4 research outputs found
Comparative analysis of human coronavirus-NL63 ORF3 protein homologues
It has been reported in some studies that the newly discovered human coronavirus NL-63 (HCoVNL63)
is one of the most common coronaviruses associated with acute respiratory infections. HCoVNL63
was first isolated in 2004 from a 7 month old infant in Holland. The HCoV-NL63 genome
encodes for one accessory protein, ORF3. This reports the computational analysis of human
coronavirus NL63 ORF3 by comparing the amino acid sequences of coronavirus ORF3-homologues.
The HCoV-NL63 ORF3 gene was found to encode a putative protein ~25.6 kDa in size. ORF3 was
predicted to contain three potential transmembrane regions. The amino acid sequence of HCoVNL63
ORF3 was shown to be most similar to HCoV 229E ORF4 (43% identity; 62% similarity).Web of Scienc
Human Coronavirus NL63 Open Reading Frame 3 encodes a virion-incorporated N-glycosylated membrane protein
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 membranespanning
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.Web of Scienc
Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines
A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission