VIROLOGY
84.63-74
(1978)
A Genetic I. Correlation ARLENE
Map of Reovirus
of Genome RNAs between
H. SHARPE,’
Serotypes
ROBERT F. RAMIG,* THOMAS BERNARD N. FIELDS
1, 2, and 3 A. MUSTOE,
AND
Department of Microbiology and Molecular Genetics, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, and Department of Medicine (Infectious Diseases), Peter Bent Brigham Hospital, Boston, Massachusetts 02115 Accepted August 16,1977 The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments Ml and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except Ml and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type U. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycineand the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.
trophoretically aberrant polypeptides in crosses between ts mutants (Cross and Fields, 1976). We recently reported that the genome RNAs and the polypeptides of the three reovirus serotypes could be distinguished following polyacrylamide-gel electrophoresis (Ramig et al., 1977). In this communication we describe experiments that permit mapping the genome of reovirus. The method is based on the prior observation that dsRNAs of the three serotypes have different patterns on polyacrylamide gels. The analysis of the RNA patterns of recombinants between serotypes allows construction of a map of the genome which correlates segments between the serotypes. Additionally, physical reassortment of segments is shown to be the mechanism of recombination,
INTRODUCTION
The genome of reovirus consists of 10 segments of double-stranded RNA (Bellamy et al., 1967; Watanabe and Graham, 1967; Shatkin et al., 1968). Genetic crosses between temperature-sensitive (ts) mutants isolated from reovirus type 3 Dearing revealed that pairwise crosses resulted in a high proportion of ts+ progeny for some pairs whereas other pairs yielded no detectable recombinants (Fields and Joklik, 1969; Fields, 1971, 1973). The “all-or-none” nature of recombination suggested that recombinants were generated by random reassortment of genome segments. Additional evidence for reassortment as the mechanism of recombination has come from the study of the segregation of elec’ Present address: Yale University School of Medicine, New Haven, Connecticut. L Address reprint requests to Dr. Ramig at the Harvard Medical School.
MATERIALS (1)
Virus.
AND
Reovirus
METHODS
type 1, strain Lang
63 004%6822/78/0841-0063$02.00 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
SHARPE ET AL.
and type 2, strain Jones were the same as medium and allowed to adsorb for 30 min described by Ramig et al. (1977). The reo- at 31” in a 5% CO, incubator. After adsorpvirus type 3, strain Dearing mutants were tion, 4.0 ml of IMEMZO medium was isolated and described by Fields and Joklik added to each plate and the plates were (1969). The following recombination group returned to 31”. At 2.5 hr postinfection the prototype mutants were used: group A, ts medium was removed from each plate and 201; group B, ts 352; group C, ts 447; group replaced with 4.0 ml of IMEMZO medium D, ts 357; group E, ts 320 and group G, ts containing 0.25 mCi of %P as orthophos453. All viruses were plaque purified and phoric acid (NEN). The plates were then passaged on L-cell monolayers twice. Sec- incubated at 31” in a 5% CO, incubator ond-passage virus was used for all genetic until they were harvested. The infected crosses. cells were harvested at 48 hr postinfection (2) Cells. Mouse L cells were main- and cytoplasmic RNA was prepared as tained in suspension culture in Joklik’s follows. The infected monolayers were modified Eagle’s minimal essential me- washed twice with cold NP40 buffer (Bodium [International Biological Laboratorun et al., 1967). The cells were then ries (IBL), Rockville, Md.] supplemented suspended in NP40 buffer using a rubber policeman and cell clumps were broken by with 5% fetal calf serum (IBL). Monkey CV-1 cells were maintained in monolayers pipetting. Nonidet P-40 (Shell Chemical in IMEMZO with zinc and insulin (IBL) Co.) was added to a final concentration of supplemented with 10% fetal calf serum 0.5% and the mixture was incubated for (IBL). 30 min at 4” (Borun et al., 1967). Nuclei (3) Genetic protocols. L cells in suspen- were removed from the cytoplasmic extract by centrifugation. The cytoplasmic sion culture were infected with a multiplicity of infection (m.0.i.) of 10 plaque- extract was adjusted to 0.2 M in NaCl, 3 forming units (PFU) each of a type 3 vol of ethanol were added, and the mixture Dearing temperature-sensitive (ts) mu- was incubated at -20” overnight to preciptant and of wild-type virus of either type 1 itate RNA. The precipitates were pelleted Lang or type 2 Jones as previously de- by centrifugation at 10,000 rpm for 30 min scribed (Fields and Joklik, 1969). After in a Sorvall SS-34 rotor. The precipitates adsorption the infected cells were incu- were dried under vacuum and dissolved in bated at 31” for 48 hr. At the end of the 0.25 ml of gel sample buffer (Laemmli, incubation period the cells were chilled 1970). and sonicated to disrupt the remaining (5) Preparation of labeled, purified, virintact cells and viral aggregates. The yield ion dsRNA . Labeled dsRNA was purified of virus from the mixedly infected cells by the method of Ramiget al. (1977) except was titered by the method of Cross and that 5 &i/ml of 32P as orthophosphoric acid (NEN) was added at 2.5 hr postinfecFields (1976). The permissive temperature for the ts mutants was 31” and the non- tion. Because poor yields of type 2 were permissive temperature was 39” (Fields obtained by this method, total 32P-labeled and Joklik, 1969). Lytic (ts+) progeny type 2 cytoplasmic dsRNA was prepared by the method of Ramig et al. (1977). The plaques were picked from plates incubated at 39” which had fewer than 10 plaques per 32P-labeled dsRNA was dissolved in 0.25 plate. These lytic plaques were passaged M Tris-acetate, pH 8.5 (Cross and Fields, once on L-cell monolayers at 37”. The dou- 1972). (6) Polyacrylamide-gel electrophoresis ble-stranded RNA (dsRNA) synthesized of dsRNA. Slab gels were prepared to a by these clones was examined as follows. final concentration of 10% acrylamide, (4) Preparation of labeled, unpurified, cytoplasmic d.sRNA. The medium was re- 0.267% bisacrylamide as described by Lacytoplasmic moved from the confluent plates of CV-1 emmli (1970). Unpurified cells (2 x lo6 cells/60 x l&mm plate) and dsRNA samples to be applied to the gels virus at an m.o.i. of 10 PFU/cell was added were boiled for 1 min within 0.5 hr prior in a total volume of 1.0 ml of Pucks’ to loading the gel. Electrophoresis was
GENETIC
MAP
carried out at a constant current of 20 mA for 18 hr. Alternatively, purified virion dsRNAs were subjected to electrophoresis in 5% gels containing the Tris-acetatebuffered system described by Cross and Fields (1972). Electrophoresis was carried out at 110 V for 24 hr on slab gels 16 cm long. After electrophoresis the gels were fixed in 50% methanol, 7% acetic acid, then dried onto filter paper in a Hoeffer Scientific Instruments gel dryer. Autoradiography was performed by placing the dried gel in contact with a sheet of NoScreen X-ray film (Kodak) for 6-24 hr. Films were processed by standard photographic procedures. LII
RESULTS
(1) Theory of the Genetic Crosses
OF REOVIRUS
65
tinguished by polyacrylamide-gel electrophoresis (Ramig et al., 1977) analysis of the dsRNAs synthesized by progeny forming lytic plaques at nonpermissive temperature allows one to distinguish between ts+ recombinant progeny and ts+ parental progeny. Furthermore, analysis of the migration rates of each of the dsRNA segments in each ts+ recombinant allows one to determine the serotype from which each segment was derived. The details of the genetic crosses from which these recombinants were derived will be described elsewhere (Ramig, Mustoe, Sharpe, and Fields, manuscript in preparation). (2) Correlation of Genome Segments between Serotypes in the Tris-Glycine Gel System
The dsRNAs synthesized by reovirus The crosses described in this communi- serotypes 1, 2, and 3 yield distinctive and cation were between a ts mutant of type 3 reproducibly different patterns when subDearing (type 3) and wild type of 1 Lang jected to polyacrylamide-gel electrophore(type 1) or type 2 Jones (type 2). Previously sis (Ramig et al., 1977). In all three cases it had been shown that multiplicity reac- the genome segments fall into three size tivation occurs between ultraviolet lightclasses, L, M, and S. However, segments irradiated reovirus of different serotypes within a size class do not correspond in (McClain and Spendlove, 1966) suggesting order of electrophoretic mobility. Examithat genetic interactions occur between nation of segment replacements (and correovirus serotypes. Thus if cells are responding deletions) in a few representamixedly infected with a type 3 ts mutant tive recombinant clones allows one to esand wild-type type 1 or type 2 at permis- tablish the correspondence between gesive temperature, four progeny types nome segments of the serotypes. Figure 1 would be expected among the progeny of shows the dsRNA patterns of the serotypes the cross: (i) the parental type 3 ts mutant, and the recombinants selected to show (ii) the parental wild-type type 1 or type that when a given L segment from type 3 2, (iii) recombinant progeny retaining the is absent, it is replaced by the correspondts lesion derived from the type 3 parent, ing segmer,b from type 1 or type 2. In and (iv) recombinant progeny having a recombinants between both type 1 and ts+ phenotype due to replacement of the type 3 and type 2 and type 3, when the Ll type 3 genome segment bearing the ts segment of type 3 is absent from a recomlesion by the corresponding genome seg- binant it is replaced by the segment havment derived from the ts+ type 1 or type 2 ing the slowest electrophoretic migration parent. Progeny types (i) and (iii) can be from type 1 or type 2 (Figs. 1C and H). selected against by harvesting lytic Likewise when the L3 segment of type 3 is plaques from progeny plated at nonper- absent it is replaced by the most rapidly missive temperature. Plating at nonper- migrating segment from type 1 or type 2 missive temperature thus leaves a mix(Figs. 1D and H). Although no examples ture of ts+ recombinant progeny and the are shown, by elimination the L segment ts+ parental type. with medium migration rate must correSince the dsRNAs synthesized by the spond in all three serotypes. Thus the L three reovirus serotypes can be easily dis- size class genome segments of all three
66
SHARPE
serotypes correspond in order of increasing electrophoretic mobility: Type
Type
Type
1
3
2
ET AL. Type
Type
2 L\
M31 /MZ
\
Ll -/-
Type
--
--A*;-------+The correspondence of M size genome segments between serotypes is shown in Fig. 2. The Ml and M2 segments of type 3 are not well separated under the conditions of these gels and run as a very tight doublet band. When Ml and/or M2 of type 3 are absent from recombinants they are replaced by the segments of slowest and of intermediate mobility in the case of recombinants with both type 1 and type 2 (Figs. 2C-E, H, and J). The correspondence of Ml and M2 of type 3 with the analogous bands of the other serotypes cannot be unambiguously established from these data. Occasionally, although not reproducibly, the Ml and M2 doublet of type 3 is well resolved. In these cases it becomes clear that when Ml of type 3 is absent from a recombinant it is replaced by the middle of the M bands from type 2 (Fig. 2H) and by the slowest of the bands from type 1 (Fig. 2D). When M2 of type 3 is absent it is replaced by the slowest M band from type 2 (Fig. 25) and by the middle M band from type 1 (Fig. 2E). (Note that in Fig. 25 the position of all the bands is slightly displaced indicating that in this sample all of the bands migrated slightly more rapidly than the analogous bands in the other lanes.) In crosses between type 1 and type 3 and between type 2 and type 3 when the most rapidly migrating of the M segments of one type is absent it is replaced by the most rapidly migrating of the M segments of the other type (Figs. 2C and I). Thus the correspondence of the M size class genome segments between the serotypes can be summarized as follows:
----i& M3 /Figure 3 shows the correspondence of genome segments among the S size class of the three serotypes. When Sl of type 3 is absent from a recombinant (Fig. 3C) it is replaced by the slowest migrating of the S segments from type 1. No electrophoretie difference is reproducibly seen between S2 of type 3 and S2 of type 1. S3 and S4 of type 1 generally migrate as a tight doublet. However, when samples ore run on a gel 16 cm long, S3 and S4 of type 1 are resolved (Fig. 4). A recombinant between S3 and S4 of types 1 and 3 (Fig. 4B) shows that when S3 of type 3 is absent it is replaced by the most rapidly migrating of the S segments from type 1. By exclusion it must follow that S4 of type 3 is replaced by the second most rapidly migrating of the S segments from type 1. Sl of type 2 is replaced by the slowest of the S bands from type 3 (Figs. 3F-I). When S2 of type 3 is absent it is replaced by the second most slowly migrating of the type 2 S RNAs (Figs. 3F and G). When S3 of type 3 is absent it is replaced by the most rapidly migrating segment from type 2 (Fig. 3H). When S4 is absent from a recombinant it is replaced by the second most rapidly migrating of the segments from type 2 (Fig. 31). The correspondence of the S size class genome segments between the serotypes can be summarized as follows: TYPe Type Type 1
3
2
GENETIC
MAP
OF REOVIRUS
67
ABCDEFG
FIG. Correspondence of L size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1 , 03) typ be3, (C) ret on nbinant No. 94, (D) recombinant No. 80, (E) type 2, (F) type 3, (G) recombinant No. 2;47, and (H) recomt kin(ant NO. 261. Electrophoresis was as described. Bands are labeled in order of el ec:trophor ,etic mobilii :Y in A, B, E, and F. Bands in C, D, G, and H are numbered according to serotype c origin. The bands 1in G were enhanced for purposes of illustration.
(3) Correlation of Genome Segments of Reovirus Type 3 between Tris-Glytine- and Tris-Acetate-Buffered Gel Systems The availability of the recombinants ex-
amined above allowed us to determine the relationships between the genome segments of type 3 as resolved in the Trisglycine gel system used here and the Trisacetate system that has been traditionally
68
SHARPE
A
B
C
D
E
ET AL.
F
G
t-l
I
J
FIG. 2. Correspondence of M size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1, (B) type 3, (Cl recombinant No. 72, (D) recombinant No. 65, (El recombinant No. 94, (F) type 2, (G) type 3, (H) recombinant No. 255, (I) recombinant No. 256, and (J) recombinant No. 231. Electrophoresis was as described. Bands are labeled in order of electrophoretic mobility in A, B, F, and G. Bands in C, D, E, H, I, and J are numbered according to serotype of origin.
used to resolve type 3 dsRNAs. The recom- of the recombinants shown in Fig. 2 were binants shown in Fig. 2 were selected for extracted from purified virions and substudy in the Tris-acetate system primarily jected to electrophoresis in the Tris-glyto resolve the ambiguity caused by the tine- and Tris-acetate-buffered gel systight Ml, M2 doublet of type 3 in the tems. The patterns obtained (not shown) Tris-glycine system. However, these re- with these purified dsRNAs in the Triscombinants could also be used to deter- glycine system were identical with those mine the relationship between other seg- shown in Fig. 2. However, when these ments in the two gel systems. The dsRNAs dsRNAs were electrophoresed in the Tris-
GENETIC A
6
C
D
MAP
69
OF REOVIRUS
E
F
.H
I
FIG. 3. Correspondence of S size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1, (B) type 3, (C) recombinant No. 80, (D) type 2, (E) type 3, (F) recombinant No. 207, (G) recombinant No. 246, (HI recombinant No. 231, and (I) recombinant No. 264. Electrophoresis was as described. Bands are labeled in order of electrophoretic mobility in A, B, D, and E. Bands in C, F, G, H, and I are numbered according to serotype of origin.
acetate system the segments did not always migrate in the same relative bsition compared to the migration in the Trisglycine system. Examination of the L segment region shows that when Ll of type 3 is absent from a recombinant it is replaced by Ll of type 1 (Figs. 5A and C) and when L3 of type 3 is absent it is replaced by L3 of type 2 (Fig. 5G). The Ll and L3 replacements are identical to those seen in the Tris-glycine system (Fig. 2) and indicate
that the relative migration positions of the type 3 L segments are the same in both gel systems. In the M segment region the resolution of type 3 bands is increased relative to the Tris-glycine system. Examination of M segments of the recombinants shows the relative order of migration of type 3 Ml and M2 is reversed compared to the Tris-glycine gel system. For example, when the slowest band of type 3 is absent
70
SHARPE
ET AL.
from a recombinant (Fig. 5C) it is replaced by the middle band from type 1. When the middle band of type 3 is absent from a recombinant (Fig. 5B) it is replaced by the slowest band from type 1. In the Trisglycine system the slowest and middle bands of type 3 were replaced by the slowest and middle bands, respectively, of type 1 (Fig. 2). When the fastest of the type 3 M bands is absent from a recombinant (Fig. 5H) it is replaced by the fastest of the M bands from type 2, a result identical to that seen with the Tris-glycine system (Fig. 2). Since the numbering system of the segments of type 3 was established using the Tris-acetate system, we will number bands in the Tris-glycine system according to that established nomenclature. As a result in the Tris-glycine system the M segments of type 3 will hereafter be assigned as M2, Ml, M3 in order of migration from slowest to fastest (see Fig. 6). Examination of the S segment region shows that the S segments of type 3 migrate in the same relative order in both gel systems. Sl of type 3 is replaced by the slowest S band from type 1 (Figs. 5A and B). S2 cannot be reproducibly resolved between serotypes in the Tris-acetate system. S3 of type 3 is replaced by the most rapidly migrating of the S segments from type 2 (Fig. 51) and S4 of type 3 is replaced by the second most rapidly migrating S segment from type 2 (Fig. 5H). These S segment replacements are identical to those seen in the Tris-glycine system (Fig. 2) and indicate that the S segments of type 3 migrate in the same relative position in both gel systems. (4) Correlation of Genome RNAs Serotypes 1,2, and 3
FIG. 4. Correspondence of S3 and 54 genome segments between serotypes 1 and 3. (A) Type 1, (B) recombinant No. 63, and (C) type 3. Electrophoresis was as described except that a 16-cm gel was run at 30-mA constant current for 18 hr. Bands of type 3 are labeled in order of electrophoretic mobil-
between
The correlation of the genome segments between the serotypes is summarized in Fig. 6. Numbering of the type 3 segments is standardized to the Tris-acetate-buffered gel system. Numbering of the segments of types 1 and 2 is according to segment ity. Bands of type 1 are labeled according to correspondence with type 3 bands. Bands in B are labeled according to parent of origin.
GENETIC
MAP
OF REOVIRUS
FIG. 5. Correspondence of the dsRNA segments between serotypes 1, 2, and 3 examined in the Trisacetate gel system. (A) Recombinant No. 72, (B) recombinant No. 65, (C) recombinant No. 94, (D) type 1, (E) type 3, (Fl type 2, (Gl recombinant No. 255, (H) recombinant No. 256, and (I) recombinant No. 231. Electrophoresis was as described. Bands of type 3 are labeled according to electrophoretic mobility. The bands of types 1 and 2 are labeled according to correspondence with type 3 bands. The bands of the recombinants are numbered according to serotype of origin. The position of the bands in channel F and the M bands in channel G were enhanced for purposes of illustration. They were clearly visible on the original gel.
72
SHARPE
ET AL.
