key: cord-0709474-gj9yzrjo
authors: Sugiyama, K.; Ishikawa, R.; Fukuhara, N.
title: Structural polypeptides of the murine coronavirus DVIM
date: 1986
journal: Arch Virol
DOI: 10.1007/bf01309893
sha: 58eda31c90335dda4f08ff69421b314648926b9f
doc_id: 709474
cord_uid: gj9yzrjo

The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.

Coronaviruses are a group of enveloped viruses which contain large single-stranded RNA genomes of messenger polarity (t2, 14, 17, 22) . However, the murine coronaviruses can be distinguished from the other coronaviruses by the presence of O-linked oligosaccharides (4, 9) Our previous studies on the morphology and some of the biological activities of the murine eoronavirus strain DVIM (diarrhoea virus of infant mice) (18, 19) have revealed the presence of characteristic short projections located on the base of 'club-shaped eoronaradiation' of the DVIM virion. We were therefore interested in studying the structural polypeptide of this virus especially in comparison with those of the well characterized murine coronaviruses.

In this report we describe the characteristic major glycopolypeptide gp 69, which is exclusively detected on the DVIM and probably corresponds to the short projections. We also describe the effect of tunicamycin on the synthesis of the various structural polypeptides and the degree of immunocross reactivity between these DVIM proteins.

The mouse enteric coronavirus (DVIM) was adapted for growth in DBT cells using Eagle's MEM and supplemented with 7 per cent (%) calf serum and 3 per cent tryptose phosphate broth (DIFCO Lab.). The virus was cloned by three consecutive plaque passages. In addition the mouse hepatitis virus strains MI-IV-2, MHV-3, MHV-4 (JHM) and MI~V-S, obtained h'om ])r. K. Fujiwara (University of Tokyo, Tokyo, Japan), as DBT cell culture suspenMons, respectively were also cloned and purified by a similar procedure as described previously (18) .

Viruses at MOI of 1.0 were allowed to adsorb to confluent monolayers of DBT cells for 90 minutes at 4 ° C, following which cells were washed twice in Dulbecco's PBS and fed with ME~ that has been supplemented with 2 per cent of diMysed calf serum. Methionine concentration in MEM was reduced to one.tenth of that in normal medium. Four hours after infection, the medium was replaced with methionine-free MEM containing 2 per cent dialysed calf serulTt and 25 vCi/ml of 35S-methionine (1200 Ci/mmol: Amersham Corp.). /)VIM was also labelled with 14C-glueosarnine (56.8 mCi/mmol: Amersham Corp.), 2-~tI-I)-mannose (22 Ci/mrnol: ICN Radio chemL cats) and 5-, 6,-aH-L-fueose (67 Ci/mmom: Amersham Corp.) at, 5 ~Ci/ml by the same procedure without a reduction of methionine, respectively. In some experiments, after virus adsorption tunicamycin (Sigma Chem. Co.) was added at a final concentration of 4 ~xg/ml. In all experiments virions were harvested 10 hours after infection, and were purified as described (18) .

Samples were analysed on 12 per cent slab gels by the method of LAE)$MLI (6) .

The proteins were treated with sample buffer, boiled for 3 minutes and separated by slab gel at 12 mA for t8 hours. Gels were then prepared for fluorography (1) and exposed to Kodak X-Omat R film at -70 ° C.

!-Iyperimmune sera against purified virions of each strain were prepared in rabbits as described previously (19) . For virus neutralization, 100 TCIDs0 of virus was incubated with fourfold dilutions of antiserum for 1 hour at 37 ° C in a final volume of 0.1 ml. The incubation mixture was then transferred to monolayers of I)BT cells in microplates using four replicas per virus-serum dilution. The test was scored for cytopathology after 48 hours at 37 ° C. For immunoprecipitation, purified 35S-methionine labelled virions were disrupted with 2 per cent Nonidet-P40 in NTE (0.1 ~i NaC1, 0.01 ~ Tris-C1, 0.001 ~ EDTA, pit 7.3) buffer and 400 ~I of this disrupted viral components was then incubated with 15 y.1 of antiserum for 60 minutes at 37 ° C and then mixed with 200 ~l of 10 per cent formalin-fixed solution of S. aureus (MILES Sei.) dissolved in NTE buffer. After incubation for 60 minutes at 37 ° C, the immunocomplexes bound to S. aureus were washed four times in NTE containing 0.1 per cent Triton-X by eentrifugation and the final pellet was suspended in the sample buffer, containing 2mereaptoethanol and boiled for 3 minutes. S. aureus was deposited by low speed eentrifugation and the supernatants were then loaded on to the gels.

