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Human Coronavirus NL63 Open Reading Frame 3 encodes a virion-incorporated N-glycosylated membrane protein

Marcel A Müller12, Lia van der Hoek3, Daniel Voss2, Oliver Bader2, Dörte Lehmann2, Axel R Schulz2, Stephan Kallies1, Tasnim Suliman4, Burtram C Fielding4, Christian Drosten1* and Matthias Niedrig2

Author Affiliations

1 University of Bonn Medical Centre, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany

2 Robert Koch-Institut, Center for Biological Safety, Nordufer 20, D-13353 Berlin, Germany

3 University of Amsterdam, Laboratory of Experimental Virology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands

4 University of the Western Cape, Department of Medical Biosciences, Private Bag X17 Bellville 7535, Republic of South Africa

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Virology Journal 2010, 7:6  doi:10.1186/1743-422X-7-6

Published: 15 January 2010



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.


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.


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.