133,276-288 (1984) VIROLOGY Variant dsRNAs Associated with Transmission-Defective Isolates of Wound Tumor Virus Represent Terminally Conserved Remnants of Genome Segments DONALD L. NUSS’ AND DENNIS SUMMERS Center for Laboratories and Research, New York State Department of Health, Albany, New York 1.%‘01 Received August 17, 1983; accepted December 22, 1985 Variant double-stranded RNAs are often associated with the genome of transmissiondefective isolates of wound tumor virus. These RNAs are replicated and packaged into virus particles in systemically infected plants and are transcribed in vitro by the virionassociated transcriptase. Direct physical evidence that the variant RNAs are remnants of particular WTV genome segments was provided by molecular hybridization studies. Subsequently, ribonuclease Tl digestion products of 3’-end-labeled genome and remnant RNAs were analyzed by one- and two-dimensional electrophoretic techniques. One-dimensional partial and complete digestion patterns were indistinguishable, indicating that the guanosine positions relative to the 3’ terminus of the corresponding strands of a particular genome segment and its remnant RNA are the same for at least 40 nucleotides from each end. Fingerprints of the 3’ terminal ribonuclease Tl-resistant fragments were identical, showing that the nucleotide composition of the 3’ terminal ends of the corresponding strands of a particular genome segment and its remnant RNA are also identical. WTV These results indicate that variant RNAs associated with transmission-defective isolates are formed by deletion of an internal portion (as much as 85%) of genomic RNA segments yielding terminally conserved genomic remnants that are functional with respect to transcription, replication, and packaging. INTRODUCTION m7G(5’)ppp(5’)Am . . . (Rhodes et cd, 1977; Nuss and Peterson, 1981b). Wound tumor virus replicates in a wide range of plant hosts and in several species of leafhoppers that act as vector (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 nonet al, pathogenic (Black, 1957; Hirumi 1967). Maintenance of WTV exclusively in the plant host by vegetative propagation of infected plants results in virus populations that are defective in transmission (Black et aZ., 1958; Reddy and Black, 1974). These virus populations are characterized by the inability to replicate either in leafhoppers or in cultured leafhopper cells that support replication of standard virus (Reddy and Black, 1974). However, trans- The genome of wound tumor virus (WTV), a member of the family Reoviridae, consists of 12 double-stranded (ds) RNA segments with a combined molecular weight of approximately 16 X lo6 (Reddy and Black, 19’73a). Purified WTV particles exhibit RNA polymerase activity (Black and Knight, 1970) that catalyzes the synthesis active of single-stranded translationally transcripts (Reddy et al, 1977; Nuss and Peterson, 1980) from each of the dsRNA genome segments (Nuss and Peterson, 1981a). Other virion-associated enzymatic activities include a mRNA guanyltransferase, a guanine-7-methyltransferase, and a mRNA 2’-O-methyltransferase that terminal are involved ends of 1 To whom reprint in modifying the 5’the transcripts to requests should 0042-6822/34 $3.00 Copyright All rights 0 1984 by Academic Press, Inc. of reproduction in any form reserved. mission-defective to induce tumors be addressed. 276 virus retains the ability in vegetatively propa- WTV TERMINALLY CONSERVED GENOME REMNANTS 277 gated, systemically infected plants (Reddy maintained in vector cell monolayers [AgaZZia ccmstrictu, line AC20 (Chiu and and Black, 1974; 1977). In 1974 Reddy and Black published an Black, 1967)] and purified by the method extensive electrophoretic analysis of the of Reddy and Black (Reddy and Black, genomes of transmission-deficient (sub- 1973b). Exvectorial WTV isolates originally vectorial) and transmission-defective (ex- obtained from L. M. Black were purified vectorial) WTV isolates (Reddy and Black, by the same procedure from root tumors freshly harvested from systemically in1974). Each isolate exhibited a distinctive electrophoretic pattern which included a fected sweet clover plants. Control and inreduction in the concentration of certain fected plants were maintained in Conviron genome segments and the appearance of El5 Chambers (Controlled Environment, variant dsRNA segments. Observing re- Inc., Pembina, N. D.) at 20”. Daily light ductions in the molar proportion of a par- cycles consisted of 8 hr of dark and 16 hr ticular genome segment and the concom- of light programmed to range from 185 to itant appearance of a variant RNA over 650 lux. When discussing exvectorial isolates of time, these investigators concluded that the variant RNAs were remnants of ge- WTV, it is helpful to define several terms nome segments and tentatively designated introduced by Black and co-workers during individual remnant RNAs as being derived their discovery and examination of these from specific genome segments. Mutation isolates. Although individual virus prepevents appeared to be restricted to only 4 arations may have consisted of mixed popof the WTV genome segments (segments ulations of transmissible (vectorial) and exvectorial virus particles, these popula1, 2, 5, and 7). Since these presumptive genome rem- tions were termed “isolates” because they nants are replicated and packaged into vi- were physically isolated from each other rus particles in systemically infected plants within a plant that was maintained by (Reddy and Black, 1974, 1977) and are vegetative propagation (Reddy and Black, transcribed in vitro by purified exvectorial 1974). That is, each virus population isolate particles (Nuss, 1983a, b), they must evolved in the isolated environment of the retain nucleotide sequences that are im- infected plant without the introduction of portant for efficient transcription, repliexogenous virus or the opportunity to repcation, and packaging of the WTV genome. licate in the insect vector. Using techniques They therefore represent potentially useful that allowed a determination of the genome tools with which to examine the role of pattern of virus particles derived from sinWTV genome structures in these events. gle plants, Reddy and Black (1977) selected In this communication we provide direct successive cuttings of systemically infected physical evidence that several variant plants containing virus populations that RNAs are derived from specific genome exhibited a progressive loss of genome segsegments and that their formation is a re- ments 2 or 5 and variant RNAs that may sult of an internal deletion event, i.e., they have been derived from these segments. In represent terminally conserved remnants this way viral isolates were obtained that of genome segments. (Some of the obser- appeared to be free of these segments: vations contained in this communication -S2(70), -S5(60), and -S5(64) [nomenclawere presented in preliminary form at the ture from Reddy and Black, 1977; the numInternational Symposium on Double- ber in parenthesis indicates the year in Stranded RNA Viruses, Frenchman’s Reef, which the vectorial parent virus was inU. S. Virgin Islands, October, 1982, and at troduced into sweet clover]. Other isolates the Banbury Conference on Plant Viruses were selected that contained 10% of the and Viroids, Cold Spring Harbor, Febru- normal complement of segment 1, 10% ary, 1983). S1(49), and 10% Sl(60) or a full complement of a presumptive remnant of segment 7 in MATERIALS AND METHODS place of the intact segment, MS7(57), where Virus preparation. Standard WTV [in- M signifies mutant. The nomenclature for oculum RB (Reddy and Black, 1972)] was these selected isolates as presented by 278 NUSS AND SUMMERS Reddy and Black (1977) will be retained even though the isolates have continued to evolve over the last 5 years in the absence of selection, as will be illustrated in this report. Purijkaticm, 3' end tkbeling, and isolation SSC), and spotted individually with the aid of a Hybri-Dot manifold (Bethesda Research Laboratories) onto nitrocellulose paper presoaked in 20X SSC. The spotted membrane was air-dried and baked under vacuum for 2 hr at 80”. of indiwidual genomic and remnant cl.9 Prehybridization and hybridization buffRNAs. Double-stranded genome RNA was ers were similar to those described by Thoreleased from purified standard or exvec- mas (1980) with the modification that 100 torial WTV particles by treatment with pg/ml of Torula RNA (Sigma Biochemical) 1% sodium dodecyl sulfate (SDS). The RNA replaced the sonicated salmon sperm DNA. was deproteinized by extraction with phe- Prehybridization was performed at 42” for nol/chloroform/isoamyl alcohol (25:24:1). 18-20 hr, while hybridization was perAlternatively, WTV genome RNA was pu- formed for 24 hr at 42” with 30,000to 60,000 rified directly from infected vector cells. cpm of heat-denatured, 3’-end-labeled Clarified lysates were adjusted to a final remnant RNA in a volume of 2.5 ml. Folconcentration of 25 mM Tris (pH 7.5), 100 lowing hybridization, the dot-blots were mM NaCI, 7.5 mlM ethylenediamine tet- washed four times, 30 min each, at room raacetate acid (EDTA), and 2.4% SDS, and temperature with 1X SSC, 0.2% SDS. Octreated with 208 pg/ml of Proteinase K for casionally, the blots were washed twice 30 min at 37” and then extracted with phe- more for 30 min each with 0.2X SSC, 0.2% nol/chloroform/isoamyl alcohol. The SDS at 68”. Blots were dried, covered with dsRNA was precipitated with ethanol, dis- Saran Wrap, and exposed to Kodak XARsolved in 10 mM Tris (pH 7.5), 10 mlMNaC1, 2 film. 10 mM MgCl,, and treated with 20 pg/ml Anal&s of d’-end-labeled RNAs. IndiDNase (Worthington) for 1 hr at 4”. After vidual 3’-end-labeled RNAs (20,000-40,000 one cycle of extraction with phenol/chlocpm) were denatured by incubation at 45” roform/isoamyl alcohol the dsRNA was in 90% Nz-flushed dimethyl sulfoxide consubjected to CF-11 chromatography, as taining 10 pg carrier tRNA for 30 min and described by Morris and Dodds (1979), to then precipitated with ethanol. The dried remove single-stranded RNA and DNA RNA pellets were dissolved in 10 ~1 of 10 degradation products. The purity of all mMTris (pH 7.4), 1 mMEDTA containing preparations was determined by electro- 0.005 units (partial digestion) or 5 units phoresis in 7.5% polyacrylamide gels (Nuss (complete digestion) of ribonuclease Tl. and Peterson, 1981a). Genome segments Incubation was at 37” for 15 min or 1 hr, were visualized by using the protocol sup- respectively. Partial alkaline hydrolysis of plied with the Bio-Rad silver staining kit end-labeled RNA was performed in half reactions containing 50 mM NaHC03, 1 (Merril et al, 1979). Purified RNA was labeled at the 3’ ter- mM EDTA (pH 9.0) at 90” for 15 and 30 mini with [5’-32P]pCp(Amersham Corp.) by min, respectively. The half reactions were the method of Bruce and Uhlenbeck (1978) pooled prior to gel electrophoresis. Olias described by Peattie (1979). To isolate gonucleotides were fractionated on a 0.75individual 3’-end-labeled dsRNA segments mm thick, 30-cm long, 20% polyacrylamide the reaction mixture was subjected to elec- gel containing 7 M urea, as described by trophoresis in a 30-cm, 7.5% polyacrylMaxam and Gilbert (1977). After electroamide gel for 5600 v-hr at room temper- phoresis, gels were exposed to Kodak XARature. Individual genome segments were 2 film at -70” using a DuPont Lightingvisualized by ethidium bromide staining, Plus intensifying screen. excised, and electroeluted. Fingerprinting of complete ribonuclease Dot hybridization, Gel-purified unlabeled Tl digest of 3’-end-labeled RNAs (50,000genome segments (40 ng in 10 ~1 of 2 mM 100,000cpm) was performed essentially as EDTA) were denatured by boiling 5 min, described by Barrel1 (1971). Electrophoquenched on ice, diluted with 9 volumes of resis on cellulose acetate strips (3 X 55 cm; 3 M NaCl, 0.3 M trisodium citrate (20x Schleicher and Schuell) was performed for WTV TERMINALLY CONSERVED 1 hr at 2500 V in 5% acetic acid/5 M urea/ 1 mM EDTA adjusted to pH 3.5 with pyridine. The oligonucleotides were transferred to plastic-backed polyethyleneimine cellulose thin layer plates (20 X 20 cm; Baker) previously washed in 2 M formic acid (pH 2.2) and chromatographed at 60” with homomixture C for 7.5-8 hr. After drying, the plates were exposed to Kodak XAR-2 film. The 3’ terminal nucleotide of individual oligonucleotides was determined by thinlayer chromatography of ribonuclease T2 digests. Oligonucleotides were eluted with 0.5 mM ammonium bicarbonate following fingerprint analysis or electroeluted from polyacrylamide gels after one-dimensional analysis, adsorbed to DEAE cellulose, and eluted with 0.5 mM ammonium bicarbonate. In both cases the ammonium bicarbonate was removed by repeated lyophilization. Ribonuclease T2 digestion was performed in 5 ~1 of 20 mM sodium citrate (pH 5.0) 1 mM EDTA with 0.1 unit of ribonuclease T2 at 37” for 2 hr. Chromatography was performed as described by Cashel et al. (1969) on polyethyleneimine cellulose thin-layer plates developed with 0.2 M KH,PO,. GENOME REMNANTS 279 A gallery of genome patterns for individual selected exvectorial isolates is presented in Fig. 1. Many of the presumptive remnant RNAs, and in one case an intact 1234567 Sl- s2s3Z' S6 s7 - S8 s9SlO SK s12 - RESULTS Genome Pattern of Exvectorial lates WTV Iso- In 1977 Ready and Black reported the selection of exvectorial isolates of WTV that lacked detectable levels of genome segments 2 or 5 or presumptive remnants of these segments, that contained 10% of the normal complement of segment 1 or that contained a presumptive remnant of segment 7 in place of the intact segment. These authors estimated that they could detect a dsRNA segment when present in as few as 0.1% of the virus particles in the population. Following the 1977 study, the selected isolates were maintained by vegetative propagation of the systemically infected plants without further selection. In 1982-1983 we reexamined the genome patterns of the isolates by 3’ end labeling of the purified genome segments with [“2PlpCp followed by gel electrophoretic analysis. FIG. 3. Gallery of genome profiles of WTV-exvec-torial isolates. Genome segments extracted from pu rified standard and selected exvectorial virus populations labeled at the 3’ ends with [5’-“P]pCp were analyzed in a 7.5% polyacrylamide gel. The genome segments are numbered (left) according to the convention established by Reddy and Black (19’73a). The dots indicate the position of presumptive remnants of genome segments observed among the genome segments of individual exvectorial virus populations. Lanes: 1, transmissible WTV; 2, MS’i’(57); 3, -S5(64): 4, -S5(60); 5, -S2(70); 6, 10% Sl(60); 7, 10% Sl(49). The nomenclature is taken from Reddy and Black (1977) as discussed in the text and designate the constitution with regard to a particular genome segment. For example -S5 and -S2 indicate “minus” segments 5 and 2 respectivey while 10% Sl indicates 10% the normal complement of segment 1 and MS7 indicates a mutated segment 7. The numbers in parenthesis indicate the year in which the vectorial parent virus was introduced into sweet clover (from Nuss, 1983a) 280 NUSS AND segment, that were undetectable in the 1977 study of the selected isolate, were present. It must be noted that many of these presumptive remnant RNAs were present in virus populations from which the exvectorial isolates were selected (Reddy and Black, 1974). The apparent increase of the presumptive remnant RNAs from less than one copy per 1000 particles in isolates examined in 1977 to the concentration observed in Fig. 1 suggests that in the absence of negative selection, these RNAs are efficiently (perhaps preferentially) replicated an/or packaged in the systemically infected plant. It was of some interest to provide phys- SUMMERS ical evidence that the presumptive remnant RNAs were derived from genome segments and to determine whether their formation involved an internal deletion or deletion of one or both termini. The following approach was considered a convenient way of distinguishing between these two mechanisms. After establishing the origin of a presumptive remnant to be a particular genome segment, 3’-end-labeled genome segments and their remnants would be denatured and digested with ribonuclease Tl. Since this nuclease cleaves specifically at the 3’ side of guanosine residues, partial digestion of the denatured dsRNAs would yield a series of 3’-end-labeled oligonucle- s4 L Sl S2 S3 & S6 S7 L S8 S9 SIO 31 S12 FIG. 2. Polyacrylamide gel electrophoretic pattern of partial ribonuclease Tl digests of [s2PfpCpend-labeled WTV genome segments Sl-S12. Lanes marked L contain the oligonucleotide ladder generated by partial alkali digestion of end-labeled genome segments 4 + 5. The migration positions of bromphenol blue (BPB) and xylene cyan01 (XC) markers are indicated. WTV TERMINALLY CONSERVED otides from each RNA strand. By analyzing these digestion products on 20% polyacrylamide sequencing gels (Maxam and Gilbert, 1977) against an alkali-generated oligonucleotide ladder, the guanosine positions relative to the 3’ terminus for each strand of a genome segment and its remnant RNA could be compared simultaneously. A similar pattern would indicate that the remnant RNAs were derived by internal deletions. This approach would be feasible only if the partial ribonuclease digestion pattern of each individual ge- GENOME REMNANTS FIG. 4. Dot hybridization of end-labeled 2d2.05 against isolated genome segments fixed to nitrocellulose. The positions of genome segments Sl-S12 are indicated by the numbers at the top. nome segment was distinct. As shown in Fig. 2, this is the case. Characterizatim 2d2 .05- -56 -57 - 56 -59 - 510 -511 -512 - 5d.26 FIG. 3. Polyacrylamide gel electrophoretic analysis of [32P]pCp-end-laheled total and isolated genomic RNAs from standard and exvectorial isolates of WTV. Lane 1, genome RNA of exvectorial isolate -S2(‘70). Lane 2, RNA 2d2.05 isolated from preparative gel of -S2(70) (see Reddy and Black, 1977 and text for nomenclatures). Lane 3, genome segments 4 + 5 also isolated from the -S2(70) preparative gel. Lane 4, genome segment 2 of standard virus. Lane 5, RNA 5d.26 isolated from a preparative gel of exvectorial isolate 10% Sl(49). Lane 6, genome RNA of 10% Sl(49). The position of genome segments and remnant RNAs are indicated in the margins (from Nuss, 1983a). 281 of Subgenomic RNAs Two presumptive remnant RNAs earlier suspected of having been derived from genome segments 1 and 2 (Reddy and Black, 19741977) were selected for analysis. The 3’-end-labeled presumptive remnant RNAs with estimated molecular weights of 2.05 x lo6 and 0.26 X lo6 (Reddy and Black, 1974) were separated from the other endlabeled WTV genomic RNAs of exvectorial isolates -S2(70) and 10% S1(49), respectively, by preparative polyacrylamide gel electrophoresis (Fig. 3). To physically determine the genome segments from which a presumptive remnant RNA was derived, the end-labeled, isolated, presumptive remnant RNAs were hybridized against individual gel-purified genome segments fixed to nitrocellulose. The presumptive remnant RNA previously reported to have originated form segment 2, the 2.05 X 106-Da RNA (Fig. 3, lane 2), hybridized to purified segment 2 and to a lesser extent to segment 4 + 5 (Fig. 4). This RNA migrates very near genome segment 4 + 5 during preparative gel electrophoresis (Fig. 3). One- and two-dimensional analysis of the ribonuclease Tl digestion products of the end-labeled presumptive remnant RNA preparation revealed a low-level of crosscontamination by end-labeled segments 4 + 5 (see Figs. 6 and 8), explaining the apparent hybridization of the end-labeled remnant RNA to segments 4 + 5. Following confirmation that the 2.05 X 10” molecular weight remnant RNA was derived from genome segment 2, it was designated 2d2.05 after the nomenclature of Reddy and Black (1974), with the mod- 282 NUSS AND SUMMERS A s4 Sl S2 S3 & S6 S7 S8 S9 SlO Sll S12 54.26 FIG. 5. Dot hybridization of end-labeled 5d.26 against isolated genome segments fixed to nitrocellulose (A). Hybridization of end-labeled 5d.26 against total genome RNA of standard virus, position 1; 10% S1(49), position 2; -S2(70), position 3; and 5d.26 RNA, position 4; is presented in B. ification that d stands for deletion rather than defective. That is, the designation 2d2.05 indicates a molecule of approximately 2.05 X lo6 molecular weight that was derived from genome segment 2 by a deletion event. The 0.26 X lo6 molecular weight presumptive remnant RNA (Fig. 3, lane 5) reported to be derived from genome segment 1 (Reddy and Black, 1977) hybridized exclusively to the unresolved mixture of genome segments 4 + 5 (Fig. 5). The origin of this remnant RNA was established as genome segment 5 by its reduced level of hybridization to genome segments 4 + 5 obtained from exvectorial isolates known to contain low levels of genome segment 5 (Nuss and Peterson, 1981a; NUSS,1983a, b) (Fig. 5). This remnant RNA was designated 5d.26. Once it was determined from which genome segments the remnant RNAs were derived, the ribonuclease Tl digestion patterns of the molecules were compared. As shown in Fig. 6, the partial ribonuclease Tl digestion patterns (lanes 2 and 3) for genome segment 2 and RNA 2d2.05 are indistinguishable for at least 40 nucleotides from each 3’ end. The one-dimensional patterns of the complete ribonuclease Tl digestion products (lanes 4 and 5) also are indistinguishable, except for the presence of oligonucleotides e and f among the remnant RNA digestion products. The 3’-terminal ends of the individual WTV genome segments were previously shown to contain equal amounts of cytidine and uridine (Lewandowski and Leppla, 1972). Oligonucleotides e and f correspond to the 3’-cytidine terminal fragments of genome segments 4 and 5, respectively, which contaminate the remnant RNA preparation. The 3’-uridine terminal fragments of segments 4 and 5 both migrate in the position of oligonucleotide b (see legend to Fig. 6). Similar results were obtained for the digestion pattern of genome segment 5 and RNA 5d.26 (Fig. 7). The analysis of these molecules was complicated by the difficulty of separating segment 5 from segment 4. This problem was resolved by hybrid-selection of end-labeled segment 5 from segment 4 by using unlabeled 5d.26 fixed to nitrocellulose. The level ,of contamination of segment 5 with segment 4 after hybridselection is indicated by the presence of oligonucleotide e. To ensure that the terminal ends of the genome segments were conserved when the remnant RNAs were formed, the 3’-terminal oligonucleotide fragments of the genome segments and the corresponding WTV TERMINALLY CONSERVED GENOME 283 REMNANTS Q FIG. 6. Polyacrylamide gel electrophoretic analysis of partial and complete ribonuclease Tl digests of [‘*P]pCp-end-labeled genome segment 2 and RNA 2d2.05. Lane 1, oligonucleotide ladder generated by partial alkaline digestion of end-labeled genome segments 4 + 5. Lane 2, partial digest of genome segment 2. Lane 3, partial digest of 2d2.05. Lanes 4 and 5, complete digests of segment 2 and 2d2.05, respectively. The migration positions of BPB and XC markers are indicated in the left margin. Indicated in the right margin is the position of 3’ terminal oligonucleotides of interest. Oligonucleotide a contains a 3’ terminal adenosine. Oligonucleotide b contains a 3’ terminal uridine and is obtained upon complete ribonuclease Tl digestion of each individual genome segment. Oligonucleotide c also contains a 3’ terminal uridine, while oligonucleotides d-f all contain a 3’ terminal cytidine. Oligonucleotide d is derived from genome segment 2, while oligonucleotide e and f are derived from genome segments 4 and 5, respectively. This information was obtained by performing one- and FIG. 7. Polyacrylamide gel electrophoretic analysis of partial and complete ribonuclease Tl digests of [arPjpCp-end-labeled, hybrid-selected genome segment 5 and RNA 5d.26. Lane 1, oligonueleotide ladder generated by partial alkaline digestion of end-labeled genome segments 4 and 5. Lane 2, partial digest of hybrid-selected genome segment 5. Lane 3, partial digestion of 5d.26. Lane 4, complete digest of hybridselected genome segment 5. Lane 5, complete digest of 5d.26. The position of oligonucleotides u-f which corresponds to the oligonucleotide described in Fig. 6 are shown on the right while the position of the BPB and XC markers are shown on the left. two-dimensional analyses of complete ribonuclease Tl digests of isolated individual genome segments including genome segments 4 and 5, which were sep-, arated by hybrid-selection. The positions of oligo-, nucleotides a, b, d, e, and f after two-dimensional analysis is shown in Fig. 8. 284 NUSS AND SUMMERS remnant RNAs were subjected to two-di- the 3’-terminal oligonucleotides of genome mensional fingerprint analysis. As ex- segments 4 + 5 (Fig. 8C). The fingerprint of RNA 5d.26 is identical to that of segpected, the fingerprints of the 3’-terminal oligonucleotides of genome segment 2 and ments 4 + 5 except that it lacks oligonuremnant 2d2.05 are identical, except for cleotide e-a 3’-terminal oligonucleotide of the presence of 3’-terminal oligonucleotide segment 4. For other members of the Reoviridae, fragments of contaminating genome segment 4 (oligonucleotide e) and segment 5 the genome segments of a particular virus (oligonucleotide f) present in the 2d2.05 have common terminal base sequences RNA preparation (Figs. 8A and B). Note (Darzynkiewica and Shatkin, 1980; Li et these oligonucleotides in the fingerprint of al, 1980; McCrae, 1981; Rao et al, 1983; FIG. 8. Fingerprint of 3’ terminal Tl ribonuclease-resistant fragments of genome segment 2 (A), RNA 2d2.05 (B), segments 4 + 5 (C), and RNA 5d.26 (D). Again oligonucleotides a, b, d, e, andf correspond to the oligonucleotides described in Fig. 6. The dotted circle indicates the position of BPB marker. The first dimension involved electrophoresis on cellulose acetate at pH 3.5, and the second dimension involved chromatography on polyethyleneimine cellulose thin-layer plates in homomixture C. WTV TERMINALLY CONSERVED Kuchino et aZ., 1982; McCrae and McCorquodale, 1983). 3’-Uridine terminal oligonucleotide b (Figs. 6-8) is generated by completed ribonuclease Tl digestion of each of the 12 WTV genome segments and apparently represents a conserved sequence common to one end of each WTV genome segment. Oligonucleotides a and c are minor oligonucleotides that are also generated upon complete ribonuclease Tl digestion of each WTV genome segment. These oligonucleotides may represent a minor degree of heterogeneity naturally existing in the population or generated during extraction of the dsRNA segments from virus particles or infected cell lysates. Sequence analysis of the 3’ and 5’ ends of both strands of each WTV genome segment is now in progress. DISCUSSION Variant dsRNAs have been observed in genome RNA preparations of other segmented dsRNA viruses: human reovirus (Ahmed and Fields, 1981;Brown et aL, 1983) and Saccharcmyces cerevisiae virus, ScV (Vodkin et aL, 1974; Tzen et aL, 1974). Base sequence analysis has established that ScV variant RNAs are derived from genome RNA by internal deletion events (Bruenn and Brennan, 1980), while the variant RNAs associated with human reovirus have not been characterized. The results for wound tumor virus presented in this report represent the first direct evidence that variant RNAs associated with the genome of a member of the family Reoviridae are terminally conserved remnants (TCRs) of genome segments. Generation of the terminally conserved remnant 2d2.05 involves the deletion of 15% of genome segment 2, while 85% of genome segment 5 is deleted to form TCR-5d.26. Genome segment 5 encodes the 76,000 molecular weight component of the outer protein coat of WTV (Nuss, 1983a). With approximately 85% of the genome deleted, it is unlikely that TCR-5d.26 would contain sequences specifying a functional polypeptide. In this regard, Reddy and Black (1977) have reported that the isolate -S2(70), which contains TCR-2d2.05 in place of seg- GENOME REMNANTS 285 ment 2, lacks any detectable product of genome segment 2, the 130,000 molecular weight component of the outer protein coat. However, the possibility that TCR-2d2.05 codes for a polypeptide that is functionally unable to associate with virus particles has not been ruled out. Polypeptides which migrate differently than the standard gene products on polyacrylamide gels and which are of a size corresponding to that predicted by the size of several remnant RNAs have been observed among the cell-free translation products of several exvectorial isolates (Nuss, unpublished observation). This is not surprising since, as indicated by the ribonuclease Tl patterns, the deletion boundaries for the TCRs analyzed in this report are located more than 40 base pairs from either end, i.e., the ribosome binding site and initiation codon could be conserved (Kozak and Shatkin, 1978). Terminally conserved remnant 5d.26 is associated with an exvectorial isolate that evolved during vegetative propagation of a sweet clover plant inoculated in 1949. The standard virus population from which genome segment 5 was prepared for comparison to TCR-5d.26 has a quite different history. The standard virus inoculum (RB) prepared in 1972 (Reddy and Black, 1972) was used directly from the freezer to infect AC20 cell monolayers in 1983. Genome RNA was then purified from the infected cells after 10 or 20 cell passages. Given the different histories of these two dsRNA segments, it is surprising that no divergence in the guanosine position relative to the 3’ termini is observed in the one-dimensional Tl digestion pattern. Similarly, the one- and two-dimensional fingerprints of TCR-2d2.05 and genome segment 2 obtained from either exvectorial isolate 10% Sl(49) or standard virus were indistinguishable. In several respects WTV exvectorial isolates resemble the defective interfering (DI) particles described for human reovirus. Both the WTV exvectorial isolates and the reovirus DI particles lack certain genome segments (Reddy and Black, 1974, 1977; Nonoyama and Graham, 1970; Nonoyama et al, 1970; Schuerch et ak, 1974; NUSS AND SUMMERS 286 Spandidos and Graham, 1976; Ahmed and Graham, 1977) and often contain variant RNAs (Reddy and Black, 1974; Ahmed and Graham 1977; Ahmed and Fields, 1981). Furthermore WTV exvectorial isolates have been reported to interfere with standard virus infection of vector cells (unpublished observation cited in Reddy and Black, 1977). Both the DI particles and WTV exvectorial