TRIS-GLYCINE TYPE
1
TYPE
3
TRIS-ACETATE TYPE
2
TYPE
TYPE
1
3
TYPE
2
FIG. 6. The map of the genome segments of reovirus serotypes 1, 2, and 3 as resolved in Tris-glycineand Tris-acetate-buffered gel-electrophoresis systems. The nomenclature of type 3 bands in the Trisacetate gel system is used as the standard system of nomenclature in both gel systems. Band identities between gel systems and correlation of bands between serotypes are as determined using hybrids between serotypes. Arrows indicate the band of type 1 or 2 that replaced the type 3 band in question to yield a viable hybrid virus.
identity as established by examination of recombinants between serotypes in the Tris-acetate- and the Tris-glycine-buffered systems. Thus, the segments of types 1 and 2 are labeled in each system according to the type 3 segment which the segment in question replaces to yield a viable recombinant. DISCUSSION
The heterogeneity seen among the genome segments of three reovirus serotypes has been used to construct a map correlating segments between serotypes. The rationale for these experiments was as follows: If a single genome segment was absent from a viable hybrid recombinant between serotypes, the function encoded by the missing segment would have to be supplied by a segment derived from the other parent in order to maintain viability. The electrophoretic heterogeneity observed among the genome segments of the three reovirus serotypes examined allowed
determination of the segment from one parent which was absent and the segment from the other parent that replaced it. In general the correlation between segments of the serotypes is that they correspond in order of increasing electrophoretie mobility when examined in a Trisglycine-buffered gel system. This simplicity is, however, not evident among the M segments of type 2 and S segments of types 1 and 2. The bands corresponding to Ml and M2 of type 2 and S3 and S4 of types 1 and 2 migrate in opposite order relative to type 3. These examples illustrate the potential problems arising in assignment of polypeptides to genome segments solely on the basis of molecular weights and coding capacities as determined on gels. In one case precise correlations cannot always be made between segments among the serotypes. This occurs in the Ml and M2 segments of type 3. This pair of bands migrates as a very tight doublet which is
GENETIC
MAP
often very poorly resolved or not resolved on these gels. These segments are clearly resolved on more traditional gel systems (Ramig et al., 1977). However, we chose to use the Tris-glycine gel system (Laemmli, 1970; Ramig et al., 1977) for analysis of the dsRNAs of recombinants because crude cytoplasmic extracts can be analyzed without extensive deproteinization of the dsRNA prior to electrophoretic analysis. This technique has made it feasible to analyze large numbers of recombinants. These recombinants have also made it possible to determine the identity of bands between gel systems where the relative mobility and positions of segments change. Although there are differences between migration in the gel systems used in the past to resolve dsRNAs of reovirus type 3 and migration in the system we use here, no problems should arise because the differences have been identified and nomenclature of the bands is consistent with that which appears in the literature. We have not defined the basis of the migration differences seen between the dsRNAs in different buffer systems. These differences are, however, highly reproducible. Similar differences have been noted when reovirus type 3 polypeptides are electrophoresed under differing ionic conditions (Ramig et al., 1977). The recombinants examined in these experiments demonstrate that recombination occurs by the mechanism of reassortment of genome segments. The results further suggest that the reassortment can occur in all possible combinations. The ability to form viable recombinants between the serotypes indicates that although there appears to have been substantial molecular divergence (as evidenced by electrophoretic heterogeneity) between the serotypes, they remain closely related. Similar results have recently been reported for influenza virus, another virus containing a segmented genome. Palese and Schulman (1976a) reported that different strains of influenza A virus had different RNA patterns upon electrophoresis. They used these differences in similar manner to identify the genome segments
73
OF REOVIRUS
coding for hemagglutinin and neuraminidase (Palese and Schulman, 197613). It is interesting to note that they found that the neuraminidase was encoded in segments having different relative positions in the two parental strains examined. This observation is similar to the situation we found for Ml and M2 and S3 and S4 of types 1, 2, and 3. More recently they have extended this method to derive a complete map of the influenza genome correlating protein products to the genome segments in which they are encoded (Richey et al ., 1976). The results reported here have made it possible to map temperature-sensitive lesions of reovirus type 3 onto genome segments and to correlate proteins with the genome segments in which they are encoded (Ramig, Mustoe, Sharpe, and Fields, manuscript in preparation). ACKNOWLEDGMENTS Supported by NIH Grant 1 ROl AI 13178-01, American Cancer Society Grant VC-195, and PHS Grant 5 T32 CA 09131-01. REFERENCES BELLAMY, A. R., SHAPIRO, L., A~~usr, J. T., and JOKLIK, W. K. (1967). Studies on reovirus RNA. I. Characterization of reovirus genome RNA. J. Mol. Biol. 29, 1-17. BORUN, T. S., SCHARFF, M. D., and ROBBINS, E. (1967). Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. CROSS, R. K., and FIELDS, B. N. (1972). Temperature-sensitive mutants of reovirus type 3: Studies on the synthesis of viral RNA. Virology 50, 799809. CROSS, R. K., and FIELDS, B. N. (1976). Use of an aberrant polypeptide as a marker in three-factor crosses: Further evidence for independent reassortment as the mechanism of recombination between temperature-sensitive mutants of reovirus type 3. Virology 74, 345-362. FIELDS, B. N. (1971). Temperature-sensitive mutants of reovirus type 3: Features of genetic recombination. Virology 46, 142-148. FIELDS, B. N. (1973). Genetic reassortment with reovirus mutants. In “Virus Research” (C. F. Fox and W. S. Robinson, eds.), pp. 461-469. Academic Press, New York. FIELDS, B. N., and JOKLIK, W. K. (1969). Isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of
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reovirus. Virology 37,335-342. LAEMMLI, U. K. (1970). Cleavage of structural
proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MCCLAIN, M. E., and SPENDLOVE, R. S. (1966). Multiplicity reactivation of reovirus particles after exposure to ultraviolet light. J. Bacterid. 92, 1422-1429. PALESE, P., and SCHULMAN, J. L. (1976a). Differences in RNA patterns of influenza A viruses. J. Viral. 17, 876-884. PALESE, P., and SCHULMAN, J. L. (1976b). Mapping of the influenza virus genome: Identification of the hemagglutinin and neuraminidase genes. Proc. Nat. Acad. Sci. USA 73, 2142-2146.
RAMIG, R. F., CROSS,R. K., and FIELDS, B. N. (1977). Genome RNAs and polypeptides of reovirus serotypes 1, 2 and 3. J. Virol. 22, 726-733. RITCHEY, M. B., PALESE, P., and SCHULMAN, J. L. (1976). Mapping of the influenza virus genome: III. Identification of genes coding for nucleoprotein, membrane protein and nonstructural protein. J. Virol. 2, 307-313. SHATKIN, A. J., SIPE, J. D., and LOH, P. (1968). Separation of 10 reovirus segments by polyacrylamide gel electrophoresis. J. Viral. 2, 982-991. WATANABE, Y., and GRAHAM, A. F. (1967). Structural units of reovirus ribonucleic acid and their possible functional significance. J. Virol. 1, 665 677.
84.63-74
(1978)
A Genetic I. Correlation ARLENE
Map of Reovirus
of Genome RNAs between
H. SHARPE,’
Serotypes
ROBERT F. RAMIG,* THOMAS BERNARD N. FIELDS
1, 2, and 3 A. MUSTOE,
AND
Department of Microbiology and Molecular Genetics, Harvard Medical School, 25 Shattuck Street, Boston, Massachusetts 02115, and Department of Medicine (Infectious Diseases), Peter Bent Brigham Hospital, Boston, Massachusetts 02115 Accepted August 16,1977 The double-stranded genome RNAs of recombinants between reovirus serotypes 1, 2, and 3 were examined by polyacrylamide-gel electrophoresis. Analysis of deletions and replacements in the recombinants allowed construction of a map of the serotypes correlating genome segments providing functions interchangeable between the serotypes. The relative migration rates of segments Ml and M2 of type 3 are reversed between the traditional Tris-acetate-buffered gel system and the Tris-glycine gel system used here. In the Tris-glycine system, the genome segments of serotype 1 correspond to type 3 in order of increasing electrophoretic mobility except for S3 and S4 which are reversed. In serotype 2 all segments except Ml and M2 and S3 and S4 correspond in order of increasing electrophoretic mobility. The migration of these two segment pairs is reversed in type 2 relative to type U. A map is presented correlating the migration of genome segments of types 1, 2, and 3 in both the Tris-glycineand the Tris-acetate-buffered systems. The nomenclature of the genome segments is standardized to that which appears in the literature. In addition, these data demonstrate that recombinants arise by physical reassortment of genome segments between parents.
trophoretically aberrant polypeptides in crosses between ts mutants (Cross and Fields, 1976). We recently reported that the genome RNAs and the polypeptides of the three reovirus serotypes could be distinguished following polyacrylamide-gel electrophoresis (Ramig et al., 1977). In this communication we describe experiments that permit mapping the genome of reovirus. The method is based on the prior observation that dsRNAs of the three serotypes have different patterns on polyacrylamide gels. The analysis of the RNA patterns of recombinants between serotypes allows construction of a map of the genome which correlates segments between the serotypes. Additionally, physical reassortment of segments is shown to be the mechanism of recombination,
INTRODUCTION
The genome of reovirus consists of 10 segments of double-stranded RNA (Bellamy et al., 1967; Watanabe and Graham, 1967; Shatkin et al., 1968). Genetic crosses between temperature-sensitive (ts) mutants isolated from reovirus type 3 Dearing revealed that pairwise crosses resulted in a high proportion of ts+ progeny for some pairs whereas other pairs yielded no detectable recombinants (Fields and Joklik, 1969; Fields, 1971, 1973). The “all-or-none” nature of recombination suggested that recombinants were generated by random reassortment of genome segments. Additional evidence for reassortment as the mechanism of recombination has come from the study of the segregation of elec’ Present address: Yale University School of Medicine, New Haven, Connecticut. L Address reprint requests to Dr. Ramig at the Harvard Medical School.
MATERIALS (1)
Virus.
AND
Reovirus
METHODS
type 1, strain Lang
63 004%6822/78/0841-0063$02.00 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
SHARPE ET AL.
and type 2, strain Jones were the same as medium and allowed to adsorb for 30 min described by Ramig et al. (1977). The reo- at 31” in a 5% CO, incubator. After adsorpvirus type 3, strain Dearing mutants were tion, 4.0 ml of IMEMZO medium was isolated and described by Fields and Joklik added to each plate and the plates were (1969). The following recombination group returned to 31”. At 2.5 hr postinfection the prototype mutants were used: group A, ts medium was removed from each plate and 201; group B, ts 352; group C, ts 447; group replaced with 4.0 ml of IMEMZO medium D, ts 357; group E, ts 320 and group G, ts containing 0.25 mCi of %P as orthophos453. All viruses were plaque purified and phoric acid (NEN). The plates were then passaged on L-cell monolayers twice. Sec- incubated at 31” in a 5% CO, incubator ond-passage virus was used for all genetic until they were harvested. The infected crosses. cells were harvested at 48 hr postinfection (2) Cells. Mouse L cells were main- and cytoplasmic RNA was prepared as tained in suspension culture in Joklik’s follows. The infected monolayers were modified Eagle’s minimal essential me- washed twice with cold NP40 buffer (Bodium [International Biological Laboratorun et al., 1967). The cells were then ries (IBL), Rockville, Md.] supplemented suspended in NP40 buffer using a rubber policeman and cell clumps were broken by with 5% fetal calf serum (IBL). Monkey CV-1 cells were maintained in monolayers pipetting. Nonidet P-40 (Shell Chemical in IMEMZO with zinc and insulin (IBL) Co.) was added to a final concentration of supplemented with 10% fetal calf serum 0.5% and the mixture was incubated for (IBL). 30 min at 4” (Borun et al., 1967). Nuclei (3) Genetic protocols. L cells in suspen- were removed from the cytoplasmic extract by centrifugation. The cytoplasmic sion culture were infected with a multiplicity of infection (m.0.i.) of 10 plaque- extract was adjusted to 0.2 M in NaCl, 3 forming units (PFU) each of a type 3 vol of ethanol were added, and the mixture Dearing temperature-sensitive (ts) mu- was incubated at -20” overnight to preciptant and of wild-type virus of either type 1 itate RNA. The precipitates were pelleted Lang or type 2 Jones as previously de- by centrifugation at 10,000 rpm for 30 min scribed (Fields and Joklik, 1969). After in a Sorvall SS-34 rotor. The precipitates adsorption the infected cells were incu- were dried under vacuum and dissolved in bated at 31” for 48 hr. At the end of the 0.25 ml of gel sample buffer (Laemmli, incubation period the cells were chilled 1970). and sonicated to disrupt the remaining (5) Preparation of labeled, purified, virintact cells and viral aggregates. The yield ion dsRNA . Labeled dsRNA was purified of virus from the mixedly infected cells by the method of Ramiget al. (1977) except was titered by the method of Cross and that 5 &i/ml of 32P as orthophosphoric acid (NEN) was added at 2.5 hr postinfecFields (1976). The permissive temperature for the ts mutants was 31” and the non- tion. Because poor yields of type 2 were permissive temperature was 39” (Fields obtained by this method, total 32P-labeled and Joklik, 1969). Lytic (ts+) progeny type 2 cytoplasmic dsRNA was prepared by the method of Ramig et al. (1977). The plaques were picked from plates incubated at 39” which had fewer than 10 plaques per 32P-labeled dsRNA was dissolved in 0.25 plate. These lytic plaques were passaged M Tris-acetate, pH 8.5 (Cross and Fields, once on L-cell monolayers at 37”. The dou- 1972). (6) Polyacrylamide-gel electrophoresis ble-stranded RNA (dsRNA) synthesized of dsRNA. Slab gels were prepared to a by these clones was examined as follows. final concentration of 10% acrylamide, (4) Preparation of labeled, unpurified, cytoplasmic d.sRNA. The medium was re- 0.267% bisacrylamide as described by Lacytoplasmic moved from the confluent plates of CV-1 emmli (1970). Unpurified cells (2 x lo6 cells/60 x l&mm plate) and dsRNA samples to be applied to the gels virus at an m.o.i. of 10 PFU/cell was added were boiled for 1 min within 0.5 hr prior in a total volume of 1.0 ml of Pucks’ to loading the gel. Electrophoresis was
GENETIC
MAP
carried out at a constant current of 20 mA for 18 hr. Alternatively, purified virion dsRNAs were subjected to electrophoresis in 5% gels containing the Tris-acetatebuffered system described by Cross and Fields (1972). Electrophoresis was carried out at 110 V for 24 hr on slab gels 16 cm long. After electrophoresis the gels were fixed in 50% methanol, 7% acetic acid, then dried onto filter paper in a Hoeffer Scientific Instruments gel dryer. Autoradiography was performed by placing the dried gel in contact with a sheet of NoScreen X-ray film (Kodak) for 6-24 hr. Films were processed by standard photographic procedures. LII
RESULTS
(1) Theory of the Genetic Crosses
OF REOVIRUS
65
tinguished by polyacrylamide-gel electrophoresis (Ramig et al., 1977) analysis of the dsRNAs synthesized by progeny forming lytic plaques at nonpermissive temperature allows one to distinguish between ts+ recombinant progeny and ts+ parental progeny. Furthermore, analysis of the migration rates of each of the dsRNA segments in each ts+ recombinant allows one to determine the serotype from which each segment was derived. The details of the genetic crosses from which these recombinants were derived will be described elsewhere (Ramig, Mustoe, Sharpe, and Fields, manuscript in preparation). (2) Correlation of Genome Segments between Serotypes in the Tris-Glycine Gel System
The dsRNAs synthesized by reovirus The crosses described in this communi- serotypes 1, 2, and 3 yield distinctive and cation were between a ts mutant of type 3 reproducibly different patterns when subDearing (type 3) and wild type of 1 Lang jected to polyacrylamide-gel electrophore(type 1) or type 2 Jones (type 2). Previously sis (Ramig et al., 1977). In all three cases it had been shown that multiplicity reac- the genome segments fall into three size tivation occurs between ultraviolet lightclasses, L, M, and S. However, segments irradiated reovirus of different serotypes within a size class do not correspond in (McClain and Spendlove, 1966) suggesting order of electrophoretic mobility. Examithat genetic interactions occur between nation of segment replacements (and correovirus serotypes. Thus if cells are responding deletions) in a few representamixedly infected with a type 3 ts mutant tive recombinant clones allows one to esand wild-type type 1 or type 2 at permis- tablish the correspondence between gesive temperature, four progeny types nome segments of the serotypes. Figure 1 would be expected among the progeny of shows the dsRNA patterns of the serotypes the cross: (i) the parental type 3 ts mutant, and the recombinants selected to show (ii) the parental wild-type type 1 or type that when a given L segment from type 3 2, (iii) recombinant progeny retaining the is absent, it is replaced by the correspondts lesion derived from the type 3 parent, ing segmer,b from type 1 or type 2. In and (iv) recombinant progeny having a recombinants between both type 1 and ts+ phenotype due to replacement of the type 3 and type 2 and type 3, when the Ll type 3 genome segment bearing the ts segment of type 3 is absent from a recomlesion by the corresponding genome seg- binant it is replaced by the segment havment derived from the ts+ type 1 or type 2 ing the slowest electrophoretic migration parent. Progeny types (i) and (iii) can be from type 1 or type 2 (Figs. 1C and H). selected against by harvesting lytic Likewise when the L3 segment of type 3 is plaques from progeny plated at nonper- absent it is replaced by the most rapidly missive temperature. Plating at nonper- migrating segment from type 1 or type 2 missive temperature thus leaves a mix(Figs. 1D and H). Although no examples ture of ts+ recombinant progeny and the are shown, by elimination the L segment ts+ parental type. with medium migration rate must correSince the dsRNAs synthesized by the spond in all three serotypes. Thus the L three reovirus serotypes can be easily dis- size class genome segments of all three
66
SHARPE
serotypes correspond in order of increasing electrophoretic mobility: Type
Type
Type
1
3
2
ET AL. Type
Type
2 L\
M31 /MZ
\
Ll -/-
Type
--
--A*;-------+The correspondence of M size genome segments between serotypes is shown in Fig. 2. The Ml and M2 segments of type 3 are not well separated under the conditions of these gels and run as a very tight doublet band. When Ml and/or M2 of type 3 are absent from recombinants they are replaced by the segments of slowest and of intermediate mobility in the case of recombinants with both type 1 and type 2 (Figs. 2C-E, H, and J). The correspondence of Ml and M2 of type 3 with the analogous bands of the other serotypes cannot be unambiguously established from these data. Occasionally, although not reproducibly, the Ml and M2 doublet of type 3 is well resolved. In these cases it becomes clear that when Ml of type 3 is absent from a recombinant it is replaced by the middle of the M bands from type 2 (Fig. 2H) and by the slowest of the bands from type 1 (Fig. 2D). When M2 of type 3 is absent it is replaced by the slowest M band from type 2 (Fig. 25) and by the middle M band from type 1 (Fig. 2E). (Note that in Fig. 25 the position of all the bands is slightly displaced indicating that in this sample all of the bands migrated slightly more rapidly than the analogous bands in the other lanes.) In crosses between type 1 and type 3 and between type 2 and type 3 when the most rapidly migrating of the M segments of one type is absent it is replaced by the most rapidly migrating of the M segments of the other type (Figs. 2C and I). Thus the correspondence of the M size class genome segments between the serotypes can be summarized as follows:
----i& M3 /Figure 3 shows the correspondence of genome segments among the S size class of the three serotypes. When Sl of type 3 is absent from a recombinant (Fig. 3C) it is replaced by the slowest migrating of the S segments from type 1. No electrophoretie difference is reproducibly seen between S2 of type 3 and S2 of type 1. S3 and S4 of type 1 generally migrate as a tight doublet. However, when samples ore run on a gel 16 cm long, S3 and S4 of type 1 are resolved (Fig. 4). A recombinant between S3 and S4 of types 1 and 3 (Fig. 4B) shows that when S3 of type 3 is absent it is replaced by the most rapidly migrating of the S segments from type 1. By exclusion it must follow that S4 of type 3 is replaced by the second most rapidly migrating of the S segments from type 1. Sl of type 2 is replaced by the slowest of the S bands from type 3 (Figs. 3F-I). When S2 of type 3 is absent it is replaced by the second most slowly migrating of the type 2 S RNAs (Figs. 3F and G). When S3 of type 3 is absent it is replaced by the most rapidly migrating segment from type 2 (Fig. 3H). When S4 is absent from a recombinant it is replaced by the second most rapidly migrating of the segments from type 2 (Fig. 31). The correspondence of the S size class genome segments between the serotypes can be summarized as follows: TYPe Type Type 1
3
2
GENETIC
MAP
OF REOVIRUS
67
ABCDEFG
FIG. Correspondence of L size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1 , 03) typ be3, (C) ret on nbinant No. 94, (D) recombinant No. 80, (E) type 2, (F) type 3, (G) recombinant No. 2;47, and (H) recomt kin(ant NO. 261. Electrophoresis was as described. Bands are labeled in order of el ec:trophor ,etic mobilii :Y in A, B, E, and F. Bands in C, D, G, and H are numbered according to serotype c origin. The bands 1in G were enhanced for purposes of illustration.
