Cores in foot-and-mouth disease virus

VIROLOGY

116, 349-353

(1982)

SHORT COMMUNICATIONS Cores

M. S.DUBRA,~J.L.LA

in Foot-and-Mouth

Disease

TORRE,E. A. SCODELLER,~.

Centro de Virolo&

Virus

D. DENOYA,ANDC.VASQUEZ

Animal, Serrano 661-1414 Capital, Argentina

Received December 3, 1980; accepted August 31, 1981 Foot-and-mouth disease virions were dissociated with ammonium acetate and observed with the electron microscope. The major products of viral disassembly were 12 S viral subunits or skullcaps and cores. Cores appeared as spherical structures and were relatively unstable upon spreading, freezing and thawing, or treatment at low pH. Upon addition of dextran sulfate to the incubation mixture, it was possible to analyze cores by ultracentrifugation on glycerol gradients. Cores sedimented at 45 S but large amounts of filamentous structures, possibly unfolded cores, were also found at slower rates. The results of this work indicated that cores are predominantly formed by genomic RNA folded in a compact spherical configuration.

Foot-and-mouth disease virus (FMDV), which belongs to the aphthovirus genus, is an acid sensitive member of the picornaviridae family (I). The virion contains 60 copies of each of four major polypeptides (VP,-,) (2-4) and the viral genome, a single-stranded RNA molecule of 2.7 x lo6 daltons (5). VP1-a (mol wt between 26,000 and 34,000) are externally located, constituting the bulk of the viral capsid (2). VP4 (mol wt 13,500) is believed to be internally located (4). In addition to the four major polypeptides (VP,-,), FMDV contains two minor polypeptides: PdO(mol wt 40,000) and Ps6(mol wt 56,000) (6). The last one represents the virus infection associated (VIA), which appears to be the RNA-dependent RNA polymerase (7). Under controlled experimental conditions, it is possible to promote a sequential and nonrandom disruption of the virions into 12 S protein subunits or skullcaps (8). These skullcaps are clusters of monomers (VP,-,) with a diameter of 12 nm. Groups of 12 skullcaps are arranged tridimensionally in a quasi-icosahedral shell (8) and the viral genome is buried within this shell. Although it has been reported from absorbance temperature measurements with whole virions that the bases of the RNA ‘To whom

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core are one-third less stacked than they would be for the same RNA in free solution (9), very little is known about the organization of the RNA molecule inside the virion. Hence, the purpose of this work was to loosen the 12 S skullcaps and to observe with the electron microscope the conformation of the genomic RNA in situ. Intact virions (Fig. 1A) were spread onto a subphase of 0.1 M ammonium acetate (pH 6.2), at room temperature; 30 set after spreading, samples for the electron microscope were taken and different steps of the viral disassembly were observed; Fig. 1B shows the presence of swollen, core structures of about 19 nm (+- 1 nm) in diameter, free (arrow) or surrounded by loose skullcaps. Ninety seconds after spreading, only scattered 12 S skullcaps were observed, suggesting that the core structures were disrupted.on the spreading surface. A similar pattern of viral disruption was obtained when virions were incubated in vitro in 0.5 1Mammonium chloride in 0.1 M Tris-HCl buffer (pH 8.5) at 37”. After 2 hr of incubation, samples were withdrawn and observed with the electron microscope: in this case, cores were also observed in close association with clusters of loosely linked 12 S skullcaps (Fig. 1C). This relationship indicates that at least some virions were disrupted on the grid

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0042-6822/82/010349-05$02.00/O Copyright All rights

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surface. In other fields of the same preparation, cores and skullcaps were observed mixed and dispersed on the grid surface (Fig. 1D). The mean diameter of 400 selected cores (Fig. 1C and D) was found to be 16 nm (+l nm). These cores had a granular structure and were never found positively stained; only a slight permeability to uranyl acetate was observed. To ascertain if the observed destabilization of virions into cores and skullcaps was due to the presence of ammonium ions, purified viral particles were incubated for 1 to 5 hr at 37” in 0.5 M potassium chloride or in 0.5 M cesium chloride 0.1 M Tris-HCl (pH 8.5). After 5 hr of incubation, less than 1% of the virions were disrupted and core structures were not found. When ammonium - incubated virions were subjected to three to five cycles of freeze and thawing or to a short treatment at low pH, an extensive disruption of cores was observed. In this case, disrupted cores appeared positively stained, indicating that uranyl acetate was able to penetrate within their structure. The stability of cores was also tested by treatment with pancreatic RNase. After 3 min of incubation at 37” with 10 pug/ml of ribonuclease A, no visible changes in the structure of cores were observed. The existence of an endonuclease located within FMDV particles has been reported (10); upon incubation of virions at 37”, this enzyme was able to promote an extensive

