Molecular cloning of the complete genome of reovirus serotype 3

VIROLOGY

133,191-196

(1934)

Molecular Cloning of the Complete

Genome of Reovirus Serotype

3

L. WILLIAM CASHDOLLAR, RICHARD CHMELO, JOSti ESPARZA, GEOFFREY R. HUDSON, AND WOLFGANG K. JOKLIK’ Department

of Microbiobg~

and Immundogg, Duke University Durham, North Carolina 27710

Medical

Center,

Received December 2, 1985; accepted December 4, 198s All 10 genes of the Dearing strain of reovirus serotype 3 have been cloned in their entirety into pBR322. This has been proved by sequencing the terminal regions (50-100 residues long) of all 10 cloned genes; all such regions were found to be identical to the corresponding terminal regions of the double-stranded RNA genes that are present in reovirus particles. The lengths of the cloned reovirus genes were estimated by comparing their electrophoretic mobilities (and those of their P&I cleavage fragments) with those of accurately known marker DNA molecules. The genes range in size from about 3825 bp for the L genes to about 1200 bp for the S4 gene.

The reovirus genome consists of 10 segments of double-stranded (ds) RNA. We have cloned all 10 genes of reovirus serotype 3 strain Dearing into pBR322, primarily for the purpose of placing them into expression vectors and isolating sufficient amounts of the proteins that they encode for studies on their structure and function. It is essential for this purpose to clone the entire genes. In the case of reovirus strain Dearing this can be readily ascertained because both ends of all 10 native genes have been sequenced (Antczak et al., 1982), and the sequences at the ends of the cloned genes can be compared with them. We have sequenced one of the cloned genes of this virus strain, gene S2, and shown that it is an intact clone (Cashdollar et al, 1982). We present evidence in this report that we have also cloned the other nine genes of reovirus serotype 3 in their complete, intact forms. MATERIALS

AND

METHODS

Preparation of full-length cDNA. The procedures used for growing reovirus serotype 3 (strain Dearing), extracting RNA from it, and transcribing it into cDNA have been described (Cashdollar et d, 1982). ’ Author addressed.

to whom requests for reprints

should be

Elimination of small transcripts in the cDNA preparation and separation of transcripts into size classes were carried out before hybridization/annealing. Singlestranded cDNA transcripts were tailed with oligo dC (Deng and Wu, 1981) and electrophoresed in 1.5% alkaline agarose gels (McDonell et al, 1977). The reason for this strategy was that attempts to tail with oligo dC cDNA molecules that had been electrophoresed in agarose gels were unsuccessful. The positions of cDNA bands in alkaline agarose gels were determined by autoradiography; the gels were sliced and cDNAs were extracted by dissolving the slices in saturated NaI (Fisher Scientific certified) and allowing the cDNA to adsorb to glass beads (Vogelstein and Gillespie 1979). The cDNA molecules were eluted from the glass beads by suspending them in 50 mM Tris-HCl (pH 9.0), 0.2 M NaCl, and incubating them at 65” for 30 min. Three cycles of elution were sufficient to elute 80% or more of cDNA from the glass beads. Cloning reovirus dsDNA. After elution from glass beads, the buffer containing the cDNA was adjusted to 80 mM Tris-HCl (pH 7.8), 250 mM NaCl, 2 mM EDTA. The samples were heated at 100’ for 3 min and quick cooled. Formamide was then added to a final concentration of 50% and the 191

0042-6822184 $3.00 Copyright All rights

0 1984 by Academic Press. Inc. of reproduction in any form rwerved.

192

CASHDOLLAR

cDNA molecules were allowed to hybridize for 24 hr at 22”. The samples were then dialyzed against 1 mMTris-HCl (pH 8.0), lyophilized, and the ds cDNA was cloned into the Pat1 site of pBR322 (Jackson et uL, 1972; Cohen et al, 1972; Deng and Wu, 1981). Plasmids containing cloned reovirus genes were identified as previously described (Cashdollar et ml, 1982). Determination of insert size. The size of the reovirus-specific insert in each clone was determined by isolating the recombinant plasmids by rapid lysis (Birnboim and Doly, 1979) and electrophoresing them in agarose gels. Precise determination of insert size was achieved by excising the inserts with PstI and electrophoresing them in agarose gels together with HaeIIIrestricted 4X 174DNA and HindIII-restricted lambda DNA (New England Biolabs) which served as size markers. Sizes were assigned to the reovirus-specific PstI restriction fragments as described by Duggleby et al. (1981). Sequencing the term&i of the cloned inserts. To determine whether entire reovirus genes had been cloned, the termini of the cloned genes were sequenced. PstI restriction fragments of the cloned genes were subcloned into Ml3 mp8 and mp9 vectors (New England Biolabs) (Messing et cd, 1981). Fragments smaller than 2000 bp were cloned directly into mp8 and mp9 vectors cleaved with PstI; larger fragments were restricted with TaqI and subcloned into mp8 which had been cleaved with AccI and PstI. Sequence analysis was performed by the dideoxy chain-terminator method of Sanger et al. (1977). RESULTS