The immunological relationship between DVIM and other well characterized strains of murine coronaviruses was examined by cross-neutralization (Table 1) . DVIM was found to be very closely related with the HMV-S strain. In addition, bilateral minor cross-reactions between DVIM and MHV-2, or MHV-3, were also observed. In contrast, DVIM showed only weak cross-reaction with MItV-4 (JItM). A strong unilateral cross-reaction was also detected between DVIM and the anti-MHV-2 serum. These results, therefore, showed that in general DVIi~{ is serologieally related to the other strains of murine coronaviruses, particularly to MHV-S. The approximate molecular weights of the virion polypeptides were determined by comparing their etectrophoretic mobilities with those of com-mercially available standards (BIO-RAD) in 7.5 per cent polyacrylamide tube gels, according to the procedure of MAIZ~L (8) . DVIM has four major polypeptides, with molecular weights of 180,000, 69,000, 58,000 and 25,000, respectively. One minor polypeptide with a molecular weight of 90,000 was detected in some preparat4ons. The smallest of the major polypeptide species produced a broad band or a group of three to four closely migrating bands. The PAGE profiles of DVIM were generally similar to those observed for other marine coronaviruses such as MHV-2, MHV-3, and MHV-4, except that one major band with a moh wt. of 69,000 was detected in DIVM virions exclusively. MHV-S strain also had a polypeptide with a moh wt. of 65,000 similar to the 69,000 of DVIM.

laC-glueosamine labelled DVIM-was used to identify glycosylated peptides. Analyses revealed that the three major polypeptides with moh wts. of 180,000, 69,000 and 25,000 were all glycosylated, and hence were designated gp 180, gp69 and gp25 respectively, whereas the other major polypeptide with a mol. wt. of 58,000, which was not glyeosylated, was designated p58 (Fig. 1 b) . It is interesting that gp180 was labelled with both, 3H-mannose and 3Hfucose, whereas the gp25 family of polypeptides were labelled with only 3tI-fucose. In addition, DVIM specific gp 69 was labelled with 8H-mannose and not with 3H-fucose. These findings, therefore, indicate that the various DVIM glyeopolypeptides differ from each other according to the composition of their sugar residue moieties.

The presence of disulfide bonds in the structural polypeptides was studied by SDS-PAGE analysis of asS-methionine labelled DVIM in the absence of 2-mereaptoethanol when compared with those of MHV-S. Under these conditions, gp 69 of DVIM could no longer be detected and instead a new species of polypeptide with a mot. wt. of 140,000 was observed (Fig. i c) . This suggested that this polypeptide is a disulfide-linked dimer of two smaller glycopolypeptides gp 69. MItV-S also had a similar disulfide-linked oligomerie species.

In order to study the serological cross-reactivity between the DVIN[ polypeptides and those of the other MHV strains, 35S-methionine labelled (Fig. 2a) . Thus using homologous antisera all the structural polypeptides of DVIM were precipitated. The minor bands, which migrated slightly faster than the p58 species, as detected by immunopreeipitation, were assumed to be degradation products, most probably of the nucleoeapsid protein. On the other hand, immunoreactions with heterologous antisera indicated that whereas there was strong cross-reactivity with p58 of DVIM, there were only faint cross-reactions with both gp 180 and gp 25, respectively. It seems, however, that gpl80 shows considerable cross-reaction with the anti-MHV-S serum. In contrast, the DVIM specific polypeptide gp69 was not immunologieally related to any of the glycoproteins of the other murine coronaviruses.

