Molecular cloning and characterization of the genome of wound tumor virus: A tumor-inducing plant reovirus

VIROLOGY

144, 398-409 (1985)

Molecular Cloning and Characterization of the Genome of Wound Tumor Virus: A Tumor-Inducing Plant Reovirus TETSUYA

Wadsworth

ASAMIZU,l DENNIS JOHN V. ANZOLA, Center

fw

Laboratories

of Health, Received

December

SUMMERS, MARY BETH MOTIKA, DONALD L. NUSS2

AND

and Research, New York New York 12201

State

Departm.ent

Albony,

6, 1984 accepted March

SO, 1985

The double-stranded RNA genome of the tumor-inducing plant pathogen, wound tumor virus, was converted to double-stranded DNA and cloned into plasmid pBR322. Multiple apparent full-length copies of 9 of the 12 wound tumor virus genome segments were identified. The entire sequence of cloned genome segment S12, the smallest of the genome segments, was determined. This genome segment was found to be 851 nucleotides in length and to possess a single long open reading frame that extends 178 codons from the first AUG triplet (residues 35-37): information sufficient to encode a protein of the size estimated for the smallest of the previously identified wound tumor virus primary gene products, Pns 12. Sequence data obtained from analysis of cloned cDNA copies of several genome segments and from direct analysis of the 3’ termini of the doublestranded genome RNAs revealed that each wound tumor virus genome segment possesses the common terminal sequences:

8 1985 Academic

(+) 5’ GGUAUU

. . . UGAU 3’

(-) 3’ CCAUAA

. . . ACUA

Press, Inc.

quently, this virus group is useful for studying, at the molecular level, a number of biologically important events related to virus transmission by insect vectors and the regulation of plant growth and development (Nuss, 1934). Wound tumor virus (WTV), the best characterized of the plant reoviruses, possesses a genome consisting of 12 ds RNA segments with a combined molecular weight of approximately 16 X lo6 (Reddy and Black, 1973a). Purified WTV particles catalyze the synthesis of 12 singlestranded transcripts (Black and Knight, 1970; Reddy et al, 1977; Nuss and Peterson, 1981a) which anneal specifically to the 12 WTV genome segments (Nuss and Peterson, 1981a). The transcripts are capped at the 5’ end (Rhodes et 4, 1977; Nuss and Peterson, 1981b) and are translationally active in vitro (Nuss and Peterson, 1980).

INTRODUCTION

Plant viruses with segmented doublestranded (ds) RNA genomes are interesting because they replicate both in a wide range of plant hosts and in a few species of leafhoppers or planthoppers that act as vectors (Black, 1965). Infection of the plant host results in a variety of disease symptoms including the formation of tumors (Black, 1965), while infection of the insect vector is generally nonsymptomatic (Black, 1957; Hirumi et uL, 1967). Conse‘On leave from Department of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugltani, Toyama 930-01, Japan. ‘Author to whom requests for reprints should be addressed at Department of Cell Biology, Roche Institute of NIoleeular Biology, Roche Research Center, Nutley, N. J. 07110. 0042-6822/85 $3.00 Copyright All rights

5’.

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

398

CLONING

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CHARACTERIZATION

Twelve virus-induced polypeptides have been identified in extracts of WTV-infected cultured vector cells (Nuss and Peterson, 1980). The same polypeptides are synthesized in cell-free systems directed by in vitro synthesized viral transcripts (Nuss and Peterson, 1980) indicating that they represent primary gene products. We recently reported that variant genome segments associated with transmission-defective populations of WTV are generated by internal deletion events resulting in terminally conserved remnants of genome segments that are functional with respect to transcription, replication, and packaging (Nuss and Summers, 1984). To further define the relationship of WTV genome structure to function, we have determined the terminal nucleotide sequences of each genome segment, constructed a cDNA library of the WTV genome, and determined the entire nucleotide sequence of cloned genome segment S12. A comparison of the nucleotide sequence data obtained for the WTV genome segments with the 5’-terminal structure reported for WTV transcripts synthesized in vitro by purified virus particles (Rhodes et &, 19’77; Nuss and Peterson, 1981b) suggests the possibility that the WTV genome may be transcribed by a mechanism that differs from that determined for other members of the reovirus group.

OF WTV

GENOME

399

isoamyl alcohol 25:24:1 and the RNA was precipitated from the aqueous phase by the addition of 2 vol of ethanol. The RNA was suspended in HzO, adjusted to 2 M LiCl, and incubated overnight at 4’. Contaminating single-stranded RNA was removed by centrifugation, and the ds RNA was collected from the supernatant by dilution with HZ0 and addition of 2 vol of ethanol. As a final step, the ds RNA preparation was passed through a Sephacryl S300 column (1.4 cm X 30 cm). The purity of all preparations was determined by gel electrophoretic analysis (Nuss and Peterson, 1981a; Nuss and Summers, 1984). Construction and Characterization W!W Genome cDNA Librar2/

of

the

Complementary DNA copies of WTV genome segments were synthesized and cloned into pBR322 essentially as described for human reovirus by Cashdollar et al. (1982) with several modifications. WTV genome RNA was not denatured before the polyadenylation step. Prior to cDNA synthesis, polyadenylated genome RNA and oligo(dT)lz_l~ primer were incubated in 90% dimethyl sulfoxide at 65“ for 20 min and precipitated by addition of NaCl, to a final concentration of 0.15 M, and 2.5 vol of ethanol. We included 4 mM sodium pyrophosphate in the reverse transcriptase reaction mixture (Murray et al., 1983). The completed reverse tranMETHODS scriptase reaction mixture was added to Virus Preparation and Isdation of a% RNA the dried, denatured polyadenylated genome RNA/primer mixture to initiate Standard WTV [inoculum RB (Reddy cDNA synthesis. Additional modifications and Black, 19’72)]was purified from acutely included (i) removal of formamide from infected vector cell monolayers [AgaZZia the self-annealing reaction by two cycles constricta, line AC20 (Chiu and Black, of ethanol precipitation and (ii) passage 196’7)] by the method of Reddy and Black of cDNA through a 1.4 X 30-cm Sephacryl (197313).Genome RNA, used for both se- S300 column equilibrated with 50 mM quence analysis and cDNA synthesis, was Tris, pH 7.8, 0.1 M NaCl, 1-mil4 EDTA, isolated by first treating purified virus and 0.2% SDS following the Klenow fragwith 200 pg/ml proteinase K in 25 mM ment-mediated repair reaction, to remove Tris (pH 7.5), 100 mM NaCl, 7.5 mM low molecular weight cDNA. The double-stranded cDNA copies were ethylenediaminetetraacetic acid (EDTA), 2.4% sodium dodecyl sulfate (SDS) for 30 tailed with oligo(dC), annealed to oligomin at 37”. The solution was then ex- (dG)-tailed, PstI-cut, pBR322 and then used to transform Eschmichia coli strain tracted twice with phenol/chloroform/

400

ASAMIZU

RR1 by standard procedures (Bolivar et ok, 1977; Sleigh et al, 1979). The resulting transformants were screened for WTV sequences by the colony hybridization method (Grunstein and Hogness, 1975; Hanahan and Meselson, 1983) using individual WTV genome RNA segments labeled with [3”P]pCp (Nuss and Summers, 1984) as probe. The size of the cDNA inserts was estimated by agarose gel electrophoresis of plasmids prepared by the method of Holmes and Quigley (1981). DNA sequence analysis was performed after subcloning into phage Ml3 (Messing et aZ., 1981) using the dideoxy chain-terminator reaction (Sanger et aL, 1977) (Amersham Kits). Phasing primers oligo(dT)r,,dAdT and oligo(dT)l,,dGdG were used to obtain terminal nucleotide sequence information in the 3’ to 5’ direction (Elleman et al, 1983).

.“,:,,(“’ SIZE (Kb)

4.4 -

SEGMENT

-

ET AL.

Labeling and Sequence Analysis of the 3’ Termini of the WTV Genome Segments Total purified WTV ds RNAs were labeled at the 3’ termini with [5’-32P]pCp and subjected to polyacrylamide gel electrophoresis to separate the individual segments followed by electroelution as previously described (Nuss and Summers, 1984). The isolated ds RNAs were denatured by incubation at 45” in 90% N2flushed dimethyl sulfoxide for 30 min and then precipitated with ethanol. The RNA pellet was dissolved in 4 ~1 of 50 mM NaHC03, 1 mM EDTA (pH 9.0), and incubated at 90” for 10 min. Analysis of the partial digestion products was performed in the first dimension by electrophoresis on cellulose acetate (3 X 55 cm; Schleicher and Schuell) for 1 hr at 2500 V in 5% acetic acid/5M urea/l mM EDTA adjusted to pH 3.5 with pyridine. The oligonucleotides were transferred to DEAEcellulose (1:7.5) thin layer plates (Analtech) and chromatographed in the second direction at 60” with homomix C (Brownlee and Sanger, 1969). After drying, the plates were exposed to Kodak XAR-2 film. RESULTS

,*A

2.3 2.0 -

0.56-

FIG. 1. Alkaline agarose gel (1.4%) (Maniatis et d, 1982) analysis of cDNA made with meltedpolyadenylated WTV genome segments as template (lane B). Assignments of cDNA bands to genome segments is based on the relative electrophoretic migration of the genome segments and the apparent size of the cDNA molecule as determined by comparison to the migration position of end-labeled Hind111 digested phage X fragments (lane A).

cDNA Synthesis and Molecular Cloning The strategy used for synthesis and cloning of cDNA copies of WTV genome segments was similar to that described by Cashdollar et aZ. (1982) for human reovirus with several modifications as described in the previous section. Analysis of the products of the reverse transcriptase reaction by alkaline agarose gel electrophoresis (Maniatis et aL, 1982) revealed the synthesis of cDNAs corresponding in size to that of the WTV genome segments (Fig. 1). However, synthesis of cDNA of discrete full-length size was dependent upon the addition of 4 mM sodium pyrophosphate to the reaction mixture (Murray et al, 1983). A transformation efficiency of 30 recombinants per nanogram of cDNA was obtained. Transformants containing WTV cDNA inserts are selected by colony hybridization using purified q-end-labeled genome

CLONING

AND

CHARACTERIZATION

OF WTV

GENOME

401

nome segments, individual 3’-end-labeled genome RNAs were subjected to partial alkaline digestion and 2-dimensional or wandering-spot analysis. The analysis of [3’-32P]pCp-labeled genome segments S3 and S9 are shown in panels A and B of Fig. 3, respectively. In each case, the right track represents the 3’-terminal sequences of the positive, or messenger sense, strand which terminates with uridine while the left track represents the 3’-terminal sequence of the negative strand which terminates with cytidine. The assignment of the wandering-spot patterns to the individual strands of a Nucleotide Sequence Analysti of WTV Ge- genome segment was performed by comnm Segnzents and Cloned cDNA paring the patterns generated by analysis of individual separated strands of several Copies genome segments with the pattern genPrior to sequence analysis of the cloned erated by analysis of both strands simulcDNA copies of wound tumor virus ge- taneously. Isolated [3’-32P]pCp-labeled ge-

segments or, for later transformations, nick-translated cDNA clones as probe. The size of the cDNA inserts was determined by electrophoretic analysis of minilysates using plasmids containing inserts of known length as markers. As indicated in Fig. 2, multiple apparent full-length copies of genome segments S12 through S4 have been obtained while only partial length clones of segments S3 through Sl have been identified. The integrity of several of the apparent full-length clones has been confirmed by analysis of the terminal nucleotide sequences.

oc-

ccc-

FIG. 2. Agarose gel (1%) (Maniatis et al, 1982) analysis of plaamids containing representative cloned eDNA copies of the WTV genome segments. OC and CCC indicate the respective positions of the open circular and covalently closed circular forms of pBR322. HindIII-digested phage XDNA and two plasmids containing inserts of known size are included as size markers. The plasmids containing inserts of genome segments S12 through S4 exhibit relative migration positions consistent with the presence of full-length copies of the respective genome segments.

402

ASAMIZU

ET AL.

FIG. 3. Two-dimensional oligonucleotide fingerprint analyses of partial alkaline digestion products of [32PlpCp-labeled genome segment S3 (A) and segment S9 (B). In each panel the left track represents the 3’-terminal sequence of the (-) strand which contains a 3’-terminal cytidine while the right track represents the 3’-terminal sequence of the (+) strand which has a 3’terminal uridine.

nome segments were denatured by treatment with 90% dimethyl sulfoxide at 65” for 15 min and electrophoresed on a 5% polyacrylamide gel with an acrylamideto-bis-acrylamide ratio of 6O:l (Maxam and Gilbert, 1980). Separated strands were located by autoradiography, collected by electroelution (Nuss and Summers, 1934), and subjected to wandering-spot analysis. Strand polarity was subsequently determined (results presented in later section) by hybridizing complementary Ml3 clones of genome segments to [“p]pCp labeled in vitro synthesized WTV transcripts. The Ml3 clone which hybridized to the transcripts was designated as having negative polarity. The polarity of the strands of the genome segment was then determined by comparing the terminal nucleotide sequences derived from the Ml3 clone with the wandering-spot patterns of isolated genome strands. Determination of 3’-terminal nucleotides of the isolated strands of Cj2PlpCp-labeled genome segments and transcripts of individual segments was performed as described previously (Nuss and Summers, 1984). Each of the 12-wound tumor virus genome segments exhibited the same 2-dimensional pattern for the four 3’-terminal nucleotides of the plus strand and the six 3’-

terminal nucleotides of the negative strand. Appropriate restriction fragments of a number of the cloned WTV genome segments were subcloned into phage Ml3 (Messing et a.& 1981) and subjected to nucleotide sequence analysis by the Sanger dideoxy method (Sanger et al, 1977). The conserved terminal nucleotide sequences that were determined by direct sequence analysis of genome RNA (Fig. 3) were also observed upon sequence analysis of three independent clones of segment S12, (Fig. 4), and one clone each of genome segments S7, S6, and S5. The combined sequence data indicate that each of the wound tumor virus genome segments possesses the sequence . . . UGAU-OH at the terminus corresponding to the 3’ end of the plus strand and . . . AAUACC-OH at the terminus corresponding to the 3’ end of the negative strand (Fig. 4). Th.eNpLcledideS~

of cloned Genome

segment 512

The entire sequence of WTV genome segment S12 was determined by the dideoxy chain-termination method (Sanger et aL, 1977) after subcloning of restriction endonuclease fragments into M13, mp8,

CLONING

AND

CHARACTERIZATION

OF WTV

A A

T

GENOME

403

B C

G

A

1 A :

T

C

G

: G T

C

i-10 G G c G A A A A A T-20

xT li c E-30 :

FIG. 4. Terminal nucleotide sequences of cloned WTV genome segment S12. Nucleotide sequences of the 3’-terminal ends of the positive and negative strands of segment S12 cloned in bacteriophage Ml3 are presented in panels A and B, respectively. The terminal sequences common to all WTV genome segments are indicated by brackets. The oligo(dA) and oligo(dC) tracks were added to the genome segment during cDNA synthesis and cloning.

and mp9 vectors (Messing et aL, 1981). The entire gene was analyzed in both directions except for two small regions for which unambiguous results were obtained in one direction from independent subclones (Fig. 5). Phasing primers 5’-(dT)10dAdT and 5’-(dT),,dGdG were used to aid in the determination of terminal nucleotide sequences. internal to the oligo(dA)/oligo(dC) stretches added during cDNA synthesis and cloning (Elleman et ak, 1983) (Fig. 5). The complete nucleotide sequence of cloned genome segment S12 is presented in Fig. 6. The cloned gene consists of 851 base pairs and possesses one long open reading frame that starts with the first AUG triplet (residues 35-37) and extends

534 nucleotides. A second open reading frame, in phase with the first, and, consisting of 40 codons, is present beginning at residues 674-676. There are four inphase termination codons, the first at residues 569-571 and the remaining three following the second open reading frame at residues 794-796,809-811, and 848-850. The last termination codon comprises part of the conserved terminal sequence. The nucleotide composition of gene S12 is 32.1% A, 26.1% T, 19.4% C, and 22.4% G. The one long open reading frame of genome segment S12 has the coding potential for a polypeptide composed of 178 amino acid residues with a calculated molecular weight of 19,171. This value is consistent with the apparent molecular

ASAMIZU

404

ET AL.

frame found in either strand. To unambiguously determine strand polarity, WTV transcripts synthesized in vitro were labeled with [32P]pCp and hybridized to the individual complementary full-length single-stranded cDNA copies of segment S12 FIG. 5. Strategy used to determine the nucleotide sequence of the cloned cDNA copy of WTV genome cloned into phage M13. The labeled transcripts hybridized to the strand that segment S12. The number of nucleotides are indicated below the line representing the clone while the contained the sequence . . . GTTCAApositions of relevant restriction sites are indicated TACCA,C,-OH and not to the strand that above the line. The gene is orientated so that the 5’ contained the sequence . . . TTCACATterminus of the plus strand is at the left. The arrows GATA,C,-OH (Fig. 7). Furthermore, the indicate the extent and orientation of the sequences transcript selected by hybridization to the determined in separate analyses. The arrows beginformer S12 strand was shown to possess ning with PP indicate the sequence determined with a 3’-terminal uridine by nearest neighbor the aid of phasing primers. analysis (data not shown). These results confirmed that the sequence 5’ GGUAUweight estimated (19,000) for the smallest UGAAC . . . UUCACAUGAU 3’, as presented in Fig. 6, is the strand of positive of the previously identified WTV primary gene products, Pns 12, the predicted prod- polarity. A similar analysis of cloned uct of segment S12 (Nuss and Peterson, genome segment S7 also revealed that 1980). As deduced from the nucleotide the strand containing the sequence 5 sequence, the polypeptide contains 74 GGUAUU . . . UGAU 3’ was the strand nonpolar hydrophobic, 56 uncharged polar, of positive polarity (Fig. 7). 16 acidic and 32 basic amino acids, resultDISCUSSION ing in a predicted net charge at pH 7.0 of +14 (Huddleston and Brownlee, 1982). We report in this paper the first sucOther distinguishing features of the poly- cessful attempt at cloning the genome of peptide include a high serine content-24 a ds RNA plant virus. Sequence analysis of 1’78amino acids-as well as alternating of the wound tumor virus ds RNA genome basic and acidic sequence domains near segments and cDNA clones revealed that the carboxyterminus (Fig. 6). each genome segment contains the comSince WTV transcripts are reported to be modified at the 5’ terminus with the mon terminal sequences: structure m’GppAm . . . (Rhodes et a& (+) 5’ GGUAUU . . . UGAU 3’ 1977; Nuss and Peterson, 1981b), it was (-) 3’ CCAUAA . . . ACUA 5’. anticipated that, similar to the situation for other members of the Reoviridae To date, multiple apparent full-length (Darzynkiewica and Shatkin, 1980; Li et cloned cDNA copies of WTV genome segal, 1980b; McCrae, 1981; Kuchino et al, ments S12-S4 have been obtained. It is 1982), the 5’ terminus of transcripts would presently unclear why only partial-length be identical to that of the plus-sense ge- clones of the largest (greater than 3500 nome RNA strand, i.e., that the sequence bp) genome segments have been obtained complimentary to the sequence shown in especially in light of the fact that apparent Fig. 6, starting with a 5’ adenosine, would full-length cDNA transcripts of the gebe the positive strand of the genome RNA. nome segments are evident when newly However, computer analysis of this pre- synthesized cDNAs are analyzed by alsumptive plus strand revealed no long kaline agarose gel electrophoresis (Fig. open reading frames. Surprisingly, anal- 1). Characterization of the partial clones ysis of the complimentary strand-that obtained for these segments may provide sequence shown in Fig. 6 beginning with some insights into this observation. a 5’-terminal guanosine-did reveal the A cloned copy of the smallest of the presence of the only long open reading WTV genome segments was selected for --

CLONING AND CHARACTERIZATION

OF WTV GENOME

405

GG;ATTGAACTGCTTGTCATTACTACTGGGAAACMET SER ASN LYS GLU SEP ASN VAL ALA LEU GLN IIIATG TCT AAT AAA GAA TCA AAC GTC GCT CTC CAG 68 TRR LRU ARG VAL TBR LYS ASP MET LPS ASP PEE LEU SE2 gIS ARG ILE VAL GLY GLU PRO ACC CTG AGA GTC ACA AAA GAT ATG AAG GAC TTT TTA AGT CAT AGA ATT GTT GGT GAA CCT

q

128 PRO ALA ASN ILE LYS ILE GLU TYR GLN LYS ILE EIS ARG TYR ARG THR CYS VAL CYS PRO CCC GCC AAT ATT AAA ATC GAG TAT CAA AAA ATA CAT AGA TAT AGG ACC TGC GTG TGT CCT 188 SER TBR GLY III.3 ILE SER GLU LEU CYS PRO SER GLY ASP LEO ILE LEU SER LEU GLY ALA AGC ACT GGA CAT ATT AGT GAA TTA TGT CCA TCA GGC GAT TTA ATA TTG TCA CTT GGT GCC 248 HIS AEG ASN VAL ILE ALA ALA ALA THR VAL TYR ASP VAL VAL LYS ASN LYS ILE LYS SER CAT CGC AAT GTT ATC GCA GCA GCA ACC GTT TAT GAT GTT GTA AAG AAT AAG ATT AAG TCT 308 THR THR SER LYS ALA GLY TBR SER SER THR LEU SER SER LEU GLY LEU SER GLY PIIE GLN ACA ACC TCG AAG GCT GGC ACA TCT TCA ACC CTC TCA AGT CTA GGT TTG TCT GGT TTT CAA 368 LYS PRO LYS ILE GLY SER LYS AAA CCT AAG ATT GGG TCA AAA

ASN AAC

00 LYS LYS TBR MET PEE SER LYS GLN ASN ASN SER TRR AAG AAA ACT ATG TTC AGT AAG CAA AAT AAT AGT ACG

ASN GLU SER ASP GLU SER GLY GLY GLU GLU GLY SER SER LEU ASN ASP LEU PRO LYS SER AAT GAP. AAT GAT TTA CCT AAG TCT --m-w-TCA GAT GAA TCT GGJ GGG GA3 GAG G&AwC&TA

0

G 488 ASP LEU ILE ASN ALA ILE MET GLU LEU ALA SER GLN GLY ARG ASN ASN SER LYS GLY LYS GAT TTG ATC AAT GCC ATT ATG GAG CTG GCA TCA CAA GGA AGA AAC AAT TCA AAA GGA AAA 548 A GLY LYS ARG GLY GLY LYS ARG JJJ CTATTTGTAGTCACAATTGTTGCCGCCATACCATTTGTAGCGTCAGT GGA AAG CGC GGT GGC AAA AGG TGA 619 699 AGAGTACTCACGCTTCTCCCGACCGCTCTGTCCTGGAGAGGGAACGCCCCTCCCTCAAAGGGTGTTCAACAGGGAAGGTT 119 AGCCGTGCTAAATGGTAIAAAGCTTTCGTCAT~ACATACTGGGATGAACTCCAGTAAAAAGCGGTTCACA~ FIG. 6. Nucleotide sequence and predicted amino acid sequence of the mRNA-sense DNA strand of cloned WTV genome segment S12. Possible initiation codons are indicated for both the long open reading frame (residues 35-37 and 89-91) and a second small (40 codons) open reading frame, in phase with the first, near the 3’ end of the molecule. In-phase termination codons are indicated by A. Basic and acidic polypeptide’domains are indicated by (-) and (---), respectively, while potential N-glycosylation sites are indicated by the symbol 0. The conserved terminal sequences found in all WTV genome segments are underlined. The numbers along the left margin corresponds to the first nucleotide in each line.

sequence analysis. The first AUG triplet (residues 35-37) of the only long open reading frame found on either strand has the sequences context AACAUGUCU, regarded as an initiation sequence of only moderate efficiency due to the pyrimidine

at position +4 (Kozak, 1981). However, the second AUG (residues 89-91), also in the same reading frame, is regarded as a stronger initiation sequence since it contains a purine at both positions -3 and +4. In addition, both of these potential

406

ASAMIZU 1

2

3

4

ZOO"2

of [3%‘]pCp-labeled, in FIG. 7. Dot hybridization WTV transcripts to full-length, opposite polarity, bacteriophage Ml3 clones of WTV genome segments S12 and S7. Each lane contained increasing concentrations of DNA as indicated at the left. Lane 1, clone of S12 with the 3’-terminal sequence. . . TTCACATGATA.C.-OH; lane 2, clone of S12 with the 3’-terminal sequence . . . GTTCAATACCA,C,-OH; lane 3, clone of S7 with the 3’terminal sequence . . . AATACCA.C,-OH; lane 4, clone S? with the If-terminal sequence . . . TGATA&-OH. Hybridization, elution of RNA, and nearest neighbor analysis (the eluted mRNA contained a 3’4erminal uridine) were performed as described in Nuss and Summers (1984).

vitro synthesized

initiation codons are followed by hydrophobic amino acid sequences. It is not presently known whether protein synthesis initiates at one or both of these AUG codons. Information concerning the distribution of carbohydrate in the polypeptide encoded by segment S12 is also currently unknown, although four potential N-glycosylation sites are present in the predicted amino acid sequence (Fig. 6). In this regard, WTV polypeptides may provide useful model molecules for comparative studies of glycosylation pathways and other post-translational events in insects and plants. The availability of sequence information for a plant member of the Reoviridae provides additional opportunities for uncovering fundamental nucleic acid and protein structure-function relationships by comparison of sequence information available for different members of the virus group. For example, a comparison of the amino acid charge distribution for

ET AL.

the proteins encoded by WTV genome segment S12 and human rotavirus segment 11 revealed a similar alternating distribution of basic and acidic sequence domains near the carboxyterminus (Fig. 5 of Imai et ak, 1983b). The cloned S2 gene of the Dearing strain of reovirus (Fig. 5 of Cashdollar et aL, 1982) contains a longer 3’-noncoding region than is usually observed for reovirus genome segments. In addition to this atypically long 3’-noncoding region, both WTV S12 and reovirus S2 genome segments have a second open reading frame within this 3’ region with the potential for encoding polypeptides of 4500 and 10,000 Da, respectively. Examination of codon usage reveals that for WTV genome segment S12, like rotavirus genome segments (Both et aL, 1982, 1983a, b; Elleman et ak, 1983; Dyall-Smith et a& 1983; Imai et aL, 1983b; Estes et ah, 1984), but unlike human reovirus genome segments (Cashdollar et uL, 1982; Richardson and Furuichi, 1983), there is a clear bias against the use of the CG dinucleotide. In addition, multiple (3 to 5) in-phase termination codons appear to be a feature found in approximately half of the genome segments that have been sequenced for this virus family (Fig. 6 and Okada et uL, 1984; Cashdollar et al, 1982; Both et al, 1982; Estes et uL, 1984). A comparison of the conserved terminal nucleotide sequences of prototype members of the Reoviridae with animal, insect, insect/animal, or insect/plant hosts reveals a number of interesting similarities (Fig. 8). Termination codons are present in the conserved terminal sequence corresponding to the 3’ end of the plus strand for.three of the five viruses. The conserved terminal sequence corresponding to the 5’ end of the plus strand is characterized by one or two C-G base pairs in the first two positions followed by a four- to six-nucleotide sequence consisting of A-U base pairs which extend into the nonconserved sequence. In most cases only one or two of the genome segments of a particular virus will contain a G-C base pair within the region immediately adjacent to this conserved terminal sequence.

CLONING AND CHARACTERIZATION OF WTV GENOME UTV

(+I 5’ (cap?) GGGAUU CCA'JAA t-1 3’

CPV

(+) 5' C-1 3'

m’Gp~~A%lAA

U CAB--

BTV

I+; ;: (cap?) GWAAA MAWL!--

Rota

(+) 5' l-1 3'

Iwo

(+) 5' C-1 3'

q‘GmvG%CUW C CGAAA ---

n’G~#?CUA

C GAL!--

I@& 3’ ACUA 5' G@@x 3' CAAOCGU 5' ACACWAC 3' UGUGAAUG 5'

u-c ACACUGG

3' 5'

UCAUC3' AGUAG 5'

FIG. 8. Conserved terminal nucleotide sequences of members of the Reoviridae. WTV = wound tumor virus, host = plant and insect, references = Rhodes et al (1977); Nuss and Peterson (1981b); this paper. CPV = cytoplasmic polyhedrosis virus, host = insect, references = Furuichi and Miura (1975) and Kuchino et al. (1982). BTV = blue tongue virus, host = insect and mammal, reference = Rao et al (1983). Rota = rotavirus, host = mammals, references = Imai et al. (1983a); McCrae and McCorguodale (1983); Both et aL (1982). Reo = human reovirus, host = mammals, references = Furuichi et al (1975a, b); Li et al (1986a); McCrae (1981).

The plus strand of cytoplasmic polyhedrosis virus (CPV), rotaviruses, and human reovirus have a 5’-terminal cap structure (Furuichi and Miura, 1975; Furuichi et al, 1975b; Imai et d., 1983a) while the nature of the 5’ terminus of WTV and Blue tongue virus (BTV) has not yet been determined. Analysis of viral transcription products have revealed that, at least in the case of human reovirus, CPV, and rotavirus, the transcripts are exact copies of the plus strand of the genome including the 5’-terminal cap (Furuichi et al, 1975a, b; Furuichi and Miura, 1975; Li et al, 1980b; Spencer and Garcia, 1984). Surprisingly, a comparison of the nucleotide sequence of WTV genome segments presented in this report (Figs. 3, 4, and 6) with the 5’-terminal structure determined for the WTV transcription products (Rhodes et c& 1977; Nuss and Peterson, 1981b) indicates that WTV transcription products may not be identical to the genome plus strand. That is, the 5’- and 3’-terminal structures of WTV transcripts are reported to be 5’ m7GpppAm . . . U 3’ (Fin. 7. Rhodes et al. 1977; Nuss and

407

Peterson, 1981b) rather than 5’ m7GpppGm . . . U 3’ expected from examination of the genome structure. It must be noted that nucleotide sequence information for the WTV genome segments was obtained by analyzing RNA and cDNA copies of RNA isolated from virus particles purified from both the insect vector and the plant host (Nuss and Summers, 19&& this report). However, information concerning the B-terminal structure of WTV transcripts (Rhodes et uL, 1977; Nuss and Peterson, 1981b) is available only for molecules synthesized in vitro by virus particles purified from the plant hosts. There is currently no information concerning the 5’-terminal structure of the plus genome strand of WTV purified from either source. A comparitive analysis of the 5’-terminal structure and nucleotide sequences of WTV genome RNA and transcripts obtained from virus preparations derived from both the insect vector and plant hosts is in progress. The information obtained should indicate whether the transcription and/ or genome replication strategy utilized by plant virus members of the Reoviridae differ significantly from that determined for other members of this virus group. The availability of cloned WTV genome segments makes possible a wide range of new approaches for studying the molecular biology of insect transmitted plant viruses. By cloning the WTV genes into expression vectors that function in procaryotes, cultured vector cells, and plant cells, it will be possible to examine WTV gene products with respect to enzymatic and biological function, regulation of biosynthesis, pathogenicity, and post-transcriptional and post-translational modifications. The ability to alter specific nucleotide sequences in the WTV genome segments will also permit the detailed analysis of genome structure-function relationships. ACKNOWLEDGMENT This work was supported in part by NIH Grant lROl-AI 17613from the National Institute of Allergy and Infectious Disease, PHPJDHHS.

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ASAMIZU REFERENCES

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