Expression and cellular localisation of porcine transmissible gastroenteritis virus N and M proteins by recombinant vaccinia viruses

Abstract

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 P 7.5K 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 ( M r) 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 endogly-cosidase H reducing the mature polypeptide of M r 29,000 to a species of M r 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 infected cells. Antisera from mice infected with recombinant viruses immunoprecipitated specific TGEV antigens from lysates of TGEV infected cells but had little significant TGEV neutralising activity in vitro.