Mengovirus Virions. growth (48-h cultures) were infected with a. cell at a density of 107 cells per ml of ABM42-

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1 JOURNAL OF VIROLOGY, Mar. 1977, p Copyright 1977 American Society for Microbiology Vol. 21, No. 3 Printed in U.S.A. Factors Affecting Composition and Thermostability of Mengovirus Virions CLIFFORD W. BOND* AND H. EARLE SWIM Department ofpathology, University ofcalifornia, San Diego, La Jolla, California 9293,* and Department of Cell Biology, University of Kentucky, Lexington, Kentucky 456 Received for publication 13 October 1976 The composition of mengovirus virions produced by infected cells varies with the incubation temperature. Virions produced at 37. or 39.5C contain four major polypeptides (a, /3, -y, and 8) and one minor polypeptide (/3'). Virions produced at 31.5C contain two additional polypeptides (D, and E). The virions of two temperature-sensitive (ts) and thermolabile mutants of mengovirus (ts25 and ts88) contain an increased amount of polypeptide /3', with a corresponding decrease in polypeptide /8 when compared with the wild-type mengovirus. Virions of cardioviruses are composed of multiple polypeptides (12). The capsid of the virion is considered to consist of 6 subunits symmetrically arranged (12, 13). Each subunit appears to be processed during viral morphogenesis by three specific cleavages of a unique precursor polypeptide (2, 3, 12, 13). This mechanism predicts that the virion should contain 6 chains each of four major structural polypeptides (12, 13) Ṫhis report demonstrates that mengovirus virions produced at 31.5C have a detectable amount of two high-molecular-weight polypeptides that are not present in virions produced at 37. or 39.5C, and that the virions of two temperature-sensitive (ts) mutants of mengovirus contain an increased amount of polypeptide,3', with a corresponding decrease in the amount of polypeptide /3. The wild type (WT) and the ts mutants of mengovirus used in this study were described previously (1). The stock of WI' used was in the ninth passage from clonal isolation. The stocks of ts25 and ts88 were in the second passage from clonal isolation. The propagation of strain L-67-S, a substrain of L929 mouse fibroblasts, in suspension culture was described previously (1). Basal medium ALM42 has the same composition as basal medium ABM42 (1), except that all of the amino acids except L-cystine-dihydrochloride, L-glutamine, L-methionine, and L-tryptophan were deleted. The balanced salts solution (AES) had the same composition as Earle salt solution (5). Medium ABM42-AES-MTH consists of 25% (vol/vol) ABM42 and 75% (vol/vol) AES supplemented with 1 mm each of morpholinopropanesulfonic acid, N-tris(hydroxymethyl)methyl aminomethanesulfonic acid, and N-2-hydroxyethylpiperzine-N'-2-ethanesulfonic acid. L-67-S cells in the logarithmic phase of growth (48-h cultures) were infected with a multiplicity of infection of 2 PFU of virus per cell at a density of 17 cells per ml of ABM42- AES-MTH at 31.5C. At 5 min postinfection (p.i.), the temperature of the cell suspension was shifted to the temperature indicated in the individual experiment. At 3 min p.i., the cell suspension was diluted to a density of 2 x 16 cells per ml with ABM42-AES-MTH, and the suspension was supplemented with 1 tkg of actinomycin D per ml of medium. At 2.5 h p.i., the cell suspensions at 37. or 39.5 C were supplemented with.3,tci of the 14C-labeled amino acid mixture (New England Nuclear Corp.) per ml of medium. At 3.5 h p.i., the cell suspensions at 31.5 C were supplemented with.3,uci of the 14C-labeled amino acid mixture per ml of medium. The suspensions incubated at 37. or 39.5 C were harvested at 12 h p.i., and those at 31.5 C were harvested 18 h p.i. The suspensions were stored frozen at -2 C. The mengovirus virions were purified as follows. The crude tissue culture fluid was thawed and clarified by centrifugation for 2 min at 1, x g at 2C. Sodium dodecyl sulfate (SDS) was added to the supernatant fraction to a concentration of.25% (wt/vol), and this fraction was layered over a pad of 28% (wt/wt) sucrose in Bu3 (5 mm NaCl, 1 mm EDTA, 1 mm Tris-hydrochloride, ph 7.4), and centrifuged in an SW27 rotor at 25, rpm for 3 h at 2C. The supernatant fraction was discarded, and the pellet was resuspended in Bu3. The suspension was layered on a sucrose gradient (15 to 28% [wt/wt] sucrose in Bu3) and centri- Downloaded from on September 8, 218 by guest

2 VOL. 21, 1977 fuged in an SW27 rotor at 25, rpm for 2 h at 4VC. The gradients were fractionated and monitored for absorbance, acid-insoluble radioactivity, and infectivity (1). Infectivity and radioactivity cosedimented with the absorbance peak. The peak tubes were pooled, diluted with Bu3, and centrifuged in an SW41 rotor at 35, rpm for 3 h at 4C. The resulting pellets were resuspended in Bu3. The virions were disrupted by the addition of.1 ml of Bu6 (6 mm Tris, 32 mm H3PO4, 1% [wt/vol] SDS, 1% [vol/vol] 2- mercaptoethanol, ph 7.4) to.9 ml of virus suspension. The lysate was heated at 1'C for 3 min to complete the disruption of the virions. Radiolabeled lysates of infected cells were prepared from cells infected with WT in medium ABM42-MTH (1), and incubated at 39.5C as described above. At 3 h p.i., a portion of cells was removed, washed once with ALM42-MTH, and suspended in ALM42-MTH supplemented with 1.5,Ci of the 14C-labeled amino acid mixture per ml of medium at a density of 4 x 16 cells per ml. At the end of the labeling period,.9 ml of the cell suspension was lysed by the addition of.1 ml of Bu6. The lysate was heated at 1C for 3 min and dialyzed extensively against Bu5 (6 mm Tris, 32 mm H3P4, 1 mm NaN3,.1% [wt/vol] SDS,.1% [vol/vol] 2-mercaptoethanol, ph 7.4). The polypeptides in disrupted virions and cell lysates were analyzed by discontinuous SDSpolyacrylamide gel electrophoresis as described by Laemmli (8). The resolving gels were 18 cm in length and contained 1% (wt/vol) acrylamide and.268% (wt/vol) N,N'-methylenebisacrylamide. The stacking gel was 1 cm in length and contained 3% (wt/vol) acrylamide and.8% (wt/vol) N,N'-methylenebisacrylamide. After electrophoresis was completed, the gels were sliced longitudinally, dried, and exposed to No-Screen X-ray film (Eastman-Kodak) as described by Fairbanks et al. (6) and Maizel (11). The resulting autoradiographs were scanned in a Gilford recording spectrophotometer Ṫhe nomenclature of Butterworth et al. (2) and Lucas-Lenard (9) was used to refer to the virion polypeptides in Fig. 1 and 3 and Tables 1 and 2, and to polypeptides in infected cells in Fig. 1. The data illustrated in Fig. 1 demonstrate that the WT mengovirus virion contains four major polypeptides (a, (3, y, and 8) and one minor polypeptide (,('). In addition, two minor polypeptides are evident in virions produced at 31.5C but are not evident in virions produced at 37. or 39.5 C. The identity of these two minor polypeptides as D, and E was confirmed NOTES 1257 by comparing the 31.5C profile with the infected-cell profile. The molecular weights of the virion polypeptides Di, E, a, /3, /3', and y were determined to be 69,, 59,, 37,, 33,, 29,, and 25,, respectively, by comparing parallel electropherograms of disrupted virions and standard proteins. Since the molecular weights of polypeptides smaller than 15, cannot be reliably determined by SDS-polyacrylamide gel electrophoresis, it was necessary to use the gel filtration method of Fish et al. (7). Disrupted virions were chromatographed on an agarose column and eluted with 6 M guanidine hydrochloride. The virion polypeptides eluted in two fractions: peak I and peak II (data not shown). The composition of each peak was determined by SDS-polyacrylamide gel electrophoresis. Peak I contained polypeptides D,, E, a, /3, /3', and y. Peak II contained only polypeptide 8. The data were analyzed by the method of Fish et al. (7), and the molecular weight of polypeptide 8 was calculated to be 7,8. This value is in agreement with the results obtained by Stoltzfus and Rueckert (14). The profiles in Fig. 1 were quantitated by integration, and the molar ratio of the polypeptides was determined with respect to polypeptide a (Table 1). The molar ratio of the virion polypeptides (a:3+,/3':-y) was found to be approximately (1:1:1). The number of chains per virion was calculated relative to a value of 6 for polypeptide a. Rueckert and his co-workers suggested that the picornavirus virion contains 6 chains each of the four major polypeptides (12, 13). The data in Table 1 indicate that two chains of polypeptide D, and two chains of polypeptide E are associated with the virions produced at 31.5C. The data illustrated in Fig. 2 demonstrate that the virions of ts25 and ts88 are substantially more thermolabile than the virions of WT. These data suggest that the mutational lesion in ts25 and ts88 may lie in a structural gene (1). The polypeptide compositions of the ts25 and ts88 virions are compared with that of WVT by SDS-polyacrylamide gel electrophoresis (Fig. 3). The proportion of /' in the virion of ts25 is greater than ts88, which is greater than WT. The differences were quantitated by integration of the profiles. The molar ratio of the polypeptides and the number of chains per virion were calculated (Table 2). The virion of ts88 contains 53 chains of /3 and 13 chains of /3'. The virion of ts25 contains 5 chains of /3 and 19 chains of /'. The virion of WT contains 6 chains of / and 7 chains of /3'. The number of Downloaded from on September 8, 218 by guest

3 1258 NOTES J. VIROL. w z UA A -i cc II I I I DISTANCE MIGRATED (Cm) FIG. 1. Electropherograms ofpolypeptides of mengovirus virions produced at 31.5, 37., and 39.5C, and an infected-cell lysate prepared at 39.5C. Virions were purified and then analyzed in parallel by SDSpolyacrylamide gel electrophoresis. Downloaded from on September 8, 218 by guest

4 VOL. 21, 1977 NOTES = 1 ġ a 4 1. z t TIME IN MINUTES AT 55 C FIG. 2. Heat stability of the virions of WT mengovirus and the ts mutants ts88 and ts25. Virus stocks were diluted fivefold into phosphate-buffered saline (4) containing 5% (vollvol) calfserum and incubated at 55TC in a constant-temperature water bath for the indicated time, and the surviving infectivity was determined at 31.5C. TABLE 1. Effect of incubation temperature on the composition of the virion of WT mengovirus Polypeptide TeMpa D E a( 3 Molar Chains/ Molar Chains/ Molar Chains/ Molar Chains/ Molar Chains/ Molar Chains/ ratio' virionc ratio vinon ratio vinon ratio virion ratio vision ratio vision a Incubation temperature of the infected cells. b Molar ratio of the virion polypeptides. The mass of the protein was assumed to be proportional to its radioactivity. The profiles of the electropherograms in Fig. 1 were integrated with a planar polarimeter. The mass ratio of each polypeptide (not shown) was calculated by dividing the area under each protein peak by the area under the entire profile. The molar ratio was calculated by dividing the mass ratio of each polypeptide by its apparent molecular weight and normalizing it with respect to a value of 1. for polypeptide a. e The relative number of polypeptide chains in the virion relative to a value of 6 for polypeptide a. The relative number of chains per virion was calculated by multiplying the molar ratio of each polypeptide by 6 and rounding off the product to the nearest whole number. TABLE 2. Polypeptide composition of the virions of WT, ts88, and ts25 produced at 31.5C Polypeptide Virus a fp Molar Chains/ Molar Chains/ Molar Chains/ Molar Chains7 ratio' virionb ratio virion ratio virion ratio virion WT ts ts a Molar ratio of the virion polypeptides relative to a normalized value of 1. for polypeptide a (see footnote b, Table 1). b Relative number of polypeptide chains in the virion relative to a number of 6 for polypeptide a (see footnote c, Table 1). Downloaded from on September 8, 218 by guest

5 126 NOTES J. VIROL. VIRIONS a V w o wild-type 6~~t 2 Downloaded from O DISTANCE MIGRATED (CM) FIG. 3. Electropherograms of the structural polypeptides of ts25, ts88, and WT mengovirus. Virions were produced at 31.5C, purified, and analyzed in parallel by SDS-polyacrylamide gel electrophoresis. chains of a and y per virion was constant for WT, ts25, and ts88. These data indicate that polypeptide (3' is related to polypeptide and may be the result of an aberrant cleavage of the precursor to polypeptide /8. Additionally, these data suggest that the variability in the thermostability of the virions of WT, ts25, and ts88 is due to alterations in the relative proportions of the related virion polypeptides A3 and A'. The bonds between /3' and its adjacent polypeptides may be weaker than the bonds between 83 and its adjacent polypeptides. The result of the decreased stability would be a virion that is less thermostable than WT. LITERATURE CITED 1. Bond, C. W., and H. E. Swim Physiological characterization of temperature-sensitive mutants of mengovirus. J. Virol. 15: Butterworth, B. E., L. Hall, C. M. Stoltzfus, and R. R. Rueckert Virus-specific proteins synthesized in encephalomyocarditis virus-infected HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 68: Butterworth, B. E., and R. R. Rueckert Gene order of encephalomyocarditis virus by studies with pactamycin. J. Virol. 9: Dulbecco, R., and M. Vogt Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99: Earle, W. R Production of malignancy in vitro. IV. The mouse fibroblast cultures and changes seen in the living cells. J. Natl. Cancer Inst. 4: on September 8, 218 by guest

6 VOL. 21, Fairbanks, G., Jr., C. Levinthal, and R. H. Reeder Analysis of 14C-labelled proteins by disc electrophoresis. Biochem. Biophys. Res. Commun. 2: Fish, W. W., K. G. Mann, and C. Tanford The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J. Biol. Chem. 244: Laemmli, U. K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: Lucas-Lenard, J Cleavage of mengovirus polyproteins in vivo. J. Virol. 14: McCahon, D., and P. D. Cooper Identification of poliovirus temperature-sensitive mutants having defects in virus structural protein. J. Gen. Virol. 6: Maizel, J. V., Jr Polyacrylamide gel electropho- NOTES 1261 resis of viral proteins, p In K. Maramorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press Inc., New York. 12. Rueckert, R Picornaviral architecture, p In K. Maramorosch and E. Kurstak (ed.), Comparative virology. Academic Press Inc., New York. 13. Rueckert, R. R., A. K. Dunker, and C. M. Stoltzfus The structure of mouse-elberfeld virus: a model. Proc. Natl. Acad. Sci. U.S.A. 62: Stoltzfus, C. M., and R. Rueckert Capsid polypeptides of mouse elberfeld virus. I. Amino acid compositions and molar ratios in the virion. J. Virol. 1: Weber, K., J. R. Pringle, and M. Osborn Measurement of molecular weights by electrophoresis on SDS-acrylamide gel, p In C. H. W. Hirs and S. N. Timasheff (ed.), Methods in enzymology, vol. 26. Academic Press Inc., New York. Downloaded from on September 8, 218 by guest