Nature of the 3′-terminal sequences of the plus and minus strands of the S1 gene of reovirus serotypes 1, 2 and 3

VIROLOGY

105,

41-51 (1980)

Nature of the 3’-Terminal Sequences of the Sl Gene of Reovirus JOSEPH

of the Plus and Minus Strands Serotypes 1, 2 and 3

K. -K. LI, JACK D. KEENE, PATRICIA AND WOLFGANG K. JOKLIK’

Department

P. SCHEIBLE,

of Microbiology and Immunology, Duke University Durham, North Carolina 27710

Medical

Center,

Accepted May 28, 1980 The 3’-terminal regions of the plus and minus strands of the Sl genes of serotypes 1, 2, and 3 of reovirus were sequenced. The Sl gene codes for polypeptide ~1, a protein with a MW of about 45,006 that elicits the principal neutralizing antibody and is the hemagglutinin, the cell attachment protein, and the primary determinant of reovirus virulence, tissue tropism, and pathogenicity. It is the most type specific of all reovirus proteins; examination of the gene that encodes it should therefore best reveal which of its sequences are conserved (constant) and which may be varied. The 3’-termini of the plus strands of the Sl genes of reovirus serotypes 1,2, and 3 share extensive homology. They share 16 of the first 20 or 21 nucleotides including the terminal sequence UCAUC-3’, as well as an additional 5-residue region near residue 40 that contains a termination codon. The extent of homology at the other end of the Sl gene (that which contains the 5’-ends of the plus strands) is less extensive; all plus strands possess the same sequence 5’-GCUAUU and serotypes 1 and 2 sl plus strands share an additional CGC. There follows a sequence with no homology until the first initiation codon is encountered at residue 14, 14, or 13, respectively. The first 20 to 30 amino acids encoded by the three Sl genes show no detectable homology, although they exhibit a similar degree of hydrophobicity. The three sl plus strands exhibit interesting secondary structure features. Few stable hairpins are present within 100 residues of either terminus; one such hairpin in the sl plus strand of serotype 2 that may possess some stability (- 13.0 kcal) encompasses three in-phase termination codons, but this structure is not present in the sl plus strands of serotypes 1 and 3. There are regions of complementarity near (but not directly at) the 5’. and 3’termini of all three species of sl plus strands that may conceivably permit circularization (-16.0 kcal). Further, the sl plus strands of serotypes 1 and 2, but not 3, possess a sequence near their 5’-terminus that is complementary to a sequence immediately adjacent to the3’-terminus of 18 S ribosomal RNA. These studies indicate that although the Sl gene codes for a very type-specific protein, the three forms of it that are present in reovirus serotypes 1, 2, and 3 nevertheless possess considerable regions of homology at their termini. These regions probably function as recognition signals for ribosomes, RNA polymeraae (transcriptase and replicase), and encapsidation. INTRODUCTION

strains of serotypes 1 and 3 are closely related (more than 80%), whereas strains of serotype 2 are related to those of serotypes 1 and 3 by no more than 10% (Martinson and Lewandowski, 19’75; Gaillard and Joklik, 1980). Antigenic analysis of the proteins coded by each of these three serotypes suggests that genetic relatedness is distributed in a very interesting manner among their genes. As expected, it is found that the antigenic determinants on most of the pairs

The three serotypes of reovirus form an interesting system for studying evolutionary relationships. As judged by the ability of their ds2 RNAs to hybridize with each other, 1 To whom requests for reprints should be addressed. 2 Abbreviations used: ds, double stranded; ss, single stranded; SDS, sodium dodecyl sulfate; STE, 100 m&f NaCl, lOmMTris-HCl(pH7.4), 5mMEDTA; EDTA, ethylenediaminetetraacetate; BPB, bromphenol blue; EtBr, ethidium bromide. 41

004%6822/80/110041-11$02.00/0 Copyright 6 IYSOby Academic Press. Inc. All rights of reproduction in any form reserved.

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LI ET AL.

of proteins encoded by them are very similar, and that only those on two proteins can be differentiated readily (that is, are type specific) (Gaillard and Joklik, 1980); but the antigenic determinants on most of the proteins encoded by serotype 2 are also very similar. This is unexpected, since the primary amino acid sequences of proteins encoded by serotype 2 on the one hand and serotypes 1 and 3 on the other must be very different. Clearly, the antigenic determinants on most of the reovirus proteins have been highly conserved. In order to investigate further how genetic relatedness is distributed among and within the genomes of these three reovirus serotypes, we have begun to sequence selected groups of their genes. We report here the 3’-terminal nucleotide sequences of the plus and minus strands of the three Sl genes. The Sl gene codes for the most type specific of all reovirus-coded proteins. This protein, which is designated crl and has a molecular weight of about 45,000, is a minor component of the reovirus outer capsid shell in which it is present to the extent of no more than 24 copies (Smith et al. 1969). Yet it fulfills a variety of crucial biologic functions: it is one of the two proteins that elicits neutralizing antibody; it is the primary determinant of type specificity (Weiner and Fields, 1977; Gaillard and Joklik, 1980; Lee et al., 1980a); it is the reovirus cell attachment protein (Lee et al., 1980b); it is the hemagglutinin (Weiner et al., 1978); and it is the primary determinant of reovirus virulence, tissue tropism, and certain aspects of cytopathogenicity (Weiner et al., 1977; Finberg et al., 1979; Babiss et al., 1979). Since this protein is so type specific, one would expect few base sequence similarities among the coding sequences of the serotype 1, 2, and 3 Sl genes; one would however, expect similarities in their terminal sequences, which should be able to recognize RNA polymerase, ribosomes, encapsidation signals, and sequences on other reovirus genes. Indeed, it is already known that the 5’-termini of the majority of reovirus mRNA species all commence with G”pppG,CUA(Kozak and Shatkin, 1977a,b; Kozak, 1977; Hastings and Millward, 1978).

METHODS

AND MATERIALS

Cells and Viruses The Dearing strain of reovirus serotype 3, the D5/Jones strain of serotype 2, and the Lang strain of serotype 1 were grown in suspension cultures of mouse L fibroblasts which were themselves grown in Eagle’s MEM (Joklik’s modification, Grand Island Biological Co.) containing 5% fetal calf serum (Smith et al., 1969). Extraction of ds RNA from Reovirus Double-stranded RNA was extracted from reovirus particles as described by McCrae and Joklik (1978), except that incubation in 1% SDS was carried out at 65” for 60 min before RNA was extracted at room temperature with phenol saturated with STE. The aqueous phase was extracted three times with chloroformisoamyl alcohol (24:l) and the RNA in it was precipitated with 2.5 vol EtOH at -20”. The RNA was passed through a Sephadex G-100 column in order to remove oligonucleotides (Bellamy and Joklik, 1967; Shatkin and Sipe, 1968; Nichols et al., 1972). Isolation of Sl Genes Individual Sl segments of ds RNA (Sl genes) were isolated by electrophoresing total genome RNA in preparative polyacrylamide slab gels (30 cm x 16 cm x 35 mm) as described by Schuerch et al. (1975) and McCrae and Joklik (1978). Briefly, about 0.8 mg of ds reovirus RNA in 200 ~1 of sample buffer (40% glycerol, 0.1% SDS, 0.05 M EDTA, and 0.5% BPB) was heated at 70” for 2 min and then applied to gels. After electrophoresis the RNA segments were visualized under uv light by staining with 0.01% EtBr or on Eastman Chromagram sheets impregnated with a fluorescent indicator. Sl segments were excised from the gels and eluted either electrophoretically (Sugden et al., 1975) or by shaking crushed gel slices for 16 hr at 37” in a small volume of elution buffer containing 0.5 M ammonium acetate, 0.3 M Mgacetate, 0.1% SDS, and 0.1 mM EDTA. Gel particles were removed by low-speed centrifugation and filtration through GF/C filters. The eluates were extracted twice

3’-TERMINAL

SEQUENCES OF THE REOVIRUS Sl GENE

43

with phenol and three times with chloroformisoamyl alcohol. The RNA was precipitated with 2.5 vol EtOH and stored at -90”. Labeling of 3’-Temini

of RNA

Sl ds RNA segments were labeled at their 3’-termini using 3’,5’-bis-cytidine[32P]diphosphate and T4 RNA ligase as described by England et al. (19’78)and Keene et al., (1978). Separation of the Plus and Minus Strands of Sl RNA Genes 3’-Terminally labeled Sl genes were hybridized as described by Ito and Joklik (1972) to a lOO-fold excess of unfractionated plus-stranded RNA synthesized in vitro by reovirus cores (Skehel and Joklik, 1969). Single-stranded RNA was separated from ds RNA by chromatography on CF-11 cellulose saturated with yeast tRNA to reduce nonspecific adsorption (Franklin, 1966). The ss RNA fraction contained the plus strands of the Sl genes, the ds RNA fraction contained their minus strands. The purity of both ss and ds RNA was confirmed by twodimensional oligonucleotide fingerprint analysis (Sanger et al., 1965; Keene et al., 1978) and by high-voltage electrophoresis of their RNase Tl limit digestion products at pH 3.5 on Whatman 3MM paper (Brownlee, 1972).

-%

FIG. 1. Autoradiograph of unfractionated reovirus serotype 3 ds RNA labeled with [“‘P]pCp, and of the isolated Sl gene. The conditions of eleetrophoresis were as described by McCrae and Joklik (1978).

et al. (1980). RNAs were subjected to partial chemical modification by treating with Me2S04 to modify G, diethylpyrocarbonate to modify A and G, hydrazine in 2.8 M NaCl to modify C and U, and hydrazine in water to modify U residues. Modified RNAs were cleaved with aniline and fragments were separated in 12 or 16% polyacrylamide-urea sequencing gels. Ladders were prepared by partial hydrolysis with 99% formamide-l mM MgCl, at 100” for 40 min. Oligonucleotide bands were visualized by autoradiography with DuPont “Lightning Plus” intensifying screens at -9O”, using either Kodak XR-5 or GAF film.

Sequence Analysis Two-dimensional oligonucleotide j?ngerprinting. 3’-Terminally labeled RNA was partially digested in 50 mM NaHCO, (pH 9)-l mM EDTA at 90” for 15 min and the products were subjected to two-dimensional oligonucleotide fingerprinting analysis RESULTS (Brownlee, 1972) using ionophoresis at pH 3.5 on Cellogel strips (Kalex Corp., Man- S’-Terminal Labeling of the Plus and hasset, N. Y.) in the first dimension and Minus Strands of Reovims Sl Genes thin-layer homochromatography on DEAEcellulose plates (Analtech Corp.) in the The isolated Sl genes of reovirus serosecond. The composition of selected oligo- types 1, 2, and 3 were labeled at their nucleotides was confirmed by elution and 3’-termini with [32P]pCpand T4 RNA ligase analysis by high-voltage electrophoresis on (England et al., 1978; Keene et al., 1978). DEAE paper at pH 3.5 and 1.7. Specific activities ranged from 1.2 to 2 x lo6 Chemical seqzlencing. Sequencing of 3’- cpm/pg RNA. Figure 1 shows that the Sl terminally labeled RNAs was performed genes were pure. Both 3’-termini were as described by Peattie (1979) and Keene labeled because (a) both the plus and minus

44

LI ET AL.

FIG. 2. Two-dimensional oligonucleotide fingerprint analysis of partial alkali digestion products of the 3’-labeled Sl gene of reovirus serotype 3. Samples A and B were analyzed with 15- and 90-min homomix digests in the second dimension, respectively.

scribed under Methods and Materials. Figure 3 shows the chromatographic separation on CF-11 cellulose of the ss RNA (which comprises the large excess of unlabeled unfractionated plus strands together with the labeled sl plus strands) and ds RNA (which consists of the labeled sl minus strands hybridized to unlabeled plus strands). As expected, the amount of radioactivity in the two fractions was equal. A second round of hybridization and/or rechromatography was performed to ensure the purity of each fraction. Two methods were used to monitor and confirm their purity. First, each of the isolated fractions was digested with Tl nuclease and the products were electrophoresed on Whatman 3MM paper at pH 3.5. The first G residue at the 3’Separation of the 3’-Terminally Labeled termini of the sl plus strands of reovirus Plus and Minus Strands of Sl RNA serotypes 1,2, and 3 is at residue 10,8, and The isolated labeled Sl genes were next 10, respectively, while that at the 3’separated into their component plus and terminus of the corresponding minus strands minus strands, using the techniques de- is at residue 2 (see below). Electrophoresis

