A Model for Vesicular Exanthema Virus, the Prototype of the Calicivirus Group

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1 d. sen. ViroL (1978), 40, Printed in Great Britain 161 A Model for Vesicular Exanthema Virus, the Prototype of the Calicivirus Group By J. N. BURROUGHS, T. R. DOEL, C. J. SMALE AND F. BROWN Animal Virus Research Institute, Pirbright, Surrey (Accepted Io February 1978) SUMMARY The structure of vesicular exanthema virus, the prototype member of the calicivirus group, has been studied in more detail. The RNA comprises 18 % of the virus particle and has a mol. wt. of about 2.8 x lo 6, based on polyacrylamide gel electrophoresis experiments in the presence of formaldehyde. The virus contains one major polypeptide, tool. wt. 70 IO 3 as determined by polyacrylamide gel electrophoresis and by chromatography on Sepharose 6B in the presence of 6 M- guanidine. Further evidence for the presence of a single major polypeptide was obtained by tryptic peptide analysis of 35S-methionine labelled virus. The mol. wt. of a protein oligomer produced by adjusting the ph of virus suspensions to 3"5 was c. 200 lo 3. On the basis of these data we propose a T = 3 model for the virus capsid incorporating 180 copies of the virus protein. INTRODUCTION Although the caliciviruses, vesicular exanthema virus (VEV), San Miguel sea lion virus (SMSV) and feline calicivirus (FCV) are tentatively classified as possible members of the family Picornaviridae (Fenner, I976), there is substantial evidence, summarized by Burroughs & Brown (I974), that they should be regarded as forming a new family, the Caliciviridae. In addition to their distinctive morphology, the caliciviruses differ from the picornaviruses in the proportion of RNA in the virus particle (Oglesby et al 1971) and in possessing a single polypeptide (J. N. Burroughs, quoted in Brown & Hull, I973; Bachrach & Hess, I973, Burroughs & Brown, I974). Recent experiments by Black & Brown (I975/76) have also shown that the translation of the RNA of VEV, the prototype member of the family, is completely different from that of the picornaviruses. In this paper additional evidence has been provided by tryptic peptide analysis for the presence of a single major polypeptide in the virus particle and a more accurate estimation of its tool. wt. has been made. The composition and mol. wt. of the virus particle have also been determined. Using this information and the production of a protein oligomer of mol. wt. approx. 200 x io 3, a model for the virus capsid is proposed. METHODS Virus growth, labelling and purification. VEV, type E54, was grown in monolayers of IBRS-2 cells (de Castro, 1964). Unlabelled virus and ah-uridine labelled virus were grown using Eagle's medium, 35S-methionine labelled virus using methionine-free Eagle's medium and 14C-amino acid labelled virus using Earle's saline. In the case of 3H-uridine labelling II-2

2 I62 I. N. BURROUGHS AND OTHERS the medium contained o-i #g/mi actinomycin D. Virus was purified by the method of Wawrzkiewicz et al. 0968). Virus RNA. The RNA was prepared by two extractions with phenol-sds, followed by three washings of the aqueous phase with ether and precipitation with 2 vol. ethanol at -20 C. The 32P-labelled vesicular stomatitis virus RNA was prepared according to the method described by Brown et al. (1967). Polyacrylamide gel electrophoresis (PAGE) Virus protein. Purified 35S-methionine or 14C-leucine labelled virus was disrupted by heating at Ioo C for IO min in 0"5 M-urea, 1% SDS and I ~o mercaptoethanol in o.oi M- phosphate buffer, ph 7"2. Because the source of SDS may affect the migration of proteins in PAGE, the BDH specially pure grade was used in all our experiments. Samples were electrophoresed for 5 h at 4 ma per gel on 7"5 ~o discontinuous gels using the method of Laemmli 0970). One mm gel slices were solubilized in Nuclear Chicago solubilizer (NCS) and counted in a Packard liquid scintillation counter. Virus RNA. Electrophoresis was performed by the method of Loening (1967). The RNA was dissolved in 50 #1 of electrophoresis buffer containing I8 ~o sucrose and o.o2 % SDS and electrophoresed in 24 % acrylamide gels containing o'4 % agarose at 5 ma per gel for 3 to 4 h. Two mm gel slices were digested in NCS and counted in a toluene scintillant in a Packard liquid scintillation counter. Gelfiltration in 6 M-guanidine hydrochloride. Phosphorylase a (mol. wt. 92"5 x IO3), bovine serum albumin (mol. wt. 66 IO3), ovalbumin (mol. wt. 46 lo3), cc-chymotrypsinogen A (mol. wt. 25"7 x io a) and horse heart cytochrome c (mol. wt Io 3) were reduced and alkylated as described by Fish et al. (1969). 35S-labelled VEV was reduced and alkylated as described by Stoltzfus & Rueckert (1972). To o'o5 ml of 35S-labelled VEV, o'35 ml protein standard solution and 25 mg sucrose were added, together with 0.2 ml calf thymus DNA (I mg/ml) and 0"05 ml phenol red solution (1 mg/ml) to act as excluded and total volume markers respectively. The mixture was then subjected to gel filtration on a 1"5 x 7 cm column of Sepharose 6B (Pharmacia) according to the method of Fish et al. (1969). One ml fractions were collected and their absorption at 28o nm measured. They were then precipitated with lo % trichloroacetic acid in the presence of bovine serum albumin carrier, dried on to glass fibre discs and counted in a Packard liquid scintillation counter. Complement fixing activity of virus fractions. The activity was measured by the method of Brooksby (I952) for foot-and-mouth disease virus, using hyperimmune sera produced in guinea pigs by the inoculation of purified virus particles. RNA estimation. Approximately 2 mg of purified virus in distilled water were dried to constant weight in pre-weighed lyophilizing tubes to an accuracy of o.ot mg and the RNA extracted and hydrolysed in t N-HC1 at IOO C for I h in the sealed tubes. The supernatant was removed after centrifugation to pellet the insoluble protein and the hydrolysis procedure repeated. The pooled supernatants, which contained guanine, adenine, cytidylic acid and uridylic acid, were analysed at 26o nm in a spectrophotometer and the weight of RNA in the known weight of virus calculated by reference to the extinction coefficient of a synthetic mixture of the purines and pyrimidine nucleotides based on the base composition of the virus RNA (Wawrzkiewicz et al. I968). Similar results were obtained when an extinction coefficient was derived from tables. Purification and tryptic peptide analysis of the major virus protein. Purified virus labelled with 3~S-methionine was disrupted with SDS and mercaptoethanol and electrophoresed in polyacrylamide gels. The major protein was isolated from the gels, carboxylmethylated and

3 Structure of VEV 163 digested with trypsin as described by Doel & Brown (1978). The tryptic peptides were dissolved in o'o4 M-pyridine acetate, ph 2.42, centrifuged at 2o ooo rev/min for I h to remove insoluble debris and loaded on to a 4o x o'9 cm column of Chromobead P ion exchange resin (Technicon Chemicals S.A., Belgium 75o) maintained at 54 C. A mixture of 3H-amino acids was incorporated with the tryptic peptides to act as internal markers. The resin was washed with 5o ml of the sample buffer containing o'5 % thiodiglycol and o'65 % (w/w) Brij 35 and the peptides and amino acids eluted with a linear gradient made from 250 g of the sample buffer and 25o g of 2 M-pyridine acetate, ph 4"98, containing thiodiglycol and Brij 35. The flow rate was approx. 5o ml/h. Fractions of 2.1 ml were collected, of which 1.8 ml was dried on to glass fibre discs and counted in a Packard scintillation counter. Electron microscopy. Drops of purified virus suspension were applied to 3 mm AEI type grids coated with a thin continuous formvar-carbon substrate and stained with 2 % PTA, ph 7-2. The grids were examined in a Siemens Elmiskop I electron microscope at 8o kv using 3o #m objective apertures. Photographic enhancement. Selected images of virus particles showing evidence of 2-, 3- or 5-fold symmetry were photographically enhanced using methods analogous to those of Markham et al. (1963). Clear patterns were obtained when particles viewed along the 2-, 3- or 5-fold axes were enhanced using 18o, 12o and 72 repeat angles respectively. A