Defective interfering RNAs and defective viruses associated with multipartite RNA viruses of plants

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1 seminars in VIROLOGY, Vol 7, 1996: pp Defective interfering RNAs and defective viruses associated with multipartite RNA viruses of plants Michael V. Graves*, Judit Pogany and Javier Romero Defective interfering (DI) RNAs and defective viruses have been described for a variety of multipartite RNA viruses of plants. At present, the DI RNAs of broad bean mottle bromovirus (BBMV) have been the most characterized defective elements of multipartite viruses. Several naturally occurring and artificial DI RNAs derived from BBMV RNA2 have been studied to determine the minimum requirements in the RNA sequence and the presence of an open reading frame (ORF) for efficient accumulation of the defective molecule. Sequence/structural elements of BBMV RNA that may be involved in DI RNA formation have been analysed as well. Additional DI RNAs found associated with members of the Bromoviridae family and other virus groups as well as defective viruses are also described. Several general features of DI RNAs and defective viruses derived from multipartite RNA viruses of plants are becoming apparent. In general, for a given virus, the defective element appears to be derived preferentially from only one of the RNA components. DI RNAs are formed via simple, single deletions that preserve some form of an ORF. Finally, when selective conditions have been altered, multipartite RNA viruses can form defective viruses quite rapidly by deleting substantial portions of the viral genome. This last point indicates that the entire viral genome is subject to continual selection pressure. Key words: defective viruses / DI RNAs / multipartite plant RNA viruses 1996 Academic Press Ltd INFECTIONS BY RNA viruses are frequently accompanied by subviral particles such as satellite RNAs, satellite viruses, defective interfering RNAs, defective viruses, and chimeric RNAs. Satellite RNAs and viruses require a complete helper virus for infectivity, but share little sequence similarity with the viral genomic From the Plant Molecular Biology Center, Northern Illinois University, DeKalb, IL, USA and Laboratory of Plant Virology, CIT-INIA, Madrid, Spain *Present address: Department of Plant Pathology, 406 Plant Sciences Hall, University of Nebraska-Lincoln, Lincoln, NE , USA 1996 Academic Press Ltd /96/ $25.00/0 RNA(s). In contrast, defective interfering RNAs (DI RNAs) are derived from the helper virus genome, but still require a complete helper virus. Furthermore, DI RNAs typically interfere with helper virus accumulation and affect the symptoms produced by the helper virus. However, there are cases in which the defective element has no apparent effect on the helper virus and is designated as a D RNA. Defective viruses are also derived from the helper virus, but the presence of the complete helper virus is only required under certain conditions (e.g. vector transmission). Chimeric RNAs are recombinant molecules that contain regions of sequence derived from the helper virus and regions derived from other sources. This review is focused on DI RNAs and defective viruses derived from plant RNA viruses with multipartite genomes. DI RNAs of plant RNA viruses with monopartite genomes are discussed elsewhere in this issue. DI RNAs are often associated with animal virus infections and they play an important role in controlling disease severity by attenuating the symptoms produced by the helper virus. 1 In contrast to animal viruses, DI RNAs were not isolated from plant virus infections for some time in spite of extensive efforts. Therefore, it was assumed that plant RNA viruses were not able to generate and/or maintain defective RNAs. This theory was proved incorrect by Hillman et al 2 when they described DI RNAs associated with tomato bushy stunt tombusvirus. Subsequently, DI RNAs have been found associated with several different plant virus groups including Carmovirus, 3 Potexvirus, 4,5 Bromovirus, 6 Tospovirus, 7,8 and Cucumovirus 9 suggesting that the existence of DI RNAs is the rule rather than the exception in viral infections. Furthermore, defective viruses associated with several plant RNA virus groups have also been described including Reovirus, 10,11 Tospovirus, 7,8,12 Furovirus, and Tobravirus The DI RNAs that have been found associated with plant RNA virus infections have been of two basic types. In the first type, the defective RNA consisted of a mosaic of the parental virus genome. Such was the 399

2 M. V. Graves et al case for the DI RNAs associated with tomato bushy stunt tombusvirus, 2 cymbidium ringspot tombusvirus, 25 cucumber necrosis tombusvirus, 26 and turnip crinkle carmovirus. 3 In the second type, the defective RNA was derived by single internal deletions and includes the D RNAs associated with clover yellow mosaic potexvirus, 4 cucumber mosaic cucumovirus, 9 alfalfa mosaic alfalmovirus, 27 and the DI RNAs of broad bean mottle bromovirus, 6 and tomato spotted wilt tospovirus. 7,8 It is interesting to note that all the defective RNAs so far isolated from multipartite RNA virus infections belong to the second group. In the first section, we review DI RNAs and will focus primarily on the Bromoviridae family, especially the DI RNAs of broad bean mottle bromovirus which are the most characterized DI RNAs of multipartite RNA plant viruses. The second section will focus upon defective viruses. Defective-interfering (DI) RNAs Family: Bromoviridae The family Bromoviridae includes the genera Bromovirus, Cucumovirus, Alfalmovirus, and Ilarvirus that all consist of tripartite, single-stranded, messenger sense RNA genomes. In general, RNAs 1 and 2 are monocistronic, encoding for the 1a and 2a proteins, respectively, that, together with host factors, form the viral replicase complex. The 1a protein contains methyl transferase and helicase domains while the 2a protein contains the RNA polymerase domain RNA 3 is dicistronic and encodes for the cell-to-cell movement protein (3a) and the coat protein (CP) that is expressed from subgenomic RNA 4. 29,31,32 Defective RNAs have been found associated with the Alfalmovirus, Bromovirus, and Cucumovirus groups. 6,9,27 These so-called naturally-occurring or wild type (wt) defective RNAs were derived from wt virus isolates and were dependent upon the presence of a complete helper virus genome for replication in planta. The defective RNAs associated with alfalfa mosaic alfalmovirus (AlMV) and cucumber mosaic cucumovirus (CMV) were derived from the RNA 3 component and had no apparent effect on symptom formation or viral RNA accumulation in the studied hosts. 9,27 The defective RNAs of broad bean mottle bromovirus (BBMV) were derived from RNA 2 and were able to interfere with viral accumulation, decreasing the concentration of the RNA 2 component in both the virion and total RNA preparations. 6 Furthermore, BBMV DI RNAs increased disease severity in pea, but not in other hosts. De-novo generation of defective RNAs has also been demonstrated for BBMV and CMV by serial passage of defective RNAfree wt virus isolates and viruses produced in vitro from cdna clones. 33,34 In addition to the naturallyoccurring and de-novo-generated defective RNAs, artificial defective RNAs have been constructed for brome mosaic bromovirus (BMV) and BBMV. (M. Babin et al, unpublished observations and refs 35-38). The wt DI RNAs isolated from BBMV were derived from RNA 2 by single deletions from the 2a ORF, whereas CMV D RNAs were derived from RNA 3 by single deletions from the 3a ORF (Figure 1, A and C). 6,9,34 The exact location of the deletion site(s) of the AlMV RNA 3 derived D RNAs is unknown. 27 Since RNA 3 is dicistronic, the D RNAs of CMV still encode for a functional CP in addition to the internally deleted, and therefore nonfunctional, 3a movement protein. 9 In contrast, BBMV DI RNAs were derived by internal deletions from the conserved region of the 2a ORF and, in some cases, the deleted region included the GDD motif characteristic of RNA polymerases 39,40, it therefore being likely that they do not encode functional polymerase proteins. This is supported by the observations of Traynor et al 30 that mutants of BMV RNA 2, with deletions in the corresponding location, did not support viral RNA replication in protoplasts in the presence of the other replicase component (1a protein). The deletions from CMV RNA 3 and BBMV RNA 2 that formed the defective RNAs occurred such that a reading frame was maintained downstream of the deletion junction. This suggests that efficient translation may be necessary for the accumulation of the defective RNAs. 6,9,34,37 Since the RNA 3 intercistronic region and CP ORF were also maintained in the CMV D RNAs, transcription of subgenomic RNA 4 may be important for CMV D RNA accumulation. Furthermore, sequences in the intercistronic region of BMV RNA 3 have been shown to be involved in plus-strand synthesis and asymmetric replication 41,42 and such sequences may also exist in CMV RNA 3. Therefore, the conservation of such sequences in the CMV D RNAs may be necessary for their efficient accumulation. Factors affecting the formation and accumulation of BBMV DI RNAs Fifteen wt and de-novo-generated BBMV RNA 400

3 Defective RNAs of multipartite plant viruses 2-derived DI RNAs have been characterized. 6,34 All of the DI RNAs arose by single deletion events except one of the de-novo-generated DI RNAs which was formed by a double deletion. Analysis of the sequences flanking the junction sites in BBMV DI RNAs revealed the presence of either short complementary and/or similar sequences. 34 To study the mechanism of DI RNA generation a 60-nt long sequence was duplicated in a DI RNA in such a way that it created either internal sequence similarity or sequence complementarity between distal parts of the molecule. 38 It was found that the artificial DI RNA containing the sequence duplication in the direct orientation was stable and did not produce additional deleted forms. In contrast, the artificial DI RNA containing the sequence duplication in reverse orientation was not detectable in systemic infections, but smaller DI RNAs, that were derived from the parental artificial DI RNA, did accumulate in several plants. The locations of the junction sites in these DI RNAs were located either very close to positions corresponding to the base of the proposed stem structure formed by the base pairing between the 60 nt sequence and its complement or were shifted to more upstream or Figure 1. Defective RNAs derived from members of the Bromoviridae. A schematic representation of BBMV and CMV defective RNAs. The 5' and 3' untranslated regions are shown by black lines. The 2a and 3a ORFs are represented by shaded boxes while the CP ORF is represented by a white box. The approximate location of the conserved GDD motif in the 2a ORF is indicated by * A scale bar (in nt) is located at the very top of the figure. (A) BBMV genomic RNA 2 (top) and wt DI RNAs (bottom). The numbered lines above the DI RNA indicate the minimum 5' and 3' sequences conserved in all the wt DI RNAs. The size range of the deletions is also given above the DI RNA. The top numbered line below the DI RNA indicates the length of the 2a ORF as compared to the entire length of the DI RNA. The bottom numbered line indicates the range in the total lengths of the wt DI RNAs. (B) BBMV artificial DI RNAs. The artificial DI RNA is labeled as in B. The data reflects only those DI RNAs that accumulated efficiently in plants. (C) CMV genomic RNA 3 (top) and wt D RNA 3β (bottom). The region deleted from the 3a ORF is indicated. 401

