. .- Err riff». ; ‘ HE‘" S L "MLWIWLW This is to certify that the dissertation entitled CLONING, SEQUENCING AND CHARACTERIZATION OF THE COAT PROTEIN GENE AND THE 3' NONCODING REGION OF RNAl AND RNAZ OF BLUEBERRY LEAF MOTTLE VIRUS. presented by JEFFERY W . BACHER has been accepted towards fulfillment of the requirements for Ph.D. degreein Horticulture (ILAFIM Major professor mew Mcllhnnlfl' .' . ‘ r In, ‘ ' ' ' 0712771 LIBRARY Michigan State - University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE c:\circ\datedm.pm3—p.1 CLONING, SEQUENCING AND CHARACTERIZATION OF THE COAT PROTEIN GENE AND THE 3' TERMINI OF RNA1 AND RNA2 OF BLUEBERRY LEAF MOTTLE VIRUS Bv Jeffery W. Bacher A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department Of Horticulture 1993 ABSTRACT CLONING, SEQUENCING AND CHARACTERIZATION OF THE COAT PROTEIN GENE AND THE 3' TERMINI OF RNA1 AND RNA2 OF BLUEBERRY LEAF MOTTLE VIRUS Bv Jeffery W. Bacher The 3’ termini of RNA1 and RNA2 Of blueberry leaf mottle virus (BBLMV) were cloned and their cDNA sequences determined. Two genes were isolated for later use in engineering resistance, the coat protein (CP) gene and the polymerase gene. The N and C termini of the CP gene were precisely located, with the resulting sequence coding for a protein of 521 amino acids with an estimated Mr of 57,542. Sequence homology between BBLMV and Other nepoviruses was investigated and compared to existing classification systems. Amino acid sequence homology with BBLMV CP was low (20-30%), but statistically significant in all comparisons. The 3’ noncoding regions of RNA1 and RNA2 of BBLMV are nearly identical with differences occurring at only four positions. The presence of this duplication (1.4kb) indicates that recombination has occurred at least once in the evolutionary history Of BBLMV. High mutation rates in RNA viruses means that strong selection pressure and/or recombination must be operating in order to maintain identity in this duplicated region. The possible involvement Of RNA recombination in maintaining identity was investigated. NO evidence Of recombination was found. If recombination was occurring in BBLMV it was below 2.3% in the overall population sampled and below 1.1% between markers in the 3' noncoding regions. The data indicate that the identity in the 3’ noncoding regions of RNA1 and RNA2 is being maintained without high levels Of recombination. The high frequency of mutations Observed in BBLMV population sampled and lack (or low levels) of recombination indicate that selection is primarily responsible for conservation of identity in the 3’ noncoding region. ACKNOWLEDGMENTS I would like to thank my major professor, James Hancock, for his guidance, his interest and enthusiasm in my research, and his totally unrealistic Optimism about life in general. I am grateful for his wiliness to change the direction of our research to pursue more "interesting" ideas. It was exciting discussing ideas about the evolution of viruses with him, and puzzling about what was going on with BBLMV and why. This is what science is really about and what an education Should be. I could not have asked for a better major professor! I would also like to thank members of my guidance committee, Rebecca Grumet, Don Ramsdell and Micheal Thomashow. l have learned a great deal about plant viruses from both Rebecca and Don have a great deal of respect and admiration for all the members of my committee. Special thanks to Don Warkentin, for his patience and time that he spent teaching me about molecular biology and plant virology. I would not have been able to accomplish what I have it had not been for him. I am grateful for having the Opportunity to know and work with an incredible group Of fellow Students... Pete Callow, Kobra Haghighi, Karen & Stan Hokanson, Theresa Acquaah, and Gerri Gillet. These people were like an extended family and made our lab a fun and exciting place to work. I cannot say enough about family... Susan, Aaron and Nora. Thank you Susan for your incredible patience and love over these past years. You have all been my foundation and are always with me in my heart. TABLE OF CONTENTS List of Tables ............................................................................... List of Figures .............................................................................. Introduction & Literature Review ..................................................... Blueberry leaf mottle disease ................................................. Nepovirus group .................................................................. Blueberry leaf mottle virus ..................................................... Conservation Of the 3’ terminus of nepoviruses ........................ Recombination in RNA viruses ................................................ Pathogen derived resistance & risks in release Of transgenics ....... Summary ........................................................................... List Of references ................................................................. SECTION I: Cloning and sequencing of the coat protein gene and the 3’ noncoding region of blueberry leaf mottle virus RNA2 ..... Introduction ....................................................................... Materials & Methods ........................................................... Results & Discussion ........................................................... List of References ............................................................... SECTION II: Near identity in the 3’ noncoding regions of blueberry leaf mottle virus RNA1 and RNA2 ......................................... vi Page viii ix 1 20 21 28 28 3O 33 42 45 Introduction ........................................................................ Materials & Methods ........................................................... Results & Discussion ........................................................... List of References ............................................................... SECTION III: Conservation of a 1.4 Kb duplication in the 3’ non- coding regions of RNA1 and RNA2 of blueberry leaf mottle virus: Possible mechanisms for maintaining identity ................ Introduction ....................................................................... Materials & Methods ........................................................... Results & Discussion .......................................................... List of References ............................................................... 45 47 49 62 65 65' 67 7o 78 Table 1. Table 2. Table 1. LIST OF TABLES SECTION I Percent amino acid identity between BBLMV coat protein and other nepovirus coat proteins .................................... Percent amino acid identity between nepovirus coat proteins. SECTION II Sequence comparison of the 3’ noncoding regions showing the percent similarity between the different nepoviruses ..... viii 4O 41 54 Figure 1. Figure 2. Figure 1. Figure 2. Figure 3. LIST OF FIGURES SECTION I Unique restriction sites for clones 24 (RNA1) and 34 (RNA2) used to distinguish between cDNA clones ............. 34 The cDNA sequence and predicted amino acid sequence Of 3’ terminus of BBLMV RNA2. The N terminal amino acid sequence of the coat protein determined by Edman degradation is underlined. The coat protein sequence is 521 amino acids long ................................................... 35-36 SECTION II The cDNA sequence and the predicted amino acid sequence Of the 3’ terminus Of BBLMV RNA1 (excluding the poly(A) tail) ........................................................... 50 Alignment of the putative RNA-dependent RNA polymerase domain of BBLMV clone 24 with GFLV RNA1 (positions 1791 to 1862), GCMV RNA1 (positions 1764 to 1835), TBRV RNA1 (positions 1780 to 1851), CPMV RNA1 (positions 1493 to 1569) and poliovirus (positions 1951 to 2146). The capital letters indicate a consensus between the BBLMV sequences and the other viral sequences. The GDD motif is common to all RNA dependent RNA polymerases ......................................... 51 cDNA sequence of RNA1 showing the 1388nt region Of identity between the 3’ termini of RNA1 and RNA2 Of blue- berry leaf mottle nepovirus (excluding the poly(A) tail). In the seven positions where differences occurred, the RNA2 sequence is shown below the sequence of RNA1. The region of Identity begins with the termination codon TAG Figure 4. Figure 5. Figure 6. Figure 7. Figure 1. for the RNA1 encoded ORF. A conserved region also found in AMV, GCMV, GFLV, RRV, TBRV and CPMV RNA M is underlined .................................................... 53 Multiple sequence alignment of the noncoding region of nine nepoviruses using GCG PRETTY program. A consensus sequence is given when a plurality of 6 out of 9 sequences exists .................................................. 56 Optimal alignment Of RRV and BBLMV noncoding regions by GCG BESTFIT program. Percent similarity is 87% over 49 nucleotides ............................................................ 56 Dendrogram showing the clustering relationships of different nepoviruses based on the nucleotide sequence of their 3’ noncoding regions (generated by GCG program PILEUP) ..................................................................... 57 Dendrogram Showing the clustering relationships of different nepoviruses based on the amino acid sequence of their 3’ noncoding regions (generated by GCG program PILEUP) ...................................................................... 58 SECTION III Consensus sequences for the 3' noncoding region Of RNA1 and RNA2 of BBLMV for the seven positions where differences were found between clones 24 and 34. Clones 24 and 34 were used originally to determine the sequence of the 3’ noncoding region Of RNA1 and RNA2 (Bacher, 1993). The marker genotypes of cDNA clones of BBLMV are aligned with consensus sequences. Bracketed [] numbers indicate coding sequence from either RNA1 =[1] or RNA2=[2]; (#) = cDNA clone number; * = muta tion; = cDNA sequence; and ..... = area not sequenced 71-73 INTRODUCTION AND LITERATURE REVIEW Blueberry leaf mottle nepovirus (BBLMV) causes a serious disease Of highbush blueberry that is Spread primarily through infected pollen. The virus has been identified in numerous cultivated and wild blueberry populations in the State of Michigan (Ramsdell & stace-Smith, 1979; Sandoval et al., 1992). Because infected native plants are often in close proximity to commercial fields and pollinating insects can travel long distances, further spread Of the virus seems likely (Hancock et al., 1993). Systemically infected plants exhibit leaf mottling, leaf distortion and general dieback Of the main stems; leading to reduced growth and productivity, and eventually death (Ramsdell & Stace- Smith, 1979). Since there are no curative measures that can taken once a plant is infected by a virus, the only alternative is to remove the diseased plant. Due to the lack Of effective control measures for BBLMV, and the unavailability of virus resistance in commercial cultivars, we began nonconventional approaches to control the virus. Pathogen derived resistance (i.e., deriving resistance genes from the pathogen’s genome) is a Strategy which has been recently used to develop resistance to many different plant viruses (Sanford & Johnston, 1985). Thus far, the engineering of viral resistance has focused primarily on the use of the 2 coat protein gene, and to a lesser extent on non-structural genes (Grumet, 1990; Hull & Davies, 1992). Transgenic plants expressing viral coat protein genes Often show a delay in disease development or are resistant to infection upon inoculation by the source virus. This type of resistance, termed coat protein mediated protection, has now been demonstrated in over 20 plant/virus systems (Hull & Davies, 1992). Another viral gene that Shows promise for use in engineering resistance is the polymerase gene. Transgenic plants expressing a non-functional form of a polymerase gene were completely resistant to infection by the source virus (Golemboski et al., 1990; Anderson et al., 1992). As a first step in engineering resistance to BBLMV in blueberries, we cloned and sequenced the coat protein gene and a portion of the polymerase gene from the BBLMV genome. During the process of cloning and sequencing genes from BBLMV, we discovered a 1.4 kb region Of identity on the 3' termini of RNA1 and RNA2. Sequence data from other nepoviruses revealed that conservation of identity in the 3' noncoding regions of the genomic RNAS is a common feature of this virus group (Greif et al., 1986, 1988; Brault et al., 1989; Serghini et al., 1990; Bertiol et al., 1991; Rott et al., 1991; Blok et al., 1992; Scott et al., 1992). Since the mutation rates are so high in RNA viruses [10'3 to 10‘5 misincorporated nucleotides per round Of replication (Domingo & Holland, 1988)), selection pressure alone may be insufficient to maintain identity in these duplicated regions. It has been suggested that the presence of duplicated regions on the 3’ terminus of some viruses is evidence for RNA recombination, 3 and that high levels of recombination may be involved in the conservation of identity in these sequences (Rott et al., 1990; Scott et al., 1991 ). If recombination is occurring at relatively high frequencies in BBLMV, then a potential risk exists for recombination in a CP transgenic plant between the mRNA of the introduced coat protein gene and an infecting virus. The exchange of genetic material through recombination could result in the evolution of new viruses which could potentially have extended host ranges and increased virulence (de Zoeten, 1991). The potential impact of release of BBLMV coat protein transgenic plants in the environment is unknown. DeZoeten (1991) has advocated the need for experimentation on which to base our risk assessment for release of transgenic plants containing viral genes. Since a goal of the blueberry breeding program is to engineer resistance to BBLMV using coat protein mediated protection, we decided to investigate whether recombination occurs in the replication of BBLMV. If recombination is a common occurrence in BBLMV, then a risk might be associated with releasing coat protein transgenic plants into the environment. The primary objectives of this research were to clone and sequence the coat protein and polymerase genes of blueberry leaf mottle virus (BBLMV) to be used by others in engineering virus resistance, and to investigate to what extent RNA recombination is involved in the conservation Of a duplicated region on the 3' termini of RNA1 and RNA2 Of BBLMV. Blueberry leaf mottle disease Blueberry leaf mottle virus (BBLMV) is the causal agent of a relatively new disease Of highbush blueberry, Vaccinium corymbosum L. (Ramsdell & Stace—Smith, 1979). Infected plants exhibit a general dieback of Stems, leaf distortion and a pronounced mottling of the leaves. Within a few years after the onset of symptoms, severe dieback of the stems results in stunted growth and reduced yield. Eventually the plants die. Currently BBLMV is found only Michigan, where it has been identified in numerous cultivated and wild blueberry populations. Since the disease is caused by a pollen—born virus, bees carrying infected pollen from nearby wild blueberry plants can transmit the virus to commercial fields (Sandoval, 1992; Boylen—Pett, 1991). Once the virus is present in a commercial field it can spread via infected pollen, as was observed in one 20 acre commercial blueberry field where the virus spread from a few bushes to infect over 50% of the field in less than 10 years (PrittS & Hancock, 1992). Unfortunately, the spread of the virus cannot be controlled by eliminating the vector, since bees are necessary for blueberry pollination and fruit set. Currently, the best approach to controlling BBLMV is to plant virus-free stock and to eradicate any infected bushes. The main disadvantages of this approach are the difficulty in detection of infected plants (there is a four year latent period before symptoms appear) and the cost and availability of virus tested Stock. Genetic based resistance is generally regarded as the best approach over the long term, but useful sources of resistance have not been 5 identified. All commercial blueberry cultivars that have been screened (Spartan, Blueray, Elliot, Bluecrop, Jersey & Rubel) were found to be susceptible to BBLMV (Sandoval, 1992). Wild blueberry plants appear to be tolerant to infection by BBLMV and may serve as a source of resistance (Hancock et al., 1993), but introgression of wild germplasm requires extensive selection and backcrossing to remove deleterious genes and to Obtain cultivars with acceptable horticultural qualities. In addition, the virus can multiply in tolerant plants which can then serve as a source of inoculum and lead to variation that could give rise to more virulent isolates. The lack useful sources of resistance genes remains a major limitation to breeding for virus resistance. Nepovirus Group. Blueberry leaf mottle virus is a putative member Of the nepovirus group whose name is an acronym Standing for @matode transmitted polyhedral virus. Definitive members of this group have three types of isometric particles 28 nm in diameter, sedimenting at 508, 90-1208 and 120-1308 and containing 0, 27— 40 and 42-46% single Stranded RNA, respectively (Harrison & Murant, 1977). All have two essential RNAS, Mr 2.4 x 106 and 1.4 to 2.2 x 106, which are encapsidated separately in particles composed of 60 subunits of a single coat polypeptide with a Mr of 55,000 to 60,000. Members of this group have a wide host range, cause ringspot and mottle symptoms, often with subsequent symptomless infection. Nepoviruses are transmitted by inoculation with sap, by nematodes and to progeny through seed and pollen. 6 Genome structure, organization and properties. The nepovirus genome consists of two single stranded, positive sense, RNAS with the larger RNA (RNA1) about 6100-8400 nucleotides and a more variable RNA2 with about 3400-7200 nucleotides (Murant et al., 1981 ). Both RNAS have 3’ poly(A) tails and a small protein (VPg) linked to their 5' terminus, which is essential for infectivity but not for in vitro translation (Mayo et al., 1979; Mayo et al., 1982). Mature proteins are released from two large polyprotein precursors corresponding to RNA1 and RNA2 by proteolytic cleavage (Demangeat et al., 1990, 1991). The 3’ noncoding regions on RNA1 and RNA2 of each distinct virus are nearly identical, but vary in length from 195 nt in arabis mosaic virus (AMV) (Bertioli et al., 1991) to 1546 nt in tomato ringspot viurs (TomRSV) (Rott et al., 1991). RNA1 carries determinants for replication (Robinson et al., 1980), processing Of viral polyproteins (Demangeat, 1990, 1991; Margis et al., 1991), host range and seed transmissibility, whereas RNA2 carries determinants for RNA encapsidation (Randles et al., 1977), for spread of the virus in the plant (Meyer et al., 1986), and nematode transmissibility; virulence depends on both RNA species (Harrison & Murant, 1977). Relationships within the group. Nepoviruses have been divided into sub— groups based on serological relationships (Francki and Hatta, 1977; Murant, 1981), by the Size of their RNA2 components (Martelli, 1975) or by the difference in sizes of their RNA1 and RNA2 components (Francki et al., 1985). Martelli assigned viruses to subgroups on the basis Of the molecular weights Of 7 their RNA2 as follows: subgroup | (Mr 1.4-1.5 x 106) includes grapevine fanleaf virus (GFLV), raspberry ringspot virus (RRV) , tobacco ringspot virus (TRSV) and arabis mosaic virus (AMV); subgroup ll (M,1.5-1.6 x 106) includes tomato black ring virus (TBRV), grapevine chrome mosaic virus (GCMV), and artichoke Italian latent virus; and subgroup lll (Mr >1.6 x 106) includes tomato ringspot virus (TomRSV), peach rosette mosaic (PRMV), cherry leaf roll virus (CLRV), and BBLMV. Francki (1 985) subdivided the nepoviruses into two groups, based on whether RNAS 1 and 2 differ in Size (difference 2 0.6 M,) or are Of a similar size (difference 5 0.4 M,). Classification based on serological relationships may be conservative and recognize as distinct only those viruses that are antigenically unrelated, with others being regarded as serotypes or strains. Using such a system of classification, Francki and Hatta (1977) recognized tobacco ringspot virus as the type member and included only six additional viruses as distinct members. Murant’s (1981) system is more liberal and applies distinctive names to those that Show a sufficiently distant serological relationship to an accepted member. Relationships with other groups. Goldbach (1987) has suggested that nepoviruses be included as part of the picornavirus-like supergroup which includes the comoviruses, potyviruses and picornaviruses (simple animal RNA viruses). Viruses in this supergroup share common characteristics like genomic structure and organization, as well as regions of sequence similarity. Nepoviruses share a number of properties with the comovirus group: the genome is bipartite, both are translated into two large polypeptides, and both 8 genomes have 5’ VPg and 3’ poly(A) tails. They differ in that the capsid protein is comprised of one polypeptide (Mr 55,000 to 60,000) for nepoviruses and two polypeptides with (Mr 24,000 and 40,000) for comoviruses and the VP9 protein is essential for nepovirus infectivity but not for comoviruses. Strawberry latent ringspot (SLRV) and cherry raspleaf virus (CRLV), both tentative members of the nepovirus group, have two capsid protein subunits (Mr 22,500 and 24,000) and may represent transition viruses in the evolution of these two virus groups (Dougherty & Hiebert, 1985). Blueberry leaf mottle nepovirus. The isolation of BBLMV, host range, purification, serology and completion of Koch’s postulates are described by Ramsdell and Stace-Smith (1979). The physical and chemical properties of the particles and RNA of BBLMV have also been determined (Ramsdell & Stace-Smith, 1981). Host range and transmission. Transmission of BBLMV in blueberry plants is via pollen, seed or rub inoculation (difficult), but not by nematodes which is the common vector of most nepoviruses (Childress & Ramsdell, 1896a,b & 1987; Sandoval, 1992). BBLMV is readily transmitted by rub inoculation to Chenopodium spp., Nicotiana c/eve/andii and other tobacco species as well as to, bean, cucumber and tomato. Chenopodium quinoa is used as a propagative host due to the extent of local and systemic infection. Serology. BBLMV was found to be distantly related to grapevine Bulgarian latent virus (GBLV); the degree of relationship was similar to that 9 which exists between grapevine fanleaf virus (GFLV) and arabis mosaic virus (AMV), and was sufficient to warrant a distinct designation rather considering BBLMV a strain of GBLV (Ramsdell and Stace-Smith, 1979). No relationship was detected between BBLMV and three other nepoviruses that have large RNA2 components, TomRSV, peach rosette mosaic virus (PRMV), and CLRV. BBLMV also does not react with antisera from arabis mosaic, artichoke Italian latent, cherry raspleaf, golden elderberry, grapevine fanleaf, raspberry ringspot, strawberry latent ringspot, tobacco ringspot, tomato blackring, tomato ringspot or walnut mosaic viruses. Two Strains of BBLMV have been described; the MI Strain found in blueberry in Michigan (Ramsdell & Stace-Smith, 1979), and the NY strain found in grapevine in New York (Uyemoto et al., 1977). Particle properties. Purified virus particles separate into three components when centrifuged through sucrose density gradients; top (empty shells), middle (RNA2) and bottom (RNA1), which sediment at 53, 120 and 1288, respectively (Ramsdell & Stace—Smith, 1981). Particles of middle and bottom components Observed by electron microscopy measure 28 nm in diameter and have a hexagonal outline. These particles are composed of a single coat protein subunit with an estimated Mr of 54,000. Incubation of BBLMV nucleic acid with DNase has no effect, but RNase will degrade it. The RNA is single stranded with the RNA1 and RNA2 components having a Mr of 2.35 and 2.15 x 106, respectively. 10 Conservation of the 3' terminus of nepoviruses. Conservation of the 3’ terminus is a common phenomenon of most multipartite viruses (Matthews, 1991). This is particularly true of the nepovirus group, where nearly identitical nucleotide sequences have been found between the 3' noncoding regions of RNA1 and RNA2 components of tomato black ring virus (Grief et al., 1986, 1988), grapevine chrome mosaic virus (Brault et al., 1989; Le Gall et al., 1989), tomato ringspot virus (Rott et al., 1991a,b) and cherry leaf roll virus (Scott et al., 1992). This marked conservation of sequences in the 3’ terminus could reflect important functions that are selectively maintained. For example, the 3’ termini of all genomic RNAS must contain the replicase recoginition Site for initiation of minus strand synthesis (Matthews, 1991). Other possible roles of the 3’ noncoding region that have been suggested for other RNA viruses include: regulatory functions, e.g., controlling the rate and timing of synthesis of genomic RNAS (Dreher & Hall, 1988); virus assembly, and signals for polyadenylation of the 3’ terminus. High mutation rates in RNA viruses means strong selection pressure must be counteracting these changes in order to maintain specific 3’ sequences. The rate of point mutations for RNA viruses has been estimated to be approximately 10‘3 to 10'5 per nucleotide per round of replication with some variation between different viruses; as contrasted with 10‘7 to 10’11 for DNA polymerases (Steinhauer & Holland, 1987). As a consequence Of these high error rates mutants arise at a high frequency in viral populations, but since most are less 11 fit than the wild type or consensus sequence they are Strongly selected against (Domingo et al., 1978). The interaction between mutation and selection results in RNA virus populations that exist as heterogeneous mixtures of related genomes that share a consensus sequence but differ from each other by one or more mutations (Holland et al., 1982; Domingo et al., 1985; Morch et al., 1988). 9 Whether selection alone can maintain identity in the 3’ noncoding region is uncertain. It has been suggested that high frequency of RNA recombination is acting in concert with selection in maintaining identity in the 3’ terminus of TmRSV (Rott et al., 1991) and CLRV (Scott et al., 1992). Recombination in RNA viruses Genetic recombination in RNA viruses has been defined as any process involving the exchange of genetic material between genomic RNA molecules (King, 1988). There are two types of RNA recombination, homologous and non-homologous. In homologous recombination, exchange in RNA sequences occurs between two parental genomes that are related and the crossover event occurs at the same location in both RNAS, thereby preserving the exact sequences in both recombinants. In non-homologous recombination, there is little or no homology between the parental molecules at or near the crossover site, often resulting in a loss or gain of genetic material, as in defective interferring (DI) RNAS. The first evidence of recombination in RNA viruses was found by Cooper 12 et al. (1968) in poliovirus. Later, Kirkegaard and Baltimore (1986) Showed that recombination in poliovirus occurs during replication, apparently by template switching during negative strand synthesis. The template switching (or copy- choice) mechanism is now widely regarded as operating in many, if not all, examples of RNA recombination. This model predicts that during viral replication the RNA polymerase may stop before reaching the 5’ end and "jump," together with the nascent RNA, to another site on the same or a different template molecule, then continue synthesis. The site of re-initiation is presumably determined by base pairing between the nascent RNA and the new template molecule. Research on RNA viruses indicates that recombination between viral RNAS can occur at relatively high frequencies. For example, in closely related Strains of polioviruses, the frequency of recombination between genetic markers only 190 bases apart, was estimated at 0.13% (Kirkegaard & Baltimore, 1986). Recombinants between isogenic Strains of another picornavirus, foot—and-mouth disease virus, were detected at a frequency of 0.92% in vitro (McCohon et al., 1977). Based on these frequencies, King (1988) estimated that 15% of the poliovirus genomes and 10-20% of the foot- and-mouth disease virus genomes, may undergo recombination in a Single growth cycle. In coronaviruses, recombination during mixed infections occurs at frequencies so high that no selection pressure was needed for the isolation of recombinants (Makino et al., 1986; Lai, 1990). Extrapolation of localized recombination frequencies to the entire genome of mouse hepatitis virus 13 (MHV), suggests an overall recombination frequency as high as 25%. Recombination sites in the mouse hepatitis virus appeared to be distributed over the entire genome, but preferred sites were found which may correspond to regions of secondary structure (Lai, 1990). It was suggested that these sites may promote pausing of the replicase, generating incomplete RNA intermediates, which led to recombination. High frequency of recombination may be further promoted by the nonprocessive nature of the coronavirus RNA polymerase. Lai hypothesized that the high frequency of recombination in coronaviruses may counteract the high error rates in RNA synthesis and allow faithful reproduction of its relatively large genome. The existence of DI RNAS derived from the genome of turnip crinkle virus (Cascone et al., 1990) and cymbidium ringspot virus (Burgyan, 1991) indicates that RNA plant viruses have a mechanism for recombination. Direct evidence of recombination in plant RNA viruses has recently been obtained for several viruses. In brome mosaic virus (BMV), a positive sense tripartite virus, deletions in the 3’ noncoding region of one of the RNA genomic components was repaired during infection, by both homologous and non-homologous recombination, with the homologous region from either of the two wild type RNA components (Bujarski & Kaesberg, 1986; Bujarski & Dzianott, 1991). In cowpea chlorotic mottle virus (CCMV), another tripartite (+) sense RNA virus, deletions in either the 3a (putative movement gene) or coat protein gene of the RNA3 component blocks systemic infection. When plants were co-inoculated with wild type RNA1 and RNA2, and both 33 and CP deletion mutants of 14 RNA3, 30 to 60% rapidly developed systemic infection (Allison et al., 1990). Hybrid TMV constructs were created in which the CAT gene was inserted between the 30K and CP genes (Dawson et al., 1989) or had two copies of the CP gene (Beck & Dawson, 1990). Both constructs replicated efficiently in inoculated leaves, however, by the time systemic infection had developed, the inserted sequences had been precisely removed, giving wild type TMV. These studies indicate that recombination within plant viruses does occur and at relatively high frequencies under conditions of high selection pressure. Indirect evidence also exists that recombination may be occurring in other plant virus groups, including the nepovirus group. For example, Rott et al., (1991) reported a 1.5 kb region of identity in RNA1 and RNA2 of TomRSV and suggested that this was evidence for RNA recombination between the two genomic RNA components. Rott further hypothesized that "in TomRSV, replication begins in cis with RNA1 and that trans replication of RNA2 occurs only following disassociation and reassociation of the initial negative-strand transcript with the corresponding region in RNA2" (i.e., that only one RNA serves as the template for the 3' terminus of both RNA1 and 2). Such a mechanism has been proposed for leader-primed generation Of subgenomic RNAS in coronaviruses (Lai et al., 1990). This model proposes that a leader RNA is transcribed from one end of the RNA template, dissociates from the template, and then rejoins the template RNA at a downstream transcription initiation sites to serve as a primer for transcription. Scott et al., (1992) found a 1.5 kb sequence homology in the 3’ terminal 15 regions of RNA1 and RNA2 of a birch isolate of cherry leaf roll nepovirus. The authors questioned why "the RNA1 and RNA2 duplication is retained during countless cycles of replication in natural and glasshouse hosts when mutation rates are so high in RNA viruses" and suggested that "this duplicated sequence is the site for high levels of recombination between RNA1 and RNA2". A region of 820 nt on the 3’ terminus of the two genome components of the bipartite virus, tobacco rattle virus, are identical (Angenent et al., 1989). King (1988) states that "it is difficult to see how that degree of conservation can be maintained by selection only, and the evidence strongly suggests that these terminal sequences are constantly being exchanged by recombination". Not all evidence supports the hypothesis that recombination is necessary to maintain identity in a duplicated region of a viral genome, at least once it has been established. For example, a viable pseudorecombinant was formed between RNA1 of the TCM strain of tobacco rattle virus (TRV) and RNA2 of the PLB Strain Of TRV, which had genome segments that differed at their 3' termini in 39 of 820 nt (Angenent et al., 1989). After 25 passages through tobacco no evidence Of recombination was found (i.e., each of the RNAS retained their strain specific 3’ sequence). Angenent hypothesized that the identical 3’ sequences on the two genome segments may result from the preference of the TRV replicase for the wild type sequence over mutant RNAS, and that this competition could lead to selection of identical sequences on both RNA molecules. 16 Pathogen Derived Resistance & Potential Risks in Release of Transgenic Plants Plant viruses cause significant losses to crops worldwide and conventional approaches to controlling them have not always been successful. Conventional control measures have been aimed at reducing the amount of virus inoculum by controlling the vector, removing virus sources, using healthy planting material, and/or providing temporal or spacial isolation by various cultural methods. Complete isolation, however, is not practical in most cases and often some economically acceptable level of infection and yield loss is the goal, as in integrated pest management. Another disadvantage Of this type of control is that it is labor intensive and therefore expensive. The use of resistant plants in controlling virus diseases overcomes many of these problems. Genetically based resistance has the advantage that, once resistant cultivars are planted, no further input is required to provide protection from targeted viruses. There are limitations to breeding for resistance, however, such as the time and expense of development, linkages to undesirable traits, incomplete levels of resistance, difficulty in transferring multigenic resistance and the lack of useful sources of resistance genes in sexually compatible species. Alternative approaches to control of plant virus diseases are being developed and applied that could overcome some Of the limitations of conventional control methods. Currently, most of these strategies are based on the concept of pathogen derived resistance (i.e., deriving resistance genes from the pathogens own genome) developed in the 19808 (Sanford & Johnston, 1985) . The basic principle of pathogen derived resistance is that 17 "in any given set of host-pathogen interactions, there are certain pathogen- encoded functions essential to the pathogen, but not the host. If one of these pathogen-specific functions is disrupted (e.g., by expressing a pathogen gene at the wrong time, in the wrong amount, or in a counter functional form), the pathogenic process should be stopped" (Grumet, 1990). Development of transformation and regeneration systems for many agronomically important plants and an increased understanding of the genome and gene functions of plant viruses has aided in the development of this concept and its application to conferring protection against plant viruses. Engineering of virus resistance has focused primarily on the use of coat protein genes, and to a lesser extent, on satellite RNAS and other non-structural genes. Reduced susceptibility to viral infection in transgenic plants expressing a virus coat protein gene was first shown in tobacco mosaic virus (Powell-Abel et al., 1986). Since that time, coat protein mediated protection has been successfully demonstrated in over 20 plant/virus systems, including 12 distinct virus groups (Reviews: Beachy et al., 1990; Grumet, 1990; Hull & Davies, 1992). While coat protein transgenic plants are usually only resistant to low levels of inoculum (comparable to natural levels), they often give some resistance to a range of viral stains or related viruses. The mechanisms responsible for coat protein mediated resistance are not well understood. A current hypothesis for rod Shaped viruses is that endogenous coat protein prevents co-translational disassembly (i.e., the disassemby of virions as they are translated) of the incoming source virus 18 during the initial stages of infection (Register & Beachy, 1988; Wu et al., 1990). This hypothesis is supported by experiments in which resistance was largely overcome when TMV CP transgenic plants were inoculated with viral RNA instead of whole virions (Beachy et al., 1990). However, this mechanism does not explain all examples of coat protein mediated resistance, since transgenic plants expressing the coat protein of potato virus X are resistant to infections by both viral RNA and whole virions (Hemenway et al., 1988). Inhibition of the synthesis or function of the replicase gene, or competition from a non-functional form of the replicase gene, are other strategies that have Shown potential in controling viruses diseases. Golemboski et al. (1990) demonstrated that tobacco plants transformed with the read- through portion of the TMV replicase gene (encodes the 54K protein) were completely resistant to infection by TMV. This resistance was not overcome by high concentrations of inoculum or by inoculation with viral RNA. Using an analogous portion of the pea early browing replicase gene, MacFarlane and Davies (1992) induced resistance in transgenic Nicotiana benthamiana plants to infection by the source virus. Extending this strategy to viruses that do not contain read-through portions of their replicases genes, Anderson et al. (1992) transformed tobacco with a modified and truncated replicase gene of cucumber mosaic virus. The modified gene had a 94 bp deletion in the conserved GDD domain of the replicase gene which caused a frame Shift, resulting in a truncated non-functional protein. The transgenic plants expressing the truncated protein were completely resistant to both virons (500 ug/ml) and viral 19 RNA (50 ug/ml). This represents an inoculation concentration 10 times higher than that tested for CMV coat protein mediated resistance (Cuozzo et al., 1988). However, plants expressing replicase genes are usually resistant only to infection from the homologous source virus. As a result of the levels of disease resistance exhibited by many transgenic plants expressing viral genes, widespread application of pathogen derived resistance is occurring and transgenic plants containing viral coat protein genes and other nonstructural genes are being developed and tested. While this approach has tremendous potential, the downside is that there may be certain risks involved in the release of transgenic plants expressing coat protein. One Of these risks is involves transcapsidation; where in mixed infections the coat protein produced by one virus might encapsidate the other infecting virus (Roschow, 1970,1977; Creamer & Falk, 1990). DeZoeten (1991) makes the point that in monocultures of coat protein transgenic plants every infection is in essence a double infection (with respect to the coat protein gene), therefore the potential for transcapsidation is high. Transcapsidation could alter the vector range of the virus leading to the creation of an apparently new disease. Another potential risk in the release of trangenic plants relates to the possibility of recombination between mRNA of the introduced viral gene and an infecting virus resulting in accelerated evolution of new viruses (de Zoeten, 1991; Palukaitis, 1991; Tolin, 1991). The likelihood of recombination occurring depends upon the frequency of recombination and how similar the 20 two virus genomes are (the greater the similarity the greater the likelihood and frequency of recombination). Summary The presence of identical 3’ noncoding regions on RNA components of BBLMV and other nepoviruses (as well as on most other multipartite RNA viruses) raises many questions. How did these duplications originate, how and why is identity maintained? Selection pressure can eliminate variants that affect fitness, but what about neutral mutations? And could selection lead to the formation of the original duplication? If so, then convergent evolution must be a common occurrence, Since this phenomena exists for so many viruses. Where does recombination fit in this scheme? Perhaps in the creation of the original duplication, but what role does recombination play in its maintenance? This research begins to answer some of these questions and in the process raises many new ones. LIST OF REFERENCES ALDAOUD, R., DAWSON, W.0., & JONES, GE. (1989). Rapid random evolution of the genetic structure of replicating tobacco mosaic virus populations. Interviro/ogy 30, 227-233. 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Plant Virology, Third Edition, pp.170—176. Academic Press |NC., London/New York. MARGIS, R., VIRY, M., PINCK, M., & PINCK, L. (1991). Cloning and in Vitro characterization of the grapevine fanleaf virus proteinase cistron. Virology 185, 779-787. MARTELLI, GO. (1975). Nematode Vectors ofP/ant Viruses, pp. 223. Edited by F. Lamberti, C. E. Taylor & J. W. Seinhorst. London & New York: Plenum Press. McCAHON, D., SLADE, W.R. PRISTON, R.A.J., & LAKE, JR. (1977). An extended genetic recombination map of foot-and-mouth disease virus. Journal of General Virology 35, 555. 26 MEYER, M., HEMMER, 0., MAYO, O. & FRITSCH, M.A. (1986). The nucleotide sequence of tomato black ring virus RNA-2. Journal of General Virology 67, 1257-1271. MURANT, AF. (1981). Nepoviruses, pp. 197-238. In: E. Kurstak (ed.), Handbook of Plant Infections and Comparative Diagnosis. Amsterdam, ElsevierlNorth—Holland. OKADA, Y., OHASHI, Y., OHNO, T., & NOZU, Y. (1986). Molecular assembly of tobacco mosaic virus in vitro. Adv. Biophys. 22, 95-149. PALUKAITIS, P. (1990). Virus-Mediated Genetic Transfer in Plants. In: M. Levin and H. Strauss (eds.), Risk Assessment in Genetic Engineering: Environmental Release of Organisms. McGraw-Hill Publishers. POWELL-ABLE, P., NELSON, R.S., DE, 8., HOFFMANN, N., ROGERS, S.G., FRALEY, R.T., & BEACHY, R.N. (1986). Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232, 738-743. PRITTS, M.P. & HANCOCK, J.F. (eds.) (1992). Highbush Blueberry Production Guide (NRAES—55). RAMSDELL, D.C. & STACE-SMITH, R. (1979). Blueberry leaf mottle, a new disease of highbush blueberry. Acta Horticulture 95, 37-45. RAMSDELL, D.C. & STAGE-SMITH, R. (1981). Physical and chemical properties of the particles Of ribonucleic acid of blueberry leaf mottle virus. Phytopatho/ogy 71 (4), 468-472. RANDLES, HARRISON, MURANT & MAYO. (1977). Journal of General Virology 36, 187. REGISTER, J.C. & BEACHY, R.N. (1988). Resistance to TMV in transgenic plants results from interference with an early event in infection. Virology 166, 524-532. ROCHOW, W.F. (1970). Barley yellow dwarf virus: phenotypic mixing and vector specificity. Science 167, 875-878. ROCHOW, W.F. (1977). Dependent virus transmission from mixed infections, Pages 253-277. In: K.F. Harris and K. Maramorosch (eds.) Aphids as Virus Vectors. Vol. 24. Academic Press, New York. ROTT, M.E., TREMAINE, J.H. & ROCHON, OM. (1991). Comparison of the 27 5' and 3’ termini of tomato ringspot virus RNA 1 and RNA 2: evidence for RNA recombination. Virology 185, 468-472. SANDOVAL, CR. (1992). Movement of Blueberry Leaf Mottle Virus (BBLMV) Within and Between Cultivated and Wild Vaccinium spp. Thesis for the degree of MS. Michigan State University, East Lansing, Michigan. SANFORD, J.C. & JOHNSTON, S.A. (1985). The concept of parasite-derived resistance: deriving resistance genes from the parasite’s own genome. Journal of Theoretical Biology 113, 395-405. SCOTT, N.W., COOPER, J.l., LIU, Y.Y. & HELLEN, C.U.T. (1992). A 1.5 kb homology in the 3’-terminal regions of RNA1 and RNA2 of birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate. Journal of General Virology, 73, 481-485. STEINHAUER, D.A. & HOLLAND, J.J. (1987). Rapid evolution of RNA viruses. Annual Review of Microbiology 41, 409-433. TOLIN, S.A. (1990). Persistence, Establishment, and Mitigation of Phytopathogenic Viruses. In: M. Levin and H. Strauss (eds.) Risk Assessment in Genetic Engineering: Environmental Release of Organisms. McGraw-Hill Publishers. UYEMOTO, J.K., TASCHENBERG, E.F., HUMMER, D.K. (1977). Isolation and identification Of a strain of grapevine Bulgarian latent virus in concord grapevine in New York State. Plant Disease Reporter 61, 949-953. WU, X., BEACHY, R.N., WILSON, T.M.A., & SHAW, JG. (1990). Inhibition of uncoating Of tobacco mosaic virus particles in protoplasts from transgenic tobacco plants that express the viral coat protein gene. Virology 179, 893- 895. ZAITLIN, M. & ISRAEL, H.W. (1975). Tobacco mosaic virus (type strain) CMl/AAB Descriptions Plant Viruses No. 151, pp1-5. SECTION I: CLONING AND SEQUENCING OF THE COAT PROTEIN GENE AND THE 3' NONCODING REGION OF BLUEBERRY LEAF MOTTLE VIRUS RNA2 Introduction Blueberry leaf mottle virus (BBLMV) is the causal agent of a relatively new viral disease of highbush blueberry, Vaccinium corymbosum L. (Ramsdell & Stace-Smith, 1979). Infected plants exhibit a general dieback of stems, leaf distortion and a pronounced mottling of the leaves. Within a few years after the onset of symptoms, severe dieback of the stems results in stunted growth and reduced yield. Eventually the plants die. Currently BBLMV is found only in Michigan, where it has been identified in numerous cultivated and wild blueberry populations (Sandoval, 1992). Since the disease is caused by a pollen—born virus, honey bees carring infected pollen from nearby wild blueberry plants can transmit the virus to commercial fields (Boylean-Pett et al., 1991; Hancock et al, 1993). The virus cannot be controlled by eliminating the vector, since honey bees are necessary for blueberry pollination and fruit set. Currently, the best approach to controlling BBLMV is to plant virus-tested Stock and to eradicate any infected bushes. However, detection of infected plants can be difficult because of the long latent period where plants remain symptomless. Genetic based resistance is generally regarded as the best approach over the long term, but useful sources of resistance have not been 28 29 identified. Commercial cultivars (Blueray, Bluecrop, Elliot, Jersey, Rubel, and Spartan) and wild blueberry species (Vaccinium corymbosum, V. angustifoliium, and V. myrtle/oidies) screened for resistance were found to be susceptible to infection by BBLMV (Sandoval, 1992). However, other forms of resistance are being developed which use the virus genome as the source of resistance genes. Pathogen derived resistance (i.e., deriving resistance genes from the pathogen’s genome) is a strategy which has been recently employed to develop useful levels of resistance to many different plant viruses (Reviews: Beachy et al., 1990; Grumet, 1990; Hull & Davies, 1992). Thus far, the engineering of this type of resistance has focused primarily on the use of the coat protein gene. Transgenic plants expressing viral coat protein genes often Show a delay in disease development or are resistant to infection upon inoculation by the source virus. This type of resistance, termed coat protein mediated protection, has now been demonstrated in over 20 plant/virus systems. As a first step in engineering resistance to BBLMV in blueberries, we have cloned and sequenced the coat protein gene of BBLMV. BBLMV, like other nepoviruses, has two Single stranded RNA components which are encapsidated in 28 nm isometric particles (Ramsdell & Stace-Smith, 1981). It’s genome is composed to two RNA species, RNA1 (~6.9 kb) and RNA2 (~6.3 kb), both of which are essential for infectivity. The RNAS are encapsidated separately in particles composed of a single coat polypeptide with an estimated Mr 54,000. AS with other nepoviruses, BBLMV genomic RNAS are believed to be translated as polyproteins, which are later 30 cleaved by a RNA1 encoded protease (Harrison & Murant, 1977). We report here on the nucleotide sequence of the 3’ terminus of BBLMV RNA2. The coat protein gene was identified and its precise location determined. A comparison was made between the coat protein sequence of BBLMV and other nepoviruses. Materials and Methods Virus purification and nucleic acid extraction. The BBLMV used in this Study was originally isolated from highbush blueberry plants (Vaccinium corymbosum L. cv. Rubel) collected in southwestern Michigan. The virus was propagated in Chenopodium quinoa and leaves were harvested 7-10 days after mechanical inoculation, then homogenized in 2 ml/gm cold 0.05 M boric acid- borax buffer containing 0.1% (w/v) of sodium thioglycollate and sodium diethyldithiocarbamate, pH7, and the extract was filtered and frozen (Ramsdell & Stace-Smith, 1981). After thawing at 4°C, the extract was centrifuged at 10,000 rpm for 15 min. Chloroform and N-butanol (each 10% v/v) were added to the supernatant and stirred for 1 hour. After low Speed centrifugation, 8% (w/v) polyethylene glycol (mol wt 6000) and 1% (w/v) NaCl were added. The mixture was centrifuged at low Speed and the pellet was resuspended in 10% of the initial volume with 0.05 M Tris-HCl buffer, pH 7.4. After an additional low speed centrifugation, the supernatant was ultracentrifuged at 38,000 rpm for 90 min. The pellet was resuspended in 0.05 M Tris-HCl buffer (pH 7.4) then centrifuged at 38,000 rpm for 90 min. through 5—30% sucrose density 31 gradients made in Tris-HCl buffer. The gradients were scanned at 254 nm and the fraction containing the virus particles was collected. The virus particles were concentrated from the sucrose by ultracentrifugation and resuspended in 0.05 M Tris-HCI buffer, pH 7.4. A mixture of 1 ml of purified virus (1mg/ml) and 1 ml dissociation buffer [1% (v/v) 2-mercaptoethanol, 4M Urea, and 1% (w/v) SDS in 0.1M sodium phosphate buffer, pH 7.2] was incubated at 50°C for 30 min. The treated sample was centrifuged at 38,000 rpm for 5 hr at 4°C through a linear-log sucrose density gradient made with RNase-free sucrose in 1 X SSC buffer, pH 7, containing 6 pg/ml purified bentonite (Fraenkel-Conrat et. al., 1961). The gradients were scanned at 254 nm and a single nucleic acid peak was collected. Sodium acetate was added to 0.15 M and the nucleic acid was precipitated with two volumes Of 100% ethanol. The mixture was centrifuged at 10,000 rpm for 10 min at —20°C, the pellet was resuspended in 1 X SSC buffer, and stored at -20°C. cDNA cloning and nucleotide sequencing. Complete separation of RNA1 and RNA2 of BBLMV is difficult due to the similarity in their molecular weights (M,2.35x108 and 2.15x106, respectively). Therefore, mixed RNA populations were used as templates for oligoldT) primed cDNA synthesis using the cDNA Synthesis System Plus Kit by Amersham (Gubler & Hoffman, 1983). Size fractionated cDNA was blunt end ligated into Bluescript KS+ vector cut with Smal, then cloned in XL1-Blue Escherichia coli cells (Sambrook et al., 1991). Clones sufficiently long to encode a coat protein of an estimated size of 1.3Kb 32 were separated into two classes (RNA1 or RNA2) based on restriction enzyme analysis. The largest representative clone from each class, clone 24 (3.1 Kb) and clone 34 (3.3 Kb), were selected. The viral origin of the clones was tested by probing a northern blot of purified BBLMV RNA with labeled cDNA clones (Sambrook et al., 1991). Subclones used to determine the cDNA sequence were obtained by Exonuclease Ill generated nested deletions and by restriction enzyme digestion. Both strands of clone 34 were sequenced by dideoxynucleotide chain termination method (Sanger, 1981) using single stranded templates prepared according to Vieria and Messing (1987). N terminal amino acid sequencing of the viral coat protein. To identify which clone encoded the coat protein gene, the N terminus of the viral coat protein was sequenced. The intact virions were denatured by combining equal volumes of the purified virus (10ul of a 1 mg/ml suspension) and dissociation buffer [1% (v/v) 2-mercaptoethanol, 4M Urea, and 1% (w/v) SDS in 0.1M sodium phosphate buffer, pH 7.2] in a microfuge tube and heating the mixture in a boiling water bath for 90 sec. The coat protein was purified on a 12% SDS-PAGE gel, Stained with coomassie blue, electroblotted onto a PVDF membrane and directly sequenced using an Applied Biosystems model 477A protein sequencer (Hunkapiller et al., 1983). Nucleotide sequence analysis. Sequence data were analyzed using the Genetics Computer Group Sequence Analysis Software Package for the VAX, Version 7.1 (Devereux et al, 1984). Alignments of the amino acid and nucleotide sequences were Obtained using the GAP and BESTFIT algorithms. 33 The sequences of the following viruses were obtained from GenBank or EMBL databases: tomato ringspot virus RNA2 (Rott et al., 1991a,b), arabis mosaic virus RNA2 (Bertioli et al., 1991), grapevine chrome mosaic virus RNA2 (Brault et al., 1989), grapevine fanleaf virus RNA2 (Serghini et al., 1990), tomato black ring virus RNA2 (Greif et al., 1986), cherry leaf roll virus RNA2 (Scott et al., 1992), and raspberry ringspot virus RNA2 (Blok et al., 1992). Results and Discussion Nucleotide sequence of BBLMVRNAZ 3’ terminus. Based on restriction enzyme analysis the cDNA clones were divided into two groups, presumably RNA1 and RNA2. The largest representative from each class, cDNA clone 24 (3.3Kb) and 34 (3.1Kb), were selected for cDNA sequencing (Figure 1). 32P labeled probes made from the 3’ ends of cDNA clones 24 and 34 hybridized to BBLMV RNA and not to total plant RNA, confirming their viral origin (data not shown). The cDNA sequence of clone 34 was determined along with the predicted amino acid sequence (Figure 2). The cDNA sequence of the 3’ portion of clone 24, determined to be from RNA1 of BBLMV, is given in Section II. Identification of coatprotein gene. The location of the coat protein gene and the protease cleavage Site from the RNA2 polyprotein precursor, were determined from the amino acid sequencing of the N-terminus of the purified coat protein. The sequence of the N-terminal 15 amino acids of the BBLMV coat protein was determined by automated Edman degradation to be 34 “It! 5111 EMA) 5 am an : : : L— Clone 24 m. 2H. 35 cm BeoRV 1'4:on Sm! PoMA) a BSKS+ El : I : :2 Clone 34 on. 1H. 2H. 3H. Figure 1. Unique restriction sites for clones 24 (RNA1) and 34 (RNA2) used to distinguish between cDNA clones. 35 1 LLunn-uu r-I-I-nl fi- l-|l-|nu Inulbblulunl HI I...“ n. T ‘ Ilf‘ll‘l 6O 1 P N M -T G R A S I P V Q T N I R N S P R 20 61 ATTGT‘ “n“ nonmnrwoaa'r'rmnr‘nnr‘rw. . ‘ .anrrA 120 21 I V D G E E I T P P R F T T C N--S G L I 40 121 l‘:“—“‘l‘:“lln“ll lflulnlfll’k Lvl All-I II I I Ankanf‘alfilll‘" TTTTTT 180 41 A D T S I A H V V Q G W V P K D A T K G 60 181 Lu;u;;1 l HI-HI-I-I | I- l 1M1 1 II-I l I I All I LTAGTTAAA 240 61 R V L E A I N L R E D I A T S D N L V K 80 241 TATGAGTGGCT'T‘Gll TTTTT TCATACATCCAGATTTGAAGTTGCGTATGACTGTCGGC 300 81 Y E W L A K G L I H P D L G 100 301 CAAAATCCCTTTGII -------AlnlhlIhlhAILAcTTTGGGCGCCTTAGT 360 101 Q N P F V G I S I G I C C D Y F G R L S 120 361 AAATATTATGAGGGCGACACTGCTTTACCTATAGAGGTATGCAATCAATTGCCCAATTTT 420 121 K Y Y E G D T A L P I E V C N Q L P N F 140 421 GTTTGCCCAATAT‘ ”“"‘ I“lbllbuflulIIUHIIlflbfllfllulbbblbbbbbbfi 480 141 V C P I S E K S V F E F D L D M S L A G 160 481 TATAACCTTTTTCAAACTTCTAAAGGCTTCGCTGATCCGGTATTATTAGTGTACATAATA 540 161 Y N L F Q T S K G F A D P V L L V Y I I 180 541 GATACTAATTCTTTACCCGCCAGTGATGAATGGGTTTACACATGTGAGGTTTGTATAAAA 600 181 D T N S L P A S D E W V Y T C E V C I K 200 601 TCTGCCTTGCATGCCACTTCTGTGGCAAATAAACCCATTCTATCGCTACCACATTTTTTT 660 201 S A L H A T S V A N K P I L S L P H F F 220 661 GACGGTCGTCTCCCACTTGACTTGTGGAGGGGACCTTTTTCTTTTGAGTTAGGTAGAAGT 720 221 D G R L P L D L W R G P F S F E L G R S 240 721 TCCAAAAGGGAGAATCACATCGGCATCAATTTTGGTAGTGCTCGTGTTGTCTCTGGGACC 780 241 K R E N H I G I G S A R V V S G T 260 781 AATACCTTTTATTCTTTTCCTGCTGCCTATACTCAGCTTTTACAGAGTGTAGGTGGTATT 840 261 N T F Y S F P A A Y T Q L L Q S V G G I 280 841 TTACATGGTACTGTCGTTCAAACTGGCAGTAAGGCTATATCTTGTGAGATGTTTCTTATC 900 281 L H G T V V Q T G S K A I S C E M F L I 300 901 CTTCAACCGGATAAGACCGCCCACAATTTAGAGCAGGCTCTCCGCCTTCCTGGTTGTCGT 960 301 L Q P D K T A H N L E Q A L R L P G C R 320 9 6 1 ATACCAACTGGGGGTGGACCA‘I‘TTTCTA‘I‘TCGTATACAGACTCCCTTCCAGCGAGAGCAA 1 02 o 321IPTGGGPFSIRIQTPFQREQ 340 1021 ATTTTTAATACCGGCGTTCAGCTGGTAATCTATGCTGTTGGGGGTCCTATGGGAGCACAA 1080 341 I F N T G V Q L V I Y A V G G P M G A Q 360 Figure 2. The cDNA sequence and predicted amino acid sequence Of 3’ terminus of BBLMV RNA2. The N terminal amino acid sequence of the coat protein determined by Edman degradaton is underlined. The coat protein sequence is 521 amino acids long. 36 GCTATATCTGCACCATATCAATATATGGTGCATTTCTCCCATATAC““"““A“ T A I P Y Q Y M V H I Q E E G D CCLCCGCLICGLLCIAI-uu- luhiLLI1nnILUuubbALbAIIIL “‘TG P P P R P I G N V L L F N W A T I S E M ACGAATCTTALLLGGIIIbflbfillbbbbbUDGHI I‘TCGTGTTGCCAGGTCAA T N L T R F Q I P A R S D L V L P G Q ACTGTCACCATGAGGCGAAATGCTTTAGCGAATCTGATAAGGTCTTGTGGGTTCTTCCGG T V T M R R N A L A N L I R S C G F F R GGCCGTGTTACATTTGTGTTCCAATGGACATTGAATGTAGCACATATTGTACCAACTGCT G R V T F V F Q W T L N V A H I V P T A ACAATGCAAATTTTAACGGCAGTTGGGCGCGTTGGCAATGCGGAGACTAATGGTTCACAA T M Q I L T R N A E T N S Q ATCCTACAAAGTTGGATTGTGCCCGTTAGTCAGGTCTTTGAGAAAGAGGTTGAAATGGAT I L Q S W I V P V F E K E V E M D CTCACCGATTATCCCGGTTTTAATACATCTGGGGGAATTGGTGCTGACCATGATCAGCCT L T F N T S G G I G A D H D Q P TACATTGACATTGCTTGTGGTAATTTTCCACAAATATTCTATATGAATATCAATGTGCGT Y I D I A C G N F Y M N I N V R GTACACCCCGGGTTTGAGCTCTATGGTAGGAGTATCACACCCCTACGCATTTAGTATGCT V H P G F E L Y * AAGTGTGTTTATAGGAACTAGAACCCTTAAGTTCTAGGAGTTGGTCTGTCCTCTCTGACA * * GGCCTTCAAAGGATAGAGAATAGCTCGAACTCTCTGTAATACGAGAGGTCCGGACCTGTA GGTCTTCCTGGCATATACCCAGGTTTTGAGATAGTAGTAAACTACTCTTCGATGTAGCGA ATCGTCGTAAATAGGACACCCTCCTAAAACGAAGCCTTAAATAGGAACTTGAAAAAGTTT CCTTTCCACTTTGTGGAGGATAGTATAAGGGACGGTGGTGCCAGCTTGATGACTGCTTAA GAGCAGGAGGTTGCTCGTTAACCTTACACGAGCTAGGACGTTCTAGTAGAGATGAGACAT CTACCTCGAAAAACGTCAGAATTACTATATGATTCAAAAGCGTGGTTTTTCCAACGTTAA CCAATGGAAACCAGGTGCACATAGGTTAGTTGTGCTGATTTGCTACCTTTTAAGAAAGGA GATTATTCTGGTGAAATTCCAGATCTATCTTAGTTTTGTTGTTTCAGTTTGATTGCAATA AACCCACATAAACTGTCGTCATTAGGACGGCATACCATTGAGCTCTTTAGGGCGCCTCTG GTTCCGTGAAATCGGTATACGTGTGAAGATTAGGGTTTGCTCGAACCATAGAGAGCTAGG TTGTTGGAGCCGAACTGAGTCCAACCGCATTTGTCAGTTTTAGATATAACTGTCCAAGGT CTACTGCTTCCGAGCCTGAAAAAATCTTAAAGCGCCCAGGCGTCCGTGACTTCACGGCAC TCGGGGACAGAGTTTAGGGAAACTCTAGAAAAATTCCCTCGCCTTTTAGTTGTGTGGCCG TGATGGACACAACTCTCTCTTCTTTCTGAGAGTGTACCGCTGTTTTAGTATCTGGTGATG ATGTAGTTTTGAAACTACCAGAGATGTCTCAGTGGAGAAGCGTCTTGCCAAACGATATTG GCTTAAGGTCTATGTGACGATAATTTGCTAGTGTACTCTAGAGAATGTGGGGTGGCACCC ACTTCTTGGATGAGGGCCGGAGATGAAAACCGGGGAGTAATAAACTCCAGCTAGCGGCAT AGGCCGACCACCGTGAGGGAGCTCACGGCGCAATTTGGACCATTTTTAGACATAAATGGC CATGTTAGTGTAGCGCTTTGCGCATGTTGAATGATAATGAACCATGCGTTGCAGCGCATG CCTTTCGAGATCGGATGTGATTACCGTGAGAAAGGGGAAACAATGCCAACATGTTCAATT CGTTGTACTATGTTTTCTTTCTTTTTGTAGACTCCTGTGAGGATTATCCAACAGCAGGTT GTGCCTTCAGTAAGCACACAAAAAGATTTCGCATTTTTCTTTGTGTTAGATAGTTTTATA TCTATAATGTCTTTATTTCAC 1140 380 1200 400 1260 420 1320 440 1380 460 1440 480 1500 500 1560 520 1620 540 1680 Figure 2. (continued) 37 S-G—L-l-A-D-T-S-l-A-H-V-V-O-G. This amino acid sequence corresponded exactly to the translated cDNA sequence of clone 34 starting at nucleotide position 109 (Figure 2). The presence of the coat protein gene on clone 34 indicates the source of the clone was RNA2, since in all other nepoviruses sequenced to datethe coat protein has been found on the 3’ end of the RNA2 component. The coat protein gene spanned 1562 nucleotide residues and terminated at a TAG stop codon at nt position 1671, followed closely by other in-frame stop codons (Figure 2). The resulting protein was 521 amino acids In length with a predicted Mr of 57,542. This is somewhat larger than the estimated Mr of 54,000, previously determined by SDS polyacrylamide gel electrophoresis (Ramsdell & Stace-Smith, 1981). Providing no processing occurs after cleavage, the cut site of the BBLMV coat protein would be (N/S) at position 108/109. This cleavage Site is unique for the nepovirus group; however, there does not seem to be much conservation of cleavage Sites among nepoviruses. The known cleavage sites for the release of other nepovirus coat proteins from polyprotein precursors are: K/A for TBRV (Demangeat et al., 1991), R/A for GCMV (Brault et al., 1989), C/A for RSV (Blok et al., 1992) and R/G for AMV (Bertioli et al., 1991) and GFLV (Serghini et al., 1990). 3’ noncoding region of RNA2. The region immediately downstream of the TAG stop codon of the coat protein gene extended about 1.4 Kb ending with a poly(A) tail. Downstream Of the coat protein Stop codon were an additional 24, 42 or 28 Stop codons in each of three reading frames, 38 respectively. All ORFS in this 3’ sequence were less than 141 nt long and are believed to be noncoding, since only a single ORF is translated from each RNA component in the other nepoviruses (Harrison & Murant, 1977). A 3’ noncoding region of this length is unusual in plant viruses, but has been reported for two other nepoviruses, TomRSV and CLRV (Rott et al., 1991a; Scott et al., 1992). The significance of this long 3’ noncoding region is not known. Coat protein comparisons with other nepoviruses. The amino acid sequence identities between the coat proteins of BBLMV and other nepoviruses were calculated (Tables 1 & 2). The statistical Significance of the alignment scores was determined by comparing the alignment score of each pair of sequences against the mean of 25 randomized comparisons, found when the second sequence was repeatedly shuffled then aligned to the first. The similarity was judged to be significant if it exceeded the randomized mean plus three standard deviations (Doolittle, 1981). The amino acid identity between the coat proteins of BBLMV and the other nepoviruses was low, ranging from 20.7% (RRV) to 29.2% (CLRV). However, in all comparisons the percent identity was statistically significant and similar to the levels of homology found between other nepoviruses (Table 2). It is interesting to note, that CLRV and TomRSV, both having along 3’ noncoding region, showed the highest similarity to BBLMV. Future efforts will be focused on using the cloned coat protein gene to determine if coat protein mediated protection will be an effective strategy for 39 developing resistance in blueberries. Initial testing of coat protein constructs will be performed in Nicotiana tobaccum, an easily transformable systemic host of BBLMV. Regeneration of Shoots from leaf disks (Callow et al., 1991) and successful agrobacterium transformation, makes the introduction of a coat protein construct in blueberries feasible. Table 1. Percent amino acid identity between BBLMV coat protein and other nepovirus coat proteins. 40 Virus % Amino Acid Alignment Randomized Identity Scorez Score :l: SD GCMV 23.0 246.0* 153 :I:4.9 GFLV 22.4 234.1 * 154 15.2 TBRV 23.9 236.8* 155 14.2 AMV 22.7 232.2* 154 $5.4 TomRSV 28.6 301.9* 165 :45 CLRV 29.2 75.7* 51 :19 RRV 20.7 214.8* 154 :4.4 GCMVY 17.0 152.8 152 :l:4.8 * Percent amino acid identity was found to statistically Significant if the alignment score exceeded the mean randomized alignment score plus three standard deviations (Doolittle, 1981). 2 Alignment score (Quality) = (1.0 X TotalMatches) - (0.9 x TotalMismatches) - (GapWeight X GapNumber) - (GapLengthWeight X TotalLength of Gaps) Y Randomized sequence 41 Table 2. Percent amino acid identity between nepovirus coat proteins. GCMV AMV GFLV RRV BBLMV CLRV TomRSV TBRV 57 22 23 23 24 24 22 GCMV 23 24 23 23 20 24 AMV 69 23 23 20 25 GFLV 23 22 21 25 RRV 21 21 24 BBLMV 29 29 CLRV 22 TomRSV -- TEVz 1 6 1 5 1 6 1 4 1 6 14 1 5 BBLMV" 16 15 16 16 18 15 18 I Bold numbers were statistically significant 2 TEV (tobacco etch virus) used for a comparison to a nonrelated virus Y Randomized sequence of BBLMV used for comparison to nonrelated sequences with similar base composition LIST OF REFERENCES BEACHY, N.R., LOESCH-FRIES, S., & TUMER, NE. (1990). Coat protein- mediated resistance against virus infection. Annual Review of Phytopatho/ogy 28,451—474. BERTIOLI, D.J., HARRIS, R.D., EDWARDS, M.L., COOPER, J.l., & HAWES, W.S. (1991). Transgenic plants and insect cells expressing the coat protein of arabis mosaic virus produce empty virus-like particles. Journal of General Virology 72, 1801-1809. BLOK, V.C., WARDELL, J., JOLLY, C.A., MANOUKIAN, A, ROBINSON, D.J., EDWARDS, M.L. & MAYO, M.A. (1992). The nucleotide sequence Of RNA2 of raspberry ringspot nepovirus. Journal of General Virology 73, 2189-2194. BOYLEN-PETT, W., RAMSDELL, D.C., HOOPINGARNER, R.A., & HANCOCK, J.F. (1991). Honeybee foraging behavior, in-hive survival of infectious, pollen-borne blueberry leaf mottle virus and transmission of the virus is highbush blueberry. Phytopatho/ogy 81, 1407-1412. BRAULT, V., HIBRAND, L., CANDRESSE, T. Le GALL, 0. & DUNEZ, J. (1989). Nucleotide sequence and genetic organization of Hungarian grapevine chrome mosaic nepovirus RNA 2. Nucleic Acids Research 17, 7809—7819. CALLOW, P., HAGHIGHI, K., GIROUX, M., & HANCOCK, J.F. (1989). In Vitro shoot regeneration of leaf tissue from micropropagated highbush blueberry. HortScience 24(2), 373-375. DEMANGEAT, G., HEMMER, 0., FRITSCH, C., Le GALL, O. & CANDRESSE, T. (1991). In Vitro processing of the RNA 2 encoded polyprotein of two nepoviruses: tomato black ring virus and grapevine chrome mosaic virus. Journal of General Virology 72, 247-252. DEVEREUX, J., HAEBERLI, P., & SMITHIES, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Research 12(1), 387-395. 42 43 DOOLITTLE, RF. 1981. Similar amino acid sequences: Chance or common ancestry? Science 214(9), 149-159. FRAENKEL-CONRAT, H., SINGER, B., TSUGlTA, A. (1961). Purification of viral RNA by means of bentonite. Virology 14, 51-58. GREIF, C., HEMMER, O. & FRITSCH, C. (1986). The nucleotide sequence of tomato black ring virus RNA 2. Journa/of General Virology 67, 1257-1271. GRUMET, R. (1990). Genetically engineered plant virus resistance. HortScience 25(5), 508-513. GUBLER, U. 8! HOFFMAN, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. HARRISON, B.D. & MURANT, AF. (1977). Nepovirus group. CMl/AAB Descriptions of Plant Viruses, no. 185. Kew, Surrey, UK. HULL, R. & DAVIES, J.W. (1992). Approaches to nonconventional control of plant virus diseases. Critical Reviews in Plant Sciences 11(1), 17-33. HUNKAPILLER, M.W., LAJUN, E., OSTRANDER, F. & HOOD, LE. (1983). Isolation of microgram quantities of protein from polyacrylamide gels for amino acid sequence analysis. Methods in Enzymo/ogy 91, RAMSDELL, D.C. & STACE—SMITH, R. (1979). Blueberry leaf mottle, a new disease of highbush blueberry. Acta Horticulture 95, 37—45. RAMSDELL, D.C. & STACE-SMITH, R. (1981). Physical and chemical properties of the particles of ribonucleic acid of blueberry leaf mottle virus. Phytopatho/ogy 71(4), 468-472. ROTT, M.E., TREMAINE, J.H. & ROCHON, D.M. (1991a). Comparison of the 5’ and 3’ termini of tomato ringspot virus RNA 1 and RNA 2: evidence for RNA recombination. Virology 185, 468-472. ROTT, M.E., TREMAINE, J.H. & ROCHON, D.M. (1991b). Nucleotide sequence of tomato ringspot virus RNA2. Journal of General Virology 72, 1505-1514. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1991). Molecular Cloning: A laboratory Manual. Cold Springs Harbor Laboratory, Cold Spring Harbor, NY. SANDOVAL, C. (1992). Movement of Blueberry Leaf Mottle Virus (BBLMV) ~r- V to. MM rm": .4 pm "pm ..II T .oa-r-a .6! “claw “WNW“ In 9309-1952? shImelaim or” (380?) .3 .HOCTIR? II .0 Jim ..5!‘ ’ 7' .r Y .—..SI '6 \l mien-I \sxeva'a‘olcn‘tioL _':_ Ann autiv unit tin-Id r wherein-- au'ri't- 1 lili'll tit-"5’ mar-I W“ in . !".-' CCU-”l . l. .'-"-'c ._ ,-r’.'_I:‘.‘. lif‘i‘fi‘lbzm‘l‘ --" -.. is. . - . I 5‘. ” 118L137: . : ‘1 1:; ‘ ‘ H "F iii 44 Within and Between Cultivated and Wild Vaccinium spp. Thesis for the degree of MS. Michigan State University, East Lansing, Michigan. SANGER, F. (1981). Determination of nucleotide sequence in DNA. Science 214, 1205-1210. SCOTT, N.W., COOPER, J.l., LIU, Y.Y. & HELLEN, C.U.T. (1992). A 1.5 kb homology in the 3’-terminal regions of RNA1 and RNA2 of birch isolate Of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate. Journal of General Virology, 73, 481-485. SERGHINI, M.A., FUCHS, M., PINCK, M., REINBOLT, J., WALTER, B. & PINCK, L. (1990). RNA 2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location. Journal of General Virology 71 , 1433-1441. VIEIRA, J. & MESSING, J. (1987). Methods in Enzymo/ogy 153, 3—1 1. SECTION II: NEAR IDENTITY IN THE 3’ NONCODING REGIONS OF BLUEBERRY LEAF MOTTLE VIRUS RNA1 AND RNA2 Introduction The 3’ terminal region of multipartite viruses is generally highly homologous. Among related viruses, there is Often more homology within the 3’ noncoding regions than within the rest of their genomes (Matthews, 1991). This conservation in the 3’ terminus is well illustrated by the nepovirus group, in which nearly identical nucleotide sequences have been found between the 3’ noncoding regions of the RNA1 and RNA2 components of tomato black ring (TBRV) virus (Grief et al., 1986,1988), grapevine chrome mosaic (GCMV) virus (Brault et al., 1989; Le Gall et al., 1989), tomato ring Spot (TmRSV) virus (Rott et al., 1991a,b) and cherry leaf roll (CLRV) virus (Scott et al., 1992). The Significance of this high level of sequence homology is unknown. Considering the estimated high error rates (10" misincorporated nucleotides per site per round of replication) of viral replicases (Domingo & Holland, 1988), selection and/or recombination must be maintaining identity. Because of the high degree of homology found in the 3’ noncoding regions of multipartite viruses, it has been suggested that 3’ sequences may be an accurate marker of genetic relatedness and could serve as an aid to identification and classification of viruses (Frenkel et al., 1989). Frenkel 45 46 compared the degree of homology between 13 strains of seven distinct potyviruses using nucleotide sequence data from their 3’ noncoding regions and coat protein genes. The homology between any pair of viruses, calculated using sequences from either the 3’ noncoding regions or the coat protein genes, was very similar (on average varied by only 2%). The homology between strains in the 3’ noncoding region ranged from 83% to 99%. In contrast, the 3’ noncoding sequences from distinct potyviruses had identities in the range of 39% to 53%, which was comparable to the identity found between viruses of unrelated plant virus groups. Blueberry leaf mottle viurus (BBLMV) is a member of the nepovirus group. All nepoviruses have bipartite genomes composed of positive sense, single stranded RNA molecules, which are encapsidated separately in polyhedral particles 28 to 30 nm in diameter (Harrison & Murant, 1977). Nepoviruses have been divided into subgroups based on serological relationships (Murant, 1981), by the size of their RNA2 components (Martelli, 1975) or by the difference in sizes of their RNA1 and RNA2 components (Francki et al., 1985). According to Martelli’s Classification subgroup | (Mr 1.4-1.5 x 10°) would include grapevine fanleaf virus (GFLV), raspberry ringspot virus (RRV) , tobacco ringspot virus (TRSV) and arabis mosaic virus (AMV); subgroup ll (Mr 1.5-1.6 x 106) would include tobacco black ring virus (TBRV), grapevine chrome mosaic virus (GCMV), and artichoke Italian latent virus (AILV); and subgroup Ill (Mr >1.6 x 106) would include tomato ringspot virus (TomRSV), peach rosette mosaic (PRMV), cherry leaf roll virus (CLRV), and BBLMV. Francki (1985) 47 subdivided the nepoviruses into two groups, based on whether RNAS 1 and 2 differ in size (difference 2 0.6 M,) or are of a similar size (difference 5 0.4 M,). How these classification