useful increase in contrast was obtained without alteration of the characteristic patterns of a- and 3-fold enhanced images by addition of mirror reversal (2-fold axis, Fig. 7J) or by using a repeat angle of 6o (3-fold axis, Fig. 7k). Physical methods RESULTS Evidence for a single major polypeptide It has been shown previously that only one major polypeptide is found when disrupted VEV is examined by electrophoresis in continuous, phosphate-buffered SDS-polyacrylamide gels and by SDS-hydroxylapatite chromatography (Burroughs & Brown, 1974). A minor polypeptide of mol. wt. about 15 lo3 is also found reproducibly in the gels but it accounts for less than 2 % of the total protein. Because it is possible that the major peak could contain more than one polypeptide unresolved by the gel system, we have attempted to resolve this peak by other PAGE systems. We have found, however, that both 8 M-urea-PAGE and discontinuous SDS-PAGE (Fig. I) show only one major peak. Additionally, chromatography on Sepharose 6B in the presence of 6 M-guanidine resolved only one peak (Fig. 2). The minor polypeptide was not observed in the present study, as it does not label with zss-methionine and migrates with the front in discontinuous SDS-PAGE. Tryptic peptide analysis Fig. 3 shows the ion exchange chromatogram of the tryptic peptides of the major virus protein and indicates 8 or 9 major and several minor peptides which contain 3~S-methionine. The peak at fraction 34 is thought to be either a heterogeneous mixture or more probably a large incompletely cleaved peptide. We find a product at this position in a large number of proteins analysed by this procedure. We have also disregarded minor peaks such as the one at fraction 213 and the shoulders on the peaks at fractions lo8 and 134 because none of the latter amount to more than 0"5 % of the total counts in the major peptides. It is possible that they are oxidized forms of the more prominent peptides. Therefore, the actual number of unique peptides which contain methionine is probably between 7 and lo. The predicted

4 I64 J. N. BURROUGHS AND OTHERS BSA Phosphorylase a ~ 1 I Ovalbumin 10? 8._= ~6 d~ 4 I l Fraction I I Fig. I. Discontinuous SDS-PAGE of ass-methionine labelled VEV-polypeptides. number of 35S-methionine peptides from amino acid composition data (Soergel et al. I976) and a mol. wt. for the major virus protein of 7000o is 8 for a single species of polypeptide and I6 for two species of polypeptide. Our data therefore favour a single species of polypeptide. Molecular weight of the major polypeptide By continuous phosphate-buffered SDS-PAGE the mol. wt. of the major polypeptide of VEV has been estimated previously to be 60 to 65 x io z (Burroughs & Brown, I974). Mol. wt. in the same range were obtained by discontinuous SDS-PAGE (Fig. r), the values tending, however, to be at the lower end of the range. This is in agreement with the results of Schaffer & Soergel (I976) who obtained higher rnol. wt. values by continuous SDS-PAGE than by discontinuous SDS-PAGE. In contrast, mol. wt. values for the major polypeptide in the range 76 to 80 io 3 were obtained by 6 M-guanidine-Sepharose chromatography (Fig. 2). Molecular weight of the virus RNA 3H-uridine labelled RNA, extracted from the virus with phenol-sds, was made 4 % in formaldehyde to reduce secondary structure and kept at 80 C for Io min. The RNA was then co-electrophoresed in 2.2 % acrylamide-o-4 % agarose gels with formaldehyde treated z2p-labelled vesicular stomatitis virus (VSV) RNA and 32p-labelled swine vesicular disease

5 i Structure of VEV ][65 6 I II III IV V VI 5 I 7 4 X " 3 E o E & 2 1 #30 40 I 50 Fraction Fig. 2. Chromatography of the disrupted 35S-methionine labelled virus on a Sepharose 6B column in the presence of 6 M-guanidine. I, Blue dextran; II, phosphorylase a; III, bovine serum albumin; IV, ovalbumin; V, c~-chymotrypsinogen A; VI, cytochrome c. 32F7 16 x =_ 8 "= 6 & 4 2 =.i' r T Fraction 18 m 16 ' 14 x.=_ 12 E 10 *6 8~ O 6.=_ E 4 ZZ 2 ~ Fig. 3. Ion exchange chromatography of the tryptic peptides of 35S-methionine labelled VEVprotein.,35S; ---, 8H amino acid markers.

6 166 J. N. BURROUGHS AND OTHERS (a) 32P-VSV RNA 32P-VSV RNA (b) z-'p_ SVDV RNA? 8 i 32P-SVDV RNA ~4 m 2 ~ Fraction Fig. 4. SDS-PAGE of a mixture of ~H-uridine labelled VEV-RNA, z~p-labelled SYDV-RNA and 32P-labelled VSV-RNA (a) after treatment with 4 70 formaldehyde; (b) untreated. The positions of the 32p peaks are shown by arrows. virus (SVDV) RNA (Fig. 4a). Electrophoresis of the untreated RNA is shown in Fig. 4(b). Because the VEV-RNA co-migrated with the SVDV-RNA when either material was used, VEV-RNA has been taken to have the same mol. wt., 2.8 x Io 6, as SVDV-RNA (see Discussion). Estimation of the RNA content of the virus Oglesby et al. (1971) determined the composition of the virus by the orcinol (Dische, I955) and Lowry reactions (Lowry et al. 1951). Because of the possible errors in using the Lowry reaction it was decided to use a direct weighing procedure. In two independent determinations the RNA content of the virus was estimated to be I7-8 ~o and i8.2 %. Because both of these values differ significantly from the value of 22 % reported by Oglesby et al. (1971), we determined the RNA content of the virus by the orcinol method of Hurlbert et al. (1954) and obtained values similar to those of Oglesby et al (1971). However, a number of substances, particularly pentoses and possibly proteins (Dische, 1955), are known to interfere with the orcinol reaction. When the major protein of VEV was purified by SDS-hydroxylapatite chromatography and tested in the orcinol reaction it gave a colour reaction equivalent to one-sixth of that given by an equal weight of the virus RNA. After allowing for the protein contribution to the orcinol reaction, a value of 18 to 19,/o was obtained. This is close to the values obtained by the first method. Production of a polypeptide oligomer from virus particles To 1 voi. of purified 35S-methionine labelled virus, 9 vol. of 0-2 M-acetate buffer, ph 3"5, were added and the solution centrifuged on a 5 to 25 Yo sucrose gradient in 0"04 M-phosphate buffer, ph 7"6, at g for 16 h at IO C. A peak of radioactivity which fixed complement with hyperimmune serum produced against virus particles was found at fractions 15 to 20 (Fig. 5) and is probably an oligomer of virus protein. Co-sedimentation of the zss-methionine

7 Structure of VEV FMDV CFA I I I' i I67 12 N Bottom Fraction Top Fig. 5. Sucrose gradient centrifugation of a mixture of 35S-methionine labelled VEV protein oligomer, bovine serum albumin, 125I-labelled IgG and the I zs protein oligomer of foot-and-mouth disease virus. CFA, complement-fixing activity. peak with bovine serum albumin, 12SI-labelled pig IgG and the IaS protein oligomer of foot-and-mouth disease virus showed that the VEV protein oligomer had a sedimentation coefficient of 6 to 7S. Polyacrylamide gel electrophoresis of a 14C-amino acid labelled preparation of the oligomer showed that it contained the major polypeptide but not the minor polypeptide found in the virus particle. A preparation of the protein oligomer labelled with 35S-methionine was treated with o.r % formaldehyde for 24 h at either ao or 37 C and then heated at Ioo C for Io min in a solution containing 1% SDS, i % 2-mercaptoethanol and 0"5 M-urea. The product was then examined by electrophoresis in a 7"5 % discontinuous SDS-polyacrylamide gel. When the oligomer was treated with formaldehyde at 20 C, most of it was disrupted to the major polypeptide (Fig. 6a). However, some material was found at a position corresponding to a mol. wt. of approx. 200 io 8. Treatment at 37 C yielded a greater proportion of fixed material with this tool. wt. (Fig. 6b). The tool. wt. of the fixed material is about three times that of the major polypeptide of the virus, suggesting that the oligomer produced by acid disruption is a trimer.

