Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.
BACKGROUND
Human pathogenic coronavirus NL63 (hCoV-NL63) is a group 1 (alpha) coronavirus commonly associated with respiratory tract infections. In addition to known non-structural and structural proteins all coronaviruses have one or more accessory proteins whose functions are mostly unknown. Our study focuses on hCoV-NL63 open reading frame 3 (ORF 3) which is a highly conserved accessory protein among coronaviruses.


RESULTS
In-silico analysis of the 225 amino acid sequence of hCoV-NL63 ORF 3 predicted a triple membrane-spanning protein. Expression in infected CaCo-2 and LLC-MK2 cells was confirmed by immunofluorescence and Western blot analysis. The protein was detected within the endoplasmatic reticulum/Golgi intermediate compartment (ERGIC) where coronavirus assembly and budding takes place. Subcellular localization studies using recombinant ORF 3 protein transfected in Huh-7 cells revealed occurrence in ERGIC, Golgi- and lysosomal compartments. By fluorescence microscopy of differently tagged envelope (E), membrane (M) and nucleocapsid (N) proteins it was shown that ORF 3 protein colocalizes extensively with E and M within the ERGIC. Using N-terminally FLAG-tagged ORF 3 protein and an antiserum specific to the C-terminus we verified the proposed topology of an extracellular N-terminus and a cytosolic C-terminus. By in-vitro translation analysis and subsequent endoglycosidase H digestion we showed that ORF 3 protein is N-glycosylated at the N-terminus. Analysis of purified viral particles revealed that ORF 3 protein is incorporated into virions and is therefore an additional structural protein.


CONCLUSIONS
This study is the first extensive expression analysis of a group 1 hCoV-ORF 3 protein. We give evidence that ORF 3 protein is a structural N-glycosylated and virion-incorporated protein.
Müller
Marcel A
MA
University of Bonn Medical Centre, Bonn, Germany.
van der Hoek
Lia
L
Voss
Daniel
D
Bader
Oliver
O
Lehmann
Dörte
D
Schulz
Axel R
AR
Kallies
Stephan
S
Suliman
Tasnim
T
Fielding
Burtram C
BC
Drosten
Christian
C
Niedrig
Matthias
M
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D017934:Coronavirus / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000737:chemistry; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry; D006801:Humans; D008247:Lysosomes / Q000737:chemistry; D008253:Macaca mulatta; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008958:Models, Molecular; D016366:Open Reading Frames; D014763:Viral Matrix Proteins / Q000235:genetics* / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000032:analysis; D014771:Virion / Q000737:chemistry*
D006023:Glycoproteins; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins