Expression and cellular localisation of porcine transmissible gastroenteritis virus N and M proteins by recombinant vaccinia viruses.
Porcine transmissible gastroenteritis virus (TGEV) nucleoprotein and integral membrane protein genes were cloned into the vaccinia virus insertion vector, pGS20, in the correct orientation for expression under the control of the vaccinia P7.5K promoter. Recombinant vaccinia viruses were generated by in vivo homologous recombination of the insertion vector with the WR strain of vaccinia virus. Nucleoprotein (N) expressed by both recombinant vaccinia virus and TGEV had a relative molecular mass (Mr) of 47,000 and was susceptible to degradation at the C-terminus yielding discrete breakdown products. The integral membrane protein (M) expressed by a recombinant vaccinia virus and TGEV was sensitive to endoglycosidase H reducing the mature polypeptide of Mr 29,000 to a species of Mr 27,000. Expression of M by recombinant vaccinia virus was inhibited during early infection due to a cryptic vaccinia virus transcriptional termination signal within the TGEV coding sequence. Indirect immunofluorescence showed that both N and M were only localised in the cell cytoplasm of either TGEV or recombinant vaccinia virus immunoprecipitated specific TGEV antigens from lysates of TGEV infected cells but had little significant TGEV neutralising activity in vitro.
Pulford
D J
DJ
A.F.R.C., Institute for Animal Health, Compton Laboratory, Berkshire, U.K.
Britton
P
P
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D004274:DNA, Recombinant / Q000235:genetics; D005260:Female; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011401:Promoter Regions, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic; D005760:Transmissible gastroenteritis virus / Q000235:genetics* / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014763:Viral Matrix Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology
D003062:Codon; D004274:DNA, Recombinant; D009698:Nucleoproteins; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase