key: cord-283998-whwksoxt
authors: Tannock, Gregory A.; Hierholzer, John C.
title: The RNA of human coronavirus OC-43
date: 1977-12-31
journal: Virology
DOI: 10.1016/0042-6822(77)90126-x
sha: 
doc_id: 283998
cord_uid: whwksoxt

Abstract A homogeneous RNA complex with a sedimentation coefficient of 70 S and an apparent molecular weight of approximately 6.1 × 106 was released from purified 32P-labeled, mouse-brain-derived OC-43 virus after treatment with 1% sodium dodecyl sulfate (SDS) for 15 min at 23°. The complex was highly susceptible to heat, releasing 4 S RNA fragments at 37° and breaking down to fragments of 4–70 S at 60°; it was also degraded by centrifugation through dimethyl sulfoxide gradients. Unlike tobacco mosaic virus or Rous sarcoma virus RNA, OC-43 RNA prepared by extraction with phenol-SDS or phenol-chloroform degraded into a range of fragments with coefficients of 15–55 S; 4 S RNA was also present as a minor component. This suggests that (a) extensive nicking of a large RNA molecule has occurred during viral growth, due to ribonucleases which are inactivated during phenol extractions; (b) heterogeneity for OC-43 RNA is not due to internal ribonuclease activity and fragments are held together by noncovalent linkages much weaker than those present in the 70 S retroviral RNA complex, or by small proteins; or, most probably, (c) a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products.

or, most probably, (cl a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products.

The Coronaviridae were described as a separate taxonomic group by Almeida et al. (1968) but, despite their ubiquity and economic importance, few biophysical characterization studies have been reported. The RNA of avian infectious bronchitis virus (IBV) was first described by Tannock (1973) , who obtained from purified virions a highly heterogeneous array of RNA fragments using a phenol-sodium dodecyl sulfate (SDS) extraction procedure. Fragments ranged in molecular weight from 0.5 x lo6 to more than 3.0 x 106; a smaller, more homogeneous class of a size similar to ribosomal 4 S RNA was also present.

More recently, Watkins et al. (1975) scribed IBV RNA, after its release from virions by SDS lysis, as a single species having a molecular weight of 9.0 x lo6 and a sedimentation coefficient of 50 S. They reported, but without supporting evidence, that by phenol extraction a range of RNA fragments could be obtained which was similar to that Tannock (1973) described. Garwes et al. (1975) examined the RNAs of two porcine coronaviruses, transmissible gastroenteritis virus (TGEV) and hemagglutinating encephalomyelitis virus (HEV), after preparation by SDS lysis. They obtained a single large RNA peak with electrophoretic mobility similar to that of 70 S Rous sarcoma virus (RSV) RNA. Like RSV RNA (Duesberg, 19681 , the RNAs of TGEV and HEV could be readily dissociated to 35 S and 4 S subunits, if prepared by SDS lysis at temperatures above 60".

In the present work, we attempt to resolve apparent differences in coronaviral RNA profiles obtained by different methods of preparation, using human coronavir-us OC-43 as the model for study. Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. ISSN 00426622 was first isolated in human tracheal organ culture, but it has been adapted to grow to high titer in suckling mouse brain (Mc-Intosh et al., 1967) . Unlike 2293, a human coronavirus strain which can be propagated only in cell culture but is extremely labile (Hierholzer, 1976) , OC-43 is a relatively stable virus and possesses a hemagglutinin (Kaye et al., 1970; Hierholzer et al., 1972) . It is, therefore, a more convenient model for study.

Solutions and reagents.

The following buffers were used: NET (0. Virus was released from cells in the suspension by freezing and thawing four times, and particulate matter was removed by centrifuging at 5,000 g in a Spinco 40 rotor for 20 min. The supernatant was retained in an ice bath while the pellet was resuspended in PBS to 20% of the original volume and recentrifuged; both supernatants were then combined for purification.

Virus was partially purified by adsorption to and elution from human "0" erythrocytes according to the method of Kaye et al. (1970) . Virus-adsorbed erythrocytes were washed three times with ice-cold PBS, and virus was finally eluted in two steps at 37" into approximately 20% of the original volume of PBS. In some early purifications, the eluate was then treated with ribonuclease and deoxyribonuclease (each at 50 pg/ml final concentration) in order to degrade any cellular nucleic acids adherent to the virus. When later work revealed that the profile of RNA extracted from virus treated in this manner was similar to that from untreated virus, we omitted the step.

Virus in the eluate was then concentrated by centrifuging through a 15% sucrose interface to a 0.75-ml cushion of 65% sucrose in NET at 43,200 g for 30 min in a Spinco SW 36 rotor. The concentrates were pooled, diluted with NET, and centrifuged to equilibrium for 15 hr at 97,lOOg through a lo-ml 25-65% sucrose gradient in NET at 7" in an SW 36 rotor. Thirtydrop fractions were collected, and aliquots of each were assayed for total radioactivity, hemagglutinin, density (from refractive index), and absorbance at 280 nm. A typical profile is seen in Fig. 1A . Two visible bands are present: (1) a lower opalescent band in the density range 1.168-1.202 g/ml containing all hemagglutinin activity and (2) an upper, more flocculent band of membranous material, having a density of 1.074-1.133 g/ml and no hemagglutinin.

Fractions 6-10 containing the lower band were then pooled, diluted with NET, and centrifuged in a velocity gradient in the SW 36 rotor for 1 hr at 43,200 g in a 9.0ml 15-65% sucrose gradient in NET. Fractions were collected and assayed for hemagglutinin and radioactivity. The profile in Fig. 1B equilibrium centrifugation may be further resolved into (1) a fast-sedimenting peak with radioactivity but little hemagglutinin, and (2) an upper heterogeneous peak of coincident hemagglutinin and radioactivity. The lower peak, which is not usually visible, probably consists of internal cell membranes and has been observed by Pocock and Garwes (1975) in the purification of TGEV. Heterogeneity in the upper peak is consistent with the high degree of pleomorphism observed for coronaviruses (Almeida et al., 1968) . Fractions from the upper peak, which were shown by electron microscopy to contain highly purified virus, were pooled, diluted to 5% sucrose with NET, and held at 4" for RNA analysis. Final virus preparations contained ap proximately 11 cpm/HA unit and purification resulted in a 1Cfold enrichment of hemagglutinin per unit of protein. RNA extraction. The following methods were used. (1) In the warm phenol-SDS (WPS) method (Tannock, 19731 , the viral suspension was shaken for 10 min at 37" with phenol containing 1% SDS. The two phases were separated by centrifugation, and the extraction was repeated. After a suitable marker RNA, 100 pg of carrier RNA, and sodium acetate to 0.15 it4 were added, 2 vol of ethanol were added and all RNAs were allowed to precipitate for 16 hr at -15". The precipitate was collected by centrifuging at 43,200 g for 30 min in an SW 36 rotor and then dissolved in a suitable volume of NET. All RNAs were stored -70" for analysis. (2) In the SDS-lysis (SL) method , SDS was added to 1% (w/v) to a purified virion suspension, and the mixture was held at 23" for 15 min. The RNAs released were mixed with suitable marker RNA and immediately analyzed in preformed sucrose gradients or in polyacrylamide gels. (3) In the phenol-chloroform (PC) method for influenza modified from Pons (1975) by using the ribonuclease inhibitor diethyl pyrocarbonate (DEP) in place of bentonite, the virus sample was mixed with 5% DEP, 0.1 vol2-mercaptoethanol, 1 vol PC extraction buffer, and 1 vol of phenol:chloroform

(1:l) and shaken for 3 min at 23". The phases were separated by centrifugation, and the extraction of the aqueous phase was repeated. Precipitation in the presence of marker and carrier RNAs and centrifugation were as described for the WPS method.

RNA sucrose gradient analysis.

prepared by phenol extraction were centrifuged through 4.4 ml of 15-30% sucrose gradients in NET in Spinco SW-50.1 rotor in a Model L-2 for 2.25 hr at 189,000 g and 7". Those prepared by SDS lysis were centrifuged in a Beckman Model L-4 ultracentrifuge for 2 hr at 189,000 g and 7" (Tannock and Griffith, 1975) .

Methods Used for Other Coronaviruses RNA was prepared from two samples of 32P-labeled OC-43 virus by the WPS and SL methods. Profiles of OC-43 and marker RNAs from each gradient after centrifugation are shown in Fig. 2 . With the WPS method ( Fig. 2A) uted broadly in the 15-50 S region of the gradient, and a smaller quantity of 4 S RNA was also present. With the SL method (Fig. 2B) , a single large homoge-OC-43 RNA, prepared by the SL method, neous RNA was obtained with a sedimenmigrates as a homogeneous complex with tation coefficient identical to the major an electrophoretic mobility similar to that (70 S) component of RSV RNA. The PC of both 45 S ribosomal precursor RNA and method was used for all subsequent ex-(not shown in this figure) 70 S RSV RNA. tractions involving phenol because of the Semilogarithmic plots of molecular weight greater RNA yields obtained (Tannock versus electrophoretic mobility from repliand Hierholzer, unpublished observation) cate experiments gave a mean apparent and the observation (Molloy et al., 1974) molecular weight of 6.1 x lo6 for this undethat phenol-chloroform mixtures congraded complex (Fig. 4) . Acrylamide gels served polyadenosine sequences in RNA. electrophoresed at 23" revealed considera-PAGE of OC-43RNA ble breakdown of the complex; this thermolability of OC-43 RNA was confirmed in The profiles obtained for OC-43 RNA by later experiments (see Fig. 5 ). No discrete sucrose-gradient centrifugation were com-viral 4 S RNA can be seen in Fig. 3A and pared with those obtained by PAGE. Figthe firm this with OC-43 RNA, preparations of OC-43 virus were treated with 1% SDS by the SL method at 23", 37", and 60". The distributions of acid-insoluble radioactivity for each gradient suggest that at 37 there was some breakdown of the 70 S complex into smaller 4 S fragments, and at 60" a more generalized breakdown to a range of intermediate species occurred (Fig. 5) (Repik and Bishop, 1973) ; 28 and 18 S ribosomal RNA, 1.75 and 0.70 x 106, respectively (Loening, 1968) ; 4 S ribosomal RNA, 0.025 x 10" (Staynov et al., 1972) . The position of the OC-43 RNA complex released by the SL method is indicated.

RNA components found at the top of gradients for total radioactivity (see Fig. 7A ). The electrophoretic profiles of OC-43 RNA, after preparation by the PC method, were determined at 5". Clearly, the PC method resulted in considerable breakdown of the large complex into a range of RNA fragments with electrophoretic mobilities between 45 S and 18 S (Fig. 3B) . A minor 4 S component was also present; it was similar to that found for IBV RNA after extraction by the WPS method (Tannock, 1973 Garwes et al. (1975) reported that RNA complexes from the porcine coronaviruses TGEV and HEV were degraded if extracted by SDS lysis at temperatures above 60", as has been noted for the RNAs of Retroviridae (Duesberg, 1968 ). To con- (Duesberg, 1968; Bishop et al., 1970a, b; Baltimore, 1974; King, 1976) . RNAs in each mixture, after preparation, were precipitated with ethanol in the presence of carrier RNA and analyzed in sucrose gradients .

The profiles of acid-insoluble radioactivity for each gradient are shown in Fig. 6 was mixed with 25 ~1 of NET containing 5 pg of ribonuclease for 30 min at 25"; the acid-insoluble radioactivity remaining was then determined after drying on paper strips. Figures 6A and 6B suggest the same heterogeneity for the major class of OC-43 RNA fragments already noted by the phenol extraction method (Figs. 2, 31 , and again confirm the presence of a minor 4 S component. Both TMV and the major 70 S RNA of RSV remain undegraded after extraction.

This suggests either (a) that the heterogeneity of the major RNA class is caused not by the activation of ribonucleases but by disruption of noncovalent linkages between RNA fragments which are much weaker than similar linkages within 70 S RSV RNA, or (b) a large RNA genome is extensively nicked by virion ribonucleases during viral growth and these enzymes are inactivated by phenol extraction. Sedimentation coefficients for the major RNAs released by phenol (15-55 S; Fig. 6B ) were less than those for the homogeneous 70 S peak obtained by the SL method (Fig. 2B) .

All 32P-labeled structures throughout the gradient in Fig. 6A are clearly RNA, as judged by their uniform susceptibility to ribonuclease.

A similar experiment was then carried out to extend this finding for 32P-labeled material released by the SL method. SL-extracted virus was centrifuged to release the RNA as a 70 S complex (Fig. 2B) , and half of each gradient fraction was treated with ribonuclease, as above. The profiles for acid-insoluble ragradient fraction were assayed for total radioactivity, and the profile is shown in A. Fractions 3-7, containing 70 S RNA, were pooled and reextracted in the presence of 0.1 ml of WPS-extracted 13Hluridine TMV RNA. All RNAs present were then reprecipitated in ethanol after 100 pg of carrier RNA was added. The precipitate was dissolved in 0.5 ml of NET and analyzed in 15-30% sucrose gradients. The profile of acid-insoluble radioactivity for and TMV (0---0) RNAs is shown in B. Fifty microliters of a preparation of 3ZP-labeled OC-43 was treated to release 70 S RNA by the SL method.

The preparation was then centrifuged through 5-20% sucrose gradients in DMSO containing 10 PM LiCl for 5 hr at 189,000 g and 22"; the profile of total radioactivity is shown in C.

TANNOCK AND HIERHOLZER dioactivity (Fig. 6C) indicate that the major 70 S complex released by SDS lysis is fully susceptible to ribonuclease, whereas the 32P-labeled material occurring at the top of the gradient remains fully resistant. The latter probably represents structural phospholipids which remain at the top of the gradient after SDS lysis and are distinct from ribonuclease-sensitive 4 S RNA obtained by the PC method (Fig. 6A ).

of OC-43 70 S RNA in the Presence of Phenol and DMSO Further evidence that OC-43 70 S RNA is a complex of fragments held together by weak, noncovalent bonds was obtained by (1) isolation of 70 S OC-43 RNA by SDS lysis and centrifugation, followed by extraction by the PC method in the presence of TMV RNA, and (2) isolation of RNA by the SL method and centrifugation in DMSO gradients. OC-43 70 S RNA was lysed with SDS and centrifuged to isolate the RNA complex as described (see Fig. 7 legend). The profile for total (acid-soluble and -insoluble) radioactivity from aliquots of each gradient fraction reveals a sharp peak of 32P radioactivity in the 70 S region (the RNA complex) and a much larger peak of low density material at the top of the gradient (Fig. 7A) . Similarly located but much smaller peaks for acid-insoluble, ribonuclease-resistant radioactivity are seen in Figs. 2B, 5A, and 6C. It therefore seems likely that SDS lysis separates RNA from large amounts of acid-soluble components, some acid-insoluble phospholipids, and perhaps other components in the outer virion coat.

Fractions 2-10 were extracted by the PC method in the presence of 13Hluridine TMV RNA, precipitated with ethanol in the presence of carrier RNA, and analyzed. The profile for acid-insoluble radioactivity reveals that such treatment completely degraded OC-43 70 S RNA, whereas TMV RNA remained intact (Fig. 7B ). This experiment thus confirms earlier evidence that the 70 S complex is held together by weak noncovalent bonds which are destroyed by phenol extraction (Figs. 2, 3) or gentle heating (Fig. 5) . The breakdown of isolated 70 S RNA by the PC extraction method is more complete than by similar extraction of purified virions. It does not appear to be due to ribonuclease activity because TMV RNA within the same mixture remains intact, although phenol extraction could denature OC-43 70 S RNA and so reveal breaks in an otherwise intact large RNA molecule which were produced by such activity.

Since DMSO has been used as a critical test for noncovalent linkages in RSV 70 S RNA (Duesberg, 1968) , we investigated its effect on 70 S OC-43 RNA prepared by SDS lysis. The profile for total radioactivity (Fig. 7C) shows a complete breakdown of the 70 S RNA complex to smaller fragments which sediment slightly faster than structural phospholipids at the top of the gradient (Fig. 7A ). Duesberg (1968) noted similar instability for RSV 70 S RNA, whose constituent RNA fragments are held together by noncovalent bonds less susceptible to phenol extraction than OC-43 RNA (Fig. 2B ).

The OC-43 RNA complex released by SDS lysis and 70 S RSV RNA have identical sedimentation coefficients. The latter is a complex of several noncovalently linked subunits (MW 2.5-3.3 x lo'? which are freed by heating or treatment with DMSO (Duesberg, 1968; King, 1976) . In addition to 70 S RNA, a range of smaller fragments, including a 4 S species, is present in phenol extracts of RSV (Robinson et al., 1965; Duesberg, 1968; Bishop et al., 1970a, b; Dahlberg et al., 1974) . The 70 S OC-43 complex may also consist of noncovalently linked RNA subunits which are disrupted by DMSO, but unlike 70 S RSV, are highly susceptible to phenol. The complex is more readily broken down by heating than are similar complexes isolated from RSV (Duesberg, 1968) and the TGEV and HEV coronaviruses . Its apparent molecular weight, determined comparatively by coelectrophoresis with other RNAs, is 6.1 x 106; this value is only considered to be approximate, however, since 2.2% gels do not adequately resolve high molecular-weight RNAs (Loening, 1969; King, 1976) .

Native OC-43 RNA is readily denatured into fragments by organic solvents and heat, much like that observed with B77 sarcoma virus RNA by Stoltzfus and Snyder (1975) . However, the sizes of the OC-43 RNA fragments from the disrupted complex seem to vary considerably. Phenol extraction of purified virions produces fragments ranging between 15 and 50 S, and a small amount of a homogeneous 4 S RNA is also present (Figs. 2, 3, 6) . A range of fragments of molecular weight 0.7-5 x lo6 and a smaller species was obtained after PAGE, similar to that described by Tanneck (1973) for IBV RNA. Coextraction experiments with TMV and RSV (Fig. 6 ) did not implicate internal ribonuclease activity as a cause of this heterogeneity.

When the 70 S complex released by SDS lysis was isolated and reextracted with phenolchloroform, only 5-10 S RNA fragments were obtained (Fig. 7) . Why fragments extracted in this manner should be smaller is unknown. Again, however, ribonucleases could not be detected, because a preparation of TMV RNA, when included in the extraction mixture, remained undegraded (Fig. 7B) . Possibly, RNA extracted by phenol from purified virions contains more secondary structure. Alternatively ribonucleases could have been present throughout viral growth, producing nicks in an otherwise intact RNA molecule and subsequently being destroyed by phenol-chloroform extraction.

The release of 4 S RNA from the 70 S complex by heating has also been observed for TGEV and HEV RNAs . Recent studies have shown that polyadenylate residues are present in 15-55 S but not in the 4 S RNAs obtained by PC extraction (Tannock and Hierholzer, in preparation) , indicating a functional difference between these RNA classes. Detailed studies are needed to determine what these functions are.

Although SDS is recognized as an effective RNA deproteinizing agent (Boatman and Kaper, 1973) , the susceptibility of the 70 S OC-43 complex to phenol suggests that small protein linkers could be required to hold the RNA fragments together and that they are not removable by SDS lysis. Evidence against this comes from cesium sulfate gradient experiments in which the buoyant densities of the OC-43 complex prepared by SDS lysis and phenol-extracted TMV RNA were identical (Tannock and Hierholzer, unpublished observations) . Additionally, Lowry protein determinations and 8% acrylamide gels (protein-stained) on purified RNA samples containing 120 pg RNA did not detect protein, implying that any protein linkers present must constitute less than 2% of the RNA structure. The complexing of a few small proteins to a large RNA molecule, however, may produce no detectable difference in buoyant density, as Sen et aZ. (1976) have noted for Rauscher murine leukemia viral RNA and phosphoprotein P12, with a type-specific RNA binding affinity.

Extensive nicking of retroviral 70 S RNA due to ribonuclease activity has been observed after extended viral growth in cell culture (Bader and Steck, 1969; King and Wells, 1976) and during virus purifcation (Scheele and Hanafusa, 1972) . The integrity of the nicked 70 S RNA is still maintained after extraction from virions by phenol, being only apparent after treatment with RNA denaturants.

Extensive changes in OC-43 RNA conformation produced by phenol suggest that, even if extensive nicking of the 70 S complex occurs during viral growth, there are marked differences in its secondary structure compared to that of 70 S retroviral RNA.

To determine whether a genuine subunit structure is present in the coronaviral complex, it would be highly desirable to examine the virion RNA present at different times after maturation in a more strictly defined milieu than that of the mouse brain. However, for human coronaviruses, the lack of convenient model systems is a severe impediment to this work.

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