isolates are transcriptionally active in vitro and in vivo (Spandidos et aL, 1976; Nuss and Peterson, 1981b; Nuss, 1983b). The possibility that the exvectorial isolates act as DI particles in the vector cell but not in the plant host is now being explored. The occurrence of homologous terminal sequences at the respective ends of genome segments appears to be a general phenomenon for viruses that have segmented single stranded or double stranded RNA genomes (Dasgupta and Kaesberg, 1977; Symons, 1979; Darzynkiewica and Shatkin, 1980; Li et al, 1980; McCrae, 1981; Kuchino et aL, 1982; Rao et al, 1983; Desselberger et aL, 1980; Iba et ok, 1982; Bruenn and Brennan, 1980; McCrae and McCorquodale, 1983). This feature has obviously led to predictions that the terminal sequences play a critical role in genome transcription, replication, and packaging. The fact that the terminal sequences of the WTV genome segments are conserved in the formation of TCRs-a situation similar to that of influenza DI RNA (Davis et czL,1980)-adds emphasis to that prediction. Presumably WTV remnant RNAs generated by deletion of one end of the genome fail to replicate or are not packaged, resulting in the loss of the complete segment, while remnant RNAs with conserved termini are perpetuated. It will be instructive to determine the minimal sequence requirements of a functional WTV terminally conserved genome remnant. ACKNOWLEDGMENT This work was supported in part by NIH Grant lROl-AI 17613from the National Institute of Allergy and Infectious Disease, PHS/DHHS. REFERENCES AHMED, R., and FIELDS, B. N. (1981). Reassortment of genome segments between reovirus defective interfering particles and infectious virus: Construction of temperature-sensitive and attenuated viruses by rescue of mutations from DI particles. Virology 111,351-363. AHMED, R., and GRAHAM,A. F. (1977). Persistent infections in L cells with temperature-sensitive mutants of reovirus. J. viral. 23, 250-262. BARRELL, B. G. (1971). Fractionation and sequence analysis of radioactive nucleotides. In “Procedures in Nucleic Acids Research” (G. L. Cantoni and D. R. Davies, eds.), Vol. 2, pp. 751-779. Harper and Row, New York. BLACK, L. M. (1957). Viruses and other pathogenic agents in plant tissue cultures. J. Nat Cancer Inst. 19, 663-685. BLACK, L. M. (1965). Physiology of virus-induced tumors in plants. In “Encyclopedia of Plant Physiology” (W. Ruhland, E. Ashby, J. Bonner, M. GeigerHuber, W. 0. James, A. Lang, D. Mullerant, and M. G. Stalfelt, eds.), Vol. 15, pp. 236-266. Springer, New York. BLACK,L. M. (1969). Insect tissue cultures as tools in plant virus research. Annu Rev. Phytopat?wl7,73100. BLACK, D. R., and KNIGHT, C. A. (1970). Ribonucleic acid transcriptase activity in purified wound tumor virus. J. l&o1 6, 194-198. BLACK.L. M., WOLCYRZ,S., and WHITCOMB,R. F. (1958). A vectorless strain of wound tumor virus, in Ab &acts, p. 255, 7th International Congress for Microbiology, Stockholm. BRUCE,A. G., and UHLENBECK,0. C. (1978). Reactions at the termini of tRNA with T4 RNA ligase. Nucleic Acids Res. 5, 3665-3677. BRUENN, J. A., and BRENNAN,V. E. (1980). Yeast viral double-stranded RNAs have h&erogenous 3’ termini. Cell 19, 923-933. BROWN, E. G., NIBERT, M. L, and FIELDS,B. N. (1983). The L2 gene of reovirus serotype 3 controls the capacity to interfere, accumulate deletions and establish persistent infection. In “Double Stranded RNA Viruses” (D. H. L. Bishop and R. W. Cornpans, eds.), pp. 274-287. Elsevier, Amsterdam. CASHEL, M., LAZZARINI, R. A., and KALBACHEN, B. (1968). An improved method for thin-layer chromatography of nucleotide mixtures containing =P labeled orthophosphate. .J. Chmndogr. 46,103-109. CHIU, R. J., and BLACK, L. M. (1967). Monolayer cultures of insect cell lines and their inoculation with a plant virus. Nature (London) 215, 1076-1078. DARZYNKIEWICZ,E., and SHATKIN, A. J. (1980). Assignment of reovirus mRNA ribosome binding sites WTV TERMINALLY CONSERVED to virion genome segments by nucleotide sequence analysis. Nucleic Acids Res. 3, 337-350. DASGUPTA, R., and KAESBERG, P. (1977). Sequence of an oligonucleotide derived from the 3’ end of each of the four brome mosaic virus RNAs. Proc. Nat. Acad Soi, USA 74.4900-4904. DAVIS, A. R., HITI, A. L., and NAYAK, D. P. (1980). Influenza defective interfering viral RNA is formed by internal deletion of genomic RNA. Proc Nat. Ad Sti USA 77,215-219. DESSELBERGER, U., RACANIELLO, V. R., ZAZRA, J. J., and PALESE, P. (1980). The 3’- and 5’-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene 8, 315-328. HIRUMI, H. GRANADOS, R. R., and MARAMOROSCH, F. (1967). Electron microscopy of a plant-pathogenic virus in the nervous system of its insect vector. J. ViroL 1, 430-444. IBA, H., WATANABE, T., EMO, Y., and OKADA, Y. (1982). Three double-stranded RNA genome segments of bacteriophage have homologous terminal sequences. FEBS Z&t. 141,111-115. KOZAK, M., and SHATKIN, A. J. (1978). Identification of features in 5’-terminal fragments from reovirus mRNA which are important for ribosome binding. Cell 13, 201-212. KUCHINO, Y., NISHIMURA, S., SMITH, R. E., and FuRUICHI, Y. (1982). Homologous terminal sequences in the double-stranded RNA genome segments of cytoplasmic polyhedrosis virus of the silkworm Bmbyx mm-i. J. Viral 44, 538-543. LEWANDOWSKI, L. J., and LEPPLA, S. H. (1972). Comparison of the 3’-termini of discrete segments of the double-stranded ribonucleic acid genomes of cytoplasmic polyhedrosis virus, wound tumor virus, and reovirus. J. ViroL 10, 965-968. LI, J. K. K., KEENE, J. D., SCHEIBLE, P. O., and JOKLIK, W. (1980). Nature of 3’-terminal sequences of the plus and minus strands of the Sl gene of reovirus serotype 1, 2, and 3. Virology 105,393-403. MAXAM, A. M., and GILBERT, W. (1977). A new method for sequencing DNA. Proc. Nat. Acad. Sci. USA 74, 560-564. MCCRAE, M. A. (1981). Terminal structure of reovirus RNA’s, J. Gen Viral. 55, 393-403. MCCRAE, M. A., and MC~ORQUODALE, J. G. (1983). Molecular biology of rotaviruses. V. Terminal structure of viral RNA species. Virology 126, 204212. MERRIL, C. R., SWITZER, R. C., and VANKEUREN, M. L. (1979). Trace polypeptides in cellular extracts and human body fluids detected by two-dimensional electrophoresis and a highly sensitive silver stain. Proc Nat. Ad SC% USA 76,4335-4339. MORRIS, T. J., and DODDS, J. A. (1979). Isolation and analysis of double-stranded RNA from virus-in- GENOME REMNANTS 287 fected plant and fungal tissue. Phytopathobgy 69, 854-858. NONOYAMA, M., and GRAHAM, A. F. (1970). Appearance of defective virions in clones of reovirus. J. ViroL 6, 693-694. NONOYAMA, M., WATANABE, Y., and GRAHAM, A. F. (1970). Defective virions in reovirus. J. Viral 6,226236. Nuss, D. L. (1983a). Molecular biology of wound tumor virus transmission: Genome segment 5 and the loss of transmissibility. In “Double Stranded RNA Viruses” (D. H. L. Bishop and R. W. Compans, eds.), pp. 415-423. Elsevier, Amsterdam. Nuss, D. L. (1983b). Characterization of subgenomic RNAs associated with exvectorial isolates of wound tumor virus. In “Plant Infections Agents: Viruses, Viroids, Virusoids, and Satellites” (H. D. Robertson, S. H. Howell, M. Zaitlin, and R. L. Malmberg, eds.), pp. 111-116. Cold Spring Harbor Laboratory, New York. Nuss, D. L., and PETERSON, A. J. (1980). Expression of wound tumor virus gene products in viva and in vitro. J. ViroL 34, 532-541. Nuss, D. L., and PETERSON, A. J. (1981a). Resolution and genome assignment of mRNA transcripts synthesized in vitro by wound tumor virus. Virology 114, 399-404. Nuss, D. L., and PETERSON, A. J. (1981b). In vitro synthesis and modification of mRNA by exvectorial isolates of wound tumor virus. J. ViroL 39,954-957. PEATTIE, D. A. (1979). Direct chemical method for sequencing RNA. Proc Nat. Acad Sci. USA 76,17601769. RAO, C. D., KIUCHI, A., and ROY, P. (1983). Homologous terminal sequences of the genome double-stranded RNAs of Bluetongue Virus. J. ViroL 46,378-383. REDDY, D. V. R., and BLACK, L. M. (1972). Increase of wound tumor virus in leafhoppers as assayed on vector cell monolayers. viro.!ogy 50, 412-421. REDDY, D. V. R., and BLACK, L. M. (1973a). Electrophoretic separation of all components of the doublestranded RNA of wound tumor virus. Virology 54, 557-562. REDDY, D. V. R., and BLACK, L. M. (1973b). Estimate of absolute specific infectivity of wound tumor virus purified with polyethylene glycol. Virology 54,150159. REDDY, D. V. R., and BLACK, L. M. (1974). Deletion mutations of the genome segments of wound tumor virus. Virology 61. 458-473. REDDY, D. V. R., RHODES, D. P., LESNAW, J. A., MACLEOD, R., BANERJEE, A. K., and BLACK, L. M. (1977). In vitro transcription of wound tumor virus RNA by virion-associated RNA transcriptase. Virology 80, 356-361. REDDY, D. V. R., and BLACK, L. M. (1977). Isolation and replication of mutant populations of wound NUSS 288 AND tumor virions lacking certain genome segments. Virobgy 80, 336-346. REDDY,D. V. R., and MACLEOD,R. (1976). Polypeptide components of wound tumor virus. View 20,274282. RHODES,D. P., REDDY,D. V. R., MACLEOD,R., BLACK, L. M., and BANERJJZE, A. K. (1977).In vitro synthesis of RNA containing 5’-terminal structure 7,G(5’)ppp(5’)Apm. . . by purified wound tumor virus. Virology 76, 554-559. SCHUERCH,A. R., MATSUHISA,T., and JOKLIK, W. K. (1974). Temperature sensitive mutants of reovirus. Intervirolcgy 3, 36-45. SPANDIDOS,D. A., and GRAHAM, A. F. (1976). Generation of defective virus after infection of newborn rats with reovirus. J. Viral. 18, 7-19. SPANDIDOS,D. A., KRYSTAL, G., and GRAHAM, A. F. SUMMERS (1976). Regulated transcriptions of the genome of defective virions and temperature-sensitive mutants of reovirus. J. Viral. 20, 234-247. SYMONS,R. H. (1979). Extensive sequence homology at the 3’-termini of four RNAs of cucumber mosaic virus. Nucleic Acids Res. 7, 825-837. THOMAS,P. S. (1980).Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat. Acad Sk USA 77, 5201-5205. TZEN,J. C., SOMERS,J. M., and MITCHELL,D. L. (1974). A ds-RNA analysis of suppressive sensitive mutants of “killer” Saccharomyces cereuisiae. Heredity 33, 132-139. VODKIN, M., KATTERMAN,F., and FINK, G. R. (1974). Yeast killer mutants with altered double-stranded ribonucleic acid. J. i3octil. 117, 681-686.