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Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

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.

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Subcellular localization of viral proteins in hCoV-NL63 infected CaCo-2 and LLC-MK2 cells by immunofluorescence assay. Confocal laser scanning microscopy on CaCo-2 (A) and LLC-MK2 cells (B) infected with hCoV-NL63. Left panels: staining with anti-ORF 3 and anti-M protein rabbit antisera (only in B) and detection by fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit antibody (green). Middle panels: detection of co-staining of the same cells with mouse-anti-ERGIC-53 mAB (Axxora) and detection with rhodamine-labelled goat-anti-mouse antibody. Yellow signals in merged pictures (right panels) show colocalization. Bars represent 20 μm.
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Figure 2: Subcellular localization of viral proteins in hCoV-NL63 infected CaCo-2 and LLC-MK2 cells by immunofluorescence assay. Confocal laser scanning microscopy on CaCo-2 (A) and LLC-MK2 cells (B) infected with hCoV-NL63. Left panels: staining with anti-ORF 3 and anti-M protein rabbit antisera (only in B) and detection by fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit antibody (green). Middle panels: detection of co-staining of the same cells with mouse-anti-ERGIC-53 mAB (Axxora) and detection with rhodamine-labelled goat-anti-mouse antibody. Yellow signals in merged pictures (right panels) show colocalization. Bars represent 20 μm.

Mentions: To analyze the expression of ORF 3 protein during viral replication, colon carcinoma cells (CaCo-2) and Rhesus monkey kidney cells (LLC-MK2) cells were infected with hCoV-NL63 and an immunofluorescence assay (IFA) was done after two and four days, respectively. A rabbit polyclonal antiserum raised against a peptide representing the C-terminal aa 211-225 of the predicted ORF 3 protein yielded fluorescence in the cytoplasm as shown in Figure 2A and 2B (upper panel). Because colocalization of SARS-CoV ORF 3a protein with the ERGIC has been reported [36,49], the same cells were counterstained with a murine monoclonal antibody against the ERGIC53 marker protein. As shown in Figure 2A and 2B (upper panel) colocalization was observed in CaCo-2 and LLC-MK2 cells. Because overlapping subcellular localization was reported for SARS-CoV proteins 3a and M [50], it was analyzed whether hCoV-NL63 ORF 3 and M proteins were located in the same compartment. As shown in Figure 2B (bottom panel), a strong colocalization was also seen for anti-NL63 M and anti-ERGIC53 signals.


Human coronavirus NL63 open reading frame 3 encodes a virion-incorporated N-glycosylated membrane protein.

Müller MA, van der Hoek L, Voss D, Bader O, Lehmann D, Schulz AR, Kallies S, Suliman T, Fielding BC, Drosten C, Niedrig M - Virol. J. (2010)

Subcellular localization of viral proteins in hCoV-NL63 infected CaCo-2 and LLC-MK2 cells by immunofluorescence assay. Confocal laser scanning microscopy on CaCo-2 (A) and LLC-MK2 cells (B) infected with hCoV-NL63. Left panels: staining with anti-ORF 3 and anti-M protein rabbit antisera (only in B) and detection by fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit antibody (green). Middle panels: detection of co-staining of the same cells with mouse-anti-ERGIC-53 mAB (Axxora) and detection with rhodamine-labelled goat-anti-mouse antibody. Yellow signals in merged pictures (right panels) show colocalization. Bars represent 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819038&req=5

Figure 2: Subcellular localization of viral proteins in hCoV-NL63 infected CaCo-2 and LLC-MK2 cells by immunofluorescence assay. Confocal laser scanning microscopy on CaCo-2 (A) and LLC-MK2 cells (B) infected with hCoV-NL63. Left panels: staining with anti-ORF 3 and anti-M protein rabbit antisera (only in B) and detection by fluorescein isothiocyanate (FITC)-labelled goat-anti-rabbit antibody (green). Middle panels: detection of co-staining of the same cells with mouse-anti-ERGIC-53 mAB (Axxora) and detection with rhodamine-labelled goat-anti-mouse antibody. Yellow signals in merged pictures (right panels) show colocalization. Bars represent 20 μm.
Mentions: To analyze the expression of ORF 3 protein during viral replication, colon carcinoma cells (CaCo-2) and Rhesus monkey kidney cells (LLC-MK2) cells were infected with hCoV-NL63 and an immunofluorescence assay (IFA) was done after two and four days, respectively. A rabbit polyclonal antiserum raised against a peptide representing the C-terminal aa 211-225 of the predicted ORF 3 protein yielded fluorescence in the cytoplasm as shown in Figure 2A and 2B (upper panel). Because colocalization of SARS-CoV ORF 3a protein with the ERGIC has been reported [36,49], the same cells were counterstained with a murine monoclonal antibody against the ERGIC53 marker protein. As shown in Figure 2A and 2B (upper panel) colocalization was observed in CaCo-2 and LLC-MK2 cells. Because overlapping subcellular localization was reported for SARS-CoV proteins 3a and M [50], it was analyzed whether hCoV-NL63 ORF 3 and M proteins were located in the same compartment. As shown in Figure 2B (bottom panel), a strong colocalization was also seen for anti-NL63 M and anti-ERGIC53 signals.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Bonn Medical Centre, Bonn, Germany.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus