key: cord-0004939-kprry0ns authors: Tannock, G. A. title: The nucleic acid of infectious bronchitis virus date: 1973 journal: Arch Gesamte Virusforsch DOI: 10.1007/bf01250421 sha: 7f46a70ff78e49f40ace25face543ea2efd8250d doc_id: 4939 cord_uid: kprry0ns The nucleic acid of infectious bronchitis virus (IBV), like that of other enveloped viruses, consists of discontinuous single stranded RNA. However, unlike many other viruses, there is extreme heterogeneity in the sizes of the RNA fragments, as revealed by centrifugation in sucrose gradients or electrophoresis in polyacrylamide gels. Two principal classes of RNA fragments are present: a. A larger class comprising 74.9–85.4 per cent of total RNA and consisting of fragments having molecular weights ranging from 0.5×10(6) to considerably greater than 3.0×10(6) daltons and, b. A smaller class comprising 9.1–19.7 per cent of total RNA with the size approximately that of ribosomal 4S RNA. All IBV RNA's were fully susceptible to ribonuclease and had a buoyant density in caesium sulphate identical to that of tobacco mosaic virus RNA. No difference in the RNA profile for IBV was observed from the use of different methods of virus purification. The single-stranded RNA's of poliovirus and tobacco mosaic virus remained undegraded after preparation in the presence of IBV. Infectious bronchitis virus (IBV), mouse hepatitis virus and certain human respiratory viruses have been classified as coronaviruses (ALMEIDA et al., 1968) . The particles of IBV are 80--120 nm in diameter and contain a thread-like internal component 7--8 nm in cross-section (APoSTOLOV et al., 1970) . They differ markedly from myxoviruses in having an envelope derived from the cytoplasmic reticulmn instead of the cell membrane (B~o~: E~ et al., 1967) and club-like projections 15--20 nm in length, projecting from the envelope, which give to the virion its characteristic halo-or corona-like appearance (BERRY et al., 1964) . No outer spiky layer containing haemagglutinin or neuraminidase subunits has been demonstrated for the IBV virion (APosToLOV et al., 1970) . The nucleic acid of IBV has not been previously isolated but, from experiments with analogue inhibitors (CuN~I~GHAM, 1963) is generally considered to be RNA. Other enveloped RNA viruses of similar size such as influenza (D~rESBEI~G and ROBIS"SO~, 1967) lymphocytic ehoriomeningitis (PEDERSON, 1970) and Rous sarcoma virus (BISHOP et al., 1970) , have been shown to contain several pieces of single-stranded RNA. The work reported here shows that the RNA of IBV is also fragmented, but the size distribution of the fragments differs from that of other groups. The following buffers were used: NET 0. Other reagents included deoxyribonuclease, pancreatic ribonuelease-A, (5• crystallised, Sigma), Biogel P-30 (Calbiochem) and diethyl pyroearbonate (Fluka). ]~ibonuclease-free sucrose (Sigma) was used in gradient preparations. Acrylamide and bisacrylamide (Eastman Organic) were purified according to LOENING (1967) , and prepared at 2.2 per cent and 0.15 per cent, respectively, in eleetrophoresis buffer. Polymerisation was accomplished with 0.2 per cent N-N-N'-N'tetramethylethylenediamine and 1 per cent ammonium persulphate. Phenol (Merck reagent grade) was twice distilled for I~NA extraction. Bentonite was prepared according to the method of F:RAENKEL-CoNI~AT et al. (1961) . Yeast carrier I~I~A (Sigma) was purified by two extractions with phenol/SDS at 37 ~ C (see below) followed by the addition of sodium acetate to 0.15 ~ and two volumes of ethanol. After 1 hour at --15 ~ C, the precipitate was collected by centrifugation, dissolved in NET, reprecipitated and adjusted to 1000 izg/ml in NET for storage at --15 ~ C. Carrier-free 32p orthophosphate was obtained from the Australian Atomic Energy Commission, Lucas Heights, Sydney and 3I-I uridine (20--30 Ci/m Mole) from the Radiochemical Centre, Amersham U.K. 3It tobacco mosaic virus, aH poliovirus and aH (BHK cell) ribosomal I~NA were prepared by the method of TANNOCK et al. (1970) , using 3I-I uridine, unlabelled purified influenza strain BEL by that of TAYLoa et al. (1969) . All preparations were equilibrated with NET buffer by passing through Biogel P-30 columns prepared in NET before use. This was carried out using 2.2 per cent polyacrylamide gels, whose preparation is described above. Fractionation was according to the method of TANNOeK et al. (1970) . Thirty 8 day old chick embryos were each inoculated with 1 millicurie of 32p orthophosphate and, after 24 hours, with 103--104 EID~0 of the Victorian S vaccine strain of IBV (35th embryo passage) by the allantoic route. The IBV preparation was obtained from Dr. W. A. Geering of Commonwealth Serum Laboratories, Melbourne. Incubation was continued for a further 48 hours at 35 ~ C before the embryos were chilled and the allantoic fluid collected. This was accomplished by a modification of the method of BLAIR and DUESBERG (1970) which was used for purification of myxoviruses. Calf serum to 5 per cent was added to chilled allantoic fluid, followed by an equal volume of neutral saturated ammonium sulphate. The mixture was stirred at 0 ~ C for 30 minutes and the precipitate then collected by centrifugation at 2000g for 20 minutes and resuspended in approximately 25 mls of NET buffer. The virus was first concentrated by centrifuging onto a 2 ml 2.3 M sucrose cushion using a Spinco SW 25. I rotor for 35 minutes at 22,000 r. p. m. It was then partially purified by further centrifuging onto a similar 0.5 ml cushion through a 1.5 ml 15 per cent sucrose interface using a Spinco SW 40 rotor at 22,000 r.p.m, for 35 minutes. Final purification was achieved by the use of isopyenie centrifugation. Material from the interface was centrifuged either (A) through a 26.6 ml 15--65 per cent sucrose gradient in NET at 22,000 r. p. m. for 16 hours using a Spinco SW 25. i rotor or, (B) through an II ml 20--40 per cent potassium tartrate gradient for 2.5 hours at 35,000 r.p.m, using a Spinco SW40 rotor. Where sucrose gradients were used, fractions of 1.0 ml were collected from the bottom and the profiles for acid-insoluble radioactivity, density and infectivity are shown in Figure 1 A. Fraction 12 representing the peak of infectivity and radioactivity had a density, determined from its refractive index, of 1.176 g/cc. Fractions 8--19, representing tile common peak of radioactivity and infectivity were pooled for use in G . A . TA~'soeI~: R N A studies. Virus a p p e a r e d as a flocculent b a n d a n d sucrose was r e m o v e d b y passing t h e pooled fractions t h r o u g h a Biogel P-30 column equilibrated w i t h N E T . The i d e n t i t y of the virus material c a n be seen from t h e electron m i e r o g r a p h in Figure 2 showing a n u m b e r of n e g a t i v e l y -s t a i n e d pleomorphie particles with characteristic d u b -l i k e projections. The m i e r o g r a p h was p r e p a r e d b y Mr. J. E. Paterson, Division of A n i m a l I-IeMth, C.S.I.t~.O,, Parkville, Vietoria using a n I-Iitaehi 11B electron mieroseope. I n a n e x p e r i m e n t to c o m p a r e the b u o y a n t densities of I B V a n d influenza virus strain B E L , 0.2 ml of partially purified I B V was mixed w i t h 0.5 ral of unlabelled B E L virus a n d 1.0 ml of N E T a n d eentrifuged as described a b o v e in p o t a s s i u m t a r t r a t e gradients. F r a c t i o n s of 0.5 ml were eolleeted a n d t h e density of each d e t e r m i n e d from its refractive index. The profiles of radioactivity, h a e m a g g l u t i n i n a n d density are s h o w n in Figure 1 B. As u n t r e a t e d I B V p r e p a r a t i o n s h a v e no h a e m a g g l u t i n i n , t h e b u o y a n t density of each virus is identical a t 1.16 g/ca. A l t h o u g h a sharper resolution of virus was afforded t h a n for sucrose gradients (Fig. 1) , t h e use of p o t a s s i u m t a r t r a t e gradients for I B V purification usuMly resulted in a considerable loss in infectivity and, except where stated, virus purified in this m a n n e r w~.~ n o t used for t~NA studies. Several unsuccessful a t t e m p t s were m a d e to f u r t h e r purify I B V b y velocity gradient, eentrifugation. Virus concentrates, either before or after isopynie eentrifugation, were centrifuged for 30 m i n u t e s at 12,000 r . p . m , t h r o u g h 26.6 ml 5 --2 0 p e r cent sucrose gradients using a Spineo SW 25.1 rotor. The gradients were p r e p a r e d in distilled water, N E T or p h o s p h a t e buffered saline, b u t on each occasion all virus in t h e suspension was deposited on the base of the tube as a fast sedimenting aggregate. I n f e c t i v i t y was d e t e r m i n e d either a) in eggs according to t h e capacity of ~BV to illduee s t u n t i n g , curling or d e a t h in infected 9 --1 0 day old chick e m b r y o s (ANON., 1963) or, b) from its capacity to inhibit ciliary activity (titres expressed as CIDs0/ml) in chicken embryo tracheal organ cultures (CHEaRY and TAYLOI~-I~oBI~SO~I, 1970). The latter procedure, while more reliable, had a sensitivity approximately one tenth that of the egg titration. The following procedure was used: Approximately 2--3 ml of purified 32p IBV in NET buffer was shaken for 10 minutes at 37 ~ C with an equal volume of phenol containing 1 per cent SDS and 0.1 per cent bentonite. After centrifugation for l0 minutes at 2000g, the upper aqueous phase was reraoved and the extraction procedure repeated. To the extract was added suitable marker or carrier I%NA's followed by two volumes of ethanol and sodium acetate to 0.15 ~. A precipitate was allowed to form at --15 ~ C for 1 hour and was collected by centrifuging at 10,000 r.p.m, for 10 minutes and was finally resuspended in either NET or electrophoresis buffer. An extract was obtained from a large batch of purified virus which had been purified in sucrose-density gradients from an initial volume of approximately 1 litre of infectious allantoie fluid. An ethanol precipitate was obtained from the extract, as described above, but without the addition of carrier or marker RNA. The precipitate was then collected and dissolved in 1 ml of NET buffer and its absorbance profile is shown in Figure 3 . Values of ODe~4/2s0 and 260/2s0 of 1.20 and 1.80, respectively, were obtained, which clearly indicates that the extracted material contained RNA. This experiment was intended to confirm the genetic material of IBV as l~NA and to characterize it. I~NA was extracted from three IBV preparations purified in either sucrose (A and C) or potassium tartarate density gradients (B). In preparation A, the initial allantoic fluid was first treated with deoxyribonuclease and ribonuclease (each at 1 ~tg per ml) for 1 hour at 25 ~ C before purification. 3I-I ribosomal marker I%NA (0.2 m]) and 100 ~g of yeast carrier RIgA were added and total lgNA's from each were precipitated, as described above, and resuspended in 0.5 ml NET buffer. Each preparation was then centrifuged through pre-formed 4.8 ml 5--20 per cent sucrose gradients (A, B and C) prepared in NET for 60 (C) or 90 minutes (A and B) at 65,000 r.p.m, using a Spinco SW65 rotor. Fractions of 0.2 ml were collected, placed onto paper strips and the acid-insoluble radioactivity present was determined. The profile for each gradient is shown in Figure 4 . As a Irrespective of the purification procedure used, the results indicate that the nucleic acid of IBV is extremely heterogeneous, being comprised of a range of species spread throughout the gradients. Because of its sensitivity to ribonuclease (C) and UV absorbance profile (Fig. 3) , the genetic material of IBV is undoubtedly P~NA and further confirmation is provided by its buoyant density in caesium sulphate (see Fig. 8 ). The pattern obtained for IBV RNA in sucrose gradients was then compared with one obtained after elcctrophoresis in polyacrylamide gels. A mixture of 32p IBV RNA, aH ribosomal RNA and 100 ~g of yeast carrier RNA was prepared as described in the previous experiment. RNA was extracted from purified IBV and the final ethanol precipitate dissolved in 50 ~l of Loening's buffer. Electrophoresis of the preparation was carried out in 2.2 per cent polyacrylamide gels at 70 volts and 6 mA per tube for 1.25 (A) and 2.5 hours (B). The resulting electropherograms in Figure 5 again indicate considerable heterogeneity for IBV RNA. Two principal classes of I~NA appear to be present : The absence of two distinct RNA classes in Figure 4 can be accounted for by the relatively poor resolving properties of sucrose gradients compared with polyaerylamide gels. In an additional two electropherograms (C and D) a similar distribution of RNA fragment sizes was apparent and figures obtained for the RNA classes are as follows : C, high molecular weight RNA fragment 75.6 per cent and low molecular weight RNA 15.8 per cent and D high molecular weight 85.4~ per cent and low molecular weight 9.1 per cent of total I~NA. Discontinuity in the t~NA of IBV may have been caused by the activity of nucleases released during extraction, or by the breakage of a large RNA molecule at specific weak points. In an examination of these possibilities, the RNA profiles of IBV and either poliovirus or tobacco mosaic virus (TMV) were compared after extraction together. Two rn~xtures consisting of 0.6 m~ of ~zp IBV and 0.6 ml of purified a) 3H poliovirus, or, b) 3I-I TMV were diluted to 3.0 ml with NET buffer. RNA was then extracted from each and precipitated with ethanol after the addition of 100 ~xg of yeast carrier RNA. Each RNA precipitate was suspended in 0.5 ml of NET and centrifuged through pre-formed 4.8 m] 5--20 per cent sucrose gradients, prepared in NET for 1 hour at 65,000 r. p. m. using a Spinco SW 65 rotor. Fractions of 0.2 ml were collected and profiles for acid-insoluble radioactivity are shown in Figure 7 . They again suggest that IBV RNA is extremely heterogeneous being spread throughout the gradient as in Figure 4 . Both poliovirus and TMV RNA appear to be present as a single undegraded species, This suggests that the extraction method used does not release virion nucleases nor degrade IBV I~NA which, therefore, is presumably present within the virion as a discontinuous structure. The buoyant density of IBV RNA was compared with that of TMV I~NA. A mixture of both RNA's was prepared as described previously and, after ethanol precipitation, was suspended in 0.4 ml of NET buffer. The preparation was then layered onto 2.5 ml of a solution of caesium sulphate (density 1.64 g/ce) and centrifuged at 33,000 r.p.m, for 65 hours at 20 ~ C using a Spinco SW39 rotor. Fractions of 0.1 ml were collected and the density of each determined from its refractive index. Each fraction was then diluted to 0.4 ml with NET and an 0.2 ml volume placed onto a paper strip for the determination of acid-insoluble radioactivity. The remaining 0.2 ml of each Iraetion was treated with 1 ~g of ribonuelease for 1 hour at 25 ~ C and radioactivity remaining then estimated in the same manner. The results shown in Figure 8 , indicate that IBV RNA has a distribution in the equilibrium gradient identical with that of TMV RNA, having a peak at 1.65 g/cc, which is characteristic of other single-stranded RNA molecules (EI~IKSON and FRANKLIN, 1966) . This peak is greatly reduced in the presence of ribonuelease. The RNA of IBV differs from all these viruses in that the great proportion of its RIgA (74.9--85.4 per cent) consists of large, extremely heterogeneous fragments. Such an lgNA profile has been obtained from all freshly prepared IBV preparations examined and from one prepared from virus after 72 hours of allantoic growth. With fowl plague RNA, prepared from virus purified after differing times of growth, differences in the sizes and amounts of various RNA classes have been described and a similar heterogeneity noted (BARRY et al., 1970) . Heterogeneity in the high molecular weight RNA of IBV may reflect a degradative process, whereby a large molecule is broken down into smaller products, either mechanieMly at random weak points in the ribonucleoprotein component of the virion or through the action of nucleuses. However, the following evidence is adduced against the observed distribution for IBV I~NA being brought about in this manner: a) The size of high molecular weight RNA fragments is such that a precursor molecule of size greater than 1 • 107 daltons would be required. Such a molecule would be larger than any known RNA and seems unlikely to be present within a virion of diameter 80--120 nm having a buoyant density of 1.16--1.17 g/ec. b) No degradation was noted in the t~NA's of poliovirus or TMV after preparation in the presence of IBV ( Figure 6 ). c) No diminution in the degree of fragmentation of RNA was observed when a freshly purified IBV preparation was shaken for 30 minutes with 3 per cent diethyl pyrocarbonate, a potent inhibitor of nuelease activity (SoLYMOSu et al., 1968) prior to lgNA extraction. The low molecular weight I~NA of IBV has a molecular size comparable with that of cellular transfer I~NA (Fig. 5A ). Further knowledge of function would be provided by data on the base composition of each class IBV RNA and the degree of methylation of component bases. The high molecular weight RNA's of IBV are comparable in size with the major 57S RNA of NDV and the 70S (non-convalently linked) RNA of RSV (Dv~sBE~C~, 1968) and AMV (RoBIc~SOS and BALUDA, 1965), which are the largest known single-stranded RNA molecules. Messenger function for high molecular weight AMV RNA has been described by RIMA~ et al. (1967) , whereas that for NDV is known to be associated not with the major 57 S I%NA component, but with shorter complementary negative strands (B~ATT and ROBINSON, 1967) . Whether high molecular weight I~NA or complementary strands act as the intraeellular messengers for IBV cannot be determined from the present study. It has been recently reported by HIER~IOLZnR et al. (1972) that at least 6 polypeptides are present within the virion of the related human coronavirus OC43, having molecular weights of 191,000, 104,000, 60,000, 47,000, 30,000 and 15,000 daltons. Occasionally, a seventh polypeptide with a molecular weight of 165,000 was also present. If values of 118.0 and 346.0 are assigned for the average molecular weights of individual amino acids and nucleotides, respectively, then an I~NA molecule of molecular weight 3.92 • 106 daltons would be required to encode the genetic information for the first six polypeptides. If the seventh is also included, then the figure is 5.37 • 106 daltons. I~NA fragments of this size range are dearly present within High Molecular Weight IBV RNA (Fig. 5 ). The pattern of IBV RNA fragment sizes, as revealed by analysis in sucrose density gradients or polyacrylamide gels, suggests that considerable heterogeneity in the I~NA content of individual virions must be present. Were the particles homogeneous, representatives of each fragment size would have to be present and the total RNA complement for each virion would be well in excess of 20 • 10 s daltons, which seems unlikely for a virion of the size and buoyant density of IBV. Differences in the size of individual virions, which are apparent in Figure 2 , are a characteristic of coronaviruses (AL~EIDA et al., 1968). Such differences may reflect differences in the I~NA content of individual particles whose buoyant densities may, in consequence, be relatively similar. Heterogeneity in particle buoyant densities is apparent after purification in sucrose-density gradients (Fig. 1A) . However, much better resolution was afforded by the use of potassium tartrate gradients (Fig. t B) and similar differences have been noted by BISHOP et al. (1970) for the purification of RSV, whose I~NA's are more uniform in size than those of RSV. I thank Miss Beverly Smyth and Mrs. Glenyse Harmer for competent technical assistance. Methods for the Examination of Poultry Biolog~es Morphology of influenza A, B, C, and infectious bronchitis virus (IBV) virions Properties of cowpea ehlorotic mottle virus, its protein and nucleic acid The characterization of influenza virus RNA Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E) The structure of infectious bronchitis virus The low molecular weight I~NA's of Rous sarcoma virus. 1. The 4 S I~NA Myxovirus ribonucleie acids Ribonueleic acid synthesis in cells infected with Newcastle disease virus Large-quantity production of chicken embryo tracheal organ cultures and use in virus and myeoplasma studies Sedimentation properties of cucumber mosaic virus and its nucleic acid Newer knowledge of infectious bronchitis virus Characterization of the products formed by the RNA polymerases of cells infected with eneephalomyoearditis virus Physical properties of l~ous sarcoma virus I~NA Rom?zso~: Isolation of the nucleic acid of Newcastle disease virus The RNA's of Influenza virus Plaque formation and isolation of pure lines with poliomyelitis viruses Symposium on replication of viral nucleic acids. 1. Formation and properties of a replicative intermediate in the biosynthesis of viral ribonueleic acid OlTA: Purification of viral RNA by means of bentonite Studies on alfalfa mosaic virus. 1. The protein and nucleic acid The fractionation of high-molecular-weight ribonuelcie acid by polyacrylamide-gel electrophoresis The determination of the molecular weight of ribonueleic acid by polyacrylamide-gel electrophoresis. The effects of changes in conformation BII~S~IEL: Properties of the ribosomal 1RNA precursor in Xenopus laevis; comparison to the precursor in mammals and in plants Density gradient eentrifugation studies on lymphocytic ehoriomeningitis virus and on viral nucleic acid Template active ]~NA isolated from an oncogenic virus DA: The nucleic acid from avian myeloblastitis virus compared with the Bryan strain of I~ous sarcoma virus A new method based on the use of diethyl pyrocarbonate as a nuclease inhibitor for the extraction of undergraded nucleic acid from plant tissues COO~ER: A re-examination of the molecular weight of poliovirus RNA The polypeptides of influenza virus. 1 Cytoplasmic synthesis and nuclear accumulation