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

Abstract

BackgroundHuman 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.ResultsIn-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.ConclusionsThis 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.

Porcine epidemic diarrhea virus ORF3 protein causes endoplasmic reticulum stress to facilitate autophagy

“…Similar with some other accessory proteins of Coronaviruses, such as severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a protein (Chan et al, 2009), and human pathogenic coronavirus NL63 (hCoV-NL63) ORF3 protein (Muller et al, 2010), the PEDV ORF3 protein has been also testified to encode an ion channel protein that regulates virus production, assembly and release. The ion-channel function is dependent on TMDs, because the ion channel activity is reduced in ORF3 protein of attenuated typical PEDV while its amino acid substitution or deletion (Wang et al, 2012).…”

Molecular Basis of Coronavirus Virulence and Vaccine Development

“…1). These genes encode transmembrane proteins that differ significantly in both their sequences and predicted structures (Muller et al, 2010;Zhang et al, 2014bZhang et al, , 2015, but may have related functions, such as ion channel activity and morphogenesis (Donaldson et al, 2008;Zhang et al, 2014bZhang et al, , 2015. In fact, the SARS-CoV 3a protein, HCoV-NL63 protein 3, and the HCoV-229E 4a protein are each able to complement in trans the absence of HCoV-OC43 ns12.9 protein .…”

Targeting B cell responses in universal influenza vaccine design

“…Besides the conserved HA stalk region, other studies have emerged to show that the conserved extracellular domain of the viral matrix 2 protein (M2e) is able to elicit cross-reactive antibodies that can confer protection in mice challenged with H5N1 or H1N1 viruses [45, 46]. …”