Comparisons of the M1 genome segments and encoded μ2 proteins of different reovirus isolates

Background The reovirus M1 genome segment encodes the μ2 protein, a structurally minor component of the viral core, which has been identified as a transcriptase cofactor, nucleoside and RNA triphosphatase, and microtubule-binding protein. The μ2 protein is the most poorly understood of the reovirus structural proteins. Genome segment sequences have been reported for 9 of the 10 genome segments for the 3 prototypic reoviruses type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), but the M1 genome segment sequences for only T1L and T3D have been previously reported. For this study, we determined the M1 nucleotide and deduced μ2 amino acid sequences for T2J, nine other reovirus field isolates, and various T3D plaque-isolated clones from different laboratories. Results Determination of the T2J M1 sequence completes the analysis of all ten genome segments of that prototype. The T2J M1 sequence contained a 1 base pair deletion in the 3' non-translated region, compared to the T1L and T3D M1 sequences. The T2J M1 gene showed ~80% nucleotide homology, and the encoded μ2 protein showed ~71% amino acid identity, with the T1L and T3D M1 and μ2 sequences, respectively, making the T2J M1 gene and μ2 proteins amongst the most divergent of all reovirus genes and proteins. Comparisons of these newly determined M1 and μ2 sequences with newly determined M1 and μ2 sequences from nine additional field isolates and a variety of laboratory T3D clones identified conserved features and/or regions that provide clues about μ2 structure and function. Conclusions The findings suggest a model for the domain organization of μ2 and provide further evidence for a role of μ2 in viral RNA synthesis. The new sequences were also used to explore the basis for M1/μ2-determined differences in the morphology of viral factories in infected cells. The findings confirm the key role of Ser/Pro208 as a prevalent determinant of differences in factory morphology among reovirus isolates and trace the divergence of this residue and its associated phenotype among the different laboratory-specific clones of type 3 Dearing.


Background
RNA viruses represent the most significant and diverse group of infectious agents for eukaryotic organisms on earth [1,2]. Virtually every RNA virus, except retroviruses, must use an RNA-dependent RNA polymerase (RdRp) to copy its RNA genome into progeny RNA, an essential step in viral replication and assembly. The virally encoded RdRp is not found in uninfected eukaryotic cells and therefore represents an attractive target for chemotherapeutic strategies to combat RNA viruses. A better understanding of the structure/function relationships of RNAvirus RdRps has been gained from recent determinations of X-ray crystal structures for several of these proteins, including the RdRps of poliovirus, hepatitis C virus, rabbit calicivirus, and mammalian orthoreovirus [3][4][5][6]. However, the diverse and complex functions and regulation of these enzymes, including their interactions with other viral proteins and cis-acting signals in the viral RNAs, determine that we have hardly scratched the surface for understanding most of them.
The nonfusogenic mammalian orthoreoviruses (reoviruses) are prototype members of the family Reoviridae, which includes segmented double-stranded RNA (dsRNA) viruses of both medical (rotavirus) and economic (orbivirus) importance (reviewed in [7][8][9]). Reoviruses have nonenveloped, double-shelled particles composed of eight different structural proteins encasing the ten dsRNA genome segments. Reovirus isolates (or "strains") can be grouped into three serotypes, represented by three commonly studied prototype isolates: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D). Sequences have been reported for all ten genome segments of T1L and T3D, as well as for nine of the ten segments of T2J (all but the M1 segment) (e.g., see [10,11]). Each of these segments encodes either one or two proteins on one of its strands, the plus strand. After cell entry, transcriptase complexes within the infecting reovirus particles synthesize and release full-length, capped plus-strand copies of each genome segment. These plus-strand RNAs are used as templates for translation by the host machinery as well as for minus-strand synthesis by the viral replicase complexes. The latter process produces the new dsRNA genome segments for packaging into progeny particles. The particle locations and functions of most of the reovirus proteins have been determined by a combination of genetic, biochemical, and biophysical techniques over the past 50 years (reviewed in [8]).
Previous studies have identified the reovirus λ3 protein, encoded by the L1 genome segment, as the viral RdRp [6,[12][13][14]. Protein λ3 is a minor component of the inner capsid, present in only 10-12 copies per particle [15]. It has been proposed to bind to the interior side of the inner capsid, near the icosahedral fivefold axes, and recent work has precisely localized it there [16,17]. In solution, purified λ3 mediates a poly(C)-dependent poly(G)-polymerase activity, but it has not been shown to use virus-specific dsRNA or plus-strand RNA as template for plus-or minusstrand RNA synthesis, respectively [14]. This lack of activity with virus-specific templates suggests that viral or cellular cofactors may be required to make λ3 fully functional. Within the viral particle, where only viral proteins are known to reside, these cofactors are presumably viral in origin. The crystal structure of λ3 has provided substantial new information about the organization of its sequences and has suggested several new hypotheses about its functions in viral RNA synthesis and the possible roles of cofactors in these functions [6]. Notably, crystallized λ3 uses short viral and nonviral oligonucleotides as templates for RNA synthesis to yield short dsRNA products [6].
The reovirus µ2 protein has been proposed as a transcriptase cofactor, but it remains the most functionally and structurally enigmatic of the eight proteins found in virions. Like λ3, µ2 is a minor component of the inner capsid, present in only 20-24 copies per particle [15]. It is thought to associate with λ3 in the particle interior, in close juxtaposition to the icosahedral fivefold axes, but has not been precisely localized there [16,17]. A recent study has shown that purified µ2 and λ3 can interact in vitro [18]. The M1 genome segment that encodes µ2 is genetically associated with viral strain differences in the in vitro transcriptase and nucleoside triphosphatase (NTPase) activities of viral particles [19,20]. Recent work with purified µ2 has shown that it can indeed function in vitro as both an NTPase and an RNA 5'-triphosphatase [18]. The µ2 protein has also been shown to bind RNA and to be involved in formation of viral inclusions, also called "factories", through microtubule binding in infected cells [18,[21][22][23]. Nevertheless, its precise function(s) in the reovirus replication cycle remain unclear. Other studies have indicated that the µ2-encoding M1 segment genetically determines the severity of cytopathic effect in mouse L929 cells, the frequency of myocarditis in infected mice, the levels of viral growth in cardiac myocytes and endothelial cells, the degree of organ-specific virulence in severe combined immunodeficiency mice, and the level of interferon induction in cardiac myocytes [24][25][26][27][28][29]. The complete sequence of the M1 segment has been reported for both T1L and T3D [23,30,31]. However, computer-based comparisons of the M1 and µ2 sequences to others in GenBank have previously failed to show significant homology to other proteins, so that no clear indications of µ2 function have come from that approach. Nevertheless, small regions of sequence similarity to NTPbinding motifs have been identified near the middle of µ2, and recent work has indicated that mutations in one of these regions indeed abrogates the triphosphatase activities of µ2 [18,20].
For this study, we performed nucleotide-sequence determinations of the M1 genome segments of reovirus T2J, nine other reovirus field isolates, and reovirus T3D clones obtained from several different laboratories. The determination of the T2J M1 sequence completes the sequence determination of all ten genome segments of that prototype strain. We reasoned that comparisons of additional M1 and µ2 sequences may reveal conserved features and/ or regions that provide clues about µ2 structure and function. The findings provide further evidence for a role of µ2 in viral RNA synthesis. We also took advantage of the newly available sequences to explore the basis for M1/µ2determined strain differences in the morphology of viral factories in reovirus-infected cells.

M1 nucleotide and µ2 amino acid sequences of reovirus T2J and nine other field isolates
We determined the nucleotide sequence of the M1 genome segment of reovirus T2J to complete the sequencing of that isolate's genome. T2J M1 was found to be 2303 base pairs in length (GenBank accession no. AF124519) ( Table 1). This is one shorter than the M1 segments of reoviruses T1L and T3D [23,30,31], due to a single base-pair deletion in T2J corresponding to position 2272 in the 3' nontranslated region of the T1L and T3D plus strands (Fig. 1, Table 1). Like those of T1L and T3D, the T2J-M1 plus strand contains a single long open reading frame, encoding a µ2 protein of 736 amino acids (Fig. 2, Table  1), having the same start and stop codons (Fig. 1), and having a 5' nontranslated region that is only 13 nucleotides in length ( Table 1). Because of the single-base deletion described above, the 3' nontranslated region of the T2J M1 plus strand is only 82 nucleotides in length, compared to 83 for T1L and T3D (Table 1). Regardless, M1 has the longest 3' nontranslated region of any of the genome segments of these viruses, the next longest being 73 nucleotides in S3 (reviewed in [32]).

Further comparisons of the M1 nucleotide sequences
The M1 genome segments of the nine other reovirus isolates examined in this study are much more closely related to those of T1L and T3D than to that of T2J (Table 2), as also clearly indicated by phylogenetic analyses (Fig. 3 and data not shown). Such greater divergence of the gene sequences of T2J has been observed to date with other segments examined from multiple reovirus field isolates [11,34,[37][38][39]. Type 2 simian virus 59 (T2S59) has the next most broadly divergent M1 sequence, but it is no more similar to the M1 sequence of T2J than it is to that of the other isolates (Table 2, Fig. 3). In sum, the results of  Alignment of the deduced µ2 amino acid sequences of T1L, T2J, T3D, and various field isolates Figure 2 Alignment of the deduced µ2 amino acid sequences of T1L, T2J, T3D, and various field isolates. The single-letter amino acid code is used, and only the T1L µ2 sequence from the Brown laboratory is shown in its entirety. For other isolates, only those amino acids that differ from this T1L sequence are shown. Clones arranged in same order as in Fig. 1; the second T1L µ2 sequence is from the Nibert and Coombs laboratories, the first T3D µ2 sequence is from the Joklik and Cashdollar laboratories, and the second T3D µ2 sequence is from the Nibert and Coombs laboratories. Amino acid positions are numbered above the sequences. Some symbols represent various nonconservative changes among the isolates: *, change involving a charged residue; § change involving an aromatic residue; †, change involving a proline residue; ‡, change involving a cysteine residue. Residue 208, which has been previously shown to affect microtubule association by µ2, is indicated by a filled diamond. Residues 410-420 and 446-449, which have been previously identified as NTP-binding motifs are indicated by filled circles. Consecutive runs of wholly conserved residues ≥ 15 amino acids in length are indicated by the lines numbered 1 to 8.  30  40  50  60  70  80  90  100  110  120   1  2  3   T1L  MAYIAVPAVVDSRSSEAIGLLESFGVDAGADANDVSYQDHDYVLDQLQYMLDGYEAGDVIDALVHKNWLHHSVYCLLPPKSQLLEYWKSNPSVIPDNVDRRLRKRLMLKKDLRKDDEYNQLARAF   T2J  V  T TKEES Q  YR  E  A  ES  M  V   T3D  A  T1C11  A  T1C29  A  T1N84  S  A  T2N84  A  T2S59  V  I  Y  A   T3C18  A  T3C44  A  T3N83  R  A   130  140  150  160  170  180  190  200  210  220  230 240 250

6
T1L SVPPDVILNISITGFINRHTIDVMPDIYDFVKPIGAVLPKGSFKSTIMRVLDSISILGVQIMPRAHVVDSDEVGEQMEPTFEHAVMEIYKGIAGVDSLDDLIKWVLNSDLIPHDDRLGQLFQAFL  630  640  650  660  670  680  690  700  710  720  730 T1L QLMQDGWCGCEDGFIPYVSIRAPRLVMEELMEKNWGQYHAQVIVTDQLVVGEPRRVSAKAVIKGNHLPVKLVSRFACFTLTAKYEMRLSCGHSTGRGAAYNARLAFRSDLA T3D  I  S  T1C11  K  I  T1C29  V  S  I  R  I  I  C  T1N84  I V  I  R  V  T2N84  I M  I  T2S59  I  I  R  L  N   T3C18  I  S  T3C44  V  S  I  R  I  I  C  T3N83 AI T1L F this study provided little or no evidence for divergence of the M1 sequences along the lines of reovirus serotype (Fig.  3), consistent with independent reassortment and evolution of the M1 and S1 segments in nature. Upon considering the sources of these isolates [34], the results similarly provided little or no evidence for divergence of the M1 sequences along the lines of host, geographic locale, or date of isolation (Fig. 3). These findings are consistent with ongoing exchange of M1 segments among reovirus strains cocirculating in different hosts and locales. Similar conclusions have been indicated by previous studies of other genome segments from multiple reovirus field isolates [11,34,[37][38][39]. The M1 nucleotide sequence of type 3 clone 12 (T3C12) is almost identical to that of the T3D clone in use in the Coombs and Nibert laboratories, with only a single silent change (U→C) at plus-strand position 1532 (i.e., 99.9+% homology). However, several of the T3C12 genome segments show distinguishable mobilities in polyacrylamide gels (data not shown), confirming that T3C12 is indeed a distinct isolate.

Further comparisons of the µ2 protein sequences
The T2J µ2 protein shares 80-81% homology with those of both T1L and T3D ( Table 2, Fig. 2). Consistent with the M1 nucleotide sequence results, this makes T2J µ2 the most divergent of all nonfusogenic mammalian orthoreovirus proteins examined to date, with the exception of the S1-encoded σ1 and σ1s proteins, which show less than 55% amino acid sequence homology between serotypes [35,36]; reviewed in [11]. In contrast, the homology between T1L and T3D µ2 approaches 99%, among the highest values seen to date between reovirus genome segments from distinct isolates [11,31,34,[37][38][39]. Also consistent with the M1 nucleotide sequence results, the µ2 proteins of the nine other reovirus isolates examined in this study are much more closely related to those of T1L and T3D than to that of T2J (Table 2, Fig. 3), affirming the divergent status of the T2J µ2 protein. The µ2 protein sequence of T3C12 is identical to that of the T3D clone in use in the Coombs and Nibert laboratories. In addition, the µ2 protein sequence of T1C29 is identical to that of T3C44. These are the first times that reovirus proteins from distinct isolates have been found to share identical amino acid sequences [11,32,34,[37][38][39], reflecting the high degree of µ2 conservation.
The encoded µ2 proteins of the twelve reovirus isolates are all calculated to have molecular masses between 83.2 and 84.0 kDa, and isoelectric points between 6.89 and 7.44 pH units (Table 1). This range of isoelectric points is the largest yet seen among reovirus proteins other than σ1s  T1L c  T2J  T3D d  T3D e  T1C11 T1C29 T1N84 T2N84 T2S59 T3C12 T3C18 T3C44 T3N83   T1L [31]; GenBank accession no. X59945). c T1L M1 and µ2 sequences for the Nibert laboratory clone [23]; GenBank accession no. AF461682) and the Coombs laboratory clone (this study). d T3D M1 and µ2 sequences for the Joklik laboratory clone [30]; GenBank accession no. M27261), and the Cashdollar laboratory clone [23]; GenBank accession no. AF461684). e T3D M1 and µ2 sequences for the Nibert laboratory clone [23]; GenBank accession no. AF461683) and the Coombs laboratory clone (this study). f Values for M1-gene sequence comparisons are shown below the diagonal, in bold; values for µ2-protein sequence comparisons are shown above the diagonal.
[11], but is largely attributable to the divergent value of T2J µ2 (others range only from 6.89 to 7.10). The substantially higher isoelectric point of T2J µ2 is explained by it containing a larger number of basic residues (excess arginine) than do the other isolates ( Table 1).
Comparisons of the twelve µ2 sequences showed eight highly conserved regions, each containing ≥ 15 consecutive residues that are identical in all of the isolates (Fig. 2). The highly conserved regions are clustered in two larger areas of µ2, spanning approximately amino acids 1-250 and amino acids 400-610. Conserved region 5 in the 400-610 area encompasses the more amino-terminal of the two NTP-binding motifs in µ2 (Fig. 2) [18,20]. The other NTP-binding motif is also wholly conserved, but within a smaller consecutive run of conserved residues. The region between the two motifs is notably variable (Fig. 2). Conserved region 5 also contains the less conservative of the two amino acid substitutions in T1L-derived temperature-sensitive (ts) mutant tsH11.2 (Pro414→His) [40]. The pattern of conserved and variable areas of µ2 was also seen by plotting scores for sequence identity in running windows over the protein length (e.g., [32]). In addition to the conserved regions described above, areas of greater than average variation are evident in this plot, spanning approximately amino acids 250-400 and 610-736 (the carboxyl terminus) (Fig. 4). The 250-400 area is notable for regularly oscillating between conserved and variable regions (Fig. 4). The two large areas of greaterthan-average sequence conservation, spanning approximately amino acids 1-250 and 400-610 (Fig. 4), are likely to be involved in the protein's primary function(s). The more variable, 250-400 area between the two conserved ones might represent a hinge or linker of mostly structural importance.
As indicated earlier, µ2 is one of the most poorly understood reovirus proteins, from both a functional and a structural point of view. For example, atomic structures are available for seven of the eight reovirus structural proteins, with µ2 being the missing one. Thus, in an effort to refine the model for µ2 structure/function relationships based on regional differences, we obtained predictions for secondary structures, hydropathy, and surface probability. PHD PredictProtein algorithms suggest that µ2 can be divided into four approximate regions characterized by different patterns of predicted secondary structures (Fig.  5C). An amino-terminal region spans to residue 157, a "variable" region spans residues 157 to 450, a "helix-rich" region spans residues 450 to 606, and a carboxyl-terminal Most parsimonious phylogenetic tree based on the M1 nucle-otide sequences of the different reoviruses  Nibert)  T3C12  T1N84  T2N84  T1C29  T3C44  T2S59  T2J 100 nucleotide differences Window-averaged scores for sequence identity among the T1L, T2J, and T3D µ2 proteins Figure 4 Window-averaged scores for sequence identity among the T1L, T2J, and T3D µ2 proteins. Identity scores averaged over running windows of 21 amino acids and centered at consecutive amino acid positions are shown. The global identity score for the three sequences is indicated by the dashed line. Two extended areas of greater-than-average sequence variation are marked with lines below the plot. Two extended areas of greater-than-average sequence conservation are marked with lines above the plot. Eight regions of ≥ 15 consecutive residues of identity among all twelve µ2 sequences from Fig. 2, as discussed in the text, are numbered above the plot. The Ser/Pro208 determinant of microtubule binding is marked with a filled diamond. The two putative NTP-binding motifs are marked with filled circles. region spans the sequences after residue 606. The aminoterminal region contains six predicted α-helices and three predicted β-strands, and is highly conserved across all twelve µ2 sequences. The "variable" region is the most structurally complex and contains numerous interspersed α-helices and β-strands. The "helix-rich" region contains seven α-helices and is highly conserved across all twelve µ2 sequences. The carboxyl-terminal region varies across all three serotypes. Overall, the µ2 protein is predicted to be 48% α-helical and 14% β-sheet in composition, making it an "α-β " protein according to the CATH designation [41]. Interestingly, most tyrosine protein kinases with SH 2 domains are also "α-β " proteins by this designation. The T1L and T3D µ2 hydropathy profiles were identical to each other. Both show numerous regions of similarity to the hydropathy profile of the T2J µ2. However, there also are several distinct differences between the T1L and T2J profiles (Fig. 5). Alterations in amino acid charge at residues 32, 430 to 432, and 673 in the T2J sequence account for the major differences in hydrophobicity between T2J and the other serotypes. In addition, the carboxyl-terminal 66 residues show multiple differences in hydropathy. The surface probability profiles of each of the three serotype's µ2 proteins are identical (Fig. 5) and show numerous regions that are highly predicted to be exposed at the surface of the protein as well as regions predicted to be buried.
The MOTIF and FingerPRINTScan programs were used to compare the highly conserved regions of µ2 with other sequences in protein data banks (ProSite, Blocks, and Pro-Domain). The results revealed that several of the conserved regions in µ2 share limited similarities with members of the DNA polymerase A family and with the SH 2 domain of tyrosine kinases. The sequence YEAgDV in µ2, located in conserved region 2 (Fig. 2), is similar to the "YAD" motif of DNA polymerase A from a number of different bacteria (e.g., YEADDV in Deinococcus radiodurans).
The YAD motif is located in the exonuclease region of DNA polymerase A, a region which also functions as an NTPase and enhances the rate of DNA polymerization [42]. The SH 2 domain of tyrosine kinases was the highest score hit for the conserved regions of µ2 with Finger-PRINTScan. Four of the five motifs in the 100 amino acid SH 2 domain matched the µ2 sequence. The SH 2 domain mediates protein-protein interactions through its capacity to bind phosphotyrosine [43]. The protein motifs found by focusing on the conserved regions of µ2 provide supportive evidence that this protein is involved in nucleotide binding and metabolism. However, the described similarities did not match with greater than 90% certainty and no other significant homologies were detected. The inability to identify higher-scoring GenBank similarities, first noted when sequences of the T3D and T1L M1 genes were reported [30,31] attests to the uniqueness of this minor core protein.

Biochemical confirmations
In an effort to provide biochemical confirmation of the predicted variation in the different isolates' µ2 proteins, we analyzed the T1L, T2J, and T3D proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Despite the slightly larger molecular mass calculated from its sequence (Table 1), T2J µ2 displayed a slightly smaller relative molecular weight on gels than T1L and T3D µ2 (Fig. 6A). This aberrant mobility may reflect the higher isoelectric point of T2J µ2 (Table 1). Polyclonal anti-µ2 antibodies that had been raised against purified T1L µ2 [44] reacted strongly with both T1L and T3D µ2, but only weakly with T2J µ2 (Fig. 6B), despite equal band loading as demonstrated by Ponceau S staining. These antibody cross-reactivities correlated well with the predicted protein homologies ( Table  2).

Factory morphologies among reovirus field isolates
We took advantage of the new M1/µ2 sequences to extend analysis of the role of µ2 in determining differences in viral factory morphology among reovirus isolates [23]. Sequence variation at µ2 residue Pro/Ser208 was previously indicated to determine the different morphologies of T1L and T3D factories: Pro208 is associated with microtubule-anchored filamentous factories, as in T1L and the Cashdollar laboratory clone of T3D, whereas Ser208 is associated with globular factories, as in the Nibert laboratory clone of T3D [23]. For the previous study we had already examined the factories of T2J and some of the nine other isolates used for M1 sequencing above. We nonetheless newly examined the factories of all ten isolates in the present study, using the same stocks used for sequencing. T3C12 was the only one of these isolates that formed globular factories; the remainder, including T2J, formed filamentous factories (Fig. 7, Table 4). This finding is consistent with the fact that T3C12 is the only one of these isolates that has a serine at µ2 residue 208, like T3D from the Nibert laboratory; the remainder, like T1L and T3D from the Cashdollar laboratory, have a proline there (Fig. 2, Table 4) [23]. Thus, although the results identify no additional µ2 residues that may influence factory morphology, they are consistent with the identification of Pro/Ser208 as a prevalent determinant of differences in this phenotype among reovirus isolates.