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degradation of the genomic RNA (11). Hence, we decided to study the structure of core particles, after degradation of the viral RNA in situ by this enzyme. When these virions with degraded RNA were incubated for 3 hr with 0.5 M ammonium chloride at 37” (pH 8.5), and observed with the electron microscope, the presence of a large number of intact core structures was detected. These experiments suggested that the integrity of cores is not dependent upon the integrity of the viral RNA, at least if the native conformation of these molecules is maintained. Attempts to isolate cores were performed as described in Fig. 2. Figure 2C shows that when cores are produced in the presence of dextran sulfate, the viral RNA sedimented as a broad peak at the 35 S position in glycerol gradients. When samples from different fractions of the gradient were observed with the electron microscope, intact cores were only seen at the 45 S region (Fig. 1E). However, other positively stained filamentous structures, possibly unfolded core structures, were also seen at the 40-30 S region of the glycerol gradient (Fig. 1F). The distribution of the [14C]uridine and ‘H-amino acid mixture labeled structures in the gradient suggested that under these experimental conditions, the RNA was associated with protein(s) at the 45 S region. Nevertheless, when this region was precipitated with acetone and run on a SDS-polyacrylamide gel, no protein label was detected (data not

FIG. 1. Observation of core structures with the electron microscope. FMDV C3 (strain Resende) was replicated in monolayers of baby hamster kidney cells (BHKzl, clone 13) obtained from The Centro Panemericano de Zoonosis, Buenos Aires, Argentina, as described elsewhere (5). The cells were infected at high multiplicity in Eagle’s medium and cell fluids were harvested at 6-9 hr postinfection (pi.). The virion-containing fractions, obtained from a preparative sucrose gradient, were pooled, divided in several samples, and pelleted (SW50 rotor, 49,000 rpm, 45 min) in NET buffer (NaCl 100 mM, EDTA 1 mM, Tris-HC150 mM, pH 7.4). The pelleted virions were resuspended as follows. (A) Control FMDV resuspended in NET buffer (pH 7.4). (B) FMDV, resuspended in a small volume of NET buffer (pH 7.4), were spread onto a subphase of 0.1 M ammonium acetate (pH 6.2) at room temperature. (C) FMDV resuspended in 0.5 M ammonium chloride, 100 mM TrisHCI buffer (pH 8.5) and incubated 2 hr at room temperature. Clusters of 12 S skullcaps are seen close to cores. (D) With the same type of destabilization, scattered 12 S skullcaps and cores can also be observed. (E) Cores obtained from the 45 S fraction of the glycerol gradient described in Fig. 2C. (F) Positively stained disrupted cores observed along the 40-30 S fractions of the same gradient (Fig. 2C). Filaments were observed extruding from disrupted cores. (A to F) Magnification X360,000. The bar represents 0.1 pm. All the samples were placed on carbon-coated grids. The samples were contrasted with 2% uranyl acetate.

