Archives of Virology 47, 279--286 (1975) © by Springer-Verlag 1975

The Ribonueleie Aeid ot Infeetious Bronehitis Virus By HILARY WATKINS, P. REEVE, and D. J. ALEXANDER1 Department of Bacteriology, University College Hospital Medical School, London, Great Britain With 5 Figures Received October 28, 1974

Summary AnMysis of the nucleic acid of infectious bronchitis virus by SDS polyacrylamide gel eleetrophoresis revealed an R N A of molecular weight 9.0 × 106 Daltons. The R N A was shown to have a sedimentation coefficient of 50.

1. Introduetion Infectious bronchitis virus (IBV) is a member of the group of RNA-containing enveloped viruses termed Coronavimses. In man, viruses of this group m a y cause common-cold-like diseases and in pigs two fatal diseases are associated with coronavirus infections. I B V infections m a y cause considerable morbidityin adult chickens, affecting egg production and growth rates, and cause high mortality in young chicks (9). In spite of the importance and wide distribution of these pathogens little is known of the virus structure. Morphological studies (3, 4, 5) have shown t h a t the virions of I B V are pleomorphic particles 80--120 nm in diameter, with an internal component 7 - - 8 nm in cross-section, bounded by an envelope membrane which in most strains has a characteristic corona of spikes. Studies on the structural polypeptides of a human coronavirus, OC43, revealed 6 or 7 polypeptides with molecular weights of 19t,000-15,000 (ll), but studies with I B V have shown a more complex picture (CoLLI~S, An~XA~])ER and HARKNESS, in preparation). A recent study of the RNA of I B V (t5) has suggested t h a t the R N A consists of a discontinuous single strand with extreme heterogeneity in the sizes of the R N A fragments as revealed by centrifugation on sucrose gradients, or polyacrylamide gel electrophoresis. 1 Poultry Department, Central Veterina~ry Laboratory, New Haw, Vgeybridge, Surrey, Great Britain.

280

HILAt~Y WATKINS, P. R.EEVE, a n d D. J . ALEXANDEI~:

I n c o n t r a s t ,we d e s c r i b e t h e e x t r a c t i o n of a single, l a r g e m o l e c u l a r w e i g h t species of t ~ N A f r o m p u r i f i e d I B V v i r i o n s , u s i n g a m e t h o d d e s c r i b e d b y K I ~ G S B V ~ r (12) for i s o l a t i o n of N e w c a s t l e d i s e a s e v i r u s R N A .

2. Materials and Methods 2.1. Radioisotopes and Other Materials T h e following r a d i o i s o t o p e s were p u r c h a s e d f r o m t h e l ~ a d i o c h e m i c a l Centre, A m e r s h a m , U . K . : Carrier free a2p o r t h o p h o s p h a t e (10 mCi/ml), ~H-L-Leucine (46 Ci/ m3~), 5-sH U r i d i n e (20---30 C i / m ~ ) . U r i d i n e 2,3 m o n o p h o s p h a t e ( U M P ) was o b t a i n e d f r o m S i g m a L t d . , r i b o n u c l e a s e was f r o m B . D . t t . L t d . , P e e l e , E n g l a n d .

2.2. Virus Preparation and Puri]ication T e n - d a y - o l d fertile c h i c k e n eggs were i n o c u l a t e d w i t h 1 0 0 - - 1 0 0 0 E L D s 0 I B V ( B e a u d e t t e strain). A p p r o x i m a t e l y 2 h o u r s later, 5 0 - 1 0 0 I~Ci ~2p a n d 2 0 - - 5 0 I~Ci 3 H - L - L e u c i n e were i n o c u l a t e d i n t o e a c h e m b r y o . I n p r e l i m i n a r y e x p e r i m e n t s e m b r y o s were i n o c u l a t e d w i t h 2 0 - - 5 0 ~Ci s H - U r i d i n e a f t e r v i r u s i n o c u l a t i o n . A f t e r 30 h o u r s i n c u b a t i o n a t 37 ° C, i n f e c t e d eggs were ehilled a n d t h e M l a n t o i e fluid h a r v e s t e d . A f t e r low s p e e d c e n t r i f u g a t i o n to r e m o v e cell debris, t h e v i r u s was c o n c e n t r a t e d b y c e n t r i f u g a t i o n a t 40,000 g for 40 m i n u t e s . T h e pellet w a s r e s u s p e n d e d in 0.01 ~ T r i s - E D T A b u f f e r p H 7.4 (TE buffer). P u r i f i c a t i o n was b y c e n t r i f u g a t i o n t h r o u g h 37 p e r c e n t (w/w) sucrose a t 40,000 g for one h o u r a n d t h e pellet w a s r e s u s p e n d e d i n T E buffer. F u r t h e r p u r i f i c a t i o n was d o n e b y isopycnie s e p a r a t i o n on l i n e a r 0 - - 6 0 p e r c e n t (w/w) sucrose g r a d i e n t s , b y e e n t r i f u g a t i o n for 2 h o u r s a t 25,000 r . p . m . , or o n d i s c o n t i n u o u s g r a d i e n t s c o n s i s t i n g of 60, 55 a n d 38 p e r c e n t sucrose. A 3 × 20 m l r o t o r was u s e d throughout,. T h e v i r u s b a n d w a s collected, d i l u t e d w i t h T E b u f f e r a n d p e l l e t e d a t 40,000 g for 40 m i n u t e s . T h e final pellet was r e s u s p e n d e d in T E b u f f e r a n d u s e d i m m e d i a t e l y or s t o r e d a t ---70 ° C.

2.3. Injectivity and Complement Fixing Activity I n f e c t i v i t y was e s t i m a t e d as 50 p e r c e n t E g g l e t h a l dose (ELDs0) in 9 to 10-day old eggs as d e s c r i b e d (2). Complement, fixing a c t i v i t y w a s m e a s u r e d b y t h e m e t h o d of B~AC~W~LL (7).

2.4. R N A Markers R i b o s o m a l R N A f r o m E. cell, a n d r a t l i v e r were e x t r a c t e d b y t h e m e t h o d of KIt~BY (13) a n d p r e c i p i t a t e d w i t h 99 p e r c e n t e t h a n o l . T h e R N A was d r i e d w i t h a n e t h e r / e t h a n o l m i x t u r e , m a d e u p to 5 m g / r n l in 0.1 ~ s o d i u m a c e t a t e b u f f e r p i t 5.2 a n d s t o r e d a t - - 2 0 ° C. T r a n s f e r (5 S) R N A w a s o b t a i n e d f r o m Miles P r o d u c t s .

2.5. S D S Sucrose Gradient Centri]ugation S a m p l e s of r a d i o d s o t o p e - l a b e l l e d I B V were m a d e I p e r c e n t w i t h r e s p e c t to SDS a n d h e a t e d for 2 m i n u t e s a t 60 ° C. S a m p l e s of v i r u s a n d E. cell r i b o s o m a l R N A were l a y e r e d o n t o p r e f o r m e d l i n e a r 1 5 - - 3 0 p e r c e n t (w/w) sucrose g r a d i e n t s c o n t a i n i n g 1 p e r c e n t SDS. R a t l i v e r r i b o s o m a l R N A a n d 5S R N A were r u n s i m u l t a n e o u s l y o n i d e n t i c a l g r a d i e n t s . C e n t r i f u g a t i o n was e i t h e r for one h o u r a t 50,000 r . p . m , i n a 3 × 6.5 m l titanim~n r o t o r or for 16 h o u r s a t 22,000 r. p. In. i n a 3 × 20 m l rotor. G r a d i e n t s were r e m o v e d b y u p w a r d d i s p l a c e m e n t u s i n g a n ISCO densi W g r a d i e n t r e m o v e r a n d t 2 or 25 d r o p f r a c t i o n s were collected. T o t a l r a d i o a c t i v i t y w a s e s t i m a t e d b y dissolving 10 or 20 izl s a m p l e s in Oxitol s c i n t i l l a t i o n fluid (1) a n d c o u n t i n g o n a n I C N T r a e e r l a b C o r u m a t i e 200 S p e c t r o m e t e r . Acid-insoluble r a d i o a c t i v i t y w a s m e a s u r e d b y p r e c i p i t a t i n g t h e r e m a i n d e r of t h e f r a c t i o n s w i t h 5 per' c e n t (w/v) t r i c h l o r a c e t i e acid (TCA) in t h e p r e s e n c e of 50 izg c a r r i e r R N A . T h e p r e c i p i t a t e s were w a s h e d w i t h 20 m l ice-cold 5 p e r c e n t TCA o n glass fibre circles ( W h a t m a n Ltd.), dried, a n d t h e radioactivity estima~d.

IBV-RNA

28i

2.6. Polyacrylamide Gel Electrophoresis (PAGE) Polyacrylamide gels 7 m m in diameter and 80 m m in length were cast in glass tubes by polymerising a solution containing: 2.2 per cent (w/v) acry]amide, 0.125 per cent (w/v) N, N-methylenebisaerylamide, 0.03 per cent (v/v) N, N, N', N'-tetramethylethylenediamine, 10 per cent (v/v) glycerol, in 0.125 ~ Tris-NiC1 buffer pI-t 7.0. Samples, or marker BNA, were made 1 per cent with SDS, 20 per cent with glycerol and 0.01 per cent with bromophenol blue and layered onto the surface of the gels. The resevoir buffer was 0.04 m Tris-acetate pH 7.8, containing 0.1 per cent SDS, 10 per cent glycerol and 2 mlv~ UMP. F~leetrophoresis was at, 5--7 mA per gel for approximately 4 hours. After fixing in ice cold 5 per cent TCA, gels were scanned on a Joyce-Loebl UV scanner, sliced, and radioactivity estimated as described (I).

3. Results

3.1. Puri[ication o / I B V P u r i f i c a t i o n of virus labelled with 3H-Uridine on linear sucrose gradients produced a single peak of r a d i o a c t i v i t y a t a n a p p r o x i m a t e d e n s i t y (D~0 o) of 1.18 g/ em 3. I n f e c t i v i t y a n d c o m p l e m e n t fixing a c t i v i t y were related to this peak, confirming t h a t I B V is a n R N A - e o n t a i n i n g virus (Figs. 1 a n d 2).

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Fig'. 1. Isopycnie eentrifugation of infectious bronchitis virus Centrifugation of partiMly purified IBV on 0--60 per cent sucrose gradients, for 2 hours at 17,000 g. Distributions of density a . . . . . . zx; protein • --®; and infectivity o - - - - - - o . Complement fixing activity is indicated by the histogram

28 2

I-III,~%l~Y WATKINS,

P. I~,EEVJ~, and D. J. ALEXANDER:

3.2. Sucrose Gradient Analysis o/ I B V RNA Virus labelled with a2p and extracted by the phenol-cresol method used by KIaBY (13) revealed a range of RNA sizes after centrifugation on 15--30 per cent sucrose gradients (data not shown) similar to those described by TA~OOK (15). In further experiments ~-irus, disrupted by SDS and smalysed by rate zonal eentrifugation on 15--30 per cent linear suerose-SDS gradients, revealed two peaks containing acid-insoluble 32p. These peaks sedimented with vMues of approximately 50S and 90S. Some 3H-leueine activity was associated with the 50S component, but a much larger peak was associated with the 90S component (Fig. 3) probably representing undegraded virus, or ribonueleoprotein.

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Fig. 2. Uptake of 3H-uridine associated with IBV Centrifugation of partially purified 3tI-uridine labelled IBV on 0--60 per cent sucrose gradients, for 2 hours at 17,000 g. Distributions of density a . . . . . . A; protein concentration (optical density at 280 nm) ; and aH-uridine radioactivity • w

Parallel experiments with unlabelled Sendai virus also showed a peak of absorbance (measuIed at 260 nm) which sedimented with a value of approximately 50S. Faster sedimenting material was also observed at the bottom of the gradient and was assumed to be partially disrupted, or whole, virus.

IBV-RNA

283

3.3. Polyacrylamide Gel Etectrophoresis o / I B V RNA Analysis of SDS-disrupted, 3~p and 3H-leucine labelled IBV by eleetrophoresis on 2.2 per cent polyacrylamide gels revealed a single major radioactive peak with a molecular weight of 9.0× 106 (approximately equivalent to 60S) (Fig. 4). In sucrose gradients two peaks of a~P activity were seen, whereas PAGE resolved only one band although some activity remained at the top of the gels. The pore size of the gel used in these experiments would have excluded lal ge molecular weight mate:riM such as the 90S component seen in sucrose gradients. The apparent discrepaney between the size of the RNA estimated by rate zonal centrifugation and PAGE was examined by analysis of the 50S component from sucrose gradients by PAGE. A peak of moleclHar weight 9.0 × 108 (A) was seen but components of lower molecular weight (B and C) were also present (Fig. 5). We consider the components B aad C to be degradation products produced during the reisolation of the 50S component from fractions of the rate zonal gradients and not subgenomie fragments as such. Sensitivity of the 50 S component of SDS disrupted IBV to ribonuclease (RNAse) was demonstrated. Part of each fraction from the 15--30 per cent sucrose gradient was precipitated with 99 per cent ethanol at - - 2 0 ° C, after addition of

7 6

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1

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10

15

Fraction No.

20

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30

Fig. 3. Rate zonal centrifugation of SDS-treated IBV Purified 32P/3tt-leueine labelled IBV in 1 per cent SDS was centrifuged on linear 15 to 30 per cent sucrose gradients containing SDS for 16 hours at 15,000 g. TCA insoluble radioactivity: ~P • .; 8tt-leucine o o

284

HILARY WATKINS, P. REEVE, and D. J. ALEXANDEI{:

100 ~xg of carrier RNA. The fractions were washed onto glass fibre circles with cold phosphate buffered saline (PBS) and the filters incubated in 2.0 ml PBS containing 10 ~tg/ml I~Nase at 28 ° C for one hour. The filters and incubation media were then washed with ice-cold 5 per cent w/v TCA and radioactivity determined. The fraction containing the 50 S component showed almost complete I~NAse sensitivity, yielding only 10 per cent of the original TCA-insoluble activity (from 550 cpm per 0.1 ml to 60 cpm per 0.1 ml).

32p o.-,o

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7 x

23

16

1

10

20

30

40

50

60

Fraction No.

Fig. l. Polyaerylamide gel eleetrophoresis of SDS-treated IBV Purified ~eP/:~H-leueine labelled IBV in 1 per cent SDS was analysed on 2.2 per cent polyaerylamide gels. Radioactivity : sep = --,, ; 31-I-leueine o. . . . o

4. Discussion

TAN~OCK (1_5) using a phenol-SDS RNA extraction technique, examined tile I~NA of IBV by sucrose gradient and PAGE analysis. His results revealed a large number of heterogeneous molecular weight components which were also obtained in our own studies using a similar technique. However, we have produced evidence, using a method involving minimum manipulation of the virus, that the t~NA genome consists of a single strand which migrates with a molecular weight of 9.0 × 106 on 2.2 per cent polyacrylamide gels or as 50S R N A on sucrose-SDS gradients. The discrepancy in value under the two systems could be accounted for if secondary structure of the R N A is involved. Further analysis of tile IBV genome

IBV-RNA

285

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A

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1

10

20

30 40 Fraction No.

50

60

Fig. 5. Polyaerylamide gel eleetrophoresis of the 50S component from SDS-suerose gradients The 32p 50S component of I B V was precipitated wi~h carrier R N A b y 99 per cent ethanol at - - 2 0 ° C a n d analysed on 2.2 per cent polyaerylamide gels

u n d e r more d e n a t u r i n g conditions, such as urea, f o r m a l d e h y d e , or DMSO r a t e zonal g r a d i e n t s m a y r e v e a l b r e a k s h i d d e n b y s e c o n d a r y s t r u c t u r e (6, 10, 12). I n studies w i t h Sendai virus (14) p a r t i a l s e c o n d a r y s t r u c t u r e of t h e R N A genome has been i n d i c a t e d using d e n a t u r i n g DMSO gradients. R e s u l t s o b t a i n e d w i t h double labelled virus do n o t preclude the p o s s i b i l i t y t h a t small a m o u n t s of p r o t e i n m a y be a s s o c i a t e d w i t h t h e R N A s t r u c t u r e u n d e r t h e conditions e m p l o y e d in this s t u d y . F u r t h e r e x a m i n a t i o n is r e q u i r e d to determ i n e t h e r e l a t i o n s h i p of t h e I~NA to the virion. W h e t h e r it exists as a nueleocapsid similar to t h e p a r a m y x o v i r u s R N A or in some o t h e r form is unknm~m a t present. Aeknowledgments

This work was a~ided b y a grant from the Agricultural Research Council (A.R.C.). H. VCL was supported b y the A.R.C. Referenees

1. ALEXANDER, D. J., and P. REEVE: The proteins of Newcastle disease virus. 1. The structural proteins. Microbios. 5, 199--212 (1972).

286

HIL~J~Y W.~I(I~S,

P. REEVE,

and D. J. ALEXANDER:

IBV-R.NA

2. ANon: Methods for the examination of poultry biologies (2nd ed.), Publication No. 1038, National Academy of Sciences, National l~eseareh Council, Washington D.C.U.S.A. (1963). 3. AI'OSTOLOV, K., T. HHH.FLEWET% and A. P. KENDALL: Morphology of influenza A, B, C and infectious bronchitis virus (IBV) virions. I n : The Biology of the Large I~NA Viruses ( B A ~ ¥ , R. D., and B. W. J. MAHY, eds.), pp. 3--26. London: Aeademic Press, 1970. 4. BE~RY, D . M . , and J. D. ALMmDA: The morphological and biological effects of various antisera on avian infectious bronchitis virus. J. gen. Virol. 3, 97--102 (1968). 5. BElfrY, D.M., J. G. CRUIOI~SHANI~, H. P. CHE, and g. J. I-I. WELLS: The structure of infectious bronchitis virus. Virology 23, 403--407 (1964). 6. BOEDTKER, H. : Dependence of the sedimentation coefficient on molecular weight of I~NA after reaction with formaldehyde. J. tool. Biol. 35, 61--70 (1968). 7. B~ACEWELL, C. D. : A direct complement fixation test for infectious bronchitis virus using heat-inactivated chicken sera. Vet. l~ec. 92, 452--454 (i973). 8. COLLINS, M.S., D.J. ALEXANDER, and J. %V. I'IAI~ICNESS : Heterogeneity of infectious bronchitis virus grown in eggs. (In preparation.) 9. CI~-~I~'GHAIVL C. H. : Avian infectious bronchitis. Adv. vet. Sci. 14, i05- -148 (1970). 10. ~ESTELAND, t2~. F., and H. BOEDTKEI~: Some physical properties of bacteriophage 1~ 17 and its ribonueleic acid. J. moL Biol. 8, 496---507 (1964). Ii. I-IIERI-IOLZER, J.C., E.C. I)ALMEII, S.G. WHIT/~'IELD, H. S. KAYE, and W. J~. DOWDLE : Protein composition of eoronavirus OC43. Virology 48, 516--527 (1972). 12. K[NGSBUI~¥, D. W. : Newcastle disease virus RNA. J. tool. Biol. 18, 195- 203 (1966). 13. KI~B~~, K. S. : Isolation and eharacterisation of ribosomM I~NA. Bioehem. J. 96, 266 269 (1965). 14. tg2OLAKOFS~¥, D., and A. BRUSCHI: Molecular weight determination of Sendal virus R N A by dimethyl sulfoxide gradient anMysis. J. ViroI. 11, 615--~620 (1973). 15. TaSrNOCK, G. A. : The nucleic acid of infectious bronchitis virus. Arch. ges. Virusforseh. 43, 259--271 (1973). Authors' address: Dr.I-I. WA~KINS, Department of Bacteriology, University College Hospital Medical School, University Street, London, W C 1 E 6 J J , Great Britain.