π 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 Ca. Co-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. Β© 2010 MΓΌller et al; licensee Bio. Med Central Ltd.
keywords
π reading frame (222)
π acid sequence (108)
π amino acid (454)
π viral particles (45)
π structural proteins (197)
π respiratory tract (344)
π highly conserved (80)
π tract infections (83)
π open reading (215)
π accessory proteins (53)
π intermediate compartment (22)
author
π€ MΓΌller, Marcel A.
π€ Van Der Hoek, Lia
π€ Voss, Daniel
π€ Bader, Oliver
π€ Lehmann, DΓΆrte
π€ Schulz, Axel R.
π€ Kallies, Stephan
π€ Suliman, Tasnim
π€ Fielding, Burtram C.
π€ Drosten, Christian
π€ Niedrig, Matthias
year
β° 2010
journal
π Virology Journal
issn
π 1743422X
volume
7
number
page
citedbycount
15
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