(3) Correlation of Genome Segments of Reovirus Type 3 between Tris-Glytine- and Tris-Acetate-Buffered Gel Systems The availability of the recombinants ex-
amined above allowed us to determine the relationships between the genome segments of type 3 as resolved in the Trisglycine gel system used here and the Trisacetate system that has been traditionally
68
SHARPE
A
B
C
D
E
ET AL.
F
G
t-l
I
J
FIG. 2. Correspondence of M size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1, (B) type 3, (Cl recombinant No. 72, (D) recombinant No. 65, (El recombinant No. 94, (F) type 2, (G) type 3, (H) recombinant No. 255, (I) recombinant No. 256, and (J) recombinant No. 231. Electrophoresis was as described. Bands are labeled in order of electrophoretic mobility in A, B, F, and G. Bands in C, D, E, H, I, and J are numbered according to serotype of origin.
used to resolve type 3 dsRNAs. The recom- of the recombinants shown in Fig. 2 were binants shown in Fig. 2 were selected for extracted from purified virions and substudy in the Tris-acetate system primarily jected to electrophoresis in the Tris-glyto resolve the ambiguity caused by the tine- and Tris-acetate-buffered gel systight Ml, M2 doublet of type 3 in the tems. The patterns obtained (not shown) Tris-glycine system. However, these re- with these purified dsRNAs in the Triscombinants could also be used to deter- glycine system were identical with those mine the relationship between other seg- shown in Fig. 2. However, when these ments in the two gel systems. The dsRNAs dsRNAs were electrophoresed in the Tris-
GENETIC A
6
C
D
MAP
69
OF REOVIRUS
E
F
.H
I
FIG. 3. Correspondence of S size dsRNA segments between serotypes 1, 2, and 3. (A) Type 1, (B) type 3, (C) recombinant No. 80, (D) type 2, (E) type 3, (F) recombinant No. 207, (G) recombinant No. 246, (HI recombinant No. 231, and (I) recombinant No. 264. Electrophoresis was as described. Bands are labeled in order of electrophoretic mobility in A, B, D, and E. Bands in C, F, G, H, and I are numbered according to serotype of origin.
acetate system the segments did not always migrate in the same relative bsition compared to the migration in the Trisglycine system. Examination of the L segment region shows that when Ll of type 3 is absent from a recombinant it is replaced by Ll of type 1 (Figs. 5A and C) and when L3 of type 3 is absent it is replaced by L3 of type 2 (Fig. 5G). The Ll and L3 replacements are identical to those seen in the Tris-glycine system (Fig. 2) and indicate
that the relative migration positions of the type 3 L segments are the same in both gel systems. In the M segment region the resolution of type 3 bands is increased relative to the Tris-glycine system. Examination of M segments of the recombinants shows the relative order of migration of type 3 Ml and M2 is reversed compared to the Tris-glycine gel system. For example, when the slowest band of type 3 is absent
70
SHARPE
ET AL.
from a recombinant (Fig. 5C) it is replaced by the middle band from type 1. When the middle band of type 3 is absent from a recombinant (Fig. 5B) it is replaced by the slowest band from type 1. In the Trisglycine system the slowest and middle bands of type 3 were replaced by the slowest and middle bands, respectively, of type 1 (Fig. 2). When the fastest of the type 3 M bands is absent from a recombinant (Fig. 5H) it is replaced by the fastest of the M bands from type 2, a result identical to that seen with the Tris-glycine system (Fig. 2). Since the numbering system of the segments of type 3 was established using the Tris-acetate system, we will number bands in the Tris-glycine system according to that established nomenclature. As a result in the Tris-glycine system the M segments of type 3 will hereafter be assigned as M2, Ml, M3 in order of migration from slowest to fastest (see Fig. 6). Examination of the S segment region shows that the S segments of type 3 migrate in the same relative order in both gel systems. Sl of type 3 is replaced by the slowest S band from type 1 (Figs. 5A and B). S2 cannot be reproducibly resolved between serotypes in the Tris-acetate system. S3 of type 3 is replaced by the most rapidly migrating of the S segments from type 2 (Fig. 51) and S4 of type 3 is replaced by the second most rapidly migrating S segment from type 2 (Fig. 5H). These S segment replacements are identical to those seen in the Tris-glycine system (Fig. 2) and indicate that the S segments of type 3 migrate in the same relative position in both gel systems. (4) Correlation of Genome RNAs Serotypes 1,2, and 3
FIG. 4. Correspondence of S3 and 54 genome segments between serotypes 1 and 3. (A) Type 1, (B) recombinant No. 63, and (C) type 3. Electrophoresis was as described except that a 16-cm gel was run at 30-mA constant current for 18 hr. Bands of type 3 are labeled in order of electrophoretic mobil-
between
The correlation of the genome segments between the serotypes is summarized in Fig. 6. Numbering of the type 3 segments is standardized to the Tris-acetate-buffered gel system. Numbering of the segments of types 1 and 2 is according to segment ity. Bands of type 1 are labeled according to correspondence with type 3 bands. Bands in B are labeled according to parent of origin.
GENETIC
MAP
OF REOVIRUS
FIG. 5. Correspondence of the dsRNA segments between serotypes 1, 2, and 3 examined in the Trisacetate gel system. (A) Recombinant No. 72, (B) recombinant No. 65, (C) recombinant No. 94, (D) type 1, (E) type 3, (Fl type 2, (Gl recombinant No. 255, (H) recombinant No. 256, and (I) recombinant No. 231. Electrophoresis was as described. Bands of type 3 are labeled according to electrophoretic mobility. The bands of types 1 and 2 are labeled according to correspondence with type 3 bands. The bands of the recombinants are numbered according to serotype of origin. The position of the bands in channel F and the M bands in channel G were enhanced for purposes of illustration. They were clearly visible on the original gel.
72
SHARPE
ET AL.
