(2) VOL. 38, 1981 REOVIRUS OUTER CAPSID POLYPEPTIDES trophoresis. Preparation of viral cytoplasmic polypeptides and immunoprecipitation. Preparation of [3S]methionine-labeled viral polypeptides was carried out by a modification of a previously described method (24). For preparative batches of viral polypeptides, 1 x 108 to 2 x 108 L-cells were infected with virus at 20 PFU/ cell as described above. Infected cells at a density of 106 cells per ml were incubated for 24 to 27 h at 330C, concentrated to 107 cells per ml, and labeled for 2 to 4 h at 330C in methionine-free medium containing 50 ,uCi of [3S]methionine per ml. Infected cells were then harvested, and extracts were prepared as described previously (24) in buffer containing 0.25 M NaCl, 0.005 M MgCl2, 0.01 M Tris-hydrochloride (pH 7.4), and 0.5% Nonidet P-40 (Particle Data Laboratories, Ltd., Elmhurst, Ill.) (NP40 buffer). Immune precipitation of viral polypeptides was carried out by a modification of a previously described procedure (6), using reovirus serotype 1, 2, or 3 hyperimmune rabbit serum instead of chicken antiserum as the first antibody and goat antirabbit globulin (GIBCO Laboratories, Grand Island, N.Y.) as the second antibody. All reovirus antiserum preparations were heat inactivated at 560C for 30 min before use. For immune precipitation, each preparation containing 108 infected cells was extracted in 10 ml of NP40 buffer, and this supernatant fraction was mixed with 200 to 400 p1 of rabbit antireovirus serum. This mixture was kept at 00C for 30 min; then 10 p1 of goat antirabbit antiserum per ml was added, and incubation was continued for 18 to 24 h at 40C. Immune precipitates were collected by centrifugation in an International centrifuge at 2,000 rpm and 40C for 10 min. Precipitates were then drained or lyophilized to dryness and suspended in Laemmli dissociation buffer (1.0 to 1.5 ml of buffer per 108 cells extracted) for preparative slab gel electrophoresis. Slab gel electrophoresis of viral polypeptides. Discontinuous preparative slab gel electrophoresis was carried out as described by Laemmli (16). Final gel concentrations were 10% acrylamide and 0.267% bisacrylamide (both from Eastman Kodak Co., Rochester, N.Y.). Viral polypeptides labeled with [3S]methionine were resolved on preparative gels at a constant current of 45 mA for 3.5 to 4.0 h. Gels were then washed for 10 min in water, dried at 800C with a gel dryer (Hoeffer Scientific Instruments, San Francisco, Calif.), and exposed to Kodak XR-5 medical X-ray film (Eastman Kodak). After electrophoresis, analytical gels were fixed in 50% methanol-7% acetic acid for 60 min, dried, and autoradiographed as described above. Alternatively, analytical gels were fluorographed by a previously described procedure (2). Tryptic peptide analysis of ["S]methionine-labeled viral polypeptides. Viral polypeptide bands were located by lining up the radioactive dye spots in a dried gel with the corresponding spots on the autoradiograph of the gel. Bands corresponding to viral polypeptides p1, ,lC, ol, and u3 were then cut out and eluted by a modification of a previously described procedure (11). Gel slices containing viral polypeptides were hydrated in 3 to 6 ml of a solution containing 0.1 M NH4HCO3 and 0.1% sodium dodecyl sulfate, the filter paper backing was removed, and the gel was minced into small (1-mm) pieces. Polypeptides were then eluted for 15 to 18 h at 370C, the supernatant was removed, and an additional 0.5 volume of 0.1 M NH4HCO3-0.1% sodium dodecyl sulfate was added. After an additional 2- to 4-h elution, the supernatants were combined and filtered through a 0.2-ym Acrodisc filter (Gelman Services, Inc., Ann Arbor, Mich.) to remove bits of acrylamide and filter paper, 200 pg of bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) was then added to each supernatant as a carrier, and the polypeptides were precipitated overnight at -20°C by adding 9 volumes of absolute ethanol. Polypeptides were collected by centrifugation at 9,000 rpm and 40C for 30 min in a Sorval RC-5 centrifuge (Du Pont Instruments, Wilmington, Del.) and an SS34 rotor. Pellets were suspended in 0.1 M NH4HCO3 and precipitated with absolute ethanol, and the protein were pelleted again by centrifugation and lyophilized to dryness. Polypeptides were suspended in 0.5 ml of 0.1 M NHIHCO3 and digested with 50 pg of L-(tosylamido-2phenyl)ethyl chloromethyl ketone-trypsin (Millipore Corp., Freehold, N.J.) for 15 h at 370C. An additional 25 pg of L-(tosylamido-2-phenyl)ethyl chloromethyl ketone-trypsin per ml was then added, and incubation was continued for another 2 h. Digests were then frozen at -70°C and lyophilized to dryness. Peptide samples were suspended in 0.5 ml of performic acid, oxidized for 1 h at 00C (5), frozen at -70°C, and lyophilized. Each digest was suspended in 50 to 100 pl of water, and insoluble material was