8 I68 J. N. BURROUGHS AND OTHERS 24 h at 20 C (a) 24 h at 37 C (b) 8 I.=. d e- Q d~ g 6 4 fl-galactosidase 1-Galactosidase BSA I 10 I L + - I I I + 2o o 3o Fraction Fig. 6. Discontinuous SDS-PAGE of the polypeptide oligomer of VEV prepared by acid treatment of the virus followed by fixation with o'i ~o formaldehyde at (a) 20 C; (b) 37 C. A structural model of the virus A mol. wt. for VEV can be determined by two independent approaches. Firstly, a mol. wt. of 2.8 x io n for the virus RNA and an RNA content of I8% indicates a tool. wt. of I5"6 x io n for the virus particle (Table I). The second approach uses the Svedberg equation: RTs RT M = - - and D = - - D(I - ~p) 67rr~N where M is the tool. wt. of the virus particle; R is the gas constant; T is the absolute temperature; D is the diffusion coefficient of the hydrated particle; p is the density and ~7 the viscosity of the solvent, water; N is Avogadro's number; s is the sedimentation coefficient of the virus particle and is taken as t83 (Schaffer & Soergel, I973); v is the partial specific volume of the virus and is derived from the ratio of RNA to protein and the partial specific volumes of RNA and protein which are 0"55 and 0"74 respectively (Markham, 1967); r is the hydrated radius of the virus particle. The mean radius from electron microscopy is I8"75 nm (Wawrzkiewicz et al. I968). We have taken the hydrated radius to be 20.6 nm by adding io % to allow for hydration. A number of well characterized viruses, for example, vaccinia and squash mosaic viruses, have hydrated diameters approximately Io% greater than their diameters from electron microscopy. M was found to be I4"8 x io 6 which is in excellent agreement with the value of I5"6 x Io 6 obtained by the first method.

9 Structure of VEV 169 Table I. Calculation of the number of polypeptides per virus particle Number of major polypeptides I Mol. wt. of major polypeptide (SDS-PAGE) 6o to 65 x io 3 0) Mol. wt. of major polypeptide (guanidine-sepharose 76 to 80 x io 3 (2) chromatography) Mol. wt. of RNA % RNA Mol. wt. of virus (calculated from % RNA) Mol. wt. of virus (using Svedberg equation) Mean Total tool. wt. of protein Number of major polypepfides per virus particle using: 2'8 X ~80 I0 6 = i2. 4 IO 6 (I) 6'2 I0 l 12" 4 X I0 e (2) 7"8 x 1o 4 Mean 2"8 X IO n 18 I I4"8 x I0 8 15"2 X " 4 X I0 e o = I80 Using the mean value of 15"2 106 for the mol. wt. of the virus, the number ofpolypeptides per virus particle can be calculated (Table I). Estimates of 16o and 2oo are obtained which are consistent with a T = 3 lattice in which there are I8o copies of the major polypeptide in each particle. From this information we have attempted to construct a model of the virus capsid using table tennis balls, as the actual shape of the morphological unit is not known. As an acid stable trimer can be produced from the virus, we have favoured a model in which each ball represents a trimer centred in the T = 3 lattice sub-triangles. A hemisphere was used to eliminate the contribution of the rear surface of the model in the photographs. The model was either photographed along the 2-, 3- and 5-fold axes against a dark background (Fig. 7 a, b, c) or illuminated so that a diffuse shadow was thrown on to a translucent screen and the image photographed from the rear (Fig. 7d, e, f). Characteristic patterns of negative staining are often seen in electron micrographs when particles are viewed down the 2- and 3-fold axes (Fig. 7g, h) and more readily when these patterns are enhanced by photographic rotational superimposition as shown in Fig. 7(j, k) (Markham et al. 1963). Such patterns are clearly seen in shadow photographs of the model (Fig. 7 d, e). An equally well defined pattern of one negatively stained area surrounded by five might be expected when particles are viewed along the 5-fold axis. Particles having such patterns are difficult to find and are typically of very low contrast (Fig. 7 i) but photographic enhancement clearly reveals the 5-fold symmetry (Fig. 71). A possible explanation is that along the 2- and 3-fold axes the morphological units are directly superimposed while along the 5-fold axis there is no such superimposition, the front and rear faces being rotated 36 with respect to one another. As a contribution to the electron microscope image from the rear of the particle is to be expected, the effect would be to enhance the contrast along the 2- and 3-fold axes, but to reduce it considerably along the 5-fold axis. Only when particles are viewed along the 5-fold axis, however, are ten evenly spaced projections seen. A similar surface appearance is also obtained in the corresponding shadow photograph. The differences in detail between the shadow photographs and the electron micrographs could be attributed to the following factors: (x) flattening of the virus particles on the grid; (2) the likelihood that the morphological units of the virus are not spherical and are not located entirely in the trimer positions but extend from the trimer to the dimer positions; (3) the bonding between the sub-units in the virus

10 I70 I. N. B U R R O U G H S AND OTHERS Fig. 7. A model for VEV. (a, b, c) are photographs of the model viewed down the 2-, 3- and 5-fold axes respectively; (d, e, f ) are corresponding shadows of the model using diffuse back lighting. To eliminate the contribution, due to superimposition, of the rear surface of the model, one hemisphere was either photographed directly or illuminated to produce shadow photographs. (g, h, i) are electron micrographs of virus particles viewed along the 2-, 3- or 5-fold axes respectively. Virus particles after photographic enhancement using rotational superimposition (j) particle g, repeat angle i8o with single mirror reversal, (k) particle h, repeat angle 60 and (0 particle i, repeat angle 72.