systems correlate to actual evolutionary relationships is not known. We report here on the nucleotide sequence of the 3’ terminus of BBLMV RNA1, and make comparisons between RNA1 and RNA2 of BBLMV and the other nepoviruses. Relationships between BBLMV and other nepoviruses, based on 3’ noncoding region and coat protein sequence data, are compared to other classification systems. Possible mechanisms for maintaining identity between RNA1 and RNA2 in nepoviruses are discussed. Materials and Methods Virus propagation and purification. The BBLMV used in this study was originally isolated from highbush blueberry plants (Vaccinium corymbosum L. cv. Rubel) and subsquently propagated in Chenopodium quinoa. The methods of virus propagation, purification and RNA extraction were as described by Ramsdell and Stace-Smith (1981). cDNA synthesis and cloning. Total viral RNA was extracted from purified virus particles as described by Ramsdell and Stace-Smith (1981) and fractionated on a linear log sucrose density gradient made with RNase-free sucrose in 1X SSC buffer, pH7, containing 6,ug/ml purified bentonite (Fraenkel— Conrat et al., 1961). Ethanol precipitated RNA1 and RNA2 mixtures were resuspended in TE and used as templates for oligoldT) primed cDNA synthesis 48 essentially as described by Gubler & Hoffman (1983). Size fractionated cDNA was blunt end ligated into Bluescript KS+ vector cut with Smal, then Cloned in XL1-Blue Escherichia coli cells (Sambrook et al., 1991). The viral orgin of the cDNA clones was confirmed by probing a Northern blot of purified BBLMV RNA with labelled cDNA clones (Sambrook et al., 1991). Nucleotide sequencing and analysis. Based on restriction enzyme analysis, cDNA clones were determined to have originated from either RNA1 or RNA2 (see Figure 1, Section 1). The largest clone from RNA1, cDNA clone number 24 (3.3kb), was selected to determine the 3’ terminal sequence of RNA1. Subclones of cDNA clone 24 were Obtained by Exonuclease Ill generated nested deletions and by restriction enzyme digestion (Sambrook et al., 1991). The sequence of both strands of the 3’ portion of clone 24 were determined by the dideoxynucleotide chain termination method (Sanger, 1981) using single stranded templates prepared according to Vieria and Messing (1987). Sequence data were analyzed using the Genetics Computer Group Sequence Analysis Software Package for the VAX, Version 7.1 (Devereux et al, 1984). Alignments of the amino acid and nucleotide sequences were obtained using the GAP and BESTFIT algorithms. Sequences of the following viruses were obtained from GenBank or EMBL databases: tomato ringspot virus RNA1 & RNA2 3’ terminus (Rott et al., 1991a), arabis mosaic virus RNA2 (Bertiol et al., 1991), grapevine chrome mosaic virus RNA1 (Le Gall et al., 1989), grapevine fanleaf virus RNA1 (Ritzenthaler et al., 1991), tomato black 49 ring virus RNA1 (Greif et al., 1988), cherry leaf roll virus RNA2 (Scott et al., 1992), raspberry ringspot virus RNA2 (Blok et al., 1992), cowpea mosaic virus RNA B (Lomonossoff & Shanks, 1983) and poliovirus type I (Kitamura et al., 1981). Results and Discussion The nucleotide sequence of both strands of the 3' terminal end of BBLMV RNA1 was determined (Figure 1). The sequence contained a single ORF, as expected for a nepovirus, and a long 3’ noncoding region of 1390 nucleotides. Identification of polymerase gene. The GDD motif, found in RNA- dependent RNA polymerases (Kamer & Argos, 1984), was located in an upstream portion Of clone 24 (Figure 2). The GDD motif and the surrounding sequences shared homology with RNA1 of GFLV, GCMV, TBRV, CPMV, and poliovirus, therefore, the origin Of clone 24 was concluded to be RNA1 of BBLMV. Comparison of the 3’noncoding regions of RNA 7 and 2 of BBLMV. The 3’ terminal sequences of BBLMV RNA1 and RNA2 were compared and found to be nearly identical for a 1390 nt Stretch with differences occurring at only seven positions (Figure 3). The region of homology began with the putative stop codon (TAG) of the RNA1 ORF and 17 nt downstream of the first stop codon of the RNA2 ORF. Upstream of the TAG sequence there was no apparent homology between RNA1 and RNA2. Downstream of the TAG 61 21 121 41 181 61 241 81 301 101 361 121 421 141 50 ATGCTTGATATTCCGGTGGAGAAAACTAAGCTCACCAGGGCAATGGCAAACCAGGGTGAG M L D I P V E K T K L T R A M A N Q G GCAGATTATAAATGCCAGGTGGCTGAACGCATTTTTGTGTGCGGCCCCAAAAGCAATTTG A D I L AGCCAGGAGCCAGCTCATTTTGTCGTTTCTCACACTGGTTCTCTTAAAAGAGGTGAAACC S Q E P A H F V V S H T G S L K R G E T GGTATTGTTGCTCCTGTCGACTTTGTTAGTGAAGGCCAGGGTCGGCTGCCAACCCAGTTG GIVAPVDFVSEGQGR QL TGGGTTAAAAAATTTCGCTCTGAATCACACACATTCAGGGTCATGATTAACGATGCCTAC W V K K F R S E S H T F R V M I N D Y Ar“"--~‘ "IlbbnlllfllIIIHUUAUIUHIDLLbDIIHCATTACTAATTGGCTTAGT T R G H S I Y F R S D P P Y I T N W L S GCCACCTCCTTTGCTCT'W‘I"l ‘ "T‘T‘“""CATTTTGGGGTTGTACCAT A T S F A L G K G M D Y K A I L G L Y H AACGTGTGCACTCCAGACGCGCAATGTTTAGATGAATATTTTGTTAGTGCGCGTTTTAGA N V C T P D A Q C L D E Y F V S A R F R AGAGCTGGATGCCTATCGGCCTCCACATATCACCAGTAGGAACTAGAACCTTTAAGTTCT T * AGGAGTTGGTCTGTCCTCTCTGACAGGCCTTCAAAGGATAGAGAATAGCTCGAACTCTCT GTAATACGAGAGGTCCGGACCTGTATGTCTTCCTGGCATATACCCAGGTTTTGAGATAGT AGTAAACTACTCTTCGATGTAGCGAATCGTCGTAAATAGGACACCCTCCTAAAACGAAGC CTTAAATAGGAACTTGAAAAAGTTTCCTTTCCACTTTGTGGAGGATAGTATAAGGGACGG TGGTGCCAGCTTGATGACTGCTTAAGAGCAGGAGGTTGCTCGTTAACCTTACACGAGCTA GGACGTTCTAGTAGAGATGAGACATCTACCTCGAAAAACGTCAGAATCACTATATGATTC AAAAGCGTGGTTTTTCCAACGTTAACCAATGGAAACCAGGTGCACATAGGTTAGTTGTGC TGATTTGCTACCIIIIAAGP“ ‘ IIHIIbIUUIUHAATTCCAGATCTATCTTAGTT TTGTTGTTTTAGTTTGATTGCAATAAACCCACATAAACTGTCGTCATTAGGACGGTATAC CATTGAGCILI1IAGGGCGLLICIGGIILLUIGAAAICGGIHIACGIGIGAAGATTAGGG TTTGCTCGAACCATAGAGAGCTAGGTTGTTGGAGCCGAACTGAGTCCAACCGCATTTGTC AGTTTTAGATATAACTGTCCAAGGTCTACTGCTTCCGAGCCTGAAAAAAATCTTAAAGCG CCCAGGCGTCCGTGACTTCACGGCACTCGGGGACAGAGTTTAGGGAAACTCTAGAAAAAT TCCCTCGCCTTTTAGTTGTGTGGCCGTGATGGACACAACTCTCTCTTCTTTCTGAGAGTG TACCGCTGTTTTAGTATCTGGTGATGATGTAGTTTTGAAACTACCAGAGATGTCTCAGTG GAGAAGCGTCTTGCCAAACGATATTGGCTTAAGGTCTATGTGACGATAATTTGCTAGTGT ACTCTAGAGAATGTGGGGTGGCACCCACTTCTTGGATGAGGGCCGGAGATGAAAACCGGG GAGTAATAAACTCCAGCTAGCGGCATAGGCCAACCACCGTGAGGGAGCTCACGGCGCAAT TTGGACCATTTTTAGACATAAATGGCCATGTTAGTGTAGCGCTTTGCGCATGTTGAATGA TAATGAACCAT 60 20 120 40 180 60 240 80 300 100 360 120 420 140 480 160 Figure 1. The cDNA sequence and the predicted amino acid sequence of the 3’ terminus of BBLMV RNA1 (excluding the poly(A) tail). 51 BBLMV 1Tv11NSifn elliRvakt 20 livYGDDnli GFLV lTvleSifk;ellmRycfkk.20 litYGDDnvf GCMV lTvvaSifn eiliRyaykt 20 lleGDDnli TBRV’ 1TvvaSvfn.eiliRyaykk 20 lleGDDnli CPMV mTvivNSifn eiliRyhykk 20 lthGDDnli POLIO gTsifNSmin nliiRtlllk 20 miaYGDDvia Consensus -T---NS ------- R ----- 20 ---YGDD--- svhpeflpyf tvaqsvmqyf svspavaswf svspsiaswf svnavvtpyf syphevdasl Figure 2. Alignment of the putatitive RNA-dependent RNA polymerase domain of BBLMV clone 24 with GFLV RNA-1 (positions 1791 to 1862), GCMV RNA-1 (positions 1764 to 1835), TBRV RNA-1 (positions 1780 to 1851 ), CPMV RNA- 1 (positions 1493 to 1569) and poliovirus (positions 1951 to 2146). The capital letters indicate a consensus between the BBLMV sequences and the other viral sequences. The GDD motif is common to all RNA-dependent RNA polymerases. 52 sequence were an additional 37, 26 or 31 stop codons in each of three reading frames, respectively. All ORFS in this 3’ region were less than 141 nt long and are believed to be noncoding, since in nepoviruses sequenced to date, only a single ORF is translated from each RNA component (Harrison & Murant, 1977). Comparisons with othernepoviruses. The nucleotide sequence similarities between the 3' noncoding region of BBLMV and the other nepoviruses are shown in Table 1. The statistical significance of the alignment scores was determined by comparing the score of each pair of sequences against the mean of 25 randomized comparisons, found when the second sequence was repeatedly shuffled and then aligned to the first. The similarity was judged to be significant if it exceeded the randomized mean plus three Standard deviations (Doolittle, 1981). Nepoviruses with a short ( <300 nt) 3’ noncoding region (i.e., GCMV, GFLV, AMV, & TBRV) all Showed significant similarity, as has been previously described (Serghini et al., 1990; Bertioli et al., 1991). However, statistically significant similarity was not found between any of the nepoviruses with the longer (>1390 nt) 3’ noncoding regions (i.e., BBLMV, CLRV & TmRSV); or between any with a long 3' vs. a short 3’ noncoding region. This lack of Similarity between distinct nepoviruses (values ranged from 37 to 47%) is comparable to that found by Frenkel (1989) in comparisons of 3’ noncoding regions between unrelated potyviruses (39 to 53%). These values are probably close to those expected from coincidental or random matching. While near identity in the 3' noncoding regions of RNA1 and RNA2 was 53 1 TAGGAACTAG AACCTTTAAG TTCTAGGAGT TGGTCTGTCC TCTCTGACAG GCCTTCAAAG 61 GATAGAGAAT AGCTCGAACT CTCTGTAATA CGAGAGGTCC GACCTGTATG TCTTCCTGGC 121 ATATACCCAG GTTTTGAGAT AGTAGTAAAC TACTCTTCGA TGTAGCGAET CGTCGTAAAT 181 AGGACACCCT CCTAAAACGA AGCCTTAAAT AGGAACTTGA AAAAGTTTCC TTTCACTTTG 241 TGGAGGATAG TATAAGGGAC GGTGGTGCCA GCTTGATGAC TGCTTAAGAG CAGGAGGTTG 301 CTCGTTAACC TTACACGAGC TAGGACGTTC TAGTAGAGAT GAGACATCTA CCTCGAAAAA 361 CGTCAGAATC ACTATATGAT TCAAAAGCGT GGTTTTTCCA ACGTTAACCA ATGGAAACCA 421 GGTGCACATI GGTTAGTTGT GCTGATTTGC TACCTTTTAA GAAAGGAGAT TATTCTGGTG 481 AAATTCCAGA TCTATCTTAG TTTTGTTGTT TTAGTTTGAT TGCAATAAAC CCACATAAAC 541 TGTCGTCATT AGGACGGTAT ACCATTGAGC TCTTTAGGGC GCCTCTGGTT CCGTGAAATC 601 GGTATACGTG TGAAGATTAG GGTTTGCTCG AACCATAGAG AGCTAGGTTG TTGGAGCCGA 661 ACTGAGTCCA ACCGCATTTG TCAGTTTTAG ATATAACTGT CCAAGGTCTA CTGCTTCCGA 721 GCCTGAAAAA AATCTTAAAG GCCCCAGGCG TCCGTGACTT CACGGCACTC GGGGACAGAG 781 TTTAGGGAAA CTCTAGAAAA ATTCCCTCGC CTTTTAGTTG TGTGGCCGTG ATGGACACAA 841 CTCTCTCTTC TTTCTGAGAG TGTACCGCTG TTTTAGTATC TGGTGATGAT GTAGTTTTGA 901 AACTACCAGA GATGTCTCAG TGGAGAAGCG TCTTGCCAAA CGATATTGGC TTAAGGTCTA 961 TGTGACGATA ATTTGCTAGT GTACTCTAGA GAATGTGGGG TGGCACCCAC TTCTTGGATG 1021 AGGGCCGGAG ATGAAAACCG GGGAGTAATA AACTCCAGCT AGCGGCATAG GCCAACCACC 1081 GTGAGGGAGC TCACGGCGCA ATTTGGACCA TTTTTAGACA TAAATGGCCA TGTTAGTGTA 1141 cccrrtcccc ATGTTGAATG ATAATGAACC ATGCGTTGCA GCGCATGCCT TTCGAGATCG 1201 GATGTGATTA CCGTGAGAAA GGGGAAACAA TGCCAACATG TTCAATTCGT TGTACTATGT 1261 TTTCTTTCTT TTTGTAGACT CCTGTGAGGA TTATCCAATA GCAGGTTGTG CCTTCAGTAA 1321 GCACACAAAA AGATTTCGCA TTTTTCTTTG TGTTAGATAG TTTTATATCT ATAATGTCTT 1381 TATTTCAC Figure 3. cDNA sequence of RNA1 showing the 1390nt region of identity between the 3’ termini of RNA1 and RNA2 of blueberry leaf mottle nepovirus (excluding the poly(A) tail). In the seven positions where differences occurred, the RNA2 sequence is shown below the sequence of RNA1. The region of identity begins with the putative termination codon TAG for the RNA1 encoded ORF. A region Showing a high degree of homology between BBLMV and other nepovuusesis undeaned. 54 Table 1. Sequence comparison of the 3' noncoding regions showing the percent similarity between the different nepoviruses. GCMV AMV GFLV RRV BBLMV CLRV TomRSV TBRV ‘ 68 56 60 41 42 38 4O GCMV‘ 57 58 36 43 39 44 AMV‘ 74 46 44 47 43 GFLV' 50 42 44 41 RRV 43 39 37 BBLMV” 37 37 CLRV” 39 TomRSVb -- ‘Nepoviruses with short l<300ntl 3’ noncoding regions. I’Nepoviruses with long (>1389nt) 3’ noncoding regions. tBold numbers indicate a significant sequence similarity matching. 