removed by centrifugation at 100,000 x g for 2 min in a microfuge (Beckman Instruments Co., Palo Alto, Calif.). Samples were stored at -20°C until use. Tryptic peptides (7,500 to 60,000 cpm/plate) were spotted 1 pl at a time onto cellulose thin-layer plates (glass; thickness, 0.1 mm; EM Laboratories, Inc.; Scientific Products, Bedford, Mass.) and resolved in the Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest supplemented with 5% fetal calf serum (Sterile Systems, Inc., Logan, Utah) or with 1% fetal calf serum plus 4% calf serum (Sterile Systems). Virus purification. Virus infections and purification of virus from infected L-cell extracts were carried out essentially as described previously (24). For preparation of [3S]methionine-labeled purified virions, 1 x 108 to 3 X 108 L-cells were concentrated to 1 x 107 cells per ml and absorbed for 30 min at room temperature with 5 PFU of reovirus serotype 1, 2, or 3 per cell. Cells were then diluted 10-fold with Eagle minimal essential medium containing 5% fetal calf serum or 1% fetal calf serum plus 4% calf serum and incubated for 18 to 24 h at 330C in suspension culture (24). Infected cells were harvested by centrifugation at 500 rpm for 5 min in an International centrifuge (refrigerated model PR-J; International Equipment Co., Needham, Mass.) and then suspended at a concentration of 108 cells per ml in Eagle minimal essential medium containing 10% of the normal amount of methionine plus 5 pCi of [3S]methionine per ml. Incubation was continued at 330C for another 24 to 48 h, and the infected cells were harvested when the density decreased to 2 x 105 to 3 x 105 cells per ml. Virions were purified from infected cells as described previously (24) and then suspended directly into Laemmli protein dissociation buffer (16) for preparative slab gel elec- 209
(3) 210 GENTSCH AND FIELDS J. VIROL. A dryness; the resulting material was then resuspended and lyophilized twice with deionized water. Tryptic peptides were resuspended in deionized water (100 to 200 ,ul), and any insoluble material was removed by centrifugation in a microfuge as described above. Tryptic peptides (2 x 105 to 5 x 105 cpm/plate) were then resolved in two dimensions as described above for [3S]methionine-containing samples. After drying, plates were fluorographed for 18 to 24 h at -70°C by using Kodak X-R5 film and ightning-Plus intensifier screens. RESULTS Preparation of eluted p1, ,ulC, al, and v3 polypeptides. Reovirus serotype 1, 2, and 3 polypeptides were labeled with [ S]methionine and immunoprecipitated from infected cells or were obtained from purified virions as described above. Polypeptides were then resolved on polyacrylamide slab gels (16) and eluted from gel slices as described above. To analyze these preparations for possible contamination with other viral or cellular polypeptides, samples of the eluted polypeptides and serotype 1, 2, and 3 polypeptides from infected cells were subjected to electrophoresis in polyacrylamide gels (Fig. 1). The eluted polypeptides showed little or no detectable contamination with other viral or cellular proteins. Tryptic peptide structures of ,1 and ,lC. The ulC polypeptide of serotype 3 reovirus is thought to be derived from Al by a proteolytic cleavage (20, 36, 37). To analyze the structural relationship be- B 3 1 2 4 5 1 2 3 4 5 1 2 3 4 5 FIG. 1. Elution profiles of reovirus polypeptides. [MSJmethionine-containing reovirus polypeptides were eluted from gel slices as described in the text, and samples ofeach were suspended in Laemmli sample buffer, resolved on 10% slab gels (16), andprocessed for fluorography (2). (A) Profiles of eluted serotype 1 .1, ul C, (l, and (3 polypeptides (lanes 2 through 5, respectively). Serotype 1 polypeptides immunoprecipitated from infected cell cytoplasm were electrophoresed in lane I as markers. (B) Elution profiles of serotype 2 il1, i1I C, al, and a3 (lanes 2 through 5, respectively), compared with serotype 2 polypeptides immunoprecipitated from infected cells (lane 1). (C) Elution profiles of serotype 3 Id, AIC, al, and a3 (lanes 2 through 5, respectively), compared with serotype 3 polypeptides immunoprecipitated from infected cells (lane 1). The profiles shown represent composite autoradiographs from polypeptides eluted at different times. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest first dimension by electrophoresis for 60 min at 700 V in 1% (vol/vol) pyridine-10% (vol/vol) glacial acetic acid buffer, pH 3.5 (8). Plates were then air dried, and tryptic peptides were resolved in the second dimension by ascending chromatography in butanol-pyridineacetic acid-water (15:10:3:12) (8). Plates were again air dried and then were impregnated with PPO (2,5-diphenyloxazole) by ascending chromatography in 10% (wt/vol) PPO-acetone (3). Plates were fluorographed at -70°C on Kodak XR-5 film as described above. Tryptic peptide analysis of "I-1abeled ul polypeptides. Peptide mapping of "nI-labeled a1 polypeptides was carried out as described by Elder and coworkers (9), with several modifications. AU operations were carried out at 4°C unless otherwise specified. Briefly, serotype 1, 2, and 3 viral polypeptides were resolved on 10% slab gels as described above, stained with a solution containing 0.1% Coomassie blue, 5% (vol/vol) methanol, and 7% (vol/vol) glacial acetic acid for 30 min, and destained with 5% methanol-7% glacial acetic acid. The (l polypeptide bands were then sliced from the wet slab gels, washed for 1 h in a solution containing 25% (vol/vol) isopropyl alcohol and 10% glacial acetic acid, and then washed for 1 h in 10% methanol. Gel slices were then frozen and lyophilized to dryness. Dried gel slices were iodinated with 300 uCi of lI (17 Cuimg; New England Nuclear Corp.) as previously described (9). Untreated "1I was removed by washing the gel slices with 10% methanol until the radioactivity (in counts per minute) being eluted was about 0.5 to 2.0% of the input "1I radioactivity. The gel slices were again lyophilized to dryness and digested with trypsin as described previously (9). The tryptic peptide supernatant was then filtered (Gelman Acrodisc 0.2-,um filters) to remove bits of acrylamide and lyophilized to
(4) VOL. 38, 1981 REOVIRUS OUTER CAPSID POLYPEPTIDES tween the ,il and ,ilC polypeptides from reovirus serotypes 1, 2, and 3 we performed a tryptic peptide analysis. Viral ,il and lAlC polypeptides labeled with [3S]methionine were digested with trypsin and subjected to two-dimensional tryptic peptide analysis as described above. Figure 2 211 shows the peptide maps of serotype 1, 2, and 3 and u1C polypeptides. Visual inspection showed that each ul and ,ulC polypeptide contained eight to nine major methionine-containing tryptic peptides, whose patterns of separation were very similar. For this paper major l1 FIG. 2. Tryptic peptide maps of the lA1 and O1C polypeptides of reovirus serotypes 1, 2, and 3. [3SJmethionine-labeled viralpolypeptides were isolated, digested with trypsin, and subjected to two-dimensional thin-layer chromatography, and the dried thin-layer plates were fluorographed at -70°C as described in the text. In these and all subsequent figures samples were spotted on the lower right corner and peptides were electrophoresed in the first dimension (from right to left) and chromatographed in the second dimension (from bottom to top). (A) Peptide map of serotype 1 A1. (B) Peptide map of serotype 2 ,1. (C) Peptide map of serotype 3 AL. (D) Peptide map of serotype 1 Al C. (E) Peptide map of serotype 2 AlI C. (F) Peptide map of serotype 3 i1 C. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest I
(5) 212 GENTSCH AND FIELDS J. VIROL. TABLE 1. Comparison of shared and unique tryptic peptides in the Al C and a3 polypeptides of serotypes 1, 2, and 3a ,ulC peptide u3 peptide Serotype 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 ++++++++-++++++++++.____ + + + + + + + + + 2 + + - - - + + + - - + + + - + + + + + + + + - + 3 + + - - - - + + + + - + - + + a +, Peptide present; -, peptide absent. ,ilC peptides 1 through 8 and a3 peptides 1, 2, 7, and 8 were common to all three serotypes. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest peptides are defined as those peptides which type 3 ,uC peptide map contained many less were consistently the most intense for different intense peptides, which in many subsequent tryptic digests of the same polypeptide. Analyses preparations were very faint. The minor (less of mixtures of the peptides of serotype 1 ,ul plus intense) peptides present in the ,lC peptide serotype 1 ,lC, serotype 2 ,tl plus serotype 2 maps (and in subsequent al and a3 tryptic pep,ulC, and serotype 3 ,ul plus serotype 3 ,ulC tide maps) probably represented, at least in part, showed that the peptide maps of ,ul and ,ulC a complex mixture of disulfide-linked peptides since oxidation of these residues with performic were essentially identical (data not shown). Different strains of the mammalian reoviruses acid is known to be incomplete (19). Therefore, differ in their sensitivities to digestion by chy- they were not considered major peptides. Long motrypsin. The M2 double-stranded RNA seg- exposures of the AlC peptide maps of all three ment, which codes for the AlC polypeptide, is serotypes indicated that almost all of the very responsible for this property (25). faint peptide spots were also shared among the To obtain information concerning the struc- serotypes. tural relationship among the ,lC polypeptides Serotype 2 and 3 ,ulC polypeptides also shared of serotypes 1, 2, and 3, we carried out tryptic peptides 1 through 8 (Fig. 2E and F). Serotype peptide analyses. To facilitate description of the 2 AlC contained one peptide (peptide 9) not structural relationship among the ,ulC polypep- present in serotype 3 ulC, whereas serotype 3 tides, we assigned a number to each major me- ,ulC contained one peptide (peptide 10) not thionine-containing peptide. We based these as- found in serotype 2 ,u1C. The shared and unique signments on comparisons of the peptide maps tryptic peptides of the ulC polypeptides are of individual ,ulC polypeptides (Fig. 2D through shown in Table 1. Although we do not know the F) and mixtures of serotype 1 plus serotype 2, number of methionines in the ulC polypeptides serotype 1 plus serotype 3, and serotype 2 plus from serotypes 1 and 2, serotype 3 strain Dearing serotype 3 ,lC polypeptide digests (data not ,ulC contains 16 methionines (23). This suggests that the eight or nine major methionine-containshown). A comparison of the tryptic peptide maps of ing peptides which we observed represent oneserotype 1 and 2 ,ulC polypeptides (Fig. 2D and half or more of the total number of such peptides E) showed eight major peptides were present in present in the ,ulC polypeptides. both serotype 1 ulC and serotype 2 MlC. The Tryptic peptide analysis of the a3 proposition of peptide 5 and, to a lesser extent, the teins. The u3 polypeptide encoded in the S4 positions of peptides 6 and 7 in serotype 1 ,ulC double-stranded RNA segment is a major virion relative to peptide 4 were found to be variable, polypeptide (26). Its role is less well defined than but they appeared as single spots in peptide the roles of the other two outer capsid polypepmaps of mixtures of ,ulC polypeptides. There- tides. Serotype 1, 2, and 3 a3 polypeptides lafore, we assumed that these peptides had the beled with [35S]methionine were digested with same amino acid compositions in all three sero- trypsin, and their peptides were mapped as detypes. Digests of serotype 2 ,lC contained one scribed above. Figure 3 shows the peptide maps major peptide (peptide 9) which was not found of individual a3 polypeptide digests (Fig. 3A in serotype 1 ,ulC digests. through C) and mixtures of a3 polypeptide diSerotype 1 and 3 ,ulC polypeptides shared gests (Fig. 3D through F). Visual inspection of peptides 1 through 8 (Fig. 2D and F). In addition, individual a3 peptide maps showed that they serotype 3 ,lC contained a peptide not found in each contained 8 to 10 major methionine-conserotype 1 ulC (peptide 10). Peptide 7 was faint taining peptides (Fig. 3A through C) and that and smeared in this preparation of serotype 3 there were both shared and unique peptides in ,tlC tryptic peptides, but was consistently pres- the a3 polypeptide of each serotype. To deterent in numerous other preparations. The sero- mine which peptides were shared and which
(6) VOL. 38, 1981 REOVIRUS OUTER CAPSID POLYPEPTIDES 213 were unique, mixtures of a3 polypeptide digests from two different serotypes were analyzed. Mixtures of serotype 1 and serotype 2(3 polypeptide digests shared peptides 1, 2, 6, 7, and 8 (Fig. 3A, B, and D). Peptides 3 through 5 were apparently unique to serotype 1 a3, whereas peptides 11 through 13 were unique to serotype 2 a3. Peptides 9 and 10 were either very faint or absent in the serotype 2 a3 peptide maps. Peptide 1 in Fig. 3A is very faint, but it was clearly present in the original fluorogram. Our analysis of the mixture of serotype 1 and serotype 3 a3 tryptic peptides showed that six peptides were shared (peptides 1, 2, and 7 through 10) (Fig. 3A, C, and E). Peptides 4 through 6 were unique to serotype 1 a3 digests, whereas peptides 14 and 15 were found in sero- type 3 a digests but not in serotype 1 a3 digests. Several serotype 3 (3 peptides (e.g., the peptide marked with an asterisk in Fig. 3C) were present as major peptides in at least one serotype 3 a3 peptide preparation, but were usually very faint and therefore were not considered major peptides. Although the spot intensity of some other peptides varied from one preparation to the next, the number and pattern of separation of the peptides were completely reproducible. Serotype 2 and serotype 3 a3 polypeptide digests aLso had five major tryptic peptides in common (peptides 1, 2, 7, 8, and 12) (Fig. 3B, C, and F). Serotype 2 a3 contained three peptides (peptides 6, 11, and 13) which were not present in serotype 3 o3 digests, whereas peptides 14 and 15 were found in serotype 3 ( digests but not in Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest FIG. 3. Trypticpeptide maps of the [3S]methionine-labeled a3polypeptides of reovirus serotypes 1, 2, and 3. Tryptic peptides were analyzed as described in the legend to Fig. I and in the text. (A) Peptide map of serotype 1 a3. (B) Peptide map of serotype 2 a3. (C) Peptide map of serotype 3 a3. (D) Peptide map of a mixture of peptides from serotype 1 a3 and serotype 2 a3. (E) Peptide map of a mixture of peptides from serotype 1 o3 and serotype 3 a3. (F) Peptide map of a mixture ofpeptides from serotype 2 a3 and serotype 3 a3.