The general strategy used to clone the genes of reovirus has been described in detail elsewhere (Cashdollar et cd, 1982). The critical step proved to be electrophoresis of ss cDNA transcripts tailed with oligo (dC) in alkaline agarose gels. The purpose of this step was to determine the size of the transcripts, to remove incomplete transcripts, and to provide an opportunity for fractionating the total transcript population into size classes corresponding to the L, M, and S gene size classes and even

ET AL.

to individual S genes. Following electrophoresis in alkaline agarose gels, cDNA was recovered as described under Materials and Methods and allowed to anneal so as to form dsDNA molecules. Figure 1 shows an electropherogram of isolated L, M, Sl, and S2 + S3 + S4 size classes of ds cDNA transcripts. Each size class is seen to be composed predominantly of the species mentioned and to be essentially free of smaller, incomplete transcripts. This fractionation procedure greatly increased the recovery of complete gene copies. Identi$catim of the Cloned Genes The identity of cloned reovirus genes in recombinant clones was determined by hy1

M

‘1

s234

4153-

21461846-

13530 1078872.

FIG. 1. Electropherogram of various size classes of reovirus ds cDNA. Reovirus cDNA tailed with oligo (dC) and labeled with [=P]dATP was electrophoresed in an alkaline agarose gel and bands corresponding to the various size classes were excised from it. Visualization was achieved by exposure of the wet gel to Kodak X-omat AR film for l-3 hr. DNA was recovered from each band as described under Materials and Methods, allowed to self-anneal, and electrophoresed in a 1.5% neutral agarose gel which was then autoradiographed (exposure 24 hr). The figures on the left represent the length in numbers of base pairs of size markers (Hoe111 fragments of 9x174 DNA and Hind111 fragments of lambda DNA end labeled with 8pp) which had been electrophoresed in the same gel.

CLONING

OF THE

REOVIRUS

GENOME

193

FIG. 2. Identification of cloned reovirus genes. Total reovirus dsRNA was electrophoresed in a 7.5% polyacrylamide gel. The RNA was transferred to diazotized aminophenylthioether paper (Alwine reovirus dsRNA to localize the genes (lanes C) and et al, 1977) and hybridized to total ‘?-labeled to various cloned DNAs labeled with zzP by nick translation (lanes LlS4).

bridizing them to authentic reovirus genes (Cashdollar et uL, 1982). Figure 2 shows Northern blots of total reovirus dsRNA after it had been electrophoresed in a 7.5% polyacrylamide gel (Schuerch et CL&1973) and hybridized to a series of recombinant clones labeled with 32Pby nick translation. The results demonstrate that DNA clones of all 10 reovirus serotype 3 genes had been obtained.

Lleterwhutim of the Sizes of Cloned Reovirus Genes Two types of analysis were carried out to determine whether the cloned genes that had been detected in the analysis shown in Fig. 2 were full size. First, the recombinant plasmids themselves were electrophoresed in agarose gels. Figure 3 shows an ethidium bromide-stained agarose gel

FIG. 3. Characterization of the recombinant plasmids containing reovirus genes by agarose gel electrophoresis. Whole plasmids (form I) were extracted from the 10 transformants known to contain reovirus genes by the analysis presented in Fig. 2, and electrophoresed in a 1% agarose gel. The gel was stained with ethidium bromide and photographed under uv light. Plasmid pBR322 was electrophoresed in the left- and right-most lanes for comparison. Ll to S4 refer to the same plasmids as were analyzed in Fig. 2.

194

CASHDOLLAR

ET AL.

in which whole plasmid DNA obtained Genes Ll, L2, L3, Sl, and S2 all have a from clones specific for the 10 reovirus single PstI site and yield two bands; gene genes had been electrophoresed. It is seen M2 has two PstI sites and yields three that the extent to which the migration bands; and the remaining genes, genes Ml, rate of each reovirus-specific recombinant M3, S3, and S4, have no PstI sites and yield plasmid was retarded relative to that of a single band. In all cases the reoviruspBR322 paralleled the expected relative specific insert was recoverable from the size of each insert (expected, that is, on recombinant plasmid by cleavage with the assumption that each gene had been PstI. cloned in full). The sizes of the excised genes/gene fragSecond, the sizes of the cloned genes were ments were estimated from their electrodetermined directly following their exci- phoretic mobilities relative to those of sion from plasmids with P&I. Figure 4 markers with the aid of a computer proshows an ethidium bromide-stained aga- gram (Duggleby et aL, 1981). The sizes that rose gel in which the cloned excised reo- were determined by this method are listed virus genes had been electrophoresed. Sev- in Table 1. In addition to the reovirus-speeral of the genes possessP&I cleavage sites cific sequences, the insert DNAs also posand therefore yield more than one band. sess several G residues at each end as a result of the cloning procedure. The exact number of G residues was determined by PST 1 sequence analysis and this number has been subtracted from the sizes reported in Table 1. The accuracy of these numbers is probably better than fl%: we have sequenced genes S2 and Sl (Cashdollar et cd, 1982; unpublished results), and their lengths are 1329 and 1416 residues, respectively, in excellent agreement with the numbers presented in Table 1. Sequencing of the Terminal Regions of the 10 Clcmed Reovirus Genes

FIG. 4. Sizing the 10 cloned reovirus genes. The plasmids shown in Figs. 2 and 3 were digested with P&I in order to excise the cloned reovirus genes. The digestion products were electrophoresed in a 1.5% agarose gel which was stained with ethidium bromide and photographed under uv light. Size markers electrophoresed in the end lanes were Hind111 restricted lambda DNA and Hoe111 restricted 4x174 DNA, the numbers along each side refer to the sizes in nucleotide base pairs of these markers. Ll to S4 refer to the P&I digests of the recombinant clones characterized in Figs. 2 and 3.

On the basis of the evidence presented so far, recombinant plasmids had been obtained that appeared to contain complete DNA copies of all 10 reovirus serotype 3 genes. In order to establish this point definitively, both termini of clones corresponding to all 10 reovirus genes were sequenced. The PstI fragments or smaller restriction fragments in the case of larger genes were subcloned into Ml3 mp8 and mp9 vectors and sequenced by the chainterminator method of Sanger et al. (1977). The sequences that corresponded to the two ends of each clone were compared with the RNA sequences at the termini of the native reovirus genes (Antczak et al, 1982). This sequencing analysis revealed that all clones possess a stretch of 9-28 G residues at each end, and that in all cases this stretch of G residues is followed by a sequence iden-

CLONING

OF THE

REOVIRUS

GENOME

195

been possible so far, because they can be compared with DNA size markers of acSIZES OF THE CLONED REOVIRUS GENES’ curately known size. This has led to the size estimates presented in Table 1. Actual Actual size size determination by sequencing of three Estimated (determined by Cloned of the genes (genes Sl, S2, and S3) suggests gene size (bp)b sequencing) (bp) that these estimates are accurate to about plus or minus 1%. Ll 3825 The availability of the cloned genes L2 3825 makes it possible to sequence the reovirus L3 3825 genes. Three serotype 3 genes, Sl (CashMl 2300 dollar, Chmelo, and Joklik, unpublished M2 2180 results), S2 (Cashdollar et a.& 1982), and M3 2240 S3 (Richardson and Furuichi, 1983), have already been sequenced; and other genes 1416” Sl 1425 are in the process of being sequenced. We 1329d s2 1335 119ge have used the techniques described in this s3 1225 s4 1200 report to clone the genes of reovirus strains belonging to serotypes 1 and 2 and have sequenced the Sl genes of these two sea Estimated by comparing the electrophoretic morotypes as well (Cashdollar, Chmelo, and bility of either cloned reovirus genes or Pstl fragments Joklik, unpublished results). The Sl genes of cloned reovirus genes with those of marker molare the genes that have diverged most execules of known size (Hue111 fragments of 9X174 DNA and H&d111 fragments of lambda DNA). tensively during the evolution of reovirus; b Probable error: fl%. as judged by the ability of the three Sl ’ Cashdollar, Chmelo, and Joklik (unpublished regenes to hybridize with each other, they sults). are not more than l-10% related to each d Cashdollar et al (1982). other (Gaillard and Joklik, 1982). From the “Richardson and Furuichi (1983). sequences of the three Sl genes, it should be possible to deduce the major structural tical to that at each end of each of the 10 features of the three al proteins. It will reovirus genes. All 10 reovirus serotype 3 be very interesting to determine the nature genes have therefore been cloned com- of the sequence divergences at the nucleopletely, without a single nucleotide missing tide and amino acid residue level, as well from either end of any of the genes. as at the quaternary structure level, given the fact that function has been highly conserved (for example, all three al proteins DISCUSSION are the viral cell attachment proteins, and all combine with the same cell surface reAll 10 genes of reovirus serotype 3 (strain Dearing) have been cloned into pBR322 ceptor (Lee et cd, 1981)). It will also now be possible to clone reowithout loss of a single base pair. The method used was efficient, since even the virus genes into expression vectors, both almost 4000-bp-long L genes were cloned prokaryotic and eukaryotic, which will in intact form. The step most critical to permit a variety of studies ranging from the successof the method was that in which examination of reovirus-coded proteins for the ss cDNA gene transcripts tailed with enzyme activities and function, to altering oligo (dc) were purified by alkaline agarose the sequences of reovirus genes and obgel electrophoresis. The method described serving the effect of such alterations on here should be applicable to cloning the translation and transcription frequency, dsRNA gene segments of other viruses. assembly during morphogenesis and enThe availability of intact DNA clones capsidation, as well as on the functions of has permitted much more accurate size de- the proteins that they encode. Such studies termination of reovirus genes than has are currently underway. TABLE

1

196

CASHDOLLAR ACKNOWLEDGMENTS

We would like to express our thanks to Sherry Larson and Pat McClellan-Green for expert technical assistance. This work was supported by research Grant ROl AI 08909 and by Program Project Grant 1POl CA 30246 from the National Institutes of Health. L. W. C. and R. C. were supported by research training Grant 5 T32 CA 09111. REFERENCES

ALWINE, J. C., KEMP, D. J., and STARK, G. R. (1977). Method for detection of specific RNAs in agarose gels by transfer to diasobenzyloxymethyl-paper and hybridization with DNA probes. Proc Nat. Acad Sci USA 74,5350-5354.

ANTCZAK,J. B., CHMELO,R., PICKUP,D. J., and JOKLIK, W. K. (1982). Sequences at both termini of the 10 genes of reovirus serotype 3 (strain Dearing). Virology 121, 307-319. BIRNBOIM,H. C., and DOLY,J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic A& Res. 7,1513-1523. CASHDOLLAR,L. W., ESPARZA, J., HUDSON, G. R., CHMELO,R., LEE, P. W. K., and JOKLIK,W. K. (1982). Cloning the double-stranded RNA genes of reovirus:sequence of the cloned S2 gene. Proc Nat. Ad

ET AL.

A computer program for determining the size of DNA restriction fragments. And Biochem. 110,4955. GAILLARD, R. K., and JOKLIK, W. K. (1982). Quantitation of the relatedness of reovirus serotypes 1, 2, and 3 at the gene level. virdogy 123,152-X4. JACKSON,D. A., SYMONS,R. H., and BERG, P. (1972). Biochemical method for inserting new genetic information into DNA of simian virus 40: Circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Esdwrichia wli Prac. Nat. Acad Sci USA 69,2904-2909.

LEE, P. W. K., HAYES, E. C., and JOKLIK, W. K. (1981). Protein ul is the reovirus cell attachment protein. virology 108,156-16.X MCDONELL,M. W., SIMON, M. D., and STUDIER,F. W. (1977).Analysis of restriction fragments of ‘I’7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Mel Bid 110, 119-146. MESSING, J., CREA, R., and SEEBURG,P. H. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res. 9, 309-321. RICHARDSON,M. A., and FURUICHI,Y. (1983). Nucleotide sequence of reovirus genome segment S3, encoding non-structural protein sigma NS. Nucl& Acids Res. 11,6399-6408.

Sci USA 79, 7644-7648. COHEN, S. N., CHANG, A. C. Y., and Hsu, L. (1972).

SANGER,F., NICKLEN, S., and CXJLSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors.

Nonchromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coZi by Rfactor DNA. Proc Nat Ad Sci USA 69, 21102114. DENG, G., and WV, R. (1981). An improved procedure for utilizing terminal transferase to add homopolymers to the 3’ termini of DNA. Nuc.!eic Acids Res. 9, 4173-4188. DUGGLEBY,R. G., KINNS, H., and ROOD,J. I. (1981).

SCHUERCH,A. R., and JOKLIK, W. K. (1973). Temperature-sensitive mutants of reovirus. IV. Evidence that anomalous electrophoretic migration behavior of certain double-stranded RNA hybrid species is mutant group specific. Fir56, 218-229. VOGELSTEIN, R., and GILLESPIE, D. (1979). Preparative and analytical purification of DNA from agarose.

Proc Nat. Ad

Sci USA 74. 5463-5467.

Proc. Nat. Acad SC% USA 76,615-619.