Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein
Date
2010Author
Müller, Marcel A.
van der Hoek, Lia
Voss, Daniel
Bader, Oliver
Lehmann, Dörte
Schulz, Axel R.
Kallies, Stephan
Suliman, Tasnim
Fielding, Burtram C.
Drosten, Christian
Niedrig, Matthias
Metadata
Show full item recordAbstract
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 membranespanning
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.