(7) 214 GENTSCH AND FIELDS al polypeptides. The resulting streaking of peptides and artifacts which were introduced prevented reliable interpretation of mixed al peptide maps. Therefore, we could not rule out the possibility that several methionine-containing tryptic peptides are shared among the al polypeptides. To analyze this possibility, the al polypeptides of serotypes 1, 2, and 3 were radioiodinated with 125I, and tryptic peptide analyses were performed as described above (Fig. 5). We found that serotype 1, 2, and 3 a digests contained 15, 13, and 13 major tyrosine-containing peptides, respectively (Fig. 5A through C). The patterns of separation suggested that there were both shared and unique tyrosine-containing peptides in each al polypeptide. To analyze this possibility, mixtures of al polypeptide digests were subjected to peptide mapping (Fig. 5D through F). Serotype al shared 10 of its 15 peptides with serotype 2 al, but only 8 of 13 peptides with serotype 3 al. Serotype 3 al shared only 6 of its 13 tyrosine-containing peptides with serotype 1 al and 8 of 13 peptides with serotype 2 al. Peptides 1, 2, 5, 15, and 17 are faint in Fig. 5A and C, but were clearly present in the original fluorographs. The shared and unique tyrosine-containing peptides of the al polypeptides are compared in Table 2. It is clear from these data that each al polypeptide had both shared and unique peptides compared with the al polypeptides of the other two serotypes. Only five peptides (peptides 1 through 9) were common to all three serotypes. Peptides 6, 11, 12, and 14 were found only in serotype 1 al, and peptides 19 through 22 were found only in serotype 3 al. DISCUSSION The aim of this study was to initiate a structural analysis of the three outer capsid polypeptides of mammalian reoviruses in order to pro- FIG. 4. Tryptic peptide maps of the [35S]methionine-labeled al polypeptides of reovirus serotypes 1, 2, and 3. Tryptic peptides were prepared and analyzed as described in the legend to Fig. 1 and in the text. (A) Peptide map of serotype 1 cl. (B) Peptide map of serotype 2 al. (C) Peptide map of serotype 3 al. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest serotype 2 a3 digests. In addition, peptides 9 and 10 were either absent or present in very low amounts in serotype 2 a3 tryptic peptide preparations. The peptides shared among the a3 polypeptides of all three serotypes and those present in only one or two serotypes are shown in Table 1. The number of major methionine-containing peptides which we observed in the a3 polypeptides of serotypes 1, 2, and 3 (9 or 10 peptides) is in good agreement with the number of methionine residues present in the a3 polypeptide of serotype 3 strain Dearing (23). Tryptic peptide structure of the reovirus al polypeptide. The reovirus HA (al polypeptide) plays several important roles in pathogenesis in experimental animals (10, 33). The structural differences in the al polypeptides from different strains or serotypes of reovirus which are responsible for these different biological activities have not been studied. To gain basic information on the structure of the al polypeptide, we utilized peptide mapping of tryptic digests of [35S]methionine-labeled reovirus al polypeptides from serotypes 1, 2 and 3. Figure 4A through C show the peptide maps of individual al polypeptide digests. A comparison of the individual peptide maps of serotype 1, 2, and 3 al polypeptides showed several differences (Fig. 4A through C). First, serotype 1 al digests contained nine major methionine-containing peptides, serotype 2 al digests contained seven major peptides, and serotype 3 al digests contained only five major peptides. Second, the patterns of separation of the peptides were different, suggesting that sequence differences exist among serotype 1, 2, and 3 al polypeptides. Because ofthe difficulty of obtaining sufficient amounts of [3S]methionine-labeled al polypeptides, thin-layer plates had to be overloaded with material (i.e., with trypsin and unlabeled bovine serum albumin carrier proteins present in tryptic peptide digests) to analyze mixtures of different J. VIROL.
