Coexpression and Association of the Spike Protein and the Membrane Protein of Mouse Hepatitis Virus

Opstelten, Dirk-Jan E.; Raamsman, M. J. B.; Wolfs, Karin; Horzinek, Marian C.; Rottier, Peter J. M.

doi:10.1007/978-1-4615-1899-0_47

Cited by 6 publications

(3 citation statements)

References 13 publications

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“…Together with the minor E protein it is responsible for the assembly of the coronavirus envelope [15][16][17]. In addition, the M protein directs the incorporation of the S protein [18,19] and the nucleocapsid [20] into the budding particle.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

et al. 2008

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Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), then cloned, sequenced, and compared with each other as well as those ten PEDV reference strains. The M protein genes of six Chinese PEDV isolates consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. The conserved intergenic motif (ATAAAC), as previously recognized in Br1/87, was found in the 5 nucleotides upstream of the initiator ATG of M protein genes of six Chinese PEDV isolates. The hexamer motif was also found in CV777, JMe2, LZC, and QH. The M protein of six isolates had three main transmembrane domains (aa20-38, aa43-65, aa75-97). The M protein of one isolate, CH/IMT/06, had one potential glycosylation site, but those of the other five isolates had two. The glycosylation sequence Asn-Phe-Thr was highly conserved in the M proteins of six PEDV isolates. The six PEDV isolates showed nucleotide sequence homology between 98.8 and 100% and deduced amino acid sequence homology between 98.2 and 100% with each other. The nucleotide and amino acid identity of M protein genes between the six PEDV isolates and ten reference PEDV strains varied from 97.2 to 99.4% and 96.9 to 100%, respectively. On the basis of the phylogenetic relationship of M protein genes, six Chinese PEDV isolates composed of a separate cluster including one Chinese strain JS-2004-02, however, not including the Chinese strain LJB/03. These results demonstrated that there was a new genotype of PEDV prevailing in China.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

et al. 2008

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“…While the budding site of several CoV has been localized at the ERGIC, the viral surface proteins can also be found in downstream compartments of the secretory pathway [8]. M localizes predominantly in the Golgi apparatus [10,11], S is found along the secretory pathway and at the plasma membrane [12,13], and E is detected in perinuclear regions, the ER and Golgi [14][15][16]. S and M are typically glycosylated and it was shown that glycosylation plays an important role in the generation of bioactive protein conformations and influences fusion activity, receptor binding, and antigenic properties of CoV [17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

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

Müller

Hoek

Voß

et al. 2010

Virol J

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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.

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“…It has been well documented that S proteins of several coronaviruses, such as mouse hepatitis virus (MHV), porcine transmissible gastroenteritis virus (TGEV), avian infectious bronchitis virus (IBV), and human severe acute respiratory syndrome coronavirus, play important roles in virus entry, neutralization, and pathogenicity [11][12][13]. The M protein of coronavirus is the most abundant component among the viral envelope proteins and plays important roles in virus assembly by interacting with S and N proteins [14][15][16][17][18][19]. The N protein of coronavirus having a RNA-binding property packages viral genomic RNA into the nucleocapsid of virus particles [20,21].…”

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confidence: 99%

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

et al. 2011

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Previously, we have reported that a serial passage of 83P-5 strain of porcine epidemic diarrhea virus (PEDV) in Vero cells resulted in a growth adaptation of the virus in cultured cells at the 22nd passage. In this study, we further maintained the 83P-5 in Vero cells up to the 100th passage and analyzed changes in the spike (S), membrane (M), and nucleocapsid (N) gene sequences and pathogenicity of the virus at the 34th, 61st, and 100th passage levels. Sequence analyses revealed a strong selection for the S gene of 83P-5 in Vero cells, and virtually all mutations occurring at the 34th and 61st passages had been carried over to the 100th-passaged virus. In contrast, the viral M and N genes showed a strong conservation during the serial passage. Pigs experimentally infected with the 34th- or 61st-passaged virus, but not the 100th-passaged virus, exhibited diarrhea, indicating an attenuation of the 83P-5 at the 100th passage. Interestingly, S protein of the attenuated 100th-passaged 83P-5 showed a remarkable sequence similarity to that of previously reported DR-13 strain of attenuated PEDV that also had been established by serial passage in Vero cells. Further studies will be required to define whether the mutations in the S gene of 83P-5 that had been selected and accumulated during the serial passages are indeed the causalities of the growth adaptation in vitro and the attenuation of virulence in vivo.

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Coexpression and Association of the Spike Protein and the Membrane Protein of Mouse Hepatitis Virus

Cited by 6 publications

References 13 publications

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China

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

Mutations in the spike gene of porcine epidemic diarrhea virus associated with growth adaptation in vitro and attenuation of virulence in vivo

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