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FIG. 2. Analysis of core structures by ultraeentrifugation. BHKai cells were infected with FMDV (type Ca, strain Resende) at high multiplicity and labeled with 3H-amino acid mixture (New England Nuclear (NEN) 15 rCi/ml) and [i”C]uridine (NEN, 10 &i/ml) in Eagle’s medium contaning 1/20th the concentration of amino acids at 120 min p.i. Cell fluids were harvested at 6-9 hr p.i., virions were repurified by ultracentrifugation to equilibrium in a performed linear eesium chloride gradient (1.3-1.6 g/ml) in NET buffer (pH 7.4) (SW41 rotor, 40,000 rpm, 5 hr, 5”). The virion-containing fractions were diluted in NET buffer (pH 7.4) and divided in different samples, each one containing 30 p of virions, and pelleted as described in Fig. 1. (A) ‘H- and 14C-labeled FMDV virions were suspended in 50 pl of ammonium acetate (pH 6.2), 0.003M DL-dithiothreitol (DTT) at room temperature for 1 min. Then, Tris-HCl (pH 8.5) was added to a final concentration of 0.1 M. The sample was diluted up to 500 ~1 with 0.1 M ammonium Tris (pH 8.5) and layered on top of the lo-30% glycerol gradient made in the same buffer and centrifuged (SW41 rotor, 40,000 rpm, 20”, 140 min). (B) Unlabeled FMDV virions obtained as in Fig. 1, were suspended in the same buffer as in (A) plus 60 pg/ml dextran sulfate (Sigma), average mol wt 500,000, and were mixed with [‘Y?]uridine 35 S viral RNA prepared by phenol-chloroform method as described (5) and incubated. (C) ‘H-14C-labeled FMDV were suspended in the same buffer as in (B) and sedimented in a lo30% glycerol gradient made in the corresponding buffer. (D) 3H-14C-labeled FMDV were suspended in the same buffer as in (C) plus 5% SDS and sedimented in a lo-30% glycerol gradient made in the corresponding buffer. Aliquots from each fraction of the gradient were TCA precipitated and counted by

shown). In addition, as can be seen in Fig. 2C, the 3H-amino acid mixture labeled proteins were found sedimenting with the main peak at the 24 S position of the gradient. Observation of this material with the electron microscope revealed the presence of skullcaps enriched in the capsid proteins (VP,-,) (data not shown). The shift in the S value of skullcaps may be due to a change in their hydrodynamic properties produced by interaction with dextran sulfate (Dubra et aL, in preparation). Partially disrupted virions were observed sedimenting near the bottom of the gradient and some clusters of disrupted viral material were observed in the top fraction of the gradient. When the incubated virions were treated for 30 set with 5% SDS at 3’7” prior to centrifugation (Fig. 2D), the viral RNA was found sedimenting at the 35 S region of the gradient. In this case no enrichment in core structures was detected at the 45 S region, suggesting that the detergent treatment might have promoted the disruption of cores with a concomitant release of the protein moiety toward the top of the gradient; additionally, very little or no label was detected at the 45 S region under these conditions. Similar results were obtained when 30 pg of virions were incubated in 0.1 M ammonium chloride, 0.1 MTris-HCl (pH 8.5), containing 60 pg/ml of dextran sulfate, average mol wt 500,000, and 0.003 M DTT for 3 min at 37”. A complete disruption of the virions could be obtained either with or without the presence of dextran sulfate; as shown in Fig. 1, cores can be produced in the absence of polianion. Dextran sulfate was necessary only for the analysis of cores on a gradient, presumably by stabilization of these structures. The spurious association between dextran sulfate, viral proteins, and RNA, leading to the for-

means of a liquid scintillation counter. BHK cytoplasmic RNA extracted as described (5) was run in a parallel gradient and used as marker for sedimentation coefficients. (0) 14C-labeled FMDV RNA, (0) 3H-labeled FMDV proteins.

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mation of artifactual core structures during incubation of virions, was ruled out by incubation of 35 S genomic RNA with unlabeled virions in ammonium salts (Fig. 2B). These results suggest that, at least under these conditions, the free RNA was not able to interact with dextran sulfate or with viral proteins to form 45 S core structures. The presence of core structures in several icosahedral viruses (12) as well as in intact polioviruses (13) and FMDV (8) has been already reported. Ribonucleoprotein strands were also seen extruding from heated poliovirus and rhinovirus particles (14). The nature of the packing of the viral RNA inside the virion is an important problem about which little is known (15). The production of cores upon incubation of intact virus with ammonium salts indicates the existence of easily disrupted bonds between the genomic RNA and the capsid proteins. As it is seen in Fig. 2C, the majority of the viral RNA sediments at the 35 S position of the gradient but part of this label is constantly found at the 45 S region (Fig. 2C) where intact cores are observed. When the incubated virions are treated with SDS prior to centrifugation, as it is explained in Fig. 2D, no cores are seen at the 45 S region of the gradient. Since no skullcaps were attached to cores at this region when observed with the electron microscope, it might be presumed some minor polypeptide(s) could be bound to the RNA. It is known that VP4 behaves as if it were internally located (16, I7), hence it might be possible that this polypeptide could be related to core structure. The small number of cores recovered from the 45 S region of the gradient is not enough to elucidate this hypothesis. Therefore, it will be necessary to stabilize the ammonium ion-derived cores by means of a reversible protein crosslinker agent, avoiding artificial binding of nonspecific capsid proteins in order to determine the possible existence of polypeptides related to core structures. The results of this work showed that core structures can be observed free of other viral structures and they probably represent discrete particle components in FMDV virions.

