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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 overexpressed hCoV-NL63 proteins in Huh-7 and HEK-293T cells. Confocal laser scanning microscopy on cells co-expressing GFP-E, GFP-M, GFP-N, respectively, together with FLAG-ORF 3. (A), Huh-7 cells. The green panels on the left show GFP fluorescence from overexpressed E, M, and N proteins. Red pictures in the next column show Cy3 fluorescence from anti-FLAG staining of overexpressed FLAG-ORF 3 fusion protein. Blue pictures show Cy5 fluorescence from staining of the ER-Golgi intermediate compartment (ERGIC) (refer to Materials and Methods section for antibodies and staining technique). Yellow areas in the right hand column represent colocalization of the GFP-proteins with FLAG-ORF 3 whereas white regions in merged pictures show colocalization of GFP proteins with FLAG-ORF 3 within the ERGIC. GFP-E and M show excessive colocalization with FLAG-ORF 3 especially within the ERGIC in both cell lines. GFP-N partially colocalizes with FLAG-ORF 3 mainly within the ERGIC. Analysis was performed with the help of a confocal laser scanning microscope (cLSM 510 Meta, Zeiss). Bars represent 20 μm. (B), to exclude altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated in HEK-293T cells using FLAG-ORF 3 in combination with HA tagged E, M and N proteins. Bars represent 10 μm.
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Figure 4: Subcellular localization of overexpressed hCoV-NL63 proteins in Huh-7 and HEK-293T cells. Confocal laser scanning microscopy on cells co-expressing GFP-E, GFP-M, GFP-N, respectively, together with FLAG-ORF 3. (A), Huh-7 cells. The green panels on the left show GFP fluorescence from overexpressed E, M, and N proteins. Red pictures in the next column show Cy3 fluorescence from anti-FLAG staining of overexpressed FLAG-ORF 3 fusion protein. Blue pictures show Cy5 fluorescence from staining of the ER-Golgi intermediate compartment (ERGIC) (refer to Materials and Methods section for antibodies and staining technique). Yellow areas in the right hand column represent colocalization of the GFP-proteins with FLAG-ORF 3 whereas white regions in merged pictures show colocalization of GFP proteins with FLAG-ORF 3 within the ERGIC. GFP-E and M show excessive colocalization with FLAG-ORF 3 especially within the ERGIC in both cell lines. GFP-N partially colocalizes with FLAG-ORF 3 mainly within the ERGIC. Analysis was performed with the help of a confocal laser scanning microscope (cLSM 510 Meta, Zeiss). Bars represent 20 μm. (B), to exclude altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated in HEK-293T cells using FLAG-ORF 3 in combination with HA tagged E, M and N proteins. Bars represent 10 μm.

Mentions: For SARS-CoV ORF 3a protein, colocalization with the structural proteins S, E, and M, but only partial colocalization with N has been suggested [36]. To investigate colocalization of NL63-ORF 3 protein with structural proteins, an expression plasmid containing ORF 3 with an N-terminal FLAG-tag epitope was co-transfected with vectors coding for green fluorescent protein (GFP) fused to hCoV-NL63 E, M and N proteins, respectively. Expression of proteins with correct molecular weights was confirmed by Western blot analysis (data not shown). The ERGIC compartment was stained in transfected cells as described above. As shown in Figure 4, GFP-E and GFP-M both showed extensive colocalization with FLAG-ORF 3 protein. Protein complexes were localized predominantly within the ERGIC, represented by white areas in Figure 4. GFP-N had primarily a cytosolic distribution but there were small areas of colocalization with FLAG-ORF 3 protein, within the ERGIC compartment. All experiments were done in Huh-7 cells supportive of hCoV-NL63 replication, but these same findings were also confirmed in another cell line, human embryonic kidney (HEK)-293T (data not shown).


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 overexpressed hCoV-NL63 proteins in Huh-7 and HEK-293T cells. Confocal laser scanning microscopy on cells co-expressing GFP-E, GFP-M, GFP-N, respectively, together with FLAG-ORF 3. (A), Huh-7 cells. The green panels on the left show GFP fluorescence from overexpressed E, M, and N proteins. Red pictures in the next column show Cy3 fluorescence from anti-FLAG staining of overexpressed FLAG-ORF 3 fusion protein. Blue pictures show Cy5 fluorescence from staining of the ER-Golgi intermediate compartment (ERGIC) (refer to Materials and Methods section for antibodies and staining technique). Yellow areas in the right hand column represent colocalization of the GFP-proteins with FLAG-ORF 3 whereas white regions in merged pictures show colocalization of GFP proteins with FLAG-ORF 3 within the ERGIC. GFP-E and M show excessive colocalization with FLAG-ORF 3 especially within the ERGIC in both cell lines. GFP-N partially colocalizes with FLAG-ORF 3 mainly within the ERGIC. Analysis was performed with the help of a confocal laser scanning microscope (cLSM 510 Meta, Zeiss). Bars represent 20 μm. (B), to exclude altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated in HEK-293T cells using FLAG-ORF 3 in combination with HA tagged E, M and N proteins. Bars represent 10 μm.
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Figure 4: Subcellular localization of overexpressed hCoV-NL63 proteins in Huh-7 and HEK-293T cells. Confocal laser scanning microscopy on cells co-expressing GFP-E, GFP-M, GFP-N, respectively, together with FLAG-ORF 3. (A), Huh-7 cells. The green panels on the left show GFP fluorescence from overexpressed E, M, and N proteins. Red pictures in the next column show Cy3 fluorescence from anti-FLAG staining of overexpressed FLAG-ORF 3 fusion protein. Blue pictures show Cy5 fluorescence from staining of the ER-Golgi intermediate compartment (ERGIC) (refer to Materials and Methods section for antibodies and staining technique). Yellow areas in the right hand column represent colocalization of the GFP-proteins with FLAG-ORF 3 whereas white regions in merged pictures show colocalization of GFP proteins with FLAG-ORF 3 within the ERGIC. GFP-E and M show excessive colocalization with FLAG-ORF 3 especially within the ERGIC in both cell lines. GFP-N partially colocalizes with FLAG-ORF 3 mainly within the ERGIC. Analysis was performed with the help of a confocal laser scanning microscope (cLSM 510 Meta, Zeiss). Bars represent 20 μm. (B), to exclude altered subcellular localization contributed by the fusion tags on the overexpressed structural proteins, experiments were repeated in HEK-293T cells using FLAG-ORF 3 in combination with HA tagged E, M and N proteins. Bars represent 10 μm.
Mentions: For SARS-CoV ORF 3a protein, colocalization with the structural proteins S, E, and M, but only partial colocalization with N has been suggested [36]. To investigate colocalization of NL63-ORF 3 protein with structural proteins, an expression plasmid containing ORF 3 with an N-terminal FLAG-tag epitope was co-transfected with vectors coding for green fluorescent protein (GFP) fused to hCoV-NL63 E, M and N proteins, respectively. Expression of proteins with correct molecular weights was confirmed by Western blot analysis (data not shown). The ERGIC compartment was stained in transfected cells as described above. As shown in Figure 4, GFP-E and GFP-M both showed extensive colocalization with FLAG-ORF 3 protein. Protein complexes were localized predominantly within the ERGIC, represented by white areas in Figure 4. GFP-N had primarily a cytosolic distribution but there were small areas of colocalization with FLAG-ORF 3 protein, within the ERGIC compartment. All experiments were done in Huh-7 cells supportive of hCoV-NL63 replication, but these same findings were also confirmed in another cell line, human embryonic kidney (HEK)-293T (data not shown).

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