T. D. K. Brown

Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation.

125I protein labelling of oligo(dT)-selected RNA from feline calicivirus (FCV)-infected cells revealed that the genomic and 2.4 kb subgenomic RNAs of FCV are linked to a 15 kDa protein (VPg). Proteinase K treatment of FCV RNA, to remove VPg, led to a decrease in the translatability of the RNA, but there was no obvious change in the site of RNA initiation. Addition of the cap analogue 7-methylGTP to in vitro translations had no effect on the translation of FCV RNA, suggesting that FCV RNA is translated by a cap-independent mechanism. Further evidence that FCV RNA is translated by an unusual mechanism was obtained by translating FCV RNA in vitro at a range of K+ concentrations. FCV RNA was able to direct translation at K+ concentrations at which cellular RNA translation was inhibited.

Coronavirus IBV: partial amino terminal sequencing of spike polypeptide S2 identifies the sequence Arg-Arg-Phe-Arg-Arg at the cleavage site of the spike precursor propolypeptide of IBV strains Beaudette and M41

Abstract The spike protein of avian infectious bronchitis coronavirus comprises two glycopolypeptides S1 and S2 derived by cleavage of a proglycopolypeptide So, the nucleotide sequence of which has recently been determined for the Beaudette strain (Binns M.M. et al., 1985, J. Gen. Virol. 66, 719–726). The order of the two glycopolypeptides within So is aminoterminus(N)-Sl-S2-carboxyterminus(C). To locate the N-terminus of S2 we have performed partial amino acid sequencing on S2 from IBV-Beaudette labelled with [3H]serine and from the related strain IBV-M41 labelled with [3H]valine, leucine and isoleucine. The residues identified and their positions relative to the N-terminus of S2 were: serine, 13; valine, 6, 12; leucine, none in the first 20 residues; isoleucine, 2, 19. These results identified the N-terminus of S2 of IBV-Beaudette as serine, 520 residues from the N-terminus of S1, excluding the signal sequence. Immediately to the N-terminal side of residue 520 So has the sequence Arg-Arg-Phe-Arg-Arg; similar basic connecting peptides are a feature of several other virus spike glycoproteins. It was deduced that for IBV-Beaudette SI comprises 519 residues (M r 57.0K) or 514 residues (56.2K) if the connecting peptide was to be removed by carboxypeptidase-like activity in vivo while S2 has 625 residues (69.2K). Nucleotide sequencing of the cleavage region of the So gene of IBV-M41 revealed the same connecting peptide as IBV-Beaudette and that the first 20 N-terminal residues of S2 of IBV-M41 were identical to those of the Beaudette strain. IBV-Beaudette grown in Vero cells had some uncleaved So; this was cleavable by 10 μg/ml of trypsin and of chymotrypsin. Partial N-terminal analysis of S1 from IBV-M41 identified leucine and valine residues at positions 2 and 9 respectively from the N-terminus. This confirms the identification made by Binns et al. (1985), of the N-terminus of S1 and the end of the signal sequence of the IBV-Beaudette spike propolypeptide. N-terminal sequencing of [3H]leucine-labelled IBV-Beaudette membrane (M) polypeptide showed leucine residues at positions 8,16 and 22 from the N-terminus; these results confirm the open reading frame identified by M.E.G. Boursnell et al. (1984, Virus Res. 1, 303–313) in the nucleotide sequence of M. The N-terminus of the nucleocapsid (n) polypeptide appeared to be blocked.

Identification of a New Membrane-associated Polypeptide Specified by the Coronavirus Infectious Bronchitis Virus

Nucleotide sequences from the third open reading frame of mRNA D (D3) of infectious bronchitis virus (IBV) were expressed in bacteria as part of a fusion protein with beta-galactosidase. Antiserum raised in rabbits against this fusion protein immunoprecipitated from IBV-infected chick kidney or Vero cells a polypeptide of 12.4K, the size expected for a D3-encoded product. The D3 polypeptide is apparently non-glycosylated, and appears to be associated with the membrane fraction of infected cells, as judged by cell fractionation and immunofluorescence.

Expression of the Infectious Bronchitis Virus Spike Protein by Recombinant Vaccinia Virus and Induction of Neutralizing Antibodies in Vaccinated Mice

A cDNA clone of the infectious bronchitis virus (IBV) spike protein gene has been recombined into vaccinia virus. Cells infected with the recombinant virus synthesized IBV spike antigen which was recognized by antibody raised against purified spike protein. Immunofluorescence showed that the IBV spike antigen was transported to the infected cell surface membrane and immunoprecipitation showed the presence of the glycosylated 180K mol. wt. polypeptide precursor of the two spike subunits S1 and S2 that comigrated with this antigen from IBV-infected cells. Vaccinated mice produced antibody that recognized the IBV spike antigen by ELISA and which neutralized IBV infectivity as shown by ciliostasis tests on tracheal organ cultures.