TRIS-GLYCINE TYPE
1
TYPE
3
TRIS-ACETATE TYPE
2
TYPE
TYPE
1
3
TYPE
2
FIG. 6. The map of the genome segments of reovirus serotypes 1, 2, and 3 as resolved in Tris-glycineand Tris-acetate-buffered gel-electrophoresis systems. The nomenclature of type 3 bands in the Trisacetate gel system is used as the standard system of nomenclature in both gel systems. Band identities between gel systems and correlation of bands between serotypes are as determined using hybrids between serotypes. Arrows indicate the band of type 1 or 2 that replaced the type 3 band in question to yield a viable hybrid virus.
identity as established by examination of recombinants between serotypes in the Tris-acetate- and the Tris-glycine-buffered systems. Thus, the segments of types 1 and 2 are labeled in each system according to the type 3 segment which the segment in question replaces to yield a viable recombinant. DISCUSSION
The heterogeneity seen among the genome segments of three reovirus serotypes has been used to construct a map correlating segments between serotypes. The rationale for these experiments was as follows: If a single genome segment was absent from a viable hybrid recombinant between serotypes, the function encoded by the missing segment would have to be supplied by a segment derived from the other parent in order to maintain viability. The electrophoretic heterogeneity observed among the genome segments of the three reovirus serotypes examined allowed
determination of the segment from one parent which was absent and the segment from the other parent that replaced it. In general the correlation between segments of the serotypes is that they correspond in order of increasing electrophoretie mobility when examined in a Trisglycine-buffered gel system. This simplicity is, however, not evident among the M segments of type 2 and S segments of types 1 and 2. The bands corresponding to Ml and M2 of type 2 and S3 and S4 of types 1 and 2 migrate in opposite order relative to type 3. These examples illustrate the potential problems arising in assignment of polypeptides to genome segments solely on the basis of molecular weights and coding capacities as determined on gels. In one case precise correlations cannot always be made between segments among the serotypes. This occurs in the Ml and M2 segments of type 3. This pair of bands migrates as a very tight doublet which is
GENETIC
MAP
often very poorly resolved or not resolved on these gels. These segments are clearly resolved on more traditional gel systems (Ramig et al., 1977). However, we chose to use the Tris-glycine gel system (Laemmli, 1970; Ramig et al., 1977) for analysis of the dsRNAs of recombinants because crude cytoplasmic extracts can be analyzed without extensive deproteinization of the dsRNA prior to electrophoretic analysis. This technique has made it feasible to analyze large numbers of recombinants. These recombinants have also made it possible to determine the identity of bands between gel systems where the relative mobility and positions of segments change. Although there are differences between migration in the gel systems used in the past to resolve dsRNAs of reovirus type 3 and migration in the system we use here, no problems should arise because the differences have been identified and nomenclature of the bands is consistent with that which appears in the literature. We have not defined the basis of the migration differences seen between the dsRNAs in different buffer systems. These differences are, however, highly reproducible. Similar differences have been noted when reovirus type 3 polypeptides are electrophoresed under differing ionic conditions (Ramig et al., 1977). The recombinants examined in these experiments demonstrate that recombination occurs by the mechanism of reassortment of genome segments. The results further suggest that the reassortment can occur in all possible combinations. The ability to form viable recombinants between the serotypes indicates that although there appears to have been substantial molecular divergence (as evidenced by electrophoretic heterogeneity) between the serotypes, they remain closely related. Similar results have recently been reported for influenza virus, another virus containing a segmented genome. Palese and Schulman (1976a) reported that different strains of influenza A virus had different RNA patterns upon electrophoresis. They used these differences in similar manner to identify the genome segments
73
OF REOVIRUS
coding for hemagglutinin and neuraminidase (Palese and Schulman, 197613). It is interesting to note that they found that the neuraminidase was encoded in segments having different relative positions in the two parental strains examined. This observation is similar to the situation we found for Ml and M2 and S3 and S4 of types 1, 2, and 3. More recently they have extended this method to derive a complete map of the influenza genome correlating protein products to the genome segments in which they are encoded (Richey et al ., 1976). The results reported here have made it possible to map temperature-sensitive lesions of reovirus type 3 onto genome segments and to correlate proteins with the genome segments in which they are encoded (Ramig, Mustoe, Sharpe, and Fields, manuscript in preparation). ACKNOWLEDGMENTS Supported by NIH Grant 1 ROl AI 13178-01, American Cancer Society Grant VC-195, and PHS Grant 5 T32 CA 09131-01. REFERENCES BELLAMY, A. R., SHAPIRO, L., A~~usr, J. T., and JOKLIK, W. K. (1967). Studies on reovirus RNA. I. Characterization of reovirus genome RNA. J. Mol. Biol. 29, 1-17. BORUN, T. S., SCHARFF, M. D., and ROBBINS, E. (1967). Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. CROSS, R. K., and FIELDS, B. N. (1972). Temperature-sensitive mutants of reovirus type 3: Studies on the synthesis of viral RNA. Virology 50, 799809. CROSS, R. K., and FIELDS, B. N. (1976). Use of an aberrant polypeptide as a marker in three-factor crosses: Further evidence for independent reassortment as the mechanism of recombination between temperature-sensitive mutants of reovirus type 3. Virology 74, 345-362. FIELDS, B. N. (1971). Temperature-sensitive mutants of reovirus type 3: Features of genetic recombination. Virology 46, 142-148. FIELDS, B. N. (1973). Genetic reassortment with reovirus mutants. In “Virus Research” (C. F. Fox and W. S. Robinson, eds.), pp. 461-469. Academic Press, New York. FIELDS, B. N., and JOKLIK, W. K. (1969). Isolation and preliminary genetic and biochemical characterization of temperature-sensitive mutants of
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reovirus. Virology 37,335-342. LAEMMLI, U. K. (1970). Cleavage of structural
proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. MCCLAIN, M. E., and SPENDLOVE, R. S. (1966). Multiplicity reactivation of reovirus particles after exposure to ultraviolet light. J. Bacterid. 92, 1422-1429. PALESE, P., and SCHULMAN, J. L. (1976a). Differences in RNA patterns of influenza A viruses. J. Viral. 17, 876-884. PALESE, P., and SCHULMAN, J. L. (1976b). Mapping of the influenza virus genome: Identification of the hemagglutinin and neuraminidase genes. Proc. Nat. Acad. Sci. USA 73, 2142-2146.
RAMIG, R. F., CROSS,R. K., and FIELDS, B. N. (1977). Genome RNAs and polypeptides of reovirus serotypes 1, 2 and 3. J. Virol. 22, 726-733. RITCHEY, M. B., PALESE, P., and SCHULMAN, J. L. (1976). Mapping of the influenza virus genome: III. Identification of genes coding for nucleoprotein, membrane protein and nonstructural protein. J. Virol. 2, 307-313. SHATKIN, A. J., SIPE, J. D., and LOH, P. (1968). Separation of 10 reovirus segments by polyacrylamide gel electrophoresis. J. Viral. 2, 982-991. WATANABE, Y., and GRAHAM, A. F. (1967). Structural units of reovirus ribonucleic acid and their possible functional significance. J. Virol. 1, 665 677.
