X-ray microanalytical investigation of the response of chicken proximal tubule cells to infection with avian infectious bronchitis virus.

The technique for X-ray microanalysis of frozen-hydrated bulk specimens was used to determine the intracellular and luminal fluid electrolyte concentrations in the proximal tubules of kidneys from chickens infected with infectious bronchitis virus. Eight days post-infection with this virus there were significant changes in the electrolyte composition when compared with values from normal control chickens. The intracellular sodium decreased from 43 to 36 mmol/l, the chloride fell from 41 to 31 mmol/l and the potassium went from 125 to 115 mmol/l. Sodium counts in the luminal fluid rose from .73 to 1.03 cps. These disturbances in electrolyte composition are consistent with alterations in sodium reabsorption in the proximal tubule due to decreased transport of sodium into the cells across the microvillus membrane. It appears that the Na-K-ATPase pump is unaffected. The results demonstrate the value of X-ray microanalysis methods for the study of electrolyte transport in pathologically affected cells and provide further information for the definition of viral-host cell interactions in the pathogenesis of viral disease. As a check on methodology two normal rat kidneys were analysed in the same way. Intracellular sodium and potassium concentrations were 22 and 138 mmol/l respectively.

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.

Coronavirus IBV glycopolypeptides: locational studies using proteases and saponin, a membrane permeabilizer.

[35S]methionine-labelled avian infectious bronchitis virus (IBV) (strain 41) and its purified protein components and virions of IBV-Beaudette were incubated with 10 proteases. Several proteases hydrolysed all or some of the membrane glycopolypeptide (M; Mr 30K) and removed about 1.3K of peptide from the amino-(N-)-terminus plus both glycans, as determined by SDS-polyacrylamide gel electrophoresis. N-terminal analysis of [3H]isoleucine-labelled M after hydrolysis by bromelain revealed that the first nine residues had been removed. After the virions had been permeabilised with saponin, a further 2.5K decrease in molecular weight was produced and this was shown to be from the carboxy-(C-)terminus. When considered with the hydropathicity plot analysis of the amino acid sequence of M (Boursnell, M.E.G. et al., 1984, Virus Res. 1, 303-313) these results suggest that as few as 9-20 N-terminal amino acid residues may protrude at the outer membrane surface and that there is a highly protease sensitive sequence of an estimated 20-25 residues at the C-terminus of M exposed in the lumen of the virion. S2 but not S1 was cleaved to a major glycopolypeptide of approximately 71K by several proteases, and to 76K by trypsin. N-terminal sequencing of the 71K glycopolypeptide revealed that it had the same N-terminus as intact S2. After hydrolysis in the presence and absence of saponin it was concluded that S2 is very sensitive to hydrolysis near its carboxy terminus at residues close to the outer membrane surface.

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.

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)-S1-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 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 S1 comprises 519 residues (Mr 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 micrograms/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.

Development of in vitro tests for detection of extraneous agents.

The use of animals in tests to detect extraneous agents is not only undesirable from the ethical viewpoint but also because of the expense and length of time involved in such tests. We have carried out tests on a variety of potential contaminating avian pathogens to determine whether tests in chicks offer any advantage over tests in embryos or cell cultures. In many cases, but not all, in vitro tests were shown to be more sensitive. The use fluorescent antibody or enzyme-linked assays serves to enhance the sensitivity of the tests. In the future it may be possible to adapt techniques such as nucleic acid hybridisation to the detection of extraneous agents.

Envelope proteins of avian infectious bronchitis virus: purification and biological properties.

Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9-11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7-11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed.

Coronavirus IBV: structural characterization of the spike protein.