ACKNOWLEDGMENTS The authors wish to thank E. Motta and A. Sagedahl for their help during the investigation. They are also indebted to M. E. Rau and G. Teglia for their excellent technical assistance. This investigation was supported by Consejo National de Investigaciones Cientificas y Tecnicas (CONICET), Fundacion para la Education la Ciencia y la Cultura (FECIC), and Junta National de Carnes (JNC), Argentina. REFERENCES 1. COOPER, P. D., BACHRACH, H. L., BROWN, F., GHENDON, Y., GIBBS, A. J., GILLESPIE, J. H., LONBERG-HOLM, K., MELNICK, J. L., MOHANTY, S. B. POVEY, R. C., RUECKERT, R. R., SCHAFFER, F. L., and TYRREL, D. A. J., Interxrirolog~ 10, 165-180 (1978). 2. BACHRACH, H. L., “Beltsville Symposia in Agricultural Research. I. Virology in Agriculture” (J. A. Romberger, ed.), pp. 3-32. Allanheld, Osmun, Montclear, 1977. 3. RUECKERT, R. R., In “Comprehensive Virology” (H. Fraenkel-Conrat and R. Wagner, eds.), Vol. 6, pp. 131-213. Plenum, New York, 1976. 4. TALBOT, P., ROWLANDS, D. J., BURROUGHS,J. N., SANGAR, D. V., and BRO~TN, F., J. Gen. Viral. 19,369-380 (1973). 5. DENOYA, C. D., SCODELLER, E. A., GIMENEZ, B. H., VASQUEZ, C., and LA TORRE, J. L., Virology 84,230-235 (1978). 6. SANGAR, D. V., ROWLANDS, D. J., CAVANAGH, H. D., and BROWN, F., J. Gen. Viral. 31, 35-46 (1976). 7. NEWMAN, J. F. E., CARTWRIGHT, B., DOEL, T. R., and BROWN, F., J. Gen Viral. 45, 497-507 (1979). 8. VASQUEZ, C., DENOYA, C. D., LA TORRE, J. L., and PALMA, E. L., Virology, 97, 195-200 (1979). 9. BACHRACH, H. L., J. Mol. Biol. 8,348-358 (1964). lo. DENOYA, C. D., SCODELLER, E. A., VASQUEZ, C., and LA TORRE, J. L., Arch. Viral. 57, 153-159 (1979). 11. DENOYA, C. D., SCODELLER, E. A., VASQUEZ, C., and LA TORRE, J. L., Virology 89,67-74 (1978). 12. FENNER, F., MCAUSLAN, B. R., MIMS, C. A., SAMBROOK, J., and WHITE, D. O., In “The Biology of Animal Viruses.” Academic Press, New York, 1974. 13. BOUBLIK, M., and DZERNIEK, R., J. Gen. Viral 31, 447-449 (1976). 1.4. MCGREGOR, S., and MAYOR, H. D., J. Gen. Viral. 10,203-207 (1971). 15. PUTNAK, J. L., and PHILLIPS, B. A., Microbial Rev. 45, 287-315 (1981). 16. ROWLANDS, D. J., CARTWRIGHT, B., and BROWN, F., J. Gen. Viral. 4, 479-487 (1969). 17. ROWLANDS, D. J., SANGAR, D. V., and BROWN, F., J. Ges. Viral. 26,227-238 (1975).