As shown in Fig. 2b a, lmost all of the strain-specific polypeptides immunoreacted with the anti-I)VIM serum in varying degrees indicating that all the structural proteins of DVIM, except gp 69, are immunologically related to those of the other MHV strains.

To identify the type of glycosidic bonds present in the structural glyeoproteins, DVIM infected DBT cells were treated with tunicamycin (4 ~g/ml) as described earlier. Viral particles isolated from tunicamycin treated cells lacked both, infectivity and haemagglutinating activity (data not shown). These viral particles were analysed together with viruses from non-treated cells on SDS-polyacrylamide gels under reducing conditions (Fig. 3) .

I~esults indicate that both gp 180 and the DVIM-specific glycopolypeptide gp 69 could no longer be detected following tunicamycin treatment of cells. In addition, it appears that gp25 consists of four polypeptides, W, X, Y and Z. Of these the small glycopolypeptide Y also disappeared completely. In contrast the synthesis of both W and X was not affected and Z was only partially a.ffected by this drug. These results, together with the ability of mannose to be incorporated into the polypeptide (Fig. 1 b) , indicated that (B) virion control Discussion The glycopeptide gp 180 appears to be equivalent to the largest glycoprotein, present in the well-characterized mouse hepatitis viruses, and we think it is a peplomerie protein in accordance with the findings of other investigators (7, 14, i5, 16, 17, 23) . Although the minor glycopolypeptide gp90 was weakly detectable in gels, it is also assumed to be a peplomerie protein and hence is probably associated with gp 180 b y noneovalent bonds (17) .

Gp 69, on the other hand, was detected exclusively on the DVIM virions as a major structural polypeptide. Removal of the large projections of DVIM, by proteolytic digestions, resulted in the loss of both gp 180 and gp90 from the intact DVIM virions. However, the viral particles still retained their haemagglutinating activity and these correlated with the presence of gp 69 (data not shown). Therefore, this suggested a close relationship between the small granular projections and the viral haemagglutinating activity. We therefore speculate that gp69 is a structural component of the small granular projections which are located close to the envelope. We presume (Fig. 1 c) that gp69 is a subunit of the larger glyeoprotein gp 140, which is bound by disulfide linkages in an oligomerie structure. Interestingly, a similar disulfide linked glyeopolypeptide has been described for the porcine haemagglutinating encephalomyelitis virus and for the bovine coronavirus, and it was suggested that this polypeptide may represent haemagglutinin (2, 5, 10, 11) . Generally, therefore, the haemagglutinating coronaviruses may share these oligomeric proteins as a distinctive structural feature in the form of small granular projections. Our studies also revealed that p58 was unaffected by protease treatment (data not shown) and hence were apparently protected by the viral envelope. This suggested that p58 was located internally and hence can be considered as a nueleoprotein as has been proposed by other workers (2, 3, 7, 14, 15, 23) .

In this study we found that the gp25 family of proteins were only slightly affected by protease treatment hence we are inclined to assume that they are embedded within the viral envelope. As was shown in Fig. 3 , this family of glyeoproteins consisted of four polypeptide species, designated W, X, ¥ any Z. In gels these polypeptides gave bands that corresponded with those reported by ROTTI~R et al. (13) and were comparable to the bands of gp26.5/E i, gp25.5/E 1, gp24.5/E i and p22/X of MHV-A59, respectively. Further, the synthesis of both W and X was not affected by tunieamycin treatment, suggesting that the carbohydrate side chains of these proteins may be linked to polypeptides through an O-glycosidic bonds (4, 9) . In fact, the evidence for the existence of this type of bonds in ])VIM was indicated by their sensitivity to alkali treatment under ~-elimination conditions (data not shown). In contrast, the synthesis of the Y protein as well as gp 180 and gp69 was completely inhibited by tunieamycin treatment. However, we failed to detect any aH-mannose following labelling, which is characteristic for the N-glyeosidic bond glycoprotein (9) , in the gp25 family. Therefore, at present we cannot define the type of glycosidic bonds in the Y protein. AeknoMed0ment

This work was supported in part by The Naito Foundation Grant No. 82~118.

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