus M Proteins; C000706237:M protein, SARS-CoV; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D008358:Mannose