Sequence of the nucleoprotein gene from a virulent British field isolate of transmissible gastroenteritis virus and its expression in Saccharomyces cerevisiae

Abstract

Subgenomic mRNA from a virulent isolate of porcine transmissible gastroenteritis virus (TGEV) was used to produce cDNA which was sequenced. Two non-overlapping open reading frames (ORFs) were identified. The largest, encoding a polypeptide of 382 amino acids (relative molecular mass (Mr) 43,483), was shown to be the viral nucleoprotein gene. The second ORF, found 3' to the larger ORF, encodes a polypeptide of 78 amino acids (Mr 9068) which has yet to be assigned to a viral product. The nucleoprotein gene was expressed in yeast cells under the control of two types of yeast promoters: the constitutive PGK promoter, and the inducible GAL1 promoter. Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of Mr 47,000, identical to the viral product, that reacted with a specific monoclonal antibody.

Sequence comparison of the 5' end of mRNA 3 from transmissible gastroenteritis virus and porcine respiratory coronavirus

“…Specific restriction fragments from TGEV cDNA clones were separated on agarose gels, purified using Geneclean (Stratech Scientific) and labelled with [~-32p]dATP (Amersham) (Maniatis et al, 1982). TGEV and PRCV subgenomic mRNA species were denatured with 6 M-glyoxal, electrophoresed into 1% agarose gels, Northern-blotted onto Biodyne membranes (P/N BNNG3R 1.2 p.m; PAL) and hybridized to 32P-labelled TGEV cDNA fragments (Britton et al, 1988a.…”

“…2) indicating that the PRCV-derived PCR fragment was 200 bp larger than expected from the mRNA analysis. The 1350 bp PRCV cDNA fragment was cloned into pUC13 and positive clones were identified using 32p_ labelled TGEV cDNA from ORF-3b as described by Britton et al (1988a). The plasmid DNA from a positive clone, pKP-1, was used for DNA sequencing.…”

“…TGEV-infected cells contain, in addition to the genomic RNA (mRNA 1), six species of subgenomic mRNA observed as a 'nested' set (mRNA 2 to 7; Britton et al, 1986;Jacobs et al, 1986). The TGEV genome, except for the polymerase gene, has been cloned and sequenced from a virulent British strain, FS772/70 (Britton et al, 1987(Britton et al, , 1988a(Britton et al, , b, 1989 and from an American avirulent strain, Purdue-ll5 (Kapke & Brian, 1986;Kapke et al, 1987Kapke et al, , 1988Jacobs et al, 1987;Laude et al, 1987;. Both TGEV and PRCV have identical leader RNA sequences .…”

Porcine respiratory coronavirus differs from transmissible gastroenteritis virus by a few genomic deletions

“…The extreme 3' non-coding region comprised an 8 nucleotide sequence conserved in coronaviruses, and ended in a poly(A) stretch. Pairwise alignment of the PRCV sequence data with those available for the three different TGEV strains Purdue 115 , FS772/70 (Britton et al, 1988a(Britton et al, , b, 1989 and Miller (Wesley et al, 1989;Wesley, 1990) revealed a very high level of homology. Thus, 96% overall homology was found between PRCV and TGEV Purdue strains at both the nucleotide and amino acid level.…”

The Polypeptide of Mr 14000 of Porcine Transmissible Gastroenteritis Virus: Gene Assignment and Intracellular Location

“…Data from the proposed amino acid sequence for ORF-4 (Britton et al, 1988) were used to synthesize three oligopeptides (produced by Dr T. Doel, AFRC Institute for Animal Health, Pirbright Laboratory, U.K.) as shown in Fig. 2.…”