11 Structure of VEV particles may be strained; (4) some degree of superimposition of the image of the rear units of the virus on that of the front units. None of these factors applies to the rigid table tennis ball model. DISCUSSION The evidence that the VEV particle contmns a single major polypeptide is now very convincing. Attempts to resolve the major peak obtained in continuous SDS-PAGE by using other physical methods, namely discontinuous SDS-PAGE and chromatography on Sepharose 6B columns in the presence of 6 M-guanidine, also failed to resolve more than one peak. Probably the most compelling evidence, however, is provided by analysis of the tryptic peptides of ass-methionine labelled virus protein. In our experiments, 7 to Io 85Smethionine containing polypeptides were obtained, in close agreement with the figure of 8 which can be calculated from the methionine content (Soergel et al. 1976) and tool. wt. (Schaffer & Soergel, I976 and the present work) of the virus polypeptide. This calculation assumes that there is a random distribution of methionine residues. The fact that the major peptides in the ion exchange chromatogram are not present in equimolat amounts does not negate this assumption. We find that a large number of peptide maps show this phenomenon which can be explained by the different susceptibilities of various peptide bonds to trypsin. Supporting evidence has also been obtained in preliminary experiments with l~5i-labelled virus protein (T. R. Doel, unpublished observations). In these experiments, ao to 25 tryptic peptides were resolved by two-dimensional chromatography electrophoresis, again in close agreement with the number of 24 calculated from the tyrosine content of the virus protein. Further evidence has also been obtained from parallel analyses of the closely related SMSV which belongs to the same family of viruses and is related serologically to VEV (Burroughs et al. 1978). Finally, attempts to dansylate the amino terminus of the major coat protein have been unsuccessful and indicate the presence of a blocked amino terminus. It would be unusual for the amino termini of two putative major coat proteins both to be blocked. Estimation of the mol. wt. of the virus protein has presented some difficulties because of its behaviour in different PAGE systems. For example, Bachrach & Hess (1973) found that the polypeptide from several ealiciviruses had a mol. wt. of 61 lo 3 in discontinuous SDS gels. In our early experiments using continuous phosphate-buffered SDS gels, a value of 68 x IO 3 was obtained (quoted in Brown & Hull, I973). Extension of this work gave values of 6o to 65 x lo 3 (Burroughs & Brown, 1974) and Schaffer & Soergel (1976) obtained values of 65 lo 3 in discontinuous gels and 7I lo 3 in continuous gels. In our present study, a value of 76 to 8o io 3 for the virus polypeptide was obtained by 6 i-guanidine-sepharose 6B chromatography. From these data it seems that a value of about 7o IO 3 is the most realistic to use for our calculations. The tool. wt. of the RNA has been estimated from a comparison of its S value with that of several picornavirus RNAs. The S value of VEV-RNA had been shown previously to be the same as that of six picornavirus RNAs after treatment with formaldehyde to reduce secondary structure (Newman et al. 1973). In the present work, the RNA was shown to co-electrophorese in polyacrylamide gels with one of these six picornavirus RNAs, namely SVDV-RNA, either with or without treatment with formaldehyde. These results show that the tool. wt. of VEV-RNA is probably similar to that of the picornavirus RNAs. The only estimates of the tool. wt. of picornavirus RNAs that have been made by methods other than sedimentation are for poliovirus RNA, which Granboulan & Girard (I969) estimated to have a tool. wt. of 2-6 lo s by measuring its length in the electron microscope, and for bovine enterovirus RNA, which Todd & Martin (I975) estimated to have a value of 2. 9 x io ~ 17 I

12 I72 J. N. BURROUGHS AND OTHERS by a method involving nuclease digestion and determination of the number of nucleotides in the molecule. Accepting these two values as the lower and upper limits for the mol. wt. of picornavirus RNAs, we have used a value of 2.8 I o ~ for VEV-RNA for our calculations. A reassessment of the RNA content of the virus has also been made. Oglesby et al. (1970 gave a value of 2I to 22 ~o for two serotypes of VEV using the orcinol method (Dische, I955). However, we have found that the protein of VEV contributed a significant orcinol reaction which must be taken into account in calculating the percentage of RNA in the virus particle. When this was done with the data from our experiments, a value of I8 to I9 % was obtained, which is in close agreement with the value we obtained by extracting the RNA from a known weight of virus and estimating the amount by measuring the absorbance of the purine bases and pyrimidine nucleotides obtained by acid hydrolysis. It was calculated from these data that there must be about 18o copies of the major polypeptide in the virus capsid (Table I). This value is at variance with that given by Schaffer & Soergel 0976) who concluded that there are probably I2O polypeptide units, present as dimers, in the virus but the discrepancy can probably be explained by the fact that these authors used a value of 22 ~o for the RNA content of the virus. Our calculation ignores the contribution of the minor polypeptide to the total tool. wt. of the virus protein. However, the total mol. wt. of the minor polypeptide would be a maximum of 200 Io a per particle which is equivalent to three copies of the major polypeptide. The protein oligomer which was isolated by acidification of the virus has a tool. wt. of 200 lo 3. This value is higher than would be expected for a protein sub-unit sedimenting at 6 to 7S. However, we favour the value of 200 x Io 3 because any configurational effects which may be influencing the sedimentation of the sub-unit in sucrose gradients will be eliminated in the presence of SDS in the polyacrylamide gel electrophoresis separation. This tool. wt. corresponds to a trimer of the polypeptide unit but although this oligomer is acid stable it may not be the dominant morphological unit. In trimeric clustering, the morphological units are clustered towards the centres of the triangles of the T = 3 lattice [Fig. 2 (b) in Klug et al. 0966)]. However, examination of electron micrographs (Fig. 7g, h, i) shows that regions of high density, i.e. low negative stain, extend from the centres of the T = 3 lattice triangles (the trimer positions) to the dimer positions at the edges of the lattice triangles, rather than being simply clustered towards the local 3-fold axes. Fig. 7 (k) supports a distribution of sub-units which is comparable with that of bacteriophage RI7 (Crowther et al. I975) in which the bulk of the sub-unit lies close to the dimer position but is extended towards the local 3-fold axis, where neighbouring sub-units are contacted, and in the case of VEV the acid stable bonds are formed. However, rotationally averaged images should be interpreted with caution, since small errors in the estimation of the centre of symmetry of a particle may over-emphasize certain morphological features. In fact, the rotationally averaged images (Fig. 7J, l) support a sub-unit distribution similar to that inferred from the unprocessed images (Fig. 7g, h, i). As in simple trimeric clustering, the polypeptide sub-units are clustered away from the 5- and local 6-fold axes, forming 32 regions which can be stained with PTA resulting in the characteristic negative-staining morphology of VEV. All the previous models for the caliciviruses have depended entirely on observations in the electron microscope. The first model was that proposed by Zwillenberg & Biirki (~ 966) for FCV. They proposed a double capsid structure, an inner capsid surrounded by an outer capsid comprising 32 morphological units. As pointed out by Peterson & Studdert 0970), such a structure was unnecessarily complex in interpreting, as an outer capsid, the surface projections seen in electron micrographs of the virus. They proposed an alternative explanation of the negative-staining morphology of FCV, with a model having 20 identical

