Intracellular processing of the porcine coronavirus transmissible gastroenteritis virus spike protein expressed by recombinant vaccinia virus.
The Spike (S) protein from a virulent British field isolate of porcine transmissible gastroenteritis virus (TGEV) FS772/70 was constructed from cDNA and inserted into the vaccinia virus (VV) thymidine kinase gene locus under the control of the VV early/late gene P7.5k promoter. Recombinant S protein was synthesized as an endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive glycoprotein with high mannose simple oligosaccharides (gp 190) that underwent post-translational modification to an Endo H-resistant glycoprotein with complex oligosaccharides (gp210). Immunofluorescence analysis demonstrated that the majority of recombinant S protein was retained at the Golgi but some S protein was expressed on the plasma membrane. Monoclonal antibodies (mAbs) raised against native S protein reacted with this recombinant S protein; also, mice infected with the recombinant vaccinia virus (rVV) expressing the S protein induced TGEV neutralizing antibodies. A truncated S protein (S delta) was also expressed in rVV-infected cells by introducing a deletion into the S protein cDNA that removed 292 amino acids from the C-terminus. The S delta protein (gp 170) was shown to be antigenically similar to TGEV S protein by immunofluorescence and immunoprecipitation tests but was retained in the endoplasmic reticulum and not expressed on the cell surface.
Pulford
D J
DJ
Division of Molecular Biology, A.F.R.C., Institute for Animal Health, Compton Laboratory, Newbury, Berkshire, United Kingdom.
Britton
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005814:Genes, Viral; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013552:Swine; D005760:Transmissible gastroenteritis virus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014616:Vaccinia virus; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000235:genetics
D000956:Antigens, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C064484:spike protein S, Transmissible gastroenteritis virus; D004247:DNA