Genome RNAs and Polypeptides of Reovirus Serotypes

Transcription

1 JOURNAL OF VIROLOGY, June 1977, p Copyright 1977 American Society for Microbiology Vol. 22, No. 3 Printed in U.S.A. Genome RNAs and Polypeptides of Reovirus Serotypes 1, 2, and 3 ROBERT F. RAMIG,* RISE K. CROSS,' AND BERNARD N. FIELDS Department of Microbiology and Molecular Genetics, Harvard Medical School, and Department ofmedicine (Infectious Disease), Peter Bent Brigham Hospital,* Boston, Massachusetts Received for publication 9 September 1976 The virus-specific double-stranded genome RNA and polypeptides present in virions and cells infected with the three mammalian reovirus serotypes have been examined by co-electrophoresis in several different polyacrylamide gel systems. The double-stranded RNA and polypeptide species previously described for type 3 Dearing were found to have corresponding species in the other serotypes examined. In each serotype several RNA and polypeptide species were found to have different electrophoretic mobilities from the corresponding RNA or polypeptide species of type 3 Dearing. The combination of electrophoretic variants among the RNAs and polypeptides of the reovirus serotypes gave electrophoretic markers in all 10 of the reovirus genes. The usefulness of these electrophoretic markers in "mapping" the reovirus genome is discussed. Mammalian reoviruses share common complement-fixing antigens (27) and are separated into three serotypes by hemagglutination inhibition (25, 26). The Dearing strain of reovirus type 3 has been extensively studied by a number of investigators (for recent reviews see references 12 and 23). The reovirus genome consists of 10 functionally as well as structurally discrete segments of double-stranded (ds) RNA, which fall into three size classes. The large size class contains three genome segments (L1 through L3), having molecular weights of approximately 2.3 x 106 to 2.5 x 106. The middle size class has three segments (Ml through M3), with molecular weights of approximately 1.4 x 106 to 1.6 x 106, and the small size class has four segments (Si through S4), with molecular weights of approximately 0.6 x 106 to 0.9 x 106 (28). Each genome segment is transcribed into single-stranded RNA, which serves as mrna for the synthesis of the viral polypeptides (2, 16). At least 11 unique virus-specified polypeptides have been detected in infected cells by high-resolution polyacrylamide gel electrophoresis in discontinuous buffer systems (5, 7). These polypeptide species fall into three size classes, large (A), medium (A), and small (a-), that are numbered in order of increasing electrophoretic mobility (7). One of the polypeptide species (,2) is derived from the primary gene product,ul by cleavage (31). Eight of the polypeptide species were found in the virion when run on gels as described by Maizel (17): Xl Present address: The Rockefeller University New York, NY through X3 and a-l in viral cores and 1, g2, a-2, and c-3 in the outer capsid (7).,t3,,u4, and a-2a are nonstructural polypeptides (7, 31). In this paper we report an analysis of the ds genome RNA and polypeptides synthesized by other reovirus serotypes, type 1 (Lang) and type 2 (Jones), as well as another strain of type 3 (Abney). Apparent differences in the molecular weights of genome segments and polypeptides of the serotypes and strains are noted, and their potential use in genetic studies is discussed. MATERIALS AND METHODS Cells. Mouse L cells were maintained in suspension culture in Joklik's modified Eagle minimal essential medium (Grand Island Biological Co., Grand Island, N.Y.) supplemented with 5% fetal calf serum (International Biological Laboratories, Inc., Rockville, Md.). Virus. The Dearing strain of reovirus type 3 was the same virus as used in previous studies from this laboratory. Seed viruses of the following serotypes and studies from this laboratory. Seed viruses of the following serotypes and strains were obtained from the National Institute of Allergy and Infectious Diseases: type 1 strain Lang (catalog no. V ), type 2 strain Jones (catalog no. V ), and type 3 strain Abney (catalog no. V ). All viruses were plaque purified and passaged on L cell monolayers twice. Second-passage virus was used for all infections. Preparation of labeled cytoplasmic ds RNA. A total of 108 cells was infected with a multiplicity of infection of 10 PFU/cell. Adsorption was carried out as described by Bellamy et al. (3), followed by 10-fold dilution into medium containing 0.25,g of actinomycin D (Sigma Chemical Co., St. Louis, Mo.) per ml. Infected cells were incubated at 330C. At 2.5 h 726

2 VOL. 22, 1977 postinfection, 50 gci of [U-'4C]uridine (New England Nuclear Corp., Boston, Mass.) was added. The infected cells were harvested at 20 h postinfection, and cytoplasmic fractions were prepared using Nonidet P-40 (Shell Chemical Corp.), as described by Borun et al. (4). RNA was extracted and deproteinized by the method of Oda and Joklik (19). Single-stranded RNA was separated from dsrna by LiCl precipitation as described by Baltimore (1). dsrna was stored at -20'C under ethanol. Preparation of virion dsrna. A total of 3 x 108 cells was infected with a multiplicity of infection of 10 PFU/cell. Adsorption was carried out as above, followed by 10-fold dilution into complete medium at 330C. The infected cells were harvested by centrifugation when the number of intact cells had fallen to approximately 0.6 the initial number. The time required to reach this point depended on the serotype and ranged from 48 to 72 h. Maximal yields were not obtained until the number of intact cells had fallen to this point. Virus was purified from infected cells by a procedure modified from Smith et al. (29). The cells were resuspended in 20 ml of homogenization medium (0.01 M Tris, 0.25 M NaCl, 0.01 M f3-mercaptoethanol) and thoroughly homogenized in a Sorvall Omnimixer. Sodium deoxycholate was added to a final concentration of 0.1% and incubated at 4VC for 30 min. One-half volume of freon was added, and the mixture was thoroughly homogenized. The resultant emulsion was separated into freon and aqueous phases by centrifugation. Virus was pelleted from the aqueous phase by centrifugation in an SW41 rotor for 1.5 h at 30,000 rpm. The virus pellet was resuspended in 2.0 ml of SSC (0.15 M NaCl plus M sodium citrate) and sonically treated to disrupt viral aggregates. The resuspended virus was further purified by centrifugation to equilibrium in a preformed CsCl gradient (p = 1.2 -* 1.4) in an SW41 rotor overnight at 24,000 rpm. The virus was made 1% with sodium dodecyl sulfate (SDS) and 0.5 M with sodium acetate, ph 5.0, and incubated at 37 C for 15 min. dsrna was deproteinized by extraction twice with chloroform-isoamyi alcohol (24:1) (19). The aqueous phase was precipitated by overnight incubation with an equal volume of ethanol. The precipitated dsrna was collected by centrifugation in a type 50 rotor for 2 h at 30,000 rpm. The precipitate was stored at -20 C under ethanol. Polyacrylamide gel analysis of dsrna. Electrophoresis of dsrna was performed as described by Cross and Fields (6), except that all components of the gel system contained 0.01 M EDTA. Alternatively, electrophoresis was by the method of Laemmli (15). Gels were dried, and autoradiography was performed. Preparation of labeled cytoplasmic polypeptides. A total of 107 cells was infected with a multiplicity of infection of 20 PFU/cell. After adsorption the infected cells were diluted to 106/ml in growth medium containing 0.25 jig of actinomycin D per ml and incubated at 31 C. At 18 h postinfection, the cells were concentrated to 107/ml in medium containing 0.05 normal amounts of methionine. A 50-MCi amount of high-specific-activity [35S]methionine (>400 Ci/mM; New England Nuclear or Amersham/ REOVIRUS SEROTYPE RNAs AND POLYPEPTIDES 727 Searle Corp., Arlington Heights, Ill.) was added, and the infected cells were returned to 31 C. At 20 h postinfection, incorporation of label was stopped by chilling, and cytoplasmic fractions were prepared as described above. Ten volumes of cold acetone were added to the cytoplasmic extracts and incubated overnight at -20 C. The precipitate was collected by centrifugation, thoroughly dried, and dissolved in gel sample buffer (15). Polyacrylamide gel electrophoresis of polypeptides. Slab gels were prepared to a final concentration of 10% acrylamide, 0.267% bisacrylamide, 0.05% N,N,N',N'-tetramethylethylenediamine, and 0.1% SDS in 0.19 M Tris-hydrochloride, ph 8.8, as described by Maizel (17) and Cross and Fields (7). Alternatively, electrophoresis was by the method described by Laemmli (15). After electrophoresis the gels were dried, and autoradiography was performed. Gel tracing. Densitometer tracings were done by cutting single lanes from autoradiograms and scanning them in a 20-cm Gilford gel scanner at 550 nm. Nomenclature of polypeptides. The polypeptides within each size class are numbered in order of increasing electrophoretic mobility (7). Polypeptide,u4 of type 3 Dearing in the Tris-glycine gel system described by Maizel corresponds to polypeptide A0 in the phosphate buffer gel system. Polypeptides al and o-2 of type 3 Dearing are reversed in the Trisglycine buffer system described by Maizel (17) relative to the phosphate buffer system (7). RESULTS Viral polypeptides. Cytoplasmic extracts of cells infected with the reovirus serotypes were subjected to electrophoresis in polyacrylamide slab gels containing a discontinuous Tris-glycine buffer and SDS, as described by Maizel (17). Ten percent acrylamide has previously been shown to allow the best resolution of all the polypeptide species of type 3 Dearing (7), and this concentration was used here. The polypeptides of all the serotypes were resolved into three size classes. The large size class of polypeptides (Xl, X2, and X3) was not well resolved and appeared to be homogeneous (Fig. 1). This class will be examined in detail below. The rates of migration of the Al and,u2 polypeptides of the serotypes were slightly, but reproducibly, different. Whereas the migration of,ul and,u2 of types 1 and 3 Abney appeared to be identical, they were different from both type 2 and type 3 Dearing. The rate of migration of polypeptide pa4 is identical for all the serotypes except type 1. The minor polypeptide species,u3 (7) was present in cells infected with some of the serotypes but was not consistently resolved and will not be commented on further. At least one of the serotypes contained a migrational variant for each of the four o- polypeptide species. Poly-

3 728 RAMIG, CROSS, AND FIELDS J. VIROL. k~2 k3 pi P2A2 P4 u412 FIG. 1. Analysis of virus-specific polypeptides from cells infected with reovirus serotypes and labeled with [35S]methionine. Electrophoresis was carried out on a 10% polyacrylamide slab gel as described by Cross and Fields (7) for 18 h at 45 V. The direction ofelectrophoresis is from left to right. Microdensitometer tracings of an autoradiogram of samples electrophoresed in adjacent lanes are shown. Vertical lines designate the position of the various polypeptides in reovirus type 3 Dearing. Arrows designate the positions ofpolypeptides present on high-background areas of the gel. peptide o-3 showed the greatest variation in migration between the serotypes. The relative amounts of a-i and a-2 of type 3 Dearing, i.e., a-l > a-2, do not agree with previously published results (31). This has been shown to be due to a reversal of relative migration rates between a-l and o-2 in the phosphatebuffered gel system as compared to the Trisglycine-buffered system described by Maizel and used here (7, 17). We were unable to reproducibly resolve polypeptides Xl and X2 under any condition of sample preparation, gel concentration, or electrophoresis by using the Tris-glycine, SDS-containing gel system described by Maizel (17). We found that we could reproducibly resolve these very large polypeptide species when we used the slightly different Tris-glycine-buffered, SDS-containing