4 M. V. Graves et al downstream regions. These results suggest that sequence complementarity and/or secondary structures formed between complementary regions can facilitate DI RNA formation in BBMV. There are at least three factors that influence the accumulation of BBMV DI RNAs in planta: (i) the presence of terminal regions, (ii) overall size, and (iii) coding capacity. 37 The BBMV wt DI RNAs retained the 5'-terminal 1152 nt and 3'-terminal 468 nt and the total length of the deleted sequences was limited to between nt (15 30% of wt RNA 2) (Figure 1A). 6 Studies using artificial BBMV DI RNAs have shown that the 5' 883 nt and 3' 387 nt are required for DI RNA accumulation in plants. Also, artificial DI RNAs had to be at least 59% (1712 nt) (Figure 1B) of the size of wt RNA 2 to accumulate to detectable levels in plants and 66% (1911 nt) for accumulation similar to the levels of the wt DI RNAs. 37 All the wt and de-novo-generated BBMV DI RNAs maintained an ORF (designated 2a) that represented at least 79% of the DI RNA molecule, suggesting that either coding capacity or the 2a protein is important for DI RNA accumulation. Artificial DI RNAs with variable coding capacities, produced by inserting frameshift mutations into the 2a ORF, revealed that those with ORFs representing less than 64% of the DI RNA accumulated to a reduced level in planta or reverted to a wt-like ORF by a single nucleotide deletion. 37 One can speculate that the unusually long 3' untranslated sequences present in these molecules are disadvantageous for their accumulation e.g. by enhancing the access of RNases to the 3' portion of the DI RNAs. All the above data demonstrate the genetic plasticity of RNA2-derived DI RNAs. Effect of the DI RNA on BBMV symptoms and RNA accumulation When DI RNAs were present, the symptoms produced by the BBMV helper virus appeared one or two days earlier than normal. Furthermore, viral RNA replication and accumulation in the inoculated and systemically infected leaves increased in the presence of DI RNAs that contained an ORF. Finally, the coding capacity of the DI RNA 2a ORF is important for DI RNA accumulation, but had no effect on the accumulation of the viral RNAs (M. Babin et al, unpublished observations). Artificial DI RNAs of BMV A set of artificial defective RNAs was constructed by deleting sequences from the 2a ORF of BMV RNA 2 35,36 and was tested in protoplasts for their effect on the replication of the genomic RNAs. The artificial DI RNAs were divided into three classes. The first group consisted of DI RNAs that were replicated efficiently and interfered with the accumulation of the helper virus. Artificial DI RNAs belonging to the second group were not replicated in protoplasts and did not influence the replication of the genomic RNAs. The third group contained one artificial DI RNA that was not replicated, but still interfered with the accumulation of the helper virus. The requirement for the terminal sequences and overall size for efficient accumulation of the artificial BMV DI RNAs in protoplasts were similar to those for BBMV DI RNAs in plants. By using replication-deficient artificial DI RNAs, it has been demonstrated that there is a correlation between the replicative ability and interference properties of the DI RNA. Defective molecules that were replicated to lower levels interfered to a lesser extent with the helper viral RNAs. 36 The elimination of the ORF from the non-replicating but interfering DI RNA did not influence its ability to interfere with the helper virus suggesting that interference was not due to the presence of the truncated 2a protein. 36 The role of the host in accumulation and systemic spread of BBMV and CMV defective RNAs The accumulation and encapsidation of BBMV DI RNAs are specific to the host. The DI RNAs did not accumulate in nor were encapsidated in BBMV local lesion hosts or in some systemic hosts (J. Romero et al, unpublished observations and ref 6). Attempts to produce DI RNAs de novo in hosts that do not encapsidate DI RNAs have been unsuccessful through 15 passages (J. Romero et al, unpublished observations). Although little is known about the sequence and/or structural requirements involved in the formation and accumulation of CMV D RNAs, it has been shown that they are maintained in a host and tissue-specific manner. 43 Among the various Nicotiana species tested, the CMV D RNAs accumulated efficiently and were encapsidated in the inoculated and systematically infected tissues. However, in another solanacious host, tomato, and in two cucurbit hosts, zucchini squash 402

5 Defective RNAs of multipartite plant viruses and muskmelon, the D RNAs only accumulated in the inoculated tissue and did not move systemically although the virus did invade the systemic tissue in the normal fashion. Furthermore, in tomato and zucchini squash, the D RNAs accumulated in both the cotyledon and leaf tissue when they were directly inoculated, but the D RNAs, accumulated only in the inoculated cotyledons and not the inoculated leaves of muskmelon. The CMV D RNAs were encapsidated in both cotyledons and leaves of zucchini squash but not in the muskmelon cotyledons. These results suggest that there is host and tissue-specific involvement in replication, cell-to-cell movement, systemic movement and encapsidation of the CMV D RNAs and that encapsidation is not sufficient for D RNA systemic spread (Graves et al, unpublished observations and ref 43). Family: Bunyaviridae Tomato spotted wilt tospovirus (TSWV) is a tripartite, ssrna virus consisting of a genome of both ambisense (S RNA) and negative polarity (L and M) RNAs 44,45 and is transmitted by thrips. 46 The virus encodes at least five protein products: the nucleocapsid and a non-structural protein encoded on the S RNA, 44 the putative viral polymerase encoded by the L RNA 45 and two viral envelope glycoproteins encoded by the M RNA. 45,47 Two different types of defective TSWV isolates have been described; those that have lost the ability to form enveloped particles, but still produce infectious nucleocapsid particles (see section on defective viruses) and those that contain DI RNAs. Resende et al, 7 reported the presence of additional RNAs associated with TSWV after serial passage. These RNAs were derived from the L segment by deletions of various lengths and were packaged by the nucleocapsid protein. Furthermore, they were found in mature virus particles and could be detected in both polarities in infected tissue. The presence of these RNAs correlated with a reduction in the accumulation of wt L RNA as well as a reduced symptom phenotype. Thus, these RNAs fit the definition of a DI RNA. Subsequent analysis of these DI RNAs 8 indicated that they were derived by deletions from the putative polymerase ORF of from 2.0 to 3.4 kb and that all maintained the 5' and 3' termini of the L RNA. Furthermore, all of the DI RNAs maintained an ORF that encoded for at least the last 717 amino acids of the wt polymerase. This suggests that, in a similar fashion as for the DI RNAs of BBMV, efficient accumulation of TSWV DI RNAs may be dependent upon the ability of the DI RNA to be translated. It is also possible that, since the DI RNAs from TSWV and BBMV likely lead to the production of a deleted viral polymerase product in planta, 6,8 the deleted protein might be necessary for DI RNA accumulation or be involved in the reduction in wt RNA accumulation and symptom alteration. Small sequence repeats (UA, UAG, CCACU) were identified at the deletion junction sites for each of the TSWV DI RNAs and computer-generated RNA folding predicted secondary structures surrounding the release and reinitiation sites for the DI RNAs. These two types of motifs may be involved in the formation of TSWV DI RNAs. 8 Defective viruses There have been reports of multipartite, plant RNA viruses that lose either entire or fragments of one or more genomic segments but are still able to produce a viable infection. In some cases, the deleted fragment was able to replace the parental, genomic component from which it was derived, but, in most cases, the appearance of a deleted segment or absence of an entire segment had no significant effect on viral replication. However, many of these deletion events correlated with the loss of vector transmission indicating that the virus was defective. While these deletion mutants are not DI RNAs, the formation of both probably follows a similar mechanism within a given virus group and is a further example of how RNA viruses can alter their genomes quite rapidly to adapt to new environmental conditions. Family: Reoviridae Wound tumor virus (WTV) contains 12 dsrna segments and is replicated by both the plant host and leafhopper vector. 48,49 Strains of WTV maintained in sweet clover for extended periods (up to 24 years) without passage through the leafhopper had lost the ability to be transmitted by the vector or to replicate in vector cell monolayers. These phenotypes correlated with the loss of specific genomic segments or parts of segments. 10,11 Subsequent characterization of vector transmission defective isolates indicated that genomic segments 2 and 5 had undergone substantial deletion (up to 85%) of the internal sequences, but maintained the 5' and 3' terminal regions necessary for transcription, replication and packaging

6 M. V. Graves et al Family: Bunyaviridae Defective forms of TSWV have been isolated after serial mechanical transmission. 7,12 These isolates no longer formed the characteristic clusters of enveloped virus particles or produced the two envelope glycoproteins, instead forming an amorphous mass of infectious nucleocapsid material. Subsequent analysis of these isolates indicated that the M RNA, which encodes for the two glycoproteins, had been partially deleted or possibly contained point mutations. 7,51 However, the mutant M RNA did not appear to have any effect on the accumulation of the L and S RNAs or on the symptoms produced. Ie 12 proposed that these morphological defective isolates would probably not be transmitted by the thrip vector based upon the assumption that, if they were transmittable, then the virus would have lost its envelope during evolution. Furovirus Group Furoviruses are fungus-borne rod-shaped viruses that contain a bipartite, message sense, ssrna genome. The group consists of the type member, soil-borne wheat mosaic furovirus (SWMV), 13,16,30,52 and two proposed members: peanut clump virus (PCV) and beet necrotic yellow vein virus (BNYVV), that consists of a quadripartite genome. 