55 found within a given nepovirus, there was little overall sequence homology between the different nepoviruses. However, a region of localized homology was found between BBLMV and the other nepoviruses from position 1289 to 1346 (Figures 3 & 4) about 40 nt upstream from the poly(A) sequence. This conserved region in BBLMV shared 15 out of the 17 nucleotides of a 3’ consensus sequence (GGACACAAAAAGA'I‘I'TT) previously identified in GFLV, GCMV, TBRV and CPMV by Serghini et al. (1990). The homology to BBLMV in the 3’ conserved region was highest with RRV, which was 87% identical over a 49 nt Stretch (Figure 5). A dendrogram (generated by GCG program PILEUP) based on the nucleotide similarities of the 3’ noncoding regions Shows the clustering relationships Of the different Nepoviruses (Figure 6). This grouping does not fit well with Martelli’s (1975) or Francki’s (1985) classification systems. The grouping of AMV/GFLV and GCMV/TBRV is consistent with both taxonomies, however, there is no clear clustering of BBLMV, TomRSV and CLRV in what would be subgroup 2 (Francki) or subgroup Ill (Martelli). This contrasts with the what we found using coat protein amino acid sequence data, where a clustering of BBLMV, TomRSV and CLRV was observed (Figure 7). RRV appears to be quite distinct from the other nepovirus, using either 3’ noncoding sequences or coat protein sequences. Blok et. al. (1992) was able to find some homology towards the N-terminus of TBRV RNA2, and to a lesser extent GFLV RNA2, using the entire amino acid sequence of the RNA2 encoded polyprotein. This relationship suggests RRV is more closely related to 56 AMV tGTtTgTCCT Tngacacac tTGCcTT... gttGGACgCA AAAAGATTTT GFLV tGTtTgTCCT Tngacacac tTGCcTa... gttGGACgCA AAAAGATTTT GCMV gGTtTgTCCT Tthtcatgt tTGCtTT... gttGGACaCA AAAAGATTTT TBRV gGTtTgTCCT Tthccthg gTGCtaT... gttGGACaCA AAAAGATTTT CPMV gagcchth TTagcagch chCcTTcag caaGGACaCA AAAAGATTTT BBLMV gGatTaTCCa acagcagth gTGCcTTcag taaGcACaCA AAAAGATTTC RRV gtTgctcCCT cTaagagch gTGCcTTtag caaGcACaCA AAAAtATgca CLRV .GTthTCaa aattcgctTa tTGtaTgagt gthGACtCA ggcAGthTT TRSV aGgthTth ngtccgtTt gTGttTcaaa acgcthttt gcAAttTTcT CON. -GT-T-TCCT TT------T- -TGC-TT ------ GGAC-CA AAAAGATTTT AMV attTTcTTTt tactgctttt ataaaTtT.. .................... GFLV tccTTTcTTt ttactgtttt gcaaaTtTat .................... GCMV ataTTTcTTa aatgttaaaa cctttctTtt ggaaaagc.. .......... TBRV ctcTTTTgTa aatgataaaa tgtttTcth aaaaagc... .......... CPMV aatTTTaTT. ........................................ BBLMV gcaTTTTTct ttgtgttaga tagttTtata tctataatgt ctttatttca RRV ttthTTTTg ttcttaagct tccctangt cgttctgtcc gaac ...... CLRV aggTTTTaTt tctttgattt agathtTac tttaagtttt ccttttacgt TRSV tttTTgTTTt attgctttcg tagthcgaa ctttgtccaa gttcataaaa CON. -— 1.1.1111. -----T-T-- Figure 4. Multiple sequence alignment of the noncoding region of nine nepoviruses using GCG PRETTY program. A consensus sequence is given when a plurality of 6 out of 9 sequences exists. 1303 AGGTCGTGCCTTTAGCAAGCA |lll lllllll llll Illllll AGGTTGTGCCTTCAGTAAGCA CACAAAAATAT...GCATTTGTTTTTGT III II lIllll I I II II llllll I II CACAAAAAGATTTCGCATTTTTCTTTGT 1351 Figure 5. Optimal alignment of RRV and BBLMV noncoding regions by GCG BESTFIT program. Percent similarity is 87% over 49 nucleotides. 57 AMV GFLV GCMV TBRV BBLMV TomRSV CLRV RRV Figure 6. Dendrogram showing the clustering relationships of different nepoviruses based on the nucleotide sequence of their 3’ noncoding regions (generated by GCG program PILEUP). 58 GCMV TBRV AMV GFLV BBLMV TomRSV CLRV RRV Figure 7. Dendrogram showing the clustering relationships of different nepoviruses based on the amino acid sequence of their coat protein genes (generated by GCG program PILEUP). 59 some of the members of subgroup || (not subgroup l as Martelli has suggested). Those nepoviruses that are serologically related, even though they may be found in very different hosts, have been classified as strains of the same virus and were placed in one subgroup (Murant, 1981). However, most nepoviruses are not serologically related, therefore, subdividing then according to their antigenic properties is not a satisfactory classification system by itself. Serologically there is little or no relationship between any of the viruses of the different subgroups of Martelli or Francki, yet nucleotide sequence data clearly shows that some relationships exist. While classification systems based on the Size of the RNA2 components are somewhat arbitary, there was some correlation with the relationships determined from sequence data. AS more sequence data become available, these subgroups will no doubt need to be modified. Maintenance of identity of the 3’ noncoding region. The marked conservation of sequences in the 3’ terminal region Of each nepovirus may reflect an important function or functions that are selectively maintained. If the highly conserved 3’ terminus represents an optimal sequence for a particular process, such as replication or encapsidation, then changes in this sequence may reduce the efficiency of the process and result in a virus that is less fit. Variant genotypes may replicate at reduced levels compared to the optimal or wild type sequence and competition could eventually eliminate them from the viral population. Selection for these functions maintains the Optimal sequence on both RNA components and, therefore, identity is maintained. 60 However, for selection to maintain identity in the 3' terminus of BBLMV, it would have to act on a very high number of individual nucleotides, all of which would have to have some affect on the fitness of the virus in order to be selected for or against. It is possible that it is not the primary sequence itself, but a secondary structure formed by this sequence that is important. Modification of the primary sequence could affect the secondary or tertiary structure of the 3’ terminus. As a consequence of these changes, the RNA molecule may not be recognized by the polymerase, resulting in the elimination of the variant from the pool of RNAS. Whether or not selection, by itself, can maintain identity in the 3’ noncoding region of BBLMV is still uncertain. It may be possible that recombination is acting in concert with selection to maintain a specific sequence. High frequency RNA recombination via template switching has been suggested as a mechanism for maintaining identity of the 3’ terminus of two other nepoviruses, TmRSV (Rott et al., 1991) and CLRV (Scott et al., 1992). Rott (1991) suggested it might be possible that replication begins in cis with RNA1 and that trans replication of RNA2 occurs only following disassociation and reassociation of the initial negative-strand transcript with the corresponding region in RNA2. Such a mechanism has been proposed for leader-primed generation of subgenomic RNAS in coronaviruses (Lai et al., 1990). Homologous recombination has been shown to occur in plant viruses such as, brome mosaic virus (Bujarski & Kaesberg, 1986), cowpea chlorotic mottle virus (Allison et at., 1990), tobacco rattle virus (Angenent et al., 1989) 61 and tobacco mosaic virus (Beck & Dawson, 1990). Recombination was probably responsible for the duplication of the 3’ terminus of BBLMV and other nepoviruses. However, what role recombination plays in the maintenance of identity in the 3’ terminus, is unknown. Recombination does compliment some of the limitations of selection, such as: (1) how new mutations are transferred from one RNA component to the other in order to maintain identity, (2) recombination swaps segments of the genome between the RNAS, slowing the accumulation of neutral mutations that would cause divergence between the two RNAS and (3) recombination does not require that all sequences correspond tO essential functions in order to be maintained, since it is essentially not a selective mechanism. Whether or not recombination is involved, selection is still required to remove or to fix particular nucleotides in an optimal 3’ sequence. LIST OF REFERENCES ALLISON, R., THOMPSON, C., AHLOUIST, P. (1990). Regeneration Of functional RNA virus genome by recombination between deletion mutants for cowpea chlorotic mottle virus 3a and coat genes for systemic infection. Proceedings of the National Academy of Sciences (USA) 87, 1820-1824. ANGENENT, G.C., POSTHUMUS, E., BREDERODE, F.T., & BOL, J.F. (1989). Genome structure of tobacco rattle virus strain PLB: Further evidence on the occurrence of RNA recombination among tobraviruses. Virology 171, 271- 274. BECK, D.L. & DAWSON, WC. (1990). Deletion of repeated sequences of tobacco mosaic virus mutants with two coat protein genes. Virology 177, 462-469. BERTIOLI, D.J., HARRIS, R.D., EDWARDS, M.L., COOPER, J.l., & HAWES, W.S. (1991). 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High error rates, population 62 63 equilibrium and evolution Of RNA replication systems, pp. 3—36. In: E. Domingo, J.J. Holland & P. Ahlquist (eds.), RNA Genetics, vol 3. CRC Press, Boca Raton, Florida. DOOLITTLE, RF. 1981. Similar amino acid sequences: Chance or common ancestry? Science 214(9), 149-159. FRANCKI, R.|.B., MILNE, R.G. & HATTA, T. (1985). Nepovirus group, pp. 23- 38. In: Atlas of Plant Viruses, Vol. II. CRC Press, Boca Raton, Florida. FRENKEL, M.J., WARD, C.W. & SHUKLA, DD. (1989). The use of 3’ noncoding nucleotide sequences in the taxonomy of Potyviruses: Application to watermelon mosaic virus 2 and soybean mosaic virus-N. Journal of General Virology 70, 2775-2783. GREIF, C., HEMMER, O. & FRITSCH, C. (1986). The nucleotide sequence of tomato black ring virus RNA 2. Journa/of General Virology 67, 1257-1271. GREIF, C., HEMMER, 0. & FRITSCH, C. (1988). Nucleotide sequence of tomato black ring virus RNA 1. Journa/of General Virology 69, 1517—1529. GUBLER, U. & HOFFMAN, B.J. (1983). A simple and very efficient method for generating cDNA libraries. Gene 25, 263-269. HARRISON, B.D. & MURANT, AF. (1977). Nepovirus group. CMl/AAB Descriptions of Plant Viruses, no. 185. Kew, Surrey, UK. KAMER, G. & ARGOS, P. (1984). Primary structural comparison of RNA- dependent polymerases from plant, animal and bacterial viruses. Nucleic Acids Research 12, 7269-7782. KITAMURA, N., SEMLER, B.L., ROTHBERG, P.G., LARSEN, G.R.,ADLER, C.J., DORNER, A.J., EMINI, E.A., HANECAK, R., LEE, J.J., VAN DER WERF, J., ANDERSON, C.W. & WIMMER, E. (1981). Primary structure, gene organization and polypeptide expression of poliovirus RNA. Nature 291, 547-553. LAI, M.M. (1990). Coronaviruses: Organization, replication and expression of genome. Annual Review of Microbiology 44, 303-333. Le GALL, 0., CANDRESSE, T., BRAULT, V. & DUNEA, J. (1989). Nucleotide sequence of Hungarian grapevine chrome mosaic nepovirus RNA1. Nucleic Acids Research 17, 7795-7807. LEMONOSSOFF, G.P. & SHANKS, M. (1983). The nucleotide sequence of 64 cowpea mosaic virus B RNA. The European Molecular Biology Organization Journal 2(12), 2253—2258. MATTHEWS, R.E.F. (1991). Plant Virology, Third Edition, pp.170-176. Academic Press Inc., London/New York. MURANT, AF. (1981). Nepoviruses. In: E. Kurstak (edl.), Handbook ofP/ant Infections and Comparative Diagnosis, pp. 197-238. Amsterdam: ElsevierlNorth-Holland. RAMSDELL, D.C. & STACE-SMITH, R. (1981). Physical and chemical properties of the particles of ribonucleic acid of blueberry leaf mottle virus. Phytopatho/ogy 71(4), 468-472. RITZENTHALER, C., VIRY, M., PINCK, M., MARGIS, R., FUCHS, M. & PINCK, L. (1991). Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA 1. Journal of General Virology 72, 2357- 2365. ROTT, M.E., TREMAINE, J.H. & ROCHON, D.M. (1991a). Comparison of the 5’ and 3’ termini of tomato ringspot virus RNA 1 and RNA 2: evidence for RNA recombination. Virology 185, 468-472. ROTT, M.E., TREMAINE, J.H. & ROCHON, D.M. (1991b). Nucleotide sequence of tomato ringspot virus RNA2. Journal of General Virology 72, 1505-1514. SAMBROOK, J., FRITSCH, E.F. & MANIATIS, T. (1991). Molecular Cloning.- A laboratory Manual. Cold Springs Harbor Laboratory, Cold Spring Harbor, N.Y. SANGER, F. (1981). Determination of nucleotide sequence in DNA. Science 214,1205-1210. SCOTT, N.W., COOPER, J.l., LIU, Y.Y. & HELLEN, C.U.T. (1992). A 1.5 kb homology in the 3'-terminal regions of RNA1 and RNA2 of birch isolate of cherry leaf roll nepovirus is also present, in part, in a rhubarb isolate. Journal of General Virology, 73, 481-485. SERGHINI, M.A., FUCHS, M., PINCK, M., REINBOLT, J., WALTER, B. & PINCK, L. (1990). RNA 2 of grapevine fanleaf virus: sequence analysis and coat protein cistron location. Journal of General Virology 71, 1433-1441. VIEIRA, J. & MESSING, J. (1987). Methods in Enzymo/ogy 153, 3-11. SECTION III: CONSERVATION OF A 1.4 KB DUPLICATION IN THE 3' NONCODING REGIONS 0F RNA1 AND RNA2 OF BLUEBERRY LEAF MOTTLE VIRUS: POSSIBLE MECHANISMS FOR MAINTAINING IDENTITY Introduction The 3' noncoding regions of blueberry leaf mottle virus (BBLMV) RNA1 and RNA2 are nearly identