key: cord-0703372-cwn1d03c
authors: Hajer, I.; Storz, J.
title: Structural polypeptides of the enteropathogenic bovine coronavirus strain LY-138
date: 1979
journal: Arch Virol
DOI: 10.1007/bf01317894
sha: ba417fb83c847c679f4c799efa6275974fad17d3
doc_id: 703372
cord_uid: cwn1d03c

The bovine coronavirus strain LY-138 was purified by differential as well as velocity and isopycnic centrifugation in sucrose or CsCl gradients. The substrate for purification was contents of the small intestine of experimentally inoculated calves. This strain is highly enteropathogenic, but it could not yet be propagated in cultured cells. Intact virions had a density of 1.245 g/cm(3) in CsCl and 1.185 g/cm(3) in sucrose. A spherical core-like structure with an average diameter of 82 nm remaining after treatment with chloroform had a density of 1.299 g/cm(3) in CsCl and 1.201 g/cm(3) in sucrose. Seven distinct bands of polypeptides and 4 shoulders were detected after electrophoresis of SDS-solubilized virions in polyacrylamide gels. The approximate molecular weights ranged from 110,000 to 36,000. Four of the bands gave a PAS positive reaction. These 4 glycoproteins and an additional protein with an approximate molecular weight of 70,000 were removed by chloroform treatment. The remaining core-like structure contained the 2 polypeptides VP3 and VP7.

Among the coronaviruses there are strains that cause intestinal infections leading to enteritis and diarrhea in swine (10, 32) , rodents (28) , dogs (2) , calves (26) and possibly man (6) . Factors determining the unique enteropathogenic properties of these coronaviral strains have not been defined (33) . Some of the enteropathogenie eoronavirus strains have been propagated in cultured cells (2, 25, 28, 30) but many others could not be adapted to cell cultures (6, 9, 28 ). An example of such a strain is the highly enteropathogenic strain LY-138 infecting calves. Several a t t e m p t s to a d a p t this strain to different types of cultured bovine fetal cells were unsuccessful (8, 9, 14) . This viral agent was m a i n t a i n e d b y oral inoculation and intestinal infection of newborn calves (8) . Electron microscopic evaluation of negatively stained preparations of purified virus particles revealed eoronaviral morphology (9) , a n d these viral preparations contained 3 antigens reacting with bovine eoronavirus antiserum (14) . U l t r a s t r u e t u r a l cytologic intestinal changes caused b y this virus were investigated (8) . Viral strain LY-138 was employed in studies on intraeellular and b o d y fluid alterations and on changes in intraeellular and extracellular ion eoneentrations t h a t occurred in the course of diarrhea following oral inoculation of calves (19, 27) .

F u r t h e r characterization of this highly virulent enteropathogen was essential. This report contains results of studies on the structure, physical properties, and polypeptide composition of the bovine eoronavirus strain LY-138 purified from intestinal infections of experimentally inoculated calves.

Contents of the small intestine of experimentally infected calves 3 days after oral inoculation served as substrates for virus purification. As a~ initial step, the virus was partially purified by sucrose velocity eentrifugation as described earlier (9) . The partially purified virus suspension or such suspensions treated to split the virions were layered onto I. I--I. 5 g/era 3 preformed linear CsCl gradients or 20--50 per cent performed linear sucrose gradients in TEN buffer (0.01 ~ tris-hydroehloride, 0.001 ~ EDTA, 0. i M NaCI). After banding by isopyenic eentrifugation at 81,000 × g for 24 hours in the SW40 rotor driven in a Beckman Model L2--65B ultracentrifuge, one ml fractions were collected. The refractive indices of band fractions were determined in a Fisher refraetometer, and the buoyant densities were calculated from a calibration of density versus refractive index chart. Each fraction was dialysed overnight in I per cent ammonium acetate at 4 ° C and concentrated to the original volume by extracting water with 40 per cent polyethylene glycol (w/v) in I per cent, ammonium acetate. Purity of the fractions was monitored by electron microscopic examination following negative staining with 1.5 per cent phosphotungstie (PTA) acid.

Purified virus suspensions were treated with 1 in 10 parts chloroform, or diethylether, 0.5 per cent Tween-80 or 0.5 per cent Nonidet P-40 (NP-40). The mixtures were shaken at room temperature for 30 minutes. After low speed centrifugation at 3000 x g for 15 minutes, 1.5 ml of the aqueous phases were layered onto a linear 20--40 per cent sucrose gradient in TEN buffer and centrifuged at 81,000 × g for 2 hours. The bands were localized by light scatter under a vertical beam of light and collected from the bottom of the tube. The band contents were dialysed overnight against I per cent ammonium acetate at 4 ° C. The dialysate was concentrated to I/5 of the original volume as described above. Samples of each band were negatively stained with 1.5 per cent PTA for EM examination.

The degree of purity and viral morphologie features of different, density gradient fractions were monitored by EM examination. Dialyzed aliquots of different fractions were each placed on 300 mesh, formvar coated, carbon-stabilized grids which had been degreased by dipping into carbon tetrachloride. Excess fluid was withdrawn by blotting with a filter paper. The grids were air-dried and a drop of 1.5 per cent PTA at p i t 6.5 was added to the grids. Excess stain was withdrawn with filter paper. The grids were dried overnight in a vacuum oven operated at room temperature and a pressure differential of 1075 g/em 3 (15 p o u n d s / s q u a r e inch). T h e specimens were e x a m i n e d with a n H i t a c h i H U -1 2 electron microscope operated a t 75,000 volts a n d a screen magnification of 40,000. F o r size m e a s u r e m e n t of viral particles magnifiestions were calibrated a g a i n s t c a r b o n -g r a t i n g replica h a v i n g 2160 lines per millimeter.

Serial two-fold dilutions were m a d e in P B S at a p i t of 7.2 from each fraction o b t a i n e d from CsC1 isopycnie e e n t r i f u g a t i o n in m i c r o t i t e r plates using mierodiluters. E a c h well t h e n received a n equal v o l u m e of 0.025 m l of 0.5 p e r cent w a s h e d mouse erythroeytes. T h e results were read after i n e u h a t i o n a t room t e m p e r a t u r e for one h o u r a n d again after o v e r n i g h t i n c u b a t i o n at 4 ° C. T h e H A titer was expressed as t h e reciprocal of t h e highest dilution causing complete h e m a g g l u t i n a t i o n . Gels were r e m o v e d from the glass tubes b y air pressure using a r u b b e r bulb. The gels were fixed o v e r n i g h t in 20 per cent sulfosalicylic acid a n d s t a i n e d for at least 12 hours with Coomassie brilliant blue. T h e gels were destained w i t h severat changes of 7 per cent acetic acid in 10 per cent m e t h a n o l . D e s t a i n e d gels were s c a n n e d w i t h a G e l m a n Scanner, a n d t h e m i g r a t i o n distance of each p o l y p e p t i d e was measured. T h e molecular weights of t h e polypeptides were d e t e r m i n e d from a s t a n d a r d curve d r a w n from m i g r a t i o n distances of m a r k e r proteins of k n o w n molecular weight. T h e m a r k e r proteins were e y t o e h r o m e C, 11,700 (Sigma Chemical Company, Saint Louis, Missouri), pepsin, 35,000 (Sigma), o v a l b u m e n , 43,000 (Sigma), lipase, 38,000 (Sigma), a n d b o v i n e serum a l b u m e n (BSA) 68,000 (Sigma).

F o r glycoprotein staining, a modifieatio~ of t h e periodic Sehiff (PAS) as ernployed b y ZACHA~I~rS a n d eoworkers (34) was used. Gels were fixed in 12.5 p e r cent trichloroacetic acid (TCA) for 30 minutes, rinsed in distilled w a t e r a n d oxidized in 1 per cent periodic acid in 3 per cent acetic acid for 1 hour. After eight 10-minute washings in distilled water, t~he gels were stained for 50 m i n u t e s in acidified 0.5 per cent. basic fuehsin-sutfite (Selfiff Reagent). T h e s t a i n was p r e p a r e d b y dissolving basic fuehsin in fresh 0.5 per cent p o t a s s i n m metasulfide a n d 0.1 ~ HCL. After t h r e e 10-minute washings w i t h freshly p r e p a r e d 0.5 per cent p o t a s s i u m metasulfide, t h e gels were d e s t a i n e d in distilled w a t e r a n d stored in 7 per cent acetic acid. Lipoproteins were s t a i n e d b y placing t h e gels in 0.7 p e r cent S u d a n Black B in polypropylene glycol for 2 hours followed b y destaining w i t h several changes of 85 per cent polypropylene glycol for 3 days (22) . After destaining, t h e gels were r e h y d r a t e d in distilled w a t e r for 3 days. Specimens for detection of proteins, lipoprotein, a n d glyeoproteins were prep a r e d a n d electrophoreseed a t t h e same time. 

Velocity centrifugation in a sucrose gradient displayed two closely placed bands 5.8 and 6.3 em from the meniscus of the gradient. Electron microscopic examination of negatively stained samples from the two bands revealed that the top band consisted of membranous fragments of viral particles and residual cellular components. The lower band contained enveloped viral particles with petal-shaped envelope projections.

When the virus band obtained at the 6.3 cm distance in sucrose velocity eentrifugation was isopycnically banded in 20--50 per cent linear sucrose gradients and 1.1--1.5 g/era 3 CsC1 gradients, single sharp bands were obtained. The bands had a density of 1.245 in CsC1 and 1.185 in sucrose. The maximum titers of hemagglutinating activity coincided with these bands as shown in Figure 1 . Electron microscopic examination of negatively stained samples from either CsC1 or sucrose isopycnic bands revealed clean fields of numerous roughly spherical particles. Elongated and other pleomorphic forms of virions were also seen. Some particles seemed to have been penetrated by the stain. Viral particles were enveloped and varied in size from 70--120 nm and possessed 15 to 20 nm petal-shaped surface projections. These projections were widely spaced and gave the typical appearance of the solar corona (Fig. 2) .

Treatment of purified virus with chloroform for 30 minutes followed by isopycnic centrifugation in CsC1 or sucrose resulted in visible bands at densities of 1.299 g/cm 3 and 1.201 g/cm a, respectively. These bands contained particles without projections and envelopes, and consequently they were considered to represent viral core-like material. These relatively homogeneous core-like structures were round. Measurements were made on 100 particles and gave a mean diameter of 82 nm. The core-like structures tended to stick to each other on the grids and formed aggregates. Although subunits appeared on the surface of the cores, no discernible s y m m e t r y in arrangement could be detected (Fig. 3) . The particles in this fraction were entire, and material protruding from t h e m was not seen. T r e a t m e n t with diethyl ether, Tween-80 and NP-40 caused disassembly oi the virus so t h a t no isopyenic bands were detected for morphological and biophysical analysis. 

Purified virions and core-like structures were solubilized by SDS, 2-mercaptoethanol and urea. The 9olypeptides were separated by polyacrytamide gel electrophoresis. The molecular weight estimates were determined in 10 per cent acrylamide gels in which plots of relative mobilities of standard proteins were linear with respect to the log of their molecular weights, Ten percent acrylamide gels gave better resolution in polypeptide separation than 7.5 per cent, gels. ]in both cases, however, it was found necessary to apply about 2 mg of protein to gels before stained polypeptide bands were detectable. A t least 7 distinct bands were visible after electrophoresis and Coomassie brilliant blue staining of gels containing solubilized polypeptides of intact virus (Fig. 4) . There were often 1 or 2 minor inconspicuous bands near the top of the gel. The viral polypeptides were designated VP1, VP2, VP3, etc. in order of increasing electrophoretic mobility of the bands. All these 7 bands were r e p e a t e d l y obtMned. F o u r shoulders (S) were observed. Three trailed VP2, VP3, and VP4, and one was in front of VP6. The a p p r o x i m a t e molecular weights of the LY-138 structural polypeptides calculated from t0 per cent acrylamide gel are summarized in Table 1 . W h e n the gels were PAS stained to reveal the c a r b o h y d r a t e containing proteins, 4 distinct reddish-pink bands were detected in gels containing polypeptides of solubilized intact virus. These bands coincided with VP 1, VP2, VP5, and V P 6 (Fig. 5) . Out of the 5 proteins split from the virion b y chloroform t r e a t m e n t only VPzi was not found to be glyeosylated. Sudan black staining of the gels did not reveal the presence of lipoproteins. P A G E -a n a l y s i s of the core-like structures in the isopycnie b a n d after chloroform t r e a t m e n t revealed t h a t only the viral proteins VP3 and V P 7 were present. Consequently, the viral structures removed, by chloroform-treatment contained V P 1, V P 2, VP 4, VP 5, and VP 6. -138 could not yet be a d a p t e d to cultured cells (9, t4) . Since this strain is highly enteropathogenic (8) and had been used in physiopathologic and u l t r a s t r u e t u r a l investigations of virus-induced enteritis and diarrhea (18, 27) , means for further identification and characterization were sought.

Studies on the morphology, antigenic structure, H A activity, morphogenesis and effect on infected intestinal epithelial cells identified some coronaviral characteristics of strain LY-138 (8, 9, 14) . The enteropathogenic properties would group this strain with others such as the virus of transmissible gastro-enteritis (TGE) of swine which differ in their resistance to a p t t of 3 from eoronaviruses associated with respiratory and other infections (25, 30) .

Intestinal contents of experimentally infected eMves were used as substrates for viral morphologic, structural and biophysieM analysis. The method used for virus purification and concentration from in vivo samples resulted in preparations containing large amounts of highly purified virus particles. The degree of virus purity was monitored by density determination, electron microscopic analysis, immunodifiusion, and hemagglutination. The morphological integrity and the hemagglutinating ability of the virus were preserved by the method of purification employed. The virions banded isopyenieally at a density of 1.245 g/cm 3 in CsC1 and 1.185 g/em ~ in sucrose. This result is in close agreement with the densitites given for eoronaviridae in the report of the study group on eoronavirus (33) .

Several past attempts to visualize the internal structure of eoronaviruses by EM examination of viruses treated with different surface active and lipid solvents were reported (1, 11, 18, 23) . All the procedures used resulted in disintegration of virus, and the internal components of eoronavirus have not been clearly characterized. Treatment of LY-138 virions with 1 in 10 parts chloroform followed by isopyenic eentrifugation removed from the virions envelope components to produce core-like, round structures which contained 2 polypeptides. The average diameter in negatively stained preparations was 82 nm, and the buoyant density of LY-138 core-like structure was 1.299 g/em 3 in CsC1 and 1.201 g/em 3 in sucrose. Following treatment of the TGE virus with t per cent NP40, GAt~wEs and eoworkers (11) found 60--70 nm structures with a CsC1 density of 1.295 g/em 3. Further analysis of the core-like structures is needed to determine whether they represent viral cores or virions merely stripped of envelope projections.

The packing arrangement of the nueleoeapsid, whether helical or compound, could not be unequivocally determined from our electron microscopic analysis. However, viral core-like structures maintained a round configuration similar to the condensing cores reaching a diameter of 60 nm that were detected in the course of studies on the morphogenesis of virions in LY-138-infected intestinal cells (9) . Obvious thread-like structures as described for infectious bronchitis virus by others were :not detected (18) . Evidence for helical symmetry was thus not found. Several eoronaviruses appear to have ribonucleoprotein cores that are formed before envelopment and that can exist without the envelope, a characteristic that was previously observed in oneornaviruses (3, 4, 7) . The possibility of a similar cubic or binal symmetry for eoronaviruses should be considered. At least 7 polypeptides were reprodueibly demonstrated from purified virions of strain LY-138 under our conditions of dissociation of purified virus by SDS, urea and mereaptoethanoi and eleetrophoretie display in polyaerylamide gels. Occasionally, 1 or 2 very minor bands appeared near the top of the gels. There were also shoulders associated with VP 2, VP 3, VP 4, and VP 6, that could represent viral or cellular polypeptides. Four of these proteins were PAS positive for carbohydrates. In addition, polypeptide analysis of LY-138 core-like structures revealed that they contained the two viral structural proteins VP3 and VPT. GARWES and PococI~ (12) detected 4 major stud 2 minor proteins of which 3 contained carbohydrates in an analysis of a TGE strain. Our results of the presence of at least 7 proteins in the virions correspond to those obtained with the human coronavirns OC43 (16) and with the virus of avian infectious bronchitis (23) . In both instances 7 polypeptides were described and 4 of these were glyeosylated. Recently, HIER~OLZE:a (15) observed that another human eoronavirus strain, 229E, had 6 glyeoproteins. In contrast, STVR~A~ (31) displayed 4 size classes of structural proteins in preparations of the murine eoronaviral strain A59 labeled with radioactive amino acids. Three of these were glycosylated. Two of these size classes consisted of a glyeoprotein with a molecular weight of 180,000 which was converted to a glyeoprotein of 90,000. Polypeptides with molecular weights close to 200,000 were not detected in our gels displaying the proteins of a virus purified from intestinal contents and analyzed speetrophotometrically.

Dissociation of the intact virions and core-like structures by SDS, urea, and mereaptoethanol followed by PAGE enabled us to locate and identify structural proteins in two viral components. This technique placed all viral glycoproteins into the components removed by chloroform treatment Which represent viral envelopes. These results were in complete agreement with previous findings on coronavirus strain 0C43 which also contained 4 glyeoproteins of which two were associated with the envelope projections (16) . It :is of interest to observe that out of the 5 proteins removed by chloroform treatment only VP4 was not a glyeoprotein. In comparison, bromelain treatment removed the 3 glyeoproteins VP 1, VP2, and VP5 from the virions of infectious bronchitis (23) . All of the 180,000 and 90,000 glycoproteins of the murine eoronavirus strain A59 were removed by protease treatment, and 20 per cent of a third glyeoprotein with 23,000 molecular weight was digested leaving a protease resistant protein of 18,000 (31).

l~eeently, infectious I~NA of the avain infectious bronchitis virus was prepared in two different laboratories (20, 29) . The molecular weight was found to be 5.5 to 5.7 × 10 a in one report (29) and 8.1 x 106 in the other (20) . If a similar genetic system functions in enteropathogenic eoronaviruses, this genetic information would be sufficient to code for more than the 7 polypeptides which were detected in the eoronavirus starin LY-138. However, another report presented evidence for a 60--~70 S RN~4 of the TGE virus. This RNA of a molecular weight of 9 X 106 dissociated into 35S and 4S subunits (13) .

It is evident that the coronavirns family may comprise presently viruses of diverse properties. I)et~iled work is justified to resolve these apparent differences.

The structure of infectious bronchitis virus

Recovery and characterization of a coronavirus from military dogs with diarrhea

Assembly of type C 0ncornaviruses: A model

Polypeptides of avian RNA tumor viruses. V. Analysis of the virus cores

The proteins of the parainfluenza virus SV5. I. Separation of virion polypeptides by polyacrylamide gel electrophoresis

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Comparison of Rous sarcoma virus specific deoxyribonucleic acid polymerases in virions of Rous sarcoma virus and in Rous sarcoma virus infected chicken cells

Light and ultrastructural pathologic changes in intestinal coronavirus infection of newborn calves

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves

A transmissible gastro-enteritis in pigs

Isolation of subviral components from transmissible gastro-enteritis virus

The polypeptide structure of transmissible gastroenteritis virus

Identification of heat-dissociable R N A complexes in two porcine coronaviruses

Antigens of bovine coronavirus strain LY-138 and their diagnostic properties. Amer

Purification and biophysical properties of human eoronavirus 229E

Protein composition of coronavirus 0C43

Purification and further characterization of an "IBV-like" virus (corenaviras)

Isolation and morphology of the internal component of human coronavirus strain 229E

Diarrheic induced changes in intracellular and extraeellular ion concentrations in neonatal calves

Genome of infectious bronchitis virus

Protein measurement with the follin phenol reagent

Manual of Histopathelogic staining methods of the Armed Forces Institute of Pathology

Polypeptides of the surface projections and the ribonucleoprotein of avian infectious bronchitis virus

SDS-acrylamide gel eleetrophoresis and its application to the proteins of poliovirus and adenovirus-infeeted human cells

Coronaviruses: A comparative review

Pathology of neonatal calf diarrhea induced by a coronavirusdike agent

Viral induced changes in intestinal transport and resultant body fluid alterations in neonatal calves

Mouse hepatitis virus infection as a highly contagious, prevalent, enteric infection of mice

Presence of infectious polyadenylated I~NA in the eoronavirus avian bronchitis virus

Characterization of a calf diarrheal eoronavirus

Characterization of a coronavirus, t. Structural proteins: Effects of preparative conditions on the migration of protein in polyaerylamide gels

l~Iorphology of transmissible gastro-enteritis virus of pigs. A possible member of eoronaviruses

Glycoprotein staining following eleetrophoresis on aerylamide gels

Authors' address: Dr. J. STOIcZ, Institu~ f(ir Virologic

Frankfurter Strasse 107, D-6300 Giessen, Federal Republic of Germany. Presently on leave from: I)epartment of Microbiology

Part of a thesis submitted by the senior author in partial fulfilhnent of the requirements for the Ph. D. Degree at Colorado State University. These investigations were supported by the Colorado Agricultural Experiment Station through Regional Project W-112, by NItt l~eseareh Grant AI-08420, by funds :from Jensen-SMsbery Laboratories, Kansas Cit.y, MO, and through a generous gift, donated anonymously to the Dr. Traey Rhodes Scholarship Fund. Published as Journal Paper No. 2382, Colorado Ag1~icultural Experiment Station.