56 RNA 1 contains a single ORF encoding the putative replicase protein of approximately 220 kda. 14,18 RNA 2 encodes the 19 kda coat protein (CP) ORF 2, that is expressed as an 84 kda CP readthrough product in SWMV and BNYVV and via a -1 frameshift or leaky scanning as a 39 kda product in PCV, 15 and at least three additional products. 14,15,18,57,58 There have been many reports of Furovirus isolates that contain RNA 2 deletion mutants that can, in some cases, replace wt RNA ORF 2, which has been shown to be involved in fungus transmission for BNYVV, 19 seems to be susceptible to deletion especially upon serial passage in the absence of the vector. Chen et al 14 proposed that a strong selective pressure must be exerted by fungal transmissibility in field isolates to maintain these sequences since they can be lost quite rapidly during mechanical transmission. Deleted forms of SWMV RNA 2 have been found in field isolates and after passage by manual inoculation or when infected wheat plants were grown at 17 C. 13,14,16-18,20 several of the isolates produced after passage by mechanical inoculation caused more severe disease symptoms than wt SWMV and contained only deleted forms of RNA 2, in addition to wt RNA 1. 13,14,16,20 The presence of the remainder of the deleted RNA 2 segments did not appear to significantly alter the replication of RNAs 1 and 2 or have any effect on viral symptoms. These results indicated that deleted forms of RNA 2 appear in infected wheat plants regardless of the mode of infection and that the deleted regions may be involved in fungus transmission since wt RNA 2 was still predominant in field isolates; fungal transmission being the primary route of virus spread in the field. 17 In-vitro translation and sequence analysis of several of the RNA 2 deletion mutants indicated that they were formed by deletions from ORF 2. 14,18,59,60 Deleted forms of RNA 2 have also been found associated with BNYVV and PCV. Two isolates of BNYVV produced RNA 2 deletion mutants that replaced wt RNA 2 upon serial mechanical passage. 19 These segments, as for the SWMV RNA 2 deletion mutants, were derived by deletions from ORF 2. Additionally, these viruses were no longer transmitted by the fungal vector indicating that the readthrough product is necessary for vector transmission. 19 Two of 20 PCV isolates examined contained only shortened RNA 2 segments that were also derived by deletions from ORF However, there was little sequence similarity between ORF 2 of PCV and ORF 2 of BNYVV and it has not been shown whether or not these defective forms of PCV can be transmitted by the vector. BNYVV contains two additional RNA segments (3 and 4) that are involved in fungus transmission and ability to infect roots BNYVV isolates that have been maintained in leaf tissue or without passage through the fungal vector show a great deal of variability in the presence and sizes of RNAs 3 and 4. 61,63,65,66 In some isolates, these two RNAs appear to have been totally lost, while in others they are still present, but in deleted forms. Furthermore, BNYVV RNAs 3 and 4, produced in vitro from cdna clones and inoculated onto Chenopodium quinoa leaves in the presence of RNAs 1 and 2, can undergo internal deletions within one to two mechanical passages. 67 In this case, one of the RNA 4 deletion mutants was derived by the precise elimination of a 15-nt sequence repeat suggesting that copy-choice switching by the replicase was involved. In one of the RNA 3 deletion mutants, the deleted sequence began with GU and ended with AG similar to a nuclear intron, however, there is no indication that BNYVV RNAs are associated with the nucleus. 67 The absence of either RNA 3 or RNA 4 or the presence of deleted forms in general had no apparent effect on the replication of 404

7 Defective RNAs of multipartite plant viruses the genomic RNAs 1 and 2 in leaves. Some of these isolates were successfully transferred back to sugar beet roots via the fungal vector. This was always associated with the reappearance of full length RNAs 3 and 4 which indicated that, in some cases, the full length segments were maintained at very low levels and confirmed their roles in fungal transmission and ability to infect roots. 63 Artificial deletion mutants produced from BNYVV RNAs Using reverse genetics, Hehn et al 58 produced deletion mutants of all four BNYVV RNAs and tested their effects on helper virus replication in protoplasts. Two deleted forms of RNA 1, one each for RNAs 3 and 4 and five for RNA 2 were constructed. The deletions were made such that the 5' and 3' terminal sequences known to be required for replication of the individual RNAs were maintained. All five of the deleted RNAs derived from RNA 2 interfered with the replication of the helper virus; reducing the level of accumulation of RNAs 1 and 2 in protoplasts by as much as 90%. However, the interference of these artificial DI RNAs was concentration-dependent and required a large excess of the artificial DI RNA relative to the helper virus RNAs for efficient interference. Also, none of the artificial DI RNAs accumulated to detectable levels in protoplasts which is similar to one of the artificial DI RNAs produced for BMV described previously. 