Factory morphologies and M1/µ2 sequences of other T3D and T3D-derived clones
T3D clones from the Nibert and Cashdollar laboratories have been shown to exhibit different factory morphologies based on differences in the microtubule-binding capacities of their µ2 proteins and the presence of either Secondary structure predictions of µ2 protein  serine or proline at µ2 residue 208 [23]. We took the opportunity in this study to examine additional T3D clones. The clones from some laboratories formed globular factories in infected cells whereas those from other laboratories or the American Type Culture Collection formed filamentous factories (Fig. 8, Table 5). T3D-derived ts mutants tsC447, tsE320, and tsG453 [45] formed filamentous factories (Fig. 8, Table 5). Other ts mutants were not examined; however, [46] have shown evidence that tsF556 [45] forms filamentous factories as well.
We additionally determined the M1 sequences of the wild-type and ts T3D clones newly tested for factory morphology. All clones with globular factories have a serine at µ2 position 208 whereas all those with filamentous factories have a proline there (Table 5). These findings provide further evidence for the influence of residue 208 on this phenotypic difference.
All wild-type T3D clones with globular factories were recently derived from a Fields laboratory parent whereas all wild-type or ts T3D clones with filamentous factories were derived from parents in other laboratories. (Although extensively characterized by both Fields (e.g., [47,48]) and Joklik (e.g., [49,50]), the original T3Dderived ts mutants in groups A through G were generated in the Joklik laboratory [45]). This correlation suggests that formation of filamentous factories is the ancestral phenotype of reovirus T3D and that the Ser208 mutation in T3D µ2 was established later, in the Fields laboratory.
As we noted in a previous study [23], several other laboratories reported evidence for filamentous T3D factories in the 1960's (e.g., [51,52]), following its isolation in 1955 [53]. Since microtubules were noted to be commonly associated with T3D factories in Fields laboratory publications from as late as 1973 [54], but not in one from 1979 [55], the µ2 Ser208 mutation was probably established in, or introduced into, that laboratory during the middle 1970's. Investigators should be alert to these different lineages of T3D and their derivatives for genetic studies. For example, reassortant 3HA1 [56] contains a T3D M1 genome segment derived from clone tsC447, and its factory phenotype is filamentous (data not shown).

Additional genome-wide comparisons of T1L, T2J, and T3D
Several types of genome-wide comparisons of T1L, T2J, and T3D have been reported previously [11]. For this study we examined the positions and types of nucleotide mismatches in these prototype isolates in order to gain a more comprehensive view of the evolutionary divergence of their protein-coding sequences. Most mismatches between T2J and either T1L or T3D segments, ~68%, are in the third codon base position, while ~21% are in the first position and ~11% are in the second position. Each of these mismatch percentages was converted to an evolutionary divergence value by multiplying mismatch percentage by 1.33 [31] (Table 3). These values have been used to argue that the homologous T1L and T3D genome segments diverged from common ancestors at different times in the past, with the M1 and L3 segments having diverged most recently and the M2, S1, S2, and S3 segments having diverged longer ago [31]. The consistently high values for divergence at third codon base positions among pairings with T2J genome segments (Table 3) indicate that all ten T2J segments diverged from common ancestors substantially before their respective T1L and T3D homologs. Relative numbers of synonymous and nonsynonymous nucleotide changes identified in pairwise comparisons of the coding sequences of these isolates (Table 3) support the same conclusion.
The types of amino acid substitutions within each of the prototype isolates' proteins were also examined. Pairwise analyses showed that most substitutions in most proteins were conservative (Table 3). Nonconservative substitutions were relatively rare in most proteins' pair-wise com- Figure 6 SDS-PAGE and immunoblot analyses of virion and core particles. Proteins from gradient-purified T1L (1), T2J (2), and T3D (3) particles were resolved in 5-15% SDS-polyacrylamide gels as detailed in Materials and methods. Gels were then fixed and stained with Coomassie Brilliant Blue R-250 and silver (A). Alternatively, proteins from the gels were transferred to nitrocellulose, probed with anti-µ2 antiserum (polyclonal antibodies raised against T1L µ2, kindly provided by E. G. Brown), and detected by chemiluminescence (B). Virion proteins are indicated to the left of panel A, except for µ2, which is indicated between the panels. parisons. For example, comparison of the T1L and T3D µ2 proteins showed none (0.0%) of the 10 amino acid substitutions were nonconservative, and most T1L:T3D comparisons gave low nonconservative substitution values ranging from 0.1-0.5% of total amino acid residues within the respective proteins. However, some genes, most notably M1, M3, and S3, demonstrated higher nonconservative variation, with values approaching 3.5% of  T1L  T1C11  T1C29   T2J  T2N84  T2S59   T3C12  T3C18  T3C44 total amino acid residues. Most of these higher nonconservative substitution values were observed when T2J proteins were compared to either T1L or T3D proteins. In addition, in many proteins, the majority of nonconservative substitutions were located within the amino-terminal portions (first ~20%) of the respective proteins (data not shown).

Viral factory morphology as demonstrated by the distribution of µNS in cells infected with various reovirus isolates
The frequencies with which different redundant codons are used to encode certain mammalian amino acids are non-random (reviewed in [57]). This phenomenon is mirrored by different abundances of the complementary tRNA molecules in mammalian cells. For example, CG pairs are underrepresented in mammalian genomes and common in their "rare" codons (see Table 6). A recent study revealed that many RNA viruses of humans display mild deviations from host codon-usage frequencies and that these deviations are more prominent among viruses with segmented genomes [57]. However, reoviruses were not included in that study. By examining reovirus isolates T1L, T2J, and T3D, for which whole-genome sequences are now available, we found that codons that qualify as rare in mammals are not rare in reovirus (Table 6). Moreover, the few codons that qualify as rare in reovirus (ACC, AGC, CCC, CGG, CUC, and GCC; data not shown) are common in mammals. The basis and significance of these deviations remain unknown, but could have impacts on the rates of translation of reovirus proteins. It is perhaps notable in this regard that the four most highly expressed reovirus proteins (µ1, σ3, µNS, and σNS) have the lowest average frequencies of codons that are rare in mammals (Table 6). Thus, incorporation of rare codons into reovirus coding sequences could be a mechanism of dampening the expression of certain viral proteins.

Sequencing the M1 genome segments
All oligonucleotide primers were obtained from Gibco/ BRL. Genomic dsRNA was extracted from gradient-purified virions with phenol/chloroform [59]. Strain identity was confirmed by resolving aliquots of each in 10% SDS-PAGE gels and comparing dsRNA band mobilities [60].
Oligonucleotide primers corresponding to either the 5' end of the plus strand or the 5' end of the minus strand were as previously described [40]. Additional oligonucleotides for sequencing were designed and obtained as needed. cDNA copies of the M1 genes of each virus were constructed by using the 5' oligonucleotide primers and reverse transcriptase (Gibco/BRL). The cDNAs were amplified by the polymerase chain reaction [61] and resolved in 0.7% agarose gels [59]. The bands corresponding to the 2.3-kb gene were then excised, purified, and eluted with Qiagen columns, using the manufacterer's instructions. Sequences of the respective cDNAs were determined in both directions by dideoxynucleotide cycle sequencing [62][63][64], using fluorescent dideoxynucleotides.
Sequences at the termini of each M1 segment were determined by one or both of two methods. For some isolates, sequences near the ends of the segment were determined by modified procedures for rapid amplification of cDNA ends (RACE) as previously described [32,65]. In addition, the sequences at the ends of all M1 segments were determined in both directions by a modification of the 3'-ligation method described by Lambden et al. [66]. Briefly, viral genes from gradient-purified virions were resolved in a 1% agarose gel, and the M segments were excised and eluted with Qiagen columns as described above. Oligonucleotide 3'L1 (5'-CCCCAACCCACTTTTTCCAT-TACGCCCCTTTCCCCC-3'; phosphorylated at the 5' end and blocked with a biotin group at the 3' end) was ligated to the 3' ends of the M segments according to the manufacterer's directions (Boehringer Mannheim) at 37°C overnight. The ligated genes were repurified by agarose gel and Qiagen columns to remove unincorporated 3'L1 oligonucleotide and precipitated overnight with ice-cold ethanol. The precipitated genes were dissolved in 4 µl of 90% dimethyl sulfoxide. cDNA copies of the ligated M1 genes were constructed by using oligonucleotide 3'L2 (5'-GGGGGAAAGGGGCGTAATGGAAAAAGTGGGTT-GGGG-3') and gene-specific internal oligonucleotide primers designed to generate a product of 0.5 to 1.2 kb in length. These constructs were amplified by PCR, purified in 1.5% agarose gels, excised, and eluted as described above. Sequences of these cDNAs were determined with gene-specific internal oligonucleotides and with oligonucleotide 3'L3 (5'-GGGGGAAAGGGGCGTAAT-3') by dideoxy-fluorescence methods.

Sequence analyses
DNA sequences were analyzed with DNASTAR, DNA Strider, BLITZ, BLAST, and CLUSTAL-W. Phylogenetic analyses were performed using the PHYLIP programs http://evolution.gs.washington.edu/phylip.html. DNA-PARS (parsimony) (Fig. 3) and DNAML (maximum likelihood) (data not shown) produced essentially identical trees. These programs were run using the Jumble option to test the trees using 50 different, randomly generated orders of adding the different sequences. In addition, DNAPENNY (parsimony by brand-and-bound algorithm) generated a tree with the same branch orders as DNAPARS and DNAML. RETREE and DRAWGRAM were used to visualize the tree and to prepare the image for publication. Protein sequence analyses were performed using the GCG programs in SeqWeb version 2 (Accelrys). Multiple sequence alignments were done with PRETTY. Determinations of molecular weights, isoelectric points, and residue counts were done with PEPTIDESORT. Determinations of percent identities in pairwise comparisons were done with GAP. Plots of sequence identity over running windows of different numbers of amino acids ( Fig. 4 and data not shown) were generated with PLOTSIMILARITY, and the image for publication was refined with Illustrator (Adobe Systems). In addition, protein sequences were analysed for conservative and nonconservative substitutions by pairwise CLUSTAL-W analyses, using BLOSUM matrix weighting [68].

Immunoblotting
Gradient-purified viral and core proteins were resolved by SDS-PAGE as described above, and sets of resolved proteins were transferred to nitrocellulose membranes with a Semi-Dry Transblot manifold (Bio-Rad Laboratories) according to the manufacturer's instructions. Transfer of all proteins was confirmed by Ponceau S staining. Nonspecific binding was blocked in TBS-T (10 mM Tris [pH 7.5], 100 mM NaCl, 0.1% Tween 20) supplemented with 5% milk proteins, and the membranes probed with polyvalent anti-µ2 antibody (a kind gift from Dr. E. G. Brown, University of Ottawa). Membranes were washed with TBS-Viral factory morphology as demonstrated by the distribu-tion of µNS in cells infected with T3D clones obtained from different laboratories or with T3D-derived ts clones Figure 8 Viral factory morphology as demonstrated by the distribution of µNS in cells infected with T3D clones obtained from different laboratories or with T3D-derived ts clones. Laboratory sources are indicated in parentheses. CV-1 cells were infected at 5 PFU/cell with the clone indicated above each panel, fixed at 18 h p.i., and immunostained with µNS-specific rabbit IgG conjugated to Alexa 488. Size bars, 10 µm.

Infections and IF microscopy
CV-1 cells were maintained in Dulbecco's modified Eagles medium (Invitrogen) containing 10% fetal bovine serum (HyClone Laboratories) and 10 µg/ml Gentamycin solu-tion (Invitrogen). Rabbit polyclonal IgG against µNS [71] was purified with protein A and conjugated to Alexa Fluor 488 or Alexa Fluor 594 using a kit obtained from Molecular Probes and titrated to optimize the signal-to-noise ratio. Cells were seeded the day before infection at a density of 1.5 × 10 4 /cm 2 in 6-well plates (9.6 cm 2 /well) containing round glass cover slips (18 mm [23]. c Origin traceable to W. K. Joklik laboratory; derived from T3D; sequences of tsC447 (GenBank accession no. AY428878), tsE320, and tsG453 are identical.  a As fraction of all codons for the particular amino acid. Bold, value higher than that in any of the indicated mammals; underlined, value more than double that in any of the indicated mammals. b Amino acid encoded by the codon c Expected frequency if codons for each amino acid are used randomly (assuming equal A, C, G, and U contents and no di-or trinucleotide bias).
adsorbed for 1 h at room temperature before fresh medium was added. Cells were further incubated for 18-24 h at 37°C before fixation for 10 min at room temperature in 2% paraformaldehyde in PBS or 3 min at -20°C in ice-cold methanol. Fixed cells were washed with PBS three times and permeabilized and blocked in PBS containing 1% bovine serum albumin and 0.1% Triton X-100. Antibody was diluted in the blocking solution and incubated with cells for 25-40 min at room temperature. After three washes in PBS, cover slips were mounted on glass slides with Prolong (Molecular Probes). Samples were examined using a Nikon TE-300 inverted microscope equipped with phase and fluorescence optics, and images were collected digitally as described elsewhere [23]. All images were processed and prepared for presentation using Photoshop (Adobe Systems).