(8) VOL. 38, 1981 REOVIRUS OUTER CAPSID POLYPEPTIDES 215 0 us i..Ih vide insight into their different biological properties. We found that the viral HA (polypeptide al) has some tryptic peptides which are unique for each serotype and others which are shared between two serotypes or among all three serotypes. These results suggest that the type-specific polypeptide al, the polypeptide that confers biological specificity for such important properties as immunological responses and specificity of binding to cell surface receptors, contains both unique and common methionine- and tyrosinecontaining tryptic peptides for each serotype. We anticipated differences in the antigenic site that reacts with neutralizing, type-specific antibodies. However, it was not clear whether the Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest FIG. 5. Tryptic peptide maps of the 'MI-labeled al polypeptides of reovirus serotypes 1, 2, and 3. Unlabeled ol polypeptides were radioiodinated in vitro with 'I and digested with trypsin, and the tryptic peptides were resolved in two dimensions as described in the legend to Fig. ) and in the text. (A) Peptide map of serotype 1 al. (B) Peptide map of serotype 2 al. (C) Peptide map of serotype 3 al. (D) Peptide map of a mixture ofpeptides from serotype 1 al and serotype 2 al. (E) Peptide map of a mixture ofpeptides from serotype 1 al and serotype 3 al. (F) Peptide map of a mixture ofpeptides from serotype 2 al and serotype 3 alc.
(9) 216 GENTSCH AND FIELDS J. VIROL. TABLE 2. Comparison of the shared and unique tryptic peptides in the al polypeptides of serotypes 1, 2, and 3a al peptide Serotype 1 2 3 a 1 2 3 4 5 + + + + + + + + + + + + + + - + Peptide present; -, 6 +a - 7 8 9 + + - + + + + + 10 + + 11 - - - 12 + 14 + - 13 + + - - - 15 16 17 18 19 20 21 22 _ + + + + - - - + + + - - + + + + + peptide absent. Peptides 1 through 4 and 9 were common to all three serotypes. cleavage of the ,ul polypeptide (20, 36, 37). We confirmed this finding for all three mammalian reovirus serotypes. The methionine-containing peptides of ,ul and ulC are essentially identical for all three serotypes. The peptide cleaved from ,lA to generate ulC was not detectable in our system. Recent studies in our laboratory have indicated that the i,lC poly7peptide determines protease sensitivity in reoviruses and plays important roles in entry and growth of reoviruses in the gastrointestinal tract and in spread through the host (25). The only differences in the methionine-containing peptides of ,ulC among the serotypes were found in peptides 9 and 10 (Table 2 and Fig. 2). Thus, the ulC polypeptide appears to be the most conserved of the reovirus outer capsid polypeptides. These results are somewhat surprising since the M2 genome segment is the site of frequent mutations (7, 25). Therefore, we expected to see considerable variability in the tryptic peptides of ,lC polypeptides. Since our method detected only peptides which contained methionine, we cannot rule out the possibility that the ,ulC polypeptide has additional regions that are more variable but do not contain methionine. We have found recently that the ,ulC polypeptides isolated from several lines of cells persistently infected with serotype 2 reovirus are missing peptide 10 and that a new peptide is present above peptide 2 (R. Ahmed and J. R. Gentsch, unpublished data). More precise studies will be necessary to determine whether these specific changes are related to biological activity. The a3 outer capsid polypeptides of the three serotypes also contain both conserved and variable peptides. The degree of conservation appears to be considerably less for a3 than for ulC, not only in the prototype strains which we examined, but also in several serotype 3 natural isolates (Gentsch and Fields, manuscript in preparation). It is not surprising that the a3 polypeptide has a variable peptide structure since it has been shown that S4, the genome segment which encodes a3, is the site of frequent mutations (1). However, as noted above, the M2 genome segment is also the site of frequent Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest al polypeptide also differed in other regions of the polypeptide outside the site that reacts with type-specific antibody. Our data suggest that although some sequences within the al polypeptide are conserved among serotypes, there are also considerable differences. It is interesting to compare these results with the results obtained for the type-specific antigen of influenza A viruses, the HA. Antigenic shifts among the human influenza A viruses involve the appearance of new epidemic strains of virus possessing different HA genes (31). Such shifts are known to have occurred three times, resulting in the appearance of three different immunological types (called Hi, H2, and H3) of virus in the population (31). The active form of the influenza HA molecule consists of two subunits (HAl and HA2) connected by a disulfide bridge (17, 35). Peptide mapping of both subunits of the HA from these viruses has indicated that they are structurally quite different (31). It has been shown that HAl is the immunologically important part of the molecule in that it is the antigen against which host neutralizing antibody is directed (3, 14, 15). On the other hand, HA2 is apparently not directly involved in the immunological response of the host to viral HA. Nucleotide and amino acid sequencing studies on the HAs of two human viruses (H2 and H3) and one avian virus (Havl) have shown that HAL is considerably more conserved than HA2 (12,21,28-30). Similarly, HAl shows many more nucleotide and amino acid changes than does HA2 during antigenic drift of a given pandemic strain (28-30). We do not know the locations of the conserved and variable peptides within the reovirus HA. However, it is possible that, similar to influenza virus, the variable peptides which we have observed in al represent the peptides found in the region of the polypeptide important in interacting with the host immune system. The conserved sequences may be found in portions of the polypeptide which are not directly involved in immunity, but possibly closely interact with other viral polypeptides during virion maturation. The ,ulC polypeptide of serotype 3 reovirus has been shown to be generated by proteolytic
(10) REOVIRUS OUTER CAPSID POLYPEPTIDES VOL. 38, 1981 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. ACKNOWLEDIGMENTS We thank Elaine Freimont and Karen Byers for excellent technical assistance, Susan Pevear and Lauri Maerov for typing, and our colleagues for many helpful discussions. This work was supported by Public Health Service grant iROl AI-13178 from the National Institutes of Health. J.R.G. was the recipient of Public Health Service Postdoctoral Research Fellowship 5F32 AI-06014-02 from the National Institute of Allergy and Infectious Disease. 16. 17. 18. 1. 2. 3. 4. LITERATURE CITED P. Ahmed, R., R. Chakraborty, and B. N. Fields. 1980. Genetic variation during lytic reovirus infection: highpassage stocks of wild-type reovirua contain temperature-sensitive mutants. J. Virol. 34:285-287. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. Brand, C. M., and J. J. Skekel. 1972. Crystalline antigen from the influenza virus envelope. Nature (London) New Biol. 238:145-147. Choppin, P. W., S. G. Lazarowitz, and A. R. Goldberg. 1975. Studies on proteolytic cleavage and glycosylation of the hemagglutinin of influenza A and B viruses, p. 105-119. In B. W. J. Mahy and R. D. Barry 19. 20. 21. (ed.), Negative strand viruses, vol. 1. Academic Press, Inc., New York. Crawford, L V., and R. F. Gesteland. 1973. Synthesis of polyoma proteins in vitro. J. Mol. Biol. 74:627-634. Cross, R. K., and B. N. Fields. 1976. Reovirus-specific polypeptides: analysis using discontinuous gel electrophoresis. J. Virol. 19:162-173. Cross, R. K., and B. N. Fields. 1976. Temperaturesensitive mutants of reovirus type 3: evidence for aberrant u1 and JL2 polypeptide species. J. Virol. 19:174-179. DeLange, R. J., L C. Williams, and R. J. Collier. 1979. The amino acid sequence of fragment A, an enzymatically active fragment of diptheria toxin. I. The tryptic peptides from the maleylated protein. J. Biol. Chem. 264:5827-5831. Elder, J. H., R. A. Pickett H, J. Hampton, and R. A. Lerner. 1977. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J. Biol. Chem. 252:6510-6515. Finberg, R., H. L. Weiner, B. N. Fields, B. Benacerraf, and S. J. Burakoff. 1979. Generation of cytolytic T lymphocytes after reovirus infection: role of 51 gene. Proc. Natl. Acad. Sci. U.S.A. 76:442-446. Gentach, J., and D. H. L. Bishop. 1978. Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein J. Virol. 28:417419. Gething, M. J., J. Bye, J. Skekel, and M. Waterfield. 1980. Cloning and DNA sequence of double-stranded copies of hemagglutinin genes from H2 and H3 strains elucidates antigenic shift and drift in human influenza virus. Nature (London) 287:301-306. Huismans, H., and W. K. Joklik. 1976. Reovirus coded polypeptides in infected cells: isolation of two native monomeric polypeptides with affinity for singlestranded and double-stranded RNA, respectively. Virology 70:411-424. Jackson, D. C., L E. Brown, D. 0. White, T. A. A. Dopheide, and C. W. Ward. 1979. Antigenic determinants of influenza virus. IV. Immunogenicity of fragments isolated from the hemagglutinin of A/Memphis/ 72. J. Immunol. 123:2610-2617. Jackson. D. C., T. A. Dopheide, R. J. Russell, D. 0. Waive, and C. W. Ward. 1979. Antigenic determinants of influenza virus hemagglutinin. II. Antigenic reactivity of the isolated N-terminal cyanogen bromide peptide of A/Memphis/72 hemagglutinin heavy chain. Virology 93:458-465. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Laver, W. G. 1971. Separation of two polypeptide chains from the hemagglutinin subunit of influenza virus. Virology 45:275-288. Laver, W. G., G. M. Air, R. G. Webster, W. Gerhard, W. C. Ward, and T. A. Dopheide. 1979. Antigenic drift in type A influenza virus: sequence differences in the hemagglutinin of Hong Kong (H3N2) variants selected with monoclonal hybridoma antibodies. Virology 98:226-237. Mahler, H. R., and E. H. Cordes. 1971. Biological chemistry. Harper & Row, Publishers, New York. McCrae, M. A., and W. K. Joklik. 1978. The nature of the polypeptide encoded by each of the 10 doublestranded RNA segments of reovirus type 3. Virology 89:578-593. Minjou, W., M. Verhoeyen, R. Devos, E. Saman, R. Fang. D. Huylebroeck, and W. Fiers. 1980. Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA. Cell 19:683-696. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest mutations (7), yet its tryptic peptide structure is highly conserved. The reason for this disparity is not known. Although it has been reported that antibody against the purified u3 polypeptide reduces the infectivity of serotype 3 reovirus (13), neither the ,llC nor the a3 polypeptide is involved directly in eliciting type-specific neutralizing antibodies by the host (34). Thus, there do not appear to be direct immunological selective pressures that could account for this variation. Although there is considerable variation in the structures of the al (type-specific antigen) and a3 polypeptides of the three serotypes, it is likely that the genes for the ,uC, al, and a3 polypeptides each were derived from common ancestral genes, which have drifted to varying degrees. It will be interesting to determine whether isolates of mammalian reoviruses from other species contain other alleles of the outer capsid genes. For example, recent study has shown that among the animal influenza A viruses, there are different alleles for at least seven of the eight influenza genes (27). We are currently analyzing this possibility with several bovine, murine, and human isolates of mammalian reoviruses. Thus, this study led us to focus our attention on certain structural components within each of the three outer capsid polypeptides. We are currently analyzing the possibility that the region of variable peptides within the ulC polypeptides may correlate with changes in ,lC-mediated biological activity and that the type-specific antigen (the al polypeptide) contains a variable region containing the antibody-binding site and a distinct conserved region. 217
(11) 218 GENTSCH AND FIELDS a 30. Waterfield, M. D., K. Espelie, K. Elder, and J. J. SkekeL 1979. Structure of the haemagglutinin of influenza virus. Br. Med. Bull. 35:57-63. 31. Webster, R. G., and W. G. Laver. 1975. Antigenic variation of influenza viruses, p. 270-314. In E. D. Kilbourne (ed.), The influenza viruses and influenza. Academic Press, Inc., New York. 32. Webster, R. G., and W. G. Laver. 1980. Determination of the number of nonoverlapping antigenic areas on Hong Kong (H2N2) influenza virus hemagglutinin with monoclonal antibodies and the selection of variants with potential epidemiological significance. Virology 104:139-148. 33. Weiner, H. L., D. Drayna, D. Averill, Jr., and B. N. Fields. 1977. Molecular basis of reovirus virulence: role of the Si gene. Proc. Natl. Acad. Sci. U.S.A. 74:57445748. 34. Weiner, H. L, and B. N. Fields. 1977. Neutralization of reovirus: the gene responsible for the neutralization antigen. J. Exp. Med. 146:1305-1310. 35. Weiner, H. L., R. F. Ramig, T. A. Mustoe, and B. N. Fields. 1978. Identification of the gene coding for the hemagglutinin of reovirus. Virology 86:581-584. 36. Zweerink, H. J., and W. K. Joklik. 1970. Studies on the intracellular synthesis ofreovirus-specified proteins. Virology 41:501-518. 37. Zweerink, H. J., M. J. McDowell, and W. K. Joklik1971. Essential and nonessential noncapsid reovirus proteins. Virology 45:716-723. Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest 22. Mustoe, T. A., R. F. Ramig, A. H. Sharpe, and B. N. Fields. 1978. Genetics of reovirus: identification of the dsRNA segments encoding the polypeptides of the u and size classes. Virology 89:594-604. 23. Pett, D. M., T. C. Vanaman, and W. K. Joklik. 1973. Studies on the amino and carboxy terminal amino acid sequences of reovirus capsid polypeptides. Virology 52: 174-180. 24. Ramig, R. F., R. K. Cross, and B. N. Fields. 1977. Genome RNAs and polypeptides of reovirus serotypes 1, 2, and 3. J. Virol. 22:726-733. 25. Rubin, D. H., and B. N. Fields. 1980. The molecular basis of reovirus virulence: the role of intestinal enzymes and the viral plC polypeptide. J. Ezp. Med. 152:853868. 26. Smith, R. E., H. J. Zweerink, and W. K. Jokiik. 1979. Polypeptide components of virions, top component and cores of reovirus type 3. Virology 39:791-810. 27. Sriram, G., W. J. Bean, Jr., V. S. Hinshaw, and R. G. Webster. 1980. Genetic diversity among avian influenza viruses. Virology 105:592-599. 28. Verhoeyen, M., R. Fang, M. Minjou, R. Devos, D. Huylebroeck, E. Saman, and W. Fiers. 1980. Antigenic drift between the haemagglutinin of the Hong Kong influenza strains A/Aichi/2/68 and A/Victoria/ 3/75. Nature (London) 286:771-776. 29. Ward, C. W., and T. A. Dopheide. 1979. Primary structure of the Hong Kong (H3) haemagglutinin. Br. Med. Bull. 35:51-56. J. VIROL.