35,36 However, at least two of the artificial DI RNAs were able to reduce the accumulation of RNAs 1 and 2 and abolish local lesion formation in Chenopodium quinoa plants. The deleted RNAs derived from RNAs 1, 3 and 4 appeared to have no effect on RNA 1 and 2 accumulation in protoplasts, but it was not clear whether any of the deleted RNAs actually accumulated to detectable levels. The interference of the RNA 2-derived DI RNAs was proposed to be the result of some unknown cis-acting sequence(s) that has a high affinity for viral replicase and is therefore able to divert it from replicating the genomic RNAs. A similar sequence was presumably not present on the RNA 1, 3 and 4 deletion mutants. 58 Tobravirus group Tobacco rattle tobravirus (TRV) contains a bipartite ssrna genome. RNA 1 encodes the putative viral replicase and cell-to-cell movement proteins and one additional cysteine rich protein with unknown function. 68 RNA 2 encodes for the CP ORF, which is located near the 5' terminus, as well as factors involved in nematode transmission. 22,69,70 RNA 2 exhibits considerable variability in size and the number of ORFs among different isolates 24 and, in some isolates, appears to become reduced in length after serial mechanical passage. 21,23 Deletion mutants of RNA 2 of TRV have been described by Hernandez et al. 22 Mutants that appeared after serial mechanical transmission of TRV RNA contained in total RNA isolated from infected tissue, were formed by single deletions that removed the CP ORF and most of the remaining internal sequences. Several of these deleted RNAs contained short, repeated sequences near the deletion junction sites that resembled the sequences found at the 5' ends of TRV genomic and subgenomic RNAs. Two of the mutants were recombinants, containing 5' sequences derived from RNA 2 and 3' sequences derived from RNA 1. One mutant was obtained after passage by sap inoculation that maintained a CP ORF and was able to outcompete RNA 2, but not RNA 1, in mixed infections. This RNA also had lost the ability to support vector transmission which may explain the observations that many TRV isolates maintained in the laboratory that have lost their vector transmissibility, maintain RNA 2 segments of a variety of different sizes. 22 Summary D RNAs, DI RNAs and defective viruses have been characterized for a growing number of plant RNA viruses with multipartite genomes (Table 1). Some of these defective elements were shown to interfere with the replication and accumulation of the helper virus, while others had little or no effect. It is not understood what feature(s) of the defective molecules determine their ability to interfere. However, all of the wt DI RNAs found associated with BBMV and TSWV described in this review were derived from the genomic segment that encodes the putative RNA polymerase protein. Sequence data available for the defective viruses, DI RNAs, and D RNAs described here revealed that most were formed by single (and, in some cases, double) deletion events. The mosaic type of defective elements often found associated with monopartite plant RNA viruses are rare among multipartite plant RNA viruses. All of the characterized D RNAs and DI RNAs of multipartite plant RNA viruses also share another common feature, the preservation of an ORF. This 405

8 M. V. Graves et al Table 1. Summary of D RNAs DI RNAs, and defective viruses of multipartite plant RNA viruses Deleted Type of defective Virus segment element Comments BBMV RNA 2 DI RNA Exacerbates symptoms in some hosts, encodes deleted polymerase protein BMV RNA 2 Artificial DI RNA CMV RNA 3 D RNA Encodes deleted 3a protein, encodes wt CP, not maintained systemically in all hosts AlMV RNA 3 D RNA TSWV L RNA DI RNA Encodes deleted polymerase protein, M RNA Defective virus Loss of viral envelope and probable loss of vector transmission WTV RNAs 2 & 5 Defective virus Loss of vector transmission SWMV RNA 2 Defective virus Some species exacerbate symptoms, probable loss of vector transmission BNYVV RNA 2 Defective virus Loss of vector transmission RNA 2 Artificial DI RNA Did not accumulate in protoplasts, inhibited local lesion formation on Chenopodium quinoa RNAs 3 & 4 Defective virus Loss of vector transmission and ability to infect roots PCV RNA 2 Defective virus Probable loss of vector transmission TRV RNA 2 Defective virus Loss of vector transmission suggests a critical role of translation in the accumulation of the single deletion type defective elements. It is also interesting to note that D RNAs and DI RNAs seem to arise preferentially from one component of a given multipartite virus and not from the other component(s). The best examples of this are from the related viruses BBMV, CMV and AlMV. All of the DI RNAs of BBMV were derived from the RNA 2 component while the D RNAs found associated with CMV and AlMV were derived from RNA 3. This might reflect features related to both defective RNA generation and post-event selection requirements. Further studies will be needed to determine whether D RNAs and DI RNAs can be generated from the other viral components and whether they can be maintained by the helper virus. In the case of the defective viruses described, the majority of the deletion events correlated with extended mechanical passage of the virus in the absence of the vector. Furthermore, the regions deleted from the viruses seemed to be those involved only in vector transmission such that an infectious, viable virus was produced. Obviously, RNA viruses are able to adapt their genomes quite rapidly to new environmental conditions even to the point of deleting large parts of or entire segments that are no longer required under the given conditions. Apparently, the entire viral genome is under continuous