The spike protein (S; surface projection) of avian infectious bronchitis virus (IBV) strain M41 comprises two glycopolypeptides, S1 (mol. wt. 90 X 10(3] and S2 (mol. wt. 84 X 10(3], in equimolar proportions. The apparent mol. wt. of S was calculated as 354 (+/- 17) X 10(3) following co-sedimentation with catalase in sucrose gradients. Incubation of radiolabelled IBV with urea resulted in the removal of most S1, but none of S2, from the virus particle. A similar result was obtained using low concentrations of SDS, although some nucleocapsid, but not matrix, protein was also released. 2% SDS alone was as effective as 2% SDS plus 2% 2-mercaptoethanol for the separation of S1 and S2 prior to SDS-polyacrylamide gel electrophoresis. Dithiothreitol did not remove S from virions but did decrease the buoyant density of the virus from 1.18 g/ml to 1.16 g/ml, and changed the configuration of S. It is concluded that IBV S protein is an oligomer comprising two copies of each of S1 and S2, although the possibility that there are three copies of each glycopolypeptide cannot be discounted. S is attached to the membrane by S2, while S1 has little or no contact with the membrane and may form the major part of the bulbous end of S. Interpeptide disulphide bonds do not occur in S, and the association of S1 and S2 is weak.

Coronavirus IBV: further evidence that the surface projections are associated with two glycopolypeptides.

The surface projections (peplomers) of avian infectious bronchitis virus (IBV) strain M41 have been separated from the nucleocapsid (N) and matrix (M) proteins by sedimentation in a sucrose gradient after virus disruption by the non-ionic detergent Nonidet P40. The peplomers comprised two glycopolypeptides of mol. wt. 90 X 10(3) (90K; S1) and 84K (S2), shown by analysis of differentially radiolabelled virus to be present in equimolar proportions. Polypeptides of 75K and 110K, which were detected by Coomassie Brilliant Blue staining in similar amounts to S1 and S2 in some unlabelled virus preparations, were absent from peplomer preparations and are probably host cell polypeptides. The S1:S2:N:M polypeptide molar ratio for IBV-M41 was approximately 1:1:6:15.

Coronavirus IBV glycopolypeptides: size of their polypeptide moieties and nature of their oligosaccharides.

Analysis of differentially radiolabelled avian infectious bronchitis virus (IBV) indicated that the matrix (M) polypeptides of mol. wt. 23 X 10(3) (23K), 26K, 28K, 30K and 34K (M23 to M34) which have been shown to give the same peptide maps, differed in their degree of glycosylation; M23 was not glycosylated while glycosylation increased with increasing mol. wt. from M26 to M34. Both glucosamine and mannose were components of M26 to M34 but [3H]fucose appeared to be associated mainly with M34. Endo-beta-N-acetylglucosaminidase H removed oligosaccharides from M28 and M30 but not M26 and M34, to give a polypeptide of 23K. The surface projection glycopolypeptides S1 (90K) and S2 (84K) incorporated 3H-labelled glucosamine and mannose but not fucose and had oligosaccharides removed by endoglycosidase H. The mol. wt. of the resultant polypeptides varied among experiments; the lowest mol. wt. observed were 64K and 61K. These results indicate (i) that the polypeptide moieties of the S polypeptides are approximately 64K and 61K, and 23K for the M polypeptide, (ii) that the oligosaccharides of the S and M polypeptides are of the high-mannose type and are linked to the polypeptides by N-glycosidic linkages, and (iii) that the M glycoprotein of IBV differs from that of murine coronaviruses and bovine coronavirus L9 which have O-linked oligosaccharides.

Coronavirus proteins: structure and function of the oligosaccharides of the avian infectious bronchitis virus glycoproteins.

The recent finding that the E1 glycoproteins of murine coronaviruses contain only O-linked oligosaccharides suggested that this unusual modification might be a distinguishing feature of coronaviruses and might play an essential role in the life cycle of this family of viruses. To examine these possibilities, we analyzed the oligosaccharide moieties of the membrane proteins of the avian coronavirus infectious bronchitis virus. In addition, we determined the effect of inhibiting the glycosylation of these proteins on viral maturation and infectivity. Infectious bronchitis virus virions contain nine proteins. Four of these proteins, GP36, GP31, GP28, and P23, are closely related structurally and appear to be homologous to the E1 proteins of murine coronaviruses. We found that the oligosaccharides of GP31 and GP28 could be removed with endoglycosidase H and that neither of these glycoproteins was detectable in tunicamycin-treated cells. These two results indicated that GP31 and GP28 contain N-linked oligosaccharides. Therefore, O-linked oligosaccharides are not a universal feature of the small coronavirus membrane glycoproteins. Tunicamycin inhibited glycosylation of all of the viral glycoproteins but did not inhibit production of virions by infectious bronchitis virus-infected cells. The virions released by these cells contained only the three non-glycosylated viral proteins P51, P23, and P14. These particles were not infectious. Therefore, it appears that glycosylated infectious bronchitis virus polypeptides are not required for particle formation. However, the viral glycoproteins are apparently indispensible for viral infectivity.

Structural analysis of virion proteins of the avian coronavirus infectious bronchitis virus.

We have found six major polypeptides in virions of the avian coronavirus infectious bronchitis virus grown in tissue culture: four glycoproteins, GP84, GP36, GP31, and GP28, and two non-glycosylated proteins, P51 and P23. In addition, we detected three minor species: two glycoproteins, GP90 and GP59, and one non-glycosylated protein, P14. Two-dimensional tryptic peptide mapping showed that GP36, GP31, GP28, and P23 comprise a group of closely related proteins which we have designated the "P23 family," but that the other proteins are distinct. Analysis by partial proteolytic digestion of P23 family, but that the other proteins are distinct. Analysis by partial proteolytic digestion of the P23 family labeled biosynthetically with [35S] methionine, and P23, labeled with [35S] formyl-methionine by in vitro translation of RNA from infected cells, revealed that the proteins of the P23 family differ in their amino-terminal domains. Similar analysis of GP31 and Gp36 labeled with [3H] mannose showed that the partial proteolytic fragments unique to these proteins were glycosylated. This suggests that differences in glycosylation in the amino-terminal domains contributes to the marked polymorphism os the P23 family. The results are discussed with respect to possible models for synthesis of the virion proteins.

Ribonucleoprotein of avian infectious bronchitis virus.

The ribonucleoprotein (RNP) of avian infectious bronchitis virus (IBV) was examined by electron microscopy after shadowing with carbon/platinum. Linear RNP strands up to 6.7 microns in length, from three IVB strains, were sensitive to both pancreatic RNase and to proteases. These strands were obtained from spontaneously disrupted complete particles but not from disrupted incomplete particles that lacked RNP. They were also released from Nonidet P40-disrupted particles and could be isolated on sucrose density gradients at a density of 1.27 g/ml. In some cases, helical RNP complexes associated with virus particles were observed that were similar to RNPs of human coronavirus strain 229E and mouse hepatitis virus strain 3.

Structural polypeptides of coronavirus IBV.

Avian infectious bronchitis virus (IBV) was grown and radiolabelled with 35S-methionine, 3H-leucine and 3H-glucosamine in de-embryonated chicken eggs. Approximately 12 different polypeptides were clearly detected by SDS-polyacrylamide gel electrophoresis of virus preparations. Growth of IBV in chorioallantoic membrane cells labelled with 35S-methionine indicated that most of these polypeptides, and additional ones, some of which were glycosylated, were host components. Five polypeptides appeared to be virus-coded, with apparent mol. wt. of 94 x 10(3), 84 x 10(3), 54 x 10(3), 30 x 10(3) and 28 x 10(3). Four of these, p94, p84, p30 and p28, were glycosylated. The virion spikes appeared to be composed of p94 and p84, while p30 and p28 were partially embedded in the virion membrane. By analogy with other reports, p54 is the nucleocapsid polypeptide.

Structural proteins and glycoproteins of infectious bronchitis virus particles labeled during growth in chick embryo cells.

Seven Australian infectious bronchitis viruses incorporated radioactive amino acid label during growth in two types of cell culture. From purified virions, four major polypeptides of molecular weights 91,000 (GP91), 51,000 (VP51), 25,000 (GP25) and 23,000 (VP23) were separated by PAGE. Two minor polypeptides of molecular weights 210,000 (GP210) and 185,000 (GP185) were also identified. 3H-glucosamine was incorporated during growth of virus into GP210, GP185, GP91 and GP25. Bromelain digestion of virions removed GP210, GP185 and GP91, leaving VP51, GP25 and VP23 in a large subviral particle. All seven viruses, which were of six serological subtypes, formed apparently identical polypeptide profiles.

Coronavirus multiplication strategy. II. Mapping the avian infectious bronchitis virus intracellular RNA species to the genome.

Avian infectious bronchitis virus, a coronavirus, directed the synthesis of six major single-stranded polyadenylated RNA species in infected chicken embryo kidney cells. These RNAs include the intracellular form of the genome (RNA F) and five smaller RNA species (RNAs A, B, C, D, and E). Species A, B, C, and D are subgenomic RNAs and together with the genome form a nested sequence set, with the sequences of each RNA contained within every larger RNA species (D. F. Stern and S. I. T. Kennedy, J. Virol 34:665-674, 1980). In the present paper we show by RNase T1 oligonucleotide fingerprinting that RNA E is also a member of the nested set. Partial alkaline fragmentation of the genome followed by sucrose fractionation, oligodeoxythymidylate-cellulose chromatography, and RNase T1 fingerprinting gave a partial 3'-to-5' oligonucleotide spot order. A comparison of the oligonucleotides of each of the five subgenomic RNAs with this spot order established that all of the RNAs are comprised of nucleotide sequences inward from the 3' end of the genome. This result is discussed in relation to the multiplication strategy both of coronaviruses and of other RNA-containing viruses.

The polypeptide composition of isolated surface projections of avian infectious bronchitis virus.

Disruption of avian infectious bronchitis virus (IBV) particles with 4% Triton X-100 and 1.0 M-KCl and centrifugation through a sucrose gradient containing 0.1% Triton X-100 and 1.0 M-KCl enabled separation of the petal-shaped surface projections. By negative-contrast electron microscopy the separated projections appeared mainly as rosettes containing 3 to 12 projections radiating from a central core, although single projections and rosettes containing up to 16 projections were seen. SDS-PAGE of these preparations revealed two polypeptides of 86,000 and 66,000 mol. wt. The larger polypeptide was glycosylated.

Two particle types of avian infectious bronchitis virus.

Two distinct types of avian infectious bronchitis virus (IBV) particles were isolated on sucrose density gradients. The higher density particles banded at 1.18 g/ml, had typical coronavirus morphology and contained all the structural polypeptides and a complete genome. The less dense particles of density 1.13 g/ml appeared to have typical coronavirus morphology, although they were much more flattened than the more dense particles. Furthermore, these particles lacked the ribonucleoprotein polypeptide and the genome, although all the other polypeptides were present in the same amounts as in the denser particles.

The polypeptides of infectious bronchitis virus (IBV-41 strain).

The Massachusetts strain of avian infectious bronchitis virus was purified from embryonated hens' eggs. Four major species of apparent mol. wt. 90 000, 52 000, 29 000 and 26 000 were resolved by SDS-polyacrylamide gel electrophoresis. Omission of reducing agent failed to resolve the 29 000 mol. wt. component. Labelling of acrylamide gels with 125I-concanavalin A indicated that polypeptides of mol. wt. 90 000, 29 000 and 26 000 were glycosylated and, in the absence of reducing agent, that the 29 000 species migrated as a dimer in the 5000 mol. wt. region. Purified IBV radio-iodinated with Bolton and Hunter reagent, which banded as a single peak of radioactivity in Metrizamide gradients, was found to contain bands of radioactivity when analysed by SDS-PAGE, corresponding to the polypeptides of mol. wt. 90 000, 52 000 and 29 000 resolved in stained gels. Disruption of IBV particles in Triton X-100 released two subviral particles, a 16 nm spike which comprised polypeptides of 90 000, 52 000 and 29 000 mol. wt. and another denser spherical particle of 25 to 45 nm which contained RNA and the 52 000 and 26 000 polypeptides.