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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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Comparison of ORF 3 protein in viral infection and overexpression by Western blot. (A), LLC-MK2 cells were inoculated with hCoV-NL63 (MOI 0.01) and analyzed by Western blot after 4 days using antibodies against the ORF 3 protein C-terminus (top) and against M (bottom). The bands named ORF 30 and M0 are corresponding to the predicted molecular weights of both proteins (26 kDa). Larger bands ORF 3 g and Mg were assumed to be the result of posttranslational modification. (B, left panel): HEK-293T cells transfected with N-terminally FLAG-tagged ORF 3 do not show signs of posttranslational modification as observed in (A). (B, right panel): overexpression of ORF 3 protein in the same system without an N-terminal fusion tag reconstitutes the additional band of higher molecular weight observed in infected cells. The "mock" lane represents a control transfected with an empty vector.
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Figure 5: Comparison of ORF 3 protein in viral infection and overexpression by Western blot. (A), LLC-MK2 cells were inoculated with hCoV-NL63 (MOI 0.01) and analyzed by Western blot after 4 days using antibodies against the ORF 3 protein C-terminus (top) and against M (bottom). The bands named ORF 30 and M0 are corresponding to the predicted molecular weights of both proteins (26 kDa). Larger bands ORF 3 g and Mg were assumed to be the result of posttranslational modification. (B, left panel): HEK-293T cells transfected with N-terminally FLAG-tagged ORF 3 do not show signs of posttranslational modification as observed in (A). (B, right panel): overexpression of ORF 3 protein in the same system without an N-terminal fusion tag reconstitutes the additional band of higher molecular weight observed in infected cells. The "mock" lane represents a control transfected with an empty vector.

Mentions: Posttranslational modification of the ORF 3 protein in hCoV-NL63-infected LLC-MK2 cells was analyzed by Western blot. The M protein which had a very similar predicted molecular mass of 26 kDa (Table 1) served as a control. As expected, the M protein and a protein corresponding to ORF 3 protein migrated at corresponding heights in Western blots (Figure 5A). Both proteins showed additional bands at slightly higher molecular mass, consistent with posttranslational modification. In contrast to virus-infected cells, cells overexpressing ORF 3 protein from plasmid with an N-terminal FLAG epitope showed only a single band in Western blot whose migration was consistent with the hypothetical unglycosylated form (Figure 5B, left panel). It was assumed that glycosylation at the predicted N-glycosylation site at position 16 (Table 1) might be ablated in the overexpressed protein, due to presence of the N-terminal epitope tag. Indeed, recombinant ORF 3 (rORF 3) protein without any tag and overexpressed in the same cells from the same vector showed both forms, identical to those observed in virus-infected cells (Figure 5B, right panel). To determine whether N-terminal glycosylation was to be expected at position 16, the membrane topology of the N-terminus was examined next.


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)

Comparison of ORF 3 protein in viral infection and overexpression by Western blot. (A), LLC-MK2 cells were inoculated with hCoV-NL63 (MOI 0.01) and analyzed by Western blot after 4 days using antibodies against the ORF 3 protein C-terminus (top) and against M (bottom). The bands named ORF 30 and M0 are corresponding to the predicted molecular weights of both proteins (26 kDa). Larger bands ORF 3 g and Mg were assumed to be the result of posttranslational modification. (B, left panel): HEK-293T cells transfected with N-terminally FLAG-tagged ORF 3 do not show signs of posttranslational modification as observed in (A). (B, right panel): overexpression of ORF 3 protein in the same system without an N-terminal fusion tag reconstitutes the additional band of higher molecular weight observed in infected cells. The "mock" lane represents a control transfected with an empty vector.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Comparison of ORF 3 protein in viral infection and overexpression by Western blot. (A), LLC-MK2 cells were inoculated with hCoV-NL63 (MOI 0.01) and analyzed by Western blot after 4 days using antibodies against the ORF 3 protein C-terminus (top) and against M (bottom). The bands named ORF 30 and M0 are corresponding to the predicted molecular weights of both proteins (26 kDa). Larger bands ORF 3 g and Mg were assumed to be the result of posttranslational modification. (B, left panel): HEK-293T cells transfected with N-terminally FLAG-tagged ORF 3 do not show signs of posttranslational modification as observed in (A). (B, right panel): overexpression of ORF 3 protein in the same system without an N-terminal fusion tag reconstitutes the additional band of higher molecular weight observed in infected cells. The "mock" lane represents a control transfected with an empty vector.
Mentions: Posttranslational modification of the ORF 3 protein in hCoV-NL63-infected LLC-MK2 cells was analyzed by Western blot. The M protein which had a very similar predicted molecular mass of 26 kDa (Table 1) served as a control. As expected, the M protein and a protein corresponding to ORF 3 protein migrated at corresponding heights in Western blots (Figure 5A). Both proteins showed additional bands at slightly higher molecular mass, consistent with posttranslational modification. In contrast to virus-infected cells, cells overexpressing ORF 3 protein from plasmid with an N-terminal FLAG epitope showed only a single band in Western blot whose migration was consistent with the hypothetical unglycosylated form (Figure 5B, left panel). It was assumed that glycosylation at the predicted N-glycosylation site at position 16 (Table 1) might be ablated in the overexpressed protein, due to presence of the N-terminal epitope tag. Indeed, recombinant ORF 3 (rORF 3) protein without any tag and overexpressed in the same cells from the same vector showed both forms, identical to those observed in virus-infected cells (Figure 5B, right panel). To determine whether N-terminal glycosylation was to be expected at position 16, the membrane topology of the N-terminus was examined next.

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