gel system described by Laemmli (15). However, even in this system, resolution was highly dependent on not overloading the gels. Figure 2 shows the X polypeptides of serotypes 1 Lang, 2 Jones, and 3 Dearing as resolved by this gel system. Because of the poor growth and labeling of type 3 Abney in our hands, it is not shown here. The Xl and X2 polypeptide species of type 1 migrate more slowly than the corresponding species of types 2 and 3 Dearing. The X1/X2 doublet of type 2 appears to be more tightly migrating than the corresponding doublet of type 3 Dearing. The X3 species migrates at equal rates in types 2 and 3 Dearing and slightly slower in type 1. The gel in Fig. 2 was run to optimize separation of the X polypeptide species; the migration of the it and a- polypeptide species in the Laemmli gel system will not be discussed. Virions of all the serotypes contain polypeptide species that correspond to those described for type 3 Dearing (data not shown). The migrational heterogeneity described for the structural polypeptides of the serotypes in cytoplasmic extracts is identical for structural polypeptides that have been assembled into virions. The nonstructural polypeptides g3, /A, and

4 VOL. 22, 1977 X-.- meow WIVIR somm" ma"am.,: j ";5. REOVIRUS SEROTYPE RNAs AND POLYPEPTIDES 729 Al A2 A cr2a are not present in detectable amounts in any of the serotype virions. Polypeptide molecular weights. Molecular weight estimates were made for the polypeptides of serotypes 1 and 2, using the molecular weights determined for type 3 Dearing in a Tris-glycine-SDS gel (5) as standards. Since these molecular weights were determined by using the gel systems described by Laemmli (15) for X and 4t species and Maizel (17) for species, all calculations were based on migration of the serotypes in adjacent tracks on a 7.5% acrylamide gel prepared as described by Laemmli or 10% acrylamide gels prepared as described by Maizel. These molecular weights are presented in Table 1. Cytoplasmic dsrna. We noted, during the examination of 35S-labeled virion polypeptides, that when whole virions were dissolved in sample buffer and subjected to electrophoresis, the stained gel not only showed the polypeptide patterns but also had a very well resolved series of bands, which seemed to correspond to the dsrna species and contained no radioactivity. Since this presented the possibility of examining reovirus dsrna without the extensive deproteinization usually required before electrophoresis, we proceeded to examine purified cywwu FIG. 2. Analysis of X polypeptide species from cells infected with reovirus serotypes and labeled with [35S]methionine. Electrophoresis was carried out on a 7.5%polyacrylamide slab gel as described by Laemmli (15) for 3 h at 50 ma. The direction of electrophoresis is from top to bottom. Serotypes: 1 Lang (A), 2 Jones (B), and 3 Dearing (C). TABLE 1. Molecular weights of reovirus serotype polypeptides a Polypep- Type 1 Lang Type 2 Jones Type 3 Deartide Tye1Ln re2jns ing xl 160, , ,000 X2 157, , ,000 X3 145, , ,000 pi 78,500 79,000 79,000 tu2 75,000 74,000 72,000,u4b 77,000 77,000 77,000 a-1 54,500 54,500 54,000 ov2 53,500 53,000 52,000 o-2a 49,000 47,000 49,000 cr3 42,500 39,000 43,000 a Molecular weights were determined after electrophoresis of polypeptides on 7.5% gels prepared as described by Laemmli (15) for the X and A polypeptide species and after electrophoresis on 10% gels prepared as described by Maizel (17) for the a- polypeptide species. The molecular weights determined for type 3 Dearing by Both et al. (5), using the same combination of gel systems for the different size classes, were used as standards in calculating molecular weights of the polypeptides of types 1 and 2. Molecular weights are not presented for type 3 Abney because of difficulties in obtaining enough material to run gels. Molecular weights are the mean of three independent determinations. b The polypeptide /i4 migrates more slowly than,u2 in gels prepared as described by Laemmli (5, 15).

5 730 RAMIG, CROSS, AND FIELDS toplasmic dsrna on Tris-glycine-SDS gels as described by Laemmli (15). The bands seen with 14C-labeled cytoplasmic dsrna were identical to the nonlabeled, stained bands seen when whole virions were dissolved in sample buffer and subjected to electrophoresis. The pattern of bands seen in the Tris-glycine gel system (Fig. 3) was slightly, but reproducibly, different from that seen when the dsrna was subjected to electrophoresis in a more traditional acetate buffer system (Fig. 4). The dsrna species of all serotypes were resolved into three size classes as previously described for type 3 Dearing. In the Laemmli gel system the Li dsrna's of all the serotypes were identical. The dsrna's of the L2 and L3 species showed variation between the serotypes (Fig. 3). Among the middle (M) size class of dsrna, migrational variants were present for all three dsrna species. The Ml dsrna's showed the greatest variation in migration rates. The small (S) size class of dsrna's also had at least one migrational variant for each of the four dsrna species. Overall, the patterns of types 1, 3 Abney, and 3 Dearing were quite similar as compared to the patterns of dsrna species obtained from cells infected with type 2. Virion dsrna. dsrna isolated from purified A B C D II 4 1,- _ -. *.:.t 9 zi ~,--Ai" J. VIROL. - si -S2 -S3 S4 FIG. 4. Analysis of [14C]uridine-labeled dsrna's extracted from purified virions of the reovirus sero- A B C D types. Electrophoresis was carried out on a 5% polyacrylamide gel as described by Cross and Fields (6) for 20 h at 50 V. Electrophoresis was from top to bottom. A, Type 1 Lang; B, type 2 Jones; C, type 3 Abney; D, type 3 Dearing. Li L2-^ L3' Ml- M2- M3- S2- - S3- S4- FIG. 3. Analysis of cytoplasmic dsrna's extracted from cells infected with the reovirus serotypes and labeled with ["4C]uridine. Electrophoresis was carried out on a 10% polyacrylamide slab gel as described by Laemmli (15) for 8 h at 40 ma. Electrophoresis was from top to bottom. A, Type 1 Lang; B, type 2 Jones; C, type 3 Abney; D, type 3 Dearing. virions of the serotypes was subjected to electrophoresis on gels containing the acetate buffer system. The pattern of bands seen in this gel system (Fig. 4) was slightly different from that seen when dsrna was subjected to electrophoresis on the Tris-glycine gel system, but the general patterns observed were the same. On gels containing the acetate buffer system, variation between serotypes was seen for all three of the large (L) dsrna species (Fig. 4). The Li species of type 1 was found to migrate significantly more rapidly in this buffer system, compared to the other serotypes, whereas no variation in migration among the Li species was detected in the Laemmli gel system. As in the Laemmli gel system, variants in migration between serotypes were observed for all three middle (M) and all four small (S) dsrna's, although the patterns of variation differed between the two gel systems. The overall patterns of dsrna's isolated from virions were similar for types 1, 3 Abney, and 3 Dearing as compared to type 2.

6 VOL. 22, 1977 Serotype dsrna molecular weights. The molecular weights given by Shatkin et al. (28) for the dsrna species of reovirus type 3 were used to calculate molecular weights of the dsrna"s of the other serotypes examined (Table 2). All calculations were based on migration in the acetate buffer system. These molecular weight estimates are intended only to illustrate the differences between corresponding dsrna species of the serotypes. DISCUSSION Examination of the polypeptides and dsrna's synthesized by reovirus type 1 Lang, type 2 Jones, type 3 Abney, and type 3 Dearing by polyacrylamide gel electrophoresis revealed substantial heterogeneity both among the corresponding gene products (polypeptides) and among the genome segments (dsrna). This heterogeneity is summarized in Table 3. The heterogeneity seen among the outer capsid polypeptides of different serotypes (especially,2 and o-3) is perhaps not surprising. It is somewhat surprising to note, however, that relatively large differences existed between the outer capsid polypeptides of the two strains, Abney and Dearing, of type 3 that were examined. The heterogeneity of the inner capsid polypeptides, especially X1 and X2, is also surprising, since the common complement-fixing antigens shared by the various reovirus serotypes are thought to reside in the core (27). Variations have been shown in the structural polypeptides of different serotypes and strains of several viruses. Examination of 16 serotypes of blue tongue virus showed that at least one of the capsid polypeptides was electrophoretically TABLE 2. Molecular weights of serotype dsrna a Mol wt (x 106) dsrna species Type 1 Lang Type 2 Jones Type 3 Abney Type 3 Dearing Li L L Ml M M Si S S S a Molecular weights calculated from molecular weights by Shatkin et al. (28) given for reovirus type 3. All calculations based on migration on a single slab gel subjected to electrophoresis using an acetate buffer system. REOVIRUS SEROTYPE RNAs AND POLYPEPTIDES 731 TABLE 3. dsrna and polypeptide heterogeneity: summary of distinguishable molecular weight differences between reovirus serotypesa dsrnab Polypeptide Li 1 = 2 = 3A = 3D X1 1 2 = 3D L2 1i2*3A= 3D X2 1 2 = 3D L3 2 # 1# 3A=3D X3 1 2 = 3D Mi 1i# 24#3A 3D Al 3D * 2# 1 = 3A M2 1i# 2 3A# 3D g2 3D #k 2# 1 = 3A M3 2 1 = 3A = 3D,4 1 2 = 3A = 3D Si 1i2 3A = 3D ol 2=3A 1 *3D S2 1 =2 3A 3D o22 1 =2#3A=3D S3 2 1#3A=3D oa2a 2 1 = 3A = 3D S4 2# 1 =3A 3D (o3 #2 3A # 3D = 1 a #, Easily detectable difference in migration between serotypes; =, No easily detectable difference between serotypes. bheterogeneity of dsrna as observed following electrophoresis in the gel system described by Laemmli (15). variant in every serotype (10). In this case, the polypeptides that showed the greatest degree of variation were those in the outer capsid of the virus. Variation has also been described for three viral polypeptides when Indiana and New Jersey serotypes of vesicular stomatitis virus were compared (30). When 53 strains of herpes simplex virus type 1 were examined, 7 of the 33 capsid polypeptides were found to have electrophoretic variants (22). There is also precedent for variation in genome structure among virus strains and serotypes within a viral group. Studies of sequence homology have shown relatively large inhomologies between the genomes of reovirus types 2 and 3, with greater sequence homology seen between types 1 and 3 (18). We note that the pattern of type 2 genome RNA segments on gels was more different from that of type 3 than was type 1. Substantial sequence inhomologies have also been shown between the genome segments of 16 serotypes of blue tongue virus (11). The genome of influenza virus, a single-stranded RNA virus with a segmented genome, has been shown to have electrophoretic heterogeneity both among viruses of types A, B, and C (24) and among strains of type A (20). Since the passage history of the virus used in these experiments was unknown, we cannot relate the variation in electrophoretic patterns of dsrna or polypeptides to factors such as passage history. We can emphasize, however, that all virus used in these experiments was removed from single-plaque isolation by only two passages and that all passages and experiments were done in L cells.

7 732 RAMIG, CROSS, AND FIELDS We have not yet defined the exact nature of the electrophoretic variation seen among either the dsrna's or the polypeptides of the reovirus serotypes. Although the gel systems used are thought to separate molecular species primarily on the basis of molecular weight, chemical modification is known to affect mobility in these systems. The gl and tt2 polypeptides of type 3 Dearing have been shown to be both glycosylated and phosphorylated (13, 14). If these polypeptides are differentially modified in the different serotypes, this could possibly account for the electrophoretic variability. Differences have been noted between the polypeptide and dsrna patterns of the serotypes, depending on the gel system used for analysis. For example, no migrational variants were noted between the X3 polypeptides species when examined in the gel system described by Cross and Fields (7) (Fig. 1), whereas variants were noted among the same polypeptide species when examined in the gel system described by Laemmli (15) (Fig. 2). Similar differences are also noted for dsrna species when migration is compared between gel systems. These differences are highly reproducible and must, therefore, be recognized as a function of the various conditions under which electrophoresis can be carried out or as different behavior of the polypeptides under differing ionic conditions. The basis of these variations between gel systems is not understood. Finally, we would like to note the potential usefulness of these electrophoretic variants in the assignment of temperature-sensitive lesions to genome segments and gene products. Electrophoretically variant polypeptides of reovirus type 3 Dearing (8) have been useful as unselected markers in three-factor crosses (9). Electrophoretic variants present in genome RNA of influenza virus have been exploited to identify the genome segments coding for hemagglutinin and neuraminidase (21). The combination of electrophoretic variants among the polypeptides and dsrna's of the reovirus serotypes has provided genetic markers for all 10 of the reovirus genes. We have found efficient reassortment of genome segments between serotypes of reovirus. In fact, the segregation of electrophoretic markers among the progeny of mixed infections between serotypes has been used to correlate genes with their products and to assign temperature-sensitive mutations to genome segments (R. F. Ramig, T. Mustoe, A. Sharpe, and B. N. Fields, submitted for publication). ACKNOWLEDGMENTS This research was supported by research grant no. VC- 195 from the American Cancer Society and Public Health J. 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