strand isolated from these genes were labeled (see below), and (b) partial alkali digests of the genes resolved into two distinct tracks when analyzed by two-dimensional oligonucleotide fingerprinting (Brownlee, 1972; Sanger et al., 1965; Keene et al., 1978) (Fig. 2). The nucleotide sequences of these two tracks were identical with those yielded by the isolated plus and minus strands (see below). Analysis of complete RNase T2 digests by high-voltage electrophoresis on Whatman 3MM paper showed that the terminal base of both plus and minus strands of all these Sl genes (and indeed of all reovirus genes) is C (see also Banerjee et al., 1971).

3’-TERMINAL

SEQUENCES OF THE REOVIRUS Sl GENE

45

responding plus strands, and also that there was no detectable contamination of one strand with the other. Second, partial alkali digests of plus and minus strands of each of the three Sl genes were subjected to two-dimensional oligonucleotide fingerprint analysis as described above (“wandering spot” analysis (Brownlee, 1972; Sanger et al., 1965; Keene et al., 1978)). The results are shown in Fig. 5. In each case only one track was obtained, not two, as yielded by ds RNA (see Fig. 2). Sequence Analysis

2 drRNe.

0l#i-&lL

2

4

6 8 IO I2 Fractmn Number

14

FIG. 3. Separation by chromatography on CF-11 cellulose of the plus and minus strands of the Sl gene of reovirus serotype 3. For experimental details, see Methods and Materials and text. (A) Separation of 3’-[32P]pCp-labeled plus and minus strands. (B and C) Rechromatography of each isolated fraction.

(Fig. 4) revealed indeed that the labeled G-containing oligonucleotide derived from digests of sl minus strands traveled more slowly than that derived from the cor-

Two-dimensional oligonucleotide jbagerprint analysis. Oligonucleotide fingerprints of partial alkali digests of the plus and minus strands of the Sl genes of reovi& serotypes 1, 2, and 3 are shown in Fig. 5. The composition of most of the spots was confirmed by elution and analysis by highvoltage electrophoresis on DEAE paper at pH 3.5 and 1.7. Chemical sequence analysis. Chemical sequencing, employing both primary and secondary loadings, was carried out on each of the six isolated Sl strands. Examples of sequencing gels are shown in Fig. 6. The results of chemical sequencing and “wandering spot” analyses agreed, except for position 11 of the minus sl strand of serotype 3, where chemical sequencing reproducibly showed a C residue, whereas the “wandering spot” analysis indicated U. The reason for this inconsistency, which was not found when the corresponding sl strands of reovirus serotypes 1 and 2 were examined, is

FIG. 4. High-voltage electrophoresis of the products of Tl nuclease digestion of the isolated plus and minus strands of the 3’-[3”P]pCp-labeled Sl gene of reovirus serotype 3. Samples of each strand (1-2 x IO3 cpm) mixed with 5 pg tRNA as carrier were precipitated with 0.3 M sodium acetate and ethanol. The pelleted precipitates were rinsed with 80% EtOH, dried, and resuspended in 20 ~120 nnl4 Tris-HCl (pH 7.4)-O. 1 mM EDTA containing 10 units of ribonuclease T,. After incubation at 37” for 30 min, the material was spotted onto a Whatman 3MM paper strip and subjected to electrophoresis in 5% acetic acid adjusted to pH 3.5 with pyridine (Brownlee, 1972) at 40 mA for 2 hr. The digestion products were located by autoradiography (12-hr exposure to Kodak X-Omat film, using a DuPont Lightning-Plus intensifying screen at -9Oq. 0 signifies the origin.

46

LI ET AL.

FIG. 5. Two-dimensional oligonucleotide fingerprint analysis (see text) of the partial alkali digestion products of the isolated plus and minus strands of the Sl genes of reovirus serotypes 1, 2, and 3 using fractions isolated by chromatography on CF-11 cellulose (see Fig. 3). Weak (90 min) homomix digests were used in the second dimension. Panels A and B: the sl plus and minus strands of serotype 1; panels C and D: the sl plus and minus strands of serotype 2; panels E and F: the sl plus and minus strands of serotype 3.

not known; it may be due to the presence of oligoadenylates which are present in reoa modified base. It should be noted that oc- virus (Bellamy and Joklik, 1967; Nichols et casionally light bands were present between al., 1972). However, they did not complicate positions 1 and 8 in the A channel of plus the interpretation of the gels, as they could strand digests. Presumably these were de- be eliminated on the basis of the “wandering rived from small amounts of contaminating spot” analysis.

3’-TERMINAL

SEQUENCES

OF THE REOVIRUS

Sl GENE

47

FIG. 5.-Continued.

The sequences determined for all six sl strands are shown in Figs. ‘7 and 8.

some 12 residues upstream. Altogether about one-half of the 40 3’-terminal residues are shared by all three sl plus strands. SevDISCUSSION eral termination codons are present in all The Sl gene of reovirus codes for the these sl plus strands; there are three such most type specific of all reovirus-coded codons in phase in serotype 1 and 2 sl plus proteins; it codes for that protein which strands, and three such codons separated controls, more than any other, how the virus by large distances and not in phase in the interacts with the various types of cells serotype 3 sl plus strands. It is not known that it encounters in the host. It therefore which of these termination codonsare actually chain terminating; it may be significant provides the best opportunity for identifying those features of the gene and of the protein that one is present in that block of homology that it encodes that must be present in order that is furthest upstream (i.e., within for them to be “reoviral” (that is, the eons&& UGAGG). No messenger RNA-processing features), and those that may be altered signals of the type AAUAAA (Proudfoot so as to provide variations in specificity and Brownlee, 1976) are present in any of (that is, the variable features). The sequences the sl plus strands. In view of the fact that that we have determined, which amount to reovirus messenger RNAs are not processed roughly 100 base pairs at each end of the Sl (or polyadenylated) (Schonberg et al., 1971; genes encoded by each of the three sero- Li et al., 1980), such absence is not surtypes, permit the following conclusions to prising. be drawn: (2) The 3’-terminal regions of the sl minus (1) The 3’-terminal regions of the sl plus strands, and therefore the 5’-terminal strands show extensive homology. Three regions of the sl plus strands, exhibit blocks of homology are discernible: a 3’- less homology. All three serotypes possess terminal block of five residues-all sl plus the same six terminal residues (5’GCUAUU) strands terminate in -UCAUC-3’; an and serotypes 1 and 2 share an additional extensive G-rich block of 11 nudeotides three residues, which is interesting because separated by 1 or 2 A or U residues; and it is serotypes 1 and 3, not 1 and 2, that another block of 5 residues-UGAGGare closely related (see above). The first

48

LI ET AL.

A GALCU

B

GALCU

C GALCU

A

FIG. 6. Autoradiograms of chemical sequencing gels of (from lett to right) the sl serotype 2 minus strand (two loadings) (A and B) and the sl serotype 1 plus strand (one loading) (C). Duration of autoradiography, 2 days. Similar gels were obtained for the sl plus and minus strands of serotype 3, the sl plus strand of serotype 2, and the sl minus strand of serotype 1.

initiation codon starts at residue 14, 14, and 13 for serotypes 1, 2 and 3 respectively. The sequence between the terminal homology region and the initiation codon is totally different for all three serotypes. Serotype

1 sl plus RNA possesses a second initiation codon just downstream from the first; but it cannot be used, as a termination codon (UAG) is encountered at residue 48 in this reading frame. The amino acid SElblNf'E

FIG. 7. The 3’4erminal sequences of the sl plus strands of reovirus serotypes 1, 2, and 3.

3’-TERMINAL

SEQUENCES OF THE REOVIRUS Sl GENE

49

2

FIG. 8. The 3’-terminal sequences of the sl minus strands of reovirus serotypes 1, 2, and 3. Also shown are the deduced 5’-terminal sequences of the corresponding plus strands (mRNA), and the amino acid sequences which they would encode.

sequences coded by the sl plus strands of the three serotypes are quite different; however, of the first 20 amino acids 9, 10, and 9 are hydrophobic for serotypes 1, 2, and 3, respectively, and 6, ‘7, and 8, respectively, are charged; but the net charge on this 20-amino acid sequence is minus 2, plus 1, and 0 for serotypes 1, 2, and 3, respectively. No significantly different codon use can be discerned for any serotype within these brief sequences. In summary, the Sl genes of reovirus serotypes 1, 2, and 3 contain regions of homology at both of their ends. The homology at the end that contains the 5’-terminus of the plus strand is most probably part of the ribosome binding site, as well as an RNA polymerase binding site (for transcribing ds RNA into ss (messenger) RNA). It is interesting in this regard that we have obtained evidence that the sequence 5’-GCUA is present in all 10 reovirus genes (Li et al., unpublished results). It should also be noted that the 5’-G-terminated oligonucleotides which make up about onehalf of the oligonucleotides present in reovirus particles (the other half being oligoadenylates) possessthe sequencesGC, GCU, GCUA, GCUA(U),-,, and GCUA(A),-, (Nichols et al., 1972). The sequence relationships between these oligonucleotides and the 5’-terminal sequences of all reovirus plus strands supports the hypothesis originally proposed that the 5%-terminated oligonucleotides are the products of abortive transcription (Nichols et al., 1972).

The homologies are more extensive at the 3’-termini of the plus strands. The terminal homology region, which encompasses 16 out of 21 or 22 residues, may well represent, at least in part, the RNA polymerase binding site for transcribing mRNA into minusstranded RNA to yield progeny ds RNA molecules, while the most distal UGAcontaining region of homology may represent a strong termination signal. Another possible function of regions of homology at either end of reovirus genes may be as elements essential for encapsidation. (3) The three sl plus strands exhibit several interesting secondary structure features (Fig. 9), which may be summarized as follows. (a) Surprisingly few hairpins can be formed at each end of the molecule; most of those that can be formed encompass no more than eight residues, with little stability. One loop that may be quite stable (- 13.0 kcal (Tinoco et al., 1973)) is one near the 3’-terminus of serotype 2 sl plus strands which encompasses all three termination codons. This feature is not present in serotypes 1 and 3 sl plus strands. (b) All sl plus strands possess regions near their 5’-termini that are homologous to regions near their 3’-termini, which may enable them to form circles. For serotype 2 sl plus strands (Fig. 9) the region of homology extends over seven base pairs, with a free energy of - 15.0 kcal. The region of homology starts five or six residues, respectively, from the 5’- and 3’-termini and

50

LI ET AL. OH

3'

tempting to speculate that a similar technique applied to serotype 1 and 2 sl messenger RNA species would yield ribosomeprotected fragments. However, it is clear that since ribosomes protect sequences that are at least 30 nucleoticles long (Kozak, 1977), the recognition between ribosomes and messenger RNA must encompass interactions that are much more extensive than the regions of 18 S ribosomal RNAmessenger RNA homology. Further studies currently underway in this laboratory are aimed at exploring the base sequence relationships of further reovirus S genes, as well as of selected M and L genes, both within the same serotype, and across serotypes.

- 15.0 KCAL

A INlTlATlON COLON

0" u c G G A u C

A

C G G C u C A C II

C” A G C : A u A A G A AGGGAGAUCU......

ACKNOWLEDGMENTS

u ;

3 TENHINATION COWNSIN PHASE

”

i c

FIG. 9. Putative secondary structure of serotype 2 sl plus RNA (mRNA). The 18 S ribosomal RNA sequence that is shown is the “consensus” sequence (Hagenbtiehle et al., 1978).

therefore does not include the regions of terminal homology. (c) The sl plus strand of serotype 2 (Fig. 9) also possesses a region near its 5’4erminus that is complementary to a region immediately adjacent to the 3’-terminus of 18 S ribosomal RNA. This region of homology encompasses six base pairs (- 15.0 kcal) which almost overlaps the sl RNA region that is homologous to the region near the 3’-terminus (Hagenbtichle et al., 1978). In messenger RNA this region may be alternatively associated with ribosomal RNA or with its own 3’-encl. Interestingly enough, this ribosomal RNA binding region is also present in serotype 1, but not in serotype 3 sl plus strands. It is conceivable that the absence of this sequence from the latter may explain in part why sl RNA ribosome binding regions were not present among the populations of such fragments isolated from serotype 3 messenger RNA molecules (Kozak, 19’77; Darzynkiewicz and Shatkin, 1980); it is

This work was supported by Research Grants ROl AI 08909 and R01 AI 16099, and by Research Training Grant T32 CA 09111 from the NIH. The expert technical assistance of Sue Hughes and Lily Lou is gratefully acknowledged. REFERENCES BABISS, L. E., LUFTIG, R. B., WEATHERBEE, J. A., WEIHING, R. R., RAY, U. R., and FIELDS, B. N. (1979). Reovirus serotypes 1 and 3 differ in their in vitro association with microtubules. J. Viral. 30, 863-874. BANEFLJEE,A. K., WARD, R., and SHATKIN, A. J. (1971). Cytosine at the 3’-termini of reovirus genome and in vitro mRNA. Nature New Btil. 232, 114115. BELLAMY, A. R., and JOKLIK, W. K. (1967). Studies on the A-rich RNA of reovirus. Proc. Nat. Acad. Sci. USA 58, 1389-1393. BROWNLEE,G. G. (1972). “Laboratory Techniques in Biochemistry and Molecular Biology: Determination of Sequences in RNA” (T. S. Work and E. Work, eds.). North-Holland/American Elsevier, New York/Amsterdam/London. DARZYNKIEWICZ, E., and SHATKIN, A. J. (1980). Assignment of reovirus mRNA ribosome binding sites to virion genome segments by nucleotide sequence analysis. Nucleic Acid Res. 8, 337-349. ENGLAND, T. E., and UHLENBECK, 0. C. (1978). 3’-Terminal labeling of RNA with T4 RNA ligase. Nature (London) 275, 560-561. FINBERG, R., WEINER, H. L., FIELDS, B. N., BENACERRAF, B., and BURAKOFF, S. J. (1979). Generation of cytolytic T lymphocytes after reovirus infection: Role of the Sl gene. Proc. Nut. Acad. Sci. USA 76, 442-446.

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SEQUENCES OF THE REOVIRUS Sl GENE

FRANKLIN, R. M. (1966). Purification and properties of the rephcative intermediate of the RNA bacteriophage R1’7. Proc. Nat. Acad. Sci. USA 55, 15051511. GAILLARD, R. K., and JOKLIK, W. K. (1980). Studies on the antigenic determinants of the proteins of the three serotypes of reovirus. Virology, in press. HAGENB~CHLE, O., SANTER, M., STEITZ, J. A., and MANS, R. J. (1978). Conservation of the primary structure at the 3’ end of the 1% rRNA from eucaryotic cells. Cell 13, 551-563. HASTINGS, K. E. M., and MILLWARD, S. (1978). Nucleotide sequences at the 5’-termini of reovirus mRNAs. J. Viral. 28, 490-498. ITO, Y., and JOKLIK, W. K. (19’72). Temperaturesensitive mutants of reovirus. I. Patterns of gene expression by mutants of groups C, D and E. Virology 50, 189-201. KEENE, J. D., SCHUBERT,M., and LAZZARINI, R. A. (1980). Intervening sequence between the leader region and the nucleocapsid gene of vesicular stomatitis virus. J. Viral. 33, 789-794. KEENE, J. D., SCHUBERT. M., LAZZARINI, R. A., and ROSENBERG,M. (1978). Nucleotide sequence homology at the 3’-termini of RNA from vesicular stomatitis virus and its defective interfering particles. Proc. Nut. Acad. Sci. USA 75, 3225-3229. KOZAK, M. (1977). Nucleotide sequences of 5’-terminal ribosome-protected initiation regions from two reovirus messages. Nafure (London) 269, 390-394. KOZAK, M., and SHATKIN, A. J. (1977a). Sequences of two 5’-terminal ribosome-protected fragments from reovirus messenger RNAs. J. Mol. Biol. 112, 75- 96. KOZAK, M., and SHATKIN, A. J. (197713).Sequences and properties of two ribosome binding sites from the small class of reovirus messenger RNA. J. Biol. Chem. 252, 6895-6908. LEE, P. W. K., HAYES, E. C., and JOKLIK, W. K. (1980a). Characterization of immunoglobulins produced by hybridomas against reovirus proteins. Virology, in press. LEE, P. W. K., HAYES, E. C., and JOKLIK, W. K. (1980b). Polypeptide 01 is the reovirus cell-attachment protein. Virology, in press. Lr, J. K., SCHEIBLE, P. P., KEENE, J. D., and JOKLIK, W. K. (1980). The plus strand of reovirus gene S2 is identical with its in vitro transcript. Virology, in press. MCCRAE, M. A., and JOKLIK, W. K. (1978). The nature of the polypeptide encoded by each of the ten doublestranded RNA segments of reovirus type 3. Virology 89, 578-593.

MARTINSON, H. G., and LEWANDOWSKI,L. J. (1975). Sequence homology studies between the doublestranded RNA genomes of cytoplasmic polyhedrosis

virus, wound tumor virus and reovirus strains I, 2 and 3. Intervirology 4, 91-98. NICHOLS, J. L., BELLAMY, A. R., and JOKLIK, W. K. (1972). Identification of the nueleotide sequences of the oligonucleotides present in reovirions. Virology 49, 562-571. PEATTIE, D. A. (1979). Direct chemical method for sequencing RNA. Proc. Nat. Acad. Ski. USA 76, 1760-1764. PROUDFOOT,N. J., and BROWNLEE, G. G. (1976). 3’ Non-coding region sequences in eukaryotic messenger RNA. Nature (London) 263,211-214. SANGER, F., BROWNLEE,G. G., and BARRELL, B. G. (1965). A two-dimensional fractionation procedure for radioactive nucleotides. J. Mol. Biol. 13, 373-398. SCHONBERG,M., SILVERSTEIN, S, C., LEVIN, D. J., and ACS, G. (1971). Asynchronous synthesis of the complementary strands of the reovirus genome. hoc. Nat. Acad. Sci. USA 68, 505- 509. SCHUERCH, A. R., MITCHELL, W. R., and JOKLIK. W. K. (1975). Isolation of intact individual species of single- and double-stranded RNA after fractionation by polyacrylamide gel electrophoresis. Amrl. Bio&em. 65, 331-345. SHATKIN, A. J., and SIPE, J. D. (1968). Single-stranded adenine-rich RNA from purified reoviruses. Proc. Nat. Acad. Sci. USA 58, 246- 250.

SKEHEL, J. J., and JOKLIK, W. K. (1969). Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores. Virology 39, 822-831. SMITH, R. E., ZWEERINK, H. J., and JOKLIK, W. K. (1969). Polypeptide components of virions, top component and cores of reovirus type 3. Virology 39, 791-810. SUGDEN, B., DETROY, B., ROBERTS, R. J., and SAMBROOK,J. (1975). Agarose gel electrophoresis equipment. Anal. Biochem. 68, 36-46. TINOCO,I., JR., BORER,P. N., DENGLER, B., LEVINE, M. D., UHLENBECK, 0. C., CROTHERS,D. M., and GRALL.A,J. (1973). Improved estimation of secondary structure in ribonucleic acids. Nature New Biol. 246, 40-41. WEINER, H. L., and FIELDS, B. N. (1977). Neutralization of reovirus: The gene responsible for the neutralization antigen. J. Ezp. Med. 146, 13051310. WEINER, H. L., RAMIG, R. F., MUSTOE, T. A., and FIELDS, B. N. (1978). Identification of the gene coding for the hemagglutinin of reovirus. Virology 86, 581-584. WEINER, H. L., DRAYNA, D., AVERILL, D. R., JR., and FIELDS, B. N. (1977). Molecular basis of reovirus virulence: Role of the Sl gene. hoc. Nat. Acad. Sci. USA 74, 5744-5748.