selection pressure. Acknowledgements These studies were supported by grants from the National Institute for Allergy and Infectious Diseases (RO1 AI26769) and the National Science Foundation (MCB ) to J.P.; by Noble Foundation to M.V.G. and by CICYT (Bio ) to J.R. References 1. Holland JJ (1990) Defective viral genomes, in Virology (Fields BN, Knipe DM, eds) pp Raven Press, New York 2. Hillman BI, Carrington JC, Morris TJ (1987) A defective interfering RNA that contains a mosaic of a plant virus genome. Cell 51: Li XH, Heaton LA, Morris TJ, Simon AE (1989) Turnip crinkle virus defective RNAs intensify viral symptoms and are generated de novo. Proc Natl Acad Sci USA 86: White KA, Bancroft JB, Mackie GA (1991) Defective RNAs of clover yellow mosaic virus encode nonstructural/coat protein fusion products. Virology 183: White KA, Bancroft JB, Mackie GA (1992) Coding capacity determines in vivo accumulation of a defective RNA of clover yellow mosaic virus. J Virol 66: Romero J, Huang Q, Pogany J, Bujarski JJ (1993) Characterization of defective interfering RNA components that increases symptom severity of broad bean mottle virus infections. Virology 194: Resende R de O, de Haan R, de Avila C, Kitajima EW, Kormelink R, Goldbach R, Peters D (1991) Generation of envelope and defective interfering RNA mutants of tomato spotted wilt virus by mechanical passage. J Gen Virol 72:

9 Defective RNAs of multipartite plant viruses 8. Resende R de O, de Haan R, van de Vossen E, de Avila AC, Goldbach R, Peters D (1992) Defective interfering L RNA segments of tomato spotted wilt virus retain both genome termini and have extensive internal deletions. J Gen Virol 73: Graves MV, Roossinck MJ (1995) Characterization of defective RNAs derived from RNA 3 of the Fny strain of cucumber mosaic cucumovirus. J Virol 69: Reddy DVR, Black LM (1974) Deletion mutations of the genome segments of wound tumor virus. Virology 61: Reddy DVR, Black LM (1977) Isolation and replication of mutant populations of wound tumor virus lacking certain genome segments. Virology 80: Ie TS (1982) A sap transmissible, defective form of tomato spotted wilt virus. J Gen Virol 59: Brakke MK (1977) Sedimentation coefficients of the virions of soil-borne wheat mosaic virus. Phytopathology 67: Chen J, MacFarlane SA, Wilson TAM (1994) Detection and sequence analysis of a spontaneous deletion mutant of soilborne wheat mosaic virus RNA2 associated with increased symptom severity. Virology 202: Manohar SK, Guilley H, Dollet M, Richards K, Jonard G (1993) Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms. Virology 195: Shirako Y, Brakke MK (1984a) Two purified RNAs of soil-borne wheat mosaic virus are needed for infection. J Gen Virol 65: Shirako Y, Brakke MK (1984b) Spontaneous deletion mutation of soil-borne wheat mosaic virus RNA II. J Gen Virol 65: Shirako Y, Ehara Y (1986) Comparison of the in vitro translation products of wild-type and a deletion mutant of soil-borne wheat mosaic virus. J Gen Virol 67: Tamada T, Kusume T (1991) Evidence that the 75K readthrough protein of beet necrotic yellow vein virus RNA-2 is essential for transmission by the fungus Polymyxa betae. J Gen Virol 72: Tsuchizaki T, Hibino H, Saito Y (1973) Comparison of soilborne wheat mosaic virus isolates from Japan and the United States. Phytopathology 63: Harrison BD, Woods RD (1966) Serotypes and particle dimensions of tobacco rattle viruses from Europe and America. Virology 28: Hernandez C, Carette JE, Brown JF, Bol JF (1996) Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2. J Virol 70: Lister RM, Bracker CE (1969) Defectiveness and dependence in three related strains of TRV. Virology 37: Mathis A, Linthorst HJM (1992) The tobraviruses, in Encyclopedia of Virology (Webster RG, Granoft A, eds). Academic Press, Ltd, London 25. Burgyan J, Grieco F, Russo M (1989) A defective interfering RNA molecule in cymbidium ringspot virus infections. J Gen Virol 70: Finnen RL, Rochon DM (1993) Sequence and structure of defective interfering RNAs associated with cucumber necrosis virus infections. J Gen Virol 74: Hajimorad MR, Kurath G, Randles JW, Francki RIB (1991) Change in phenotype and encapsidated RNA segments of an isolate of alfalfa mosaic virus: influence of host passage. J Gen Virol 72: 28. Kroner PA, Young BM, Ahlquist P (1990) Analysis of the role of brome mosaic virus 1a protein domains in RNA replication, using linker insertion mutagenesis. J Virol 64: Palukaitis P, Roossinck MJ, Dietzgen RG, Francki RIB (1992) Cucumber mosaic virus, in Advances in Virus Research, Vol 41, (Maramorosch K, Murphy FA, Shatkin AJ, eds) pp Academic Press, Inc 30. Traynor P, Young BM, Ahlquist P (1991) Deletion analysis of brome mosaic virus 2a protein: effects on RNA replication and systemic spread. J Virol 65: Ahlquist P (1992) Bromovirus RNA replication and transcription. Curr Opin Genet Dev 2: Mise K, Allison RF, Janda M, Ahlquist P (1993) Bromovirus movement protein genes play a crucial role in host specificity. J Virol 67: Graves MV, Roossinck MJ, Bujarski JJ (1996a) De novo generation of defective RNAs from cucumber mosaic cucumovirus. 15th Annual Meeting, American Society for Virology, University of Western Ontario, London, Ontario, Canada 34. Pogany J, Romero J, Huang Q, Sgro J-Y, Shang H, Bujarski JJ (1995a) De novo generation of defective interfering-like RNAs in broad bean mottle bromovirus. Virology 212: Marsh LE, Pogue GP, Connell JP, Hall TC (1991b) Artificial defective interfering RNAs derived from brome mosaic virus. J Gen Virol 72: Marsh LE, Pogue GP, Szybiak U, Connell JP, Hall TC (1991c) Non-replicating deletion mutants of brome mosaic virus RNA-2 interfere with viral replication. J Gen Virol 72: Pogany J, Romero J, Bujarski JJ (1995b) Deletion analysis of defective interfering (DI) RNAs associated with broad bean mottle bromovirus (BBMV). 14th Annual Meeting, American Society for Virology, University of Texas, Austin, Texas, USA 38. Pogany J, Bujarski JJ (1996) Complementary sequences facilitate deletions in defective (D) RNAs associated with broad bean mottle bromovirus (BBMV). 15th Annual Meeting, American Society for Virology, University of Western Ontario, London, Ontario, Canada 39. Kamer G, Argos P (1984) Primary structural comparison of RNA dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Res 12: Koonin EV (1991) The phylogeny of RNA-dependent RNA polymerases of positive-strand RNA viruses. J Gen Virol 72: French R, Ahlquist P (1988) Inercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J Virol 5: Marsh LE, Huntley CC, Pogue GP, Connell JP, Hall TC (1991a) Regulation of ( + ):( )-strand asymmetry in replication of brome mosaic virus RNA. Virology 182: Graves MV, Roossinck MJ (1995) Host specific maintenance of a cucumovirus defective RNA. 14th Annual Meeting, American Society for Virology, University of Texas, Austin, Texas, USA 44. De Haan P, Wagemakers L, Peters D, Goldbach R (1990) The S RNA segment of tomato spotted wilt virus has an ambisense character. J Gen Virol 71: De Haan P, Kormelink R, Resende R De O, van Poelwijk F, Peters D, Goldbach R (1991) Tomato spotted wilt virus L RNA encodes putative RNA polymerase. J Gen Virol 72: Cho JJ, Mau RFL, Hamasaki RT, Gonsalves D (1988) Detection of tomato spotted wilt virus in individual thrips bt enzymelinked immunosorbent assay. Phytopathology 78: De Haan P, Wagemakers L, Peters D, Goldbach R (1989) Molecular cloning and terminal sequence determination of the S and the M RNAs of tomato spotted wilt virus. J Gen Virol 70: Black LM, Brakke MK (1952) Multiplication of wound tumor virus in an insect vector. Phytopathology 42: Brakke MK, Vatter AE, Black LM (1954) Size and shape of wound tumor virus. Brookhaven Symp Biol 6: Nuss DL, Summers D (1984) Variant dsrnas associated with transmission-defective isolates of wound tumor virus represent terminally conserved remnants of genome segments. Virology 133:

10 M. V. Graves et al 51. Verkleij FN, Peters D (1983) Characterization of a defective form of tomato spotted wilt virus. J Gen Virol 64: Brunt AA, Richards KE (1989) Biology and molecular biology of furoviruses. Adv Virus Res 36: Thouvenal JC, Dollet M, Fauquet C (1976) Some properties of peanut clump virus, a newly discovered virus. Ann Appl Biol 84: Thouvenel JC, Fauquet C (1981a) Further properties of peanut clump virus and studies on its natural transmission. Ann Appl Biol 97: Thouvenel JC, Fauquet C (1981b) Peanut clump virus, in CMI/ AAB: Description of Plant Viruses Vol Richards K, Jonard G, Guilley H, Ziegler V, Putz C (1985) In vitro translation of beet necrotic yellow vein virus RNA and studies of sequence homology among the RNA species using cloned cdna probes. J Gen Virol 66: Gilmer S, Bouzoubaa S, Hehn A, Guilley H, Richards K, Jonard G (1992) Efficient cell-to-cell movement of beet necrotic yellow vein virus requires 3' proximal genes located on RNA-2. Virology 189: Hehn A, Bouzoubaa S, Jonard G, Guilley H, Richards KE (1994) Artificial defective interfering RNAs derived from RNA 2 of beet necrotic yellow vein virus. Arch Virol 135: Hsu YH, Brakke MK (1985) Cell-free translation of soil-borne wheat mosaic virus RNAs. Virology 143: Shirako Y, Ali I, Wilson TMA (1990) Nucleotide sequence of soil-borne wheat mosaic virus RNA II. Phytopathology 80: Koenig R, Burgermeister W, Weich H, Sebald W, Kothe C (1986) Uniform RNA patterns of beet necrotic yellow vein virus in sugarbeet roots, but not in leaves from several plant species. J Gen Virol 67: Koenig R, Burgermeister W (1989) Mechanical inoculation of sugar beet roots with isolates of beet necrotic yellow vein virus having different RNA compositions. J Phytopath 124: Lemaire O, Merdinoglu D, Valentin P, Putz C, Ziegler-Graff V, Guilley H, Jonard G, Richards K (1988) Effect of beet necrotic yellow vein virus RNA composition on transmission by Polymyxa betae. Virology 162: Tamada T, Abe H (1989) Evidence that beet necrotic yellow vein virus RNA-4 is essential for efficient transmission by the fungus Polymyxa betae. J Gen Virol 70: Bouzoubaa S, Guilley H, Jonard G, Richards K, Putz C (1985) Nucleotide sequence analysis of RNA-3 and RNA-4 of beet necrotic yellow vein virus, isolates F2 and G1. J Gen Virol 66: Kuszala M, Ziegler V, Bouzoubaa S, Richards K, Putz C, Guilley H, Jonard G (1986) Beet necrotic yellow vein virus: different isolates are serologically similar but differ in RNA composition. Ann Appl Biol 109: Bouzoubaa S, Niesbach-Klösgen U, Jupin I, Guilley H, Richards K, Jonard G (1991) Shortened forms of beet necrotic yellow vein virus RNA-3 and -4: internal deletions and a subgenomic RNA. J Gen Virol 72: Hamilton WDO, Boccara M, Robinson DJ, Baulcombe DC (1987) The complete nucleotide sequence of tobacco rattle virus RNA 1. J Gen Virol 68: MacFarlane SA, Brown JF, Bol JF (1995) The transmission by nematodes of tobraviruses is not determined exclusively by the virus coat protein. Eur J Plant Pathol 101: Ploeg AT, Robinson DJ, Brown DJF (1993) RNA 2 of tobacco rattle virus encodes the determinants of transmissability by trichodroid nematodes. J Gen Virol 74: