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

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• 作者：Marcel A Müller (1) (2)
  Lia van der Hoek (3)
  Daniel Voss (2)
  Oliver Bader (2)
  D?rte Lehmann (2)
  Axel R Schulz (2)
  Stephan Kallies (1)
  Tasnim Suliman (4)
  Burtram C Fielding (4)
  Christian Drosten (1)
  Matthias Niedrig (2)
  
• 刊名：Virology Journal
• 出版年：2010
• 出版时间：December 2010
• 年：2010
• 卷：7
• 期：1
• 全文大小：2461KB
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• 作者单位：Marcel A Müller (1) (2)
  Lia van der Hoek (3)
  Daniel Voss (2)
  Oliver Bader (2)
  D?rte Lehmann (2)
  Axel R Schulz (2)
  Stephan Kallies (1)
  Tasnim Suliman (4)
  Burtram C Fielding (4)
  Christian Drosten (1)
  Matthias Niedrig (2)
  
  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. Laboratory of Experimental Virology, University of Amsterdam, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands
  4. Department of Medical Biosciences, University of the Western Cape, Private Bag X17, Bellville, 7535, Republic of South Africa
  
• ISSN：1743-422X

文摘

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.