13 Structure of VEV 173 morphological units. Another model of FCV had been proposed by Almeida et al. (I968), who suggested a structure composed of 3z morphological units, 2o of which would contain 6 structural units and I2 of which would contain 5 structural units, i.e. a T = 3 lattice, the structural units forming a 'negamere' type of morphological unit. This third model is in closest agreement with that we describe for VEV in this paper. Our model, however, does not require such an idea as the 'negamere' but is based on the concepts of Caspar & Klug 0962), Klug et al. (r966) and Finch & Holmes 0967). It describes the structure of VEV in terms of polypeptide subunits forming bridges between the dimer and trimer positions of the T = 3 lattice. We wish to thank Miss Jenny Ryder and Mr P. A. Wallbridge for the photographs and Dr T. J. R. Harris for the gift of 3zP-labelled SVDV. REFERENCES ALMEIDA, J. D., WATERSON, A. P., PRYDIE, J. & FLETCHER, E. W. L. (1968)- The structure of a feline picornavirus and its relevance to cubic viruses in general. Archiv fiir die gesamte Virusforschung 25, lo aaci-irach, rt. L. & HESS, W. R. (I973)- Animal picornaviruses with a single major species of capsid protein. Biochemical and Biophysical Research Communications 55, BLACK, D. N. & BROWN, F. (1975/76). A major difference in the strategy of the calici- and picornaviruses and its significance in classification. Intervirology 6, 57-6o. BROOKSBY, J.B. (1952). The technique of complement-fixation in foot-and-mouth disease research. Agricultural Research Council Report Series, No. 12. BROWN, F. & HULL, R. (1973). Comparative virology of the small RNA viruses. Journal of General Virology 20, BROWN, r., MARTIN, S. J., CARTWRXOHT, B. & CRICK, J. (I967). The ribonucleic acids of the infective and interfering components of vesicular stomatitis virus. Journal of General Virology x, BtrRROUCHS, 1. N. & BROWN, r. (1974). Physico-chemical evidence for the re-classification of the caliciviruses. Journal of General Virology 22, BUI~OUCI-IS, N., DOEL, T. & BROWN, V. (1978). Relationship of San Miguel sea lion virus to other members of the calicivirus group, lntervirology (in the press). CASPAR, O. L. D. & KLt7~, A. (1962). Physical principles in the construction of regular viruses. CoM Spring Harbor Symposium on Quantitative Biology 27, caowxner, R. A., AMOS, L. A. & PINCH, J. T. (I 975). Three-dimensional image reconstructions of bacteriophages RI7 and f2. Journal of Molecular Biology 98, DE CASTRO, M. P. (I964). Compartamento do virus aftoso em cultura de celulas: susceptibilidade da linhagem de celulas swinas IB-RS-2. Archivos do Instituto biologico, Sao Paulo 3I, DlSCHE, Z. (1955). Color reactions of nucleic acid components. In The Nucleic Acids, vol. I, p Edited by E. Chargaff and J. N. Davidson. New York and London: Academic Press. DOEL, T. R. & BROWN, r. (I978). Tryptic peptide analysis of the structural proteins of vesicular stomatitis virus. Journal of General Virology 38, I. PENNER, P. (1976). Classification and nomenclature of viruses. Second report of the international committee on taxonomy of viruses. Intervirology 7, 1-I 16. rlncn, J. T. & holmes, K. C. (1967). Structural studies of viruses. In Methods in Virology, vol. 3, PP Edited by K. Maramorosch and H. Koprowski. London and New York: Academic Press. fish, w. w., MANN, K. 6. & TANrORD, C. (I969). The estimation of polypeptide chain molecular weights by gel filtration in 6 M-guanidine hydrochloride. Journal of Biological Chemistry 244, GRANBOULAN, N. & GIRARd, M. (I969). Molecular weight of poliovirus ribonucleic acid. Journal of Virology 4, I-IIJR.LBERT, R. B., SCHMITZ, H., BRUMM, A. F. & POTTER, V. R. (I954). Nucleotide metabolism. II. Chromatographic separation of acid-soluble nucleotides. Journal of Biological Chemistry ~o9, KLUG, A., rinci-t, 1. T., LEBERMAN, R. & LONGLEY, W. (1966). Design and structure of regular virus particles. In Ciba Foundation Symposium: Princip&s of Biomolecular Organization, pp. 158-I89. London: Churchill. LAE~MLt, 17. K. (I970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, LOENING, tr. E. (I967). The fractionation of high molecular weight RNA by polyacrylamide gel electrophoresis. Biochemical Journal xo2, LOWRY, O. H., ROSEBROLIGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry x93,

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