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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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Topology of recombinant FLAG-tagged ORF 3 protein. Recombinant N-terminal tagged FLAG-ORF 3 protein was transiently expressed in HEK-293T cells and localization was analyzed by confocal laser scanning microscopy (cLSM 510 Meta, Zeiss). FLAG-ORF 3 protein was stained with rabbit-anti-ORF 3 recognizing the C-terminus and mouse-anti-FLAG for detection of the FLAG-tagged N-terminus (upper panel). Permeabilized cells (+TritonX100) show colocalized signals mainly in perinuclear regions for protein ORF 3 C-terminus and N-terminus whereas without permeabilization (-TritonX100) only FLAG-tagged N-terminus of protein ORF 3 could be detected at the plasma membrane (lower panel). Bars represent 10 μm.
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Figure 6: Topology of recombinant FLAG-tagged ORF 3 protein. Recombinant N-terminal tagged FLAG-ORF 3 protein was transiently expressed in HEK-293T cells and localization was analyzed by confocal laser scanning microscopy (cLSM 510 Meta, Zeiss). FLAG-ORF 3 protein was stained with rabbit-anti-ORF 3 recognizing the C-terminus and mouse-anti-FLAG for detection of the FLAG-tagged N-terminus (upper panel). Permeabilized cells (+TritonX100) show colocalized signals mainly in perinuclear regions for protein ORF 3 C-terminus and N-terminus whereas without permeabilization (-TritonX100) only FLAG-tagged N-terminus of protein ORF 3 could be detected at the plasma membrane (lower panel). Bars represent 10 μm.

Mentions: Based on our in-silico analyses and in agreement with reports on SARS-CoV ORF 3a protein [36], we hypothesized that the hCoV-NL63 ORF 3 protein N-terminus reached the ER lumen and was eventually exposed on the cell surface. For confirmation, N-terminally FLAG-tagged ORF 3 protein was overexpressed in HEK-293T cells and stained by IFA using monoclonal antibodies against the FLAG tag, or alternatively, a polyclonal antibody against a peptide representing the ORF 3 protein C-terminus. As shown in Figure 6, a perinuclear distribution of fluorescence was observed with both antibodies in permeabilized cells. In non-permeabilized cells, only the anti-FLAG antibody yielded fluorescence at cell surfaces. Unfortunately, there was no complete overlap of signals from both antibodies in fully permeabilized cells in merged fluorescence pictures, most likely due to additional non-specific recognition of non-viral epitopes by the polyclonal antibody against the ORF 3 protein C-terminus. For this reason a clear intracellular localization of the C-terminus in relation to the ER/Golgi membrane could not be formally determined. However, it could be concluded that the N-terminus of the ORF 3 protein was facing towards the extracellular space.


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)

Topology of recombinant FLAG-tagged ORF 3 protein. Recombinant N-terminal tagged FLAG-ORF 3 protein was transiently expressed in HEK-293T cells and localization was analyzed by confocal laser scanning microscopy (cLSM 510 Meta, Zeiss). FLAG-ORF 3 protein was stained with rabbit-anti-ORF 3 recognizing the C-terminus and mouse-anti-FLAG for detection of the FLAG-tagged N-terminus (upper panel). Permeabilized cells (+TritonX100) show colocalized signals mainly in perinuclear regions for protein ORF 3 C-terminus and N-terminus whereas without permeabilization (-TritonX100) only FLAG-tagged N-terminus of protein ORF 3 could be detected at the plasma membrane (lower panel). Bars represent 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Topology of recombinant FLAG-tagged ORF 3 protein. Recombinant N-terminal tagged FLAG-ORF 3 protein was transiently expressed in HEK-293T cells and localization was analyzed by confocal laser scanning microscopy (cLSM 510 Meta, Zeiss). FLAG-ORF 3 protein was stained with rabbit-anti-ORF 3 recognizing the C-terminus and mouse-anti-FLAG for detection of the FLAG-tagged N-terminus (upper panel). Permeabilized cells (+TritonX100) show colocalized signals mainly in perinuclear regions for protein ORF 3 C-terminus and N-terminus whereas without permeabilization (-TritonX100) only FLAG-tagged N-terminus of protein ORF 3 could be detected at the plasma membrane (lower panel). Bars represent 10 μm.
Mentions: Based on our in-silico analyses and in agreement with reports on SARS-CoV ORF 3a protein [36], we hypothesized that the hCoV-NL63 ORF 3 protein N-terminus reached the ER lumen and was eventually exposed on the cell surface. For confirmation, N-terminally FLAG-tagged ORF 3 protein was overexpressed in HEK-293T cells and stained by IFA using monoclonal antibodies against the FLAG tag, or alternatively, a polyclonal antibody against a peptide representing the ORF 3 protein C-terminus. As shown in Figure 6, a perinuclear distribution of fluorescence was observed with both antibodies in permeabilized cells. In non-permeabilized cells, only the anti-FLAG antibody yielded fluorescence at cell surfaces. Unfortunately, there was no complete overlap of signals from both antibodies in fully permeabilized cells in merged fluorescence pictures, most likely due to additional non-specific recognition of non-viral epitopes by the polyclonal antibody against the ORF 3 protein C-terminus. For this reason a clear intracellular localization of the C-terminus in relation to the ER/Golgi membrane could not be formally determined. However, it could be concluded that the N-terminus of the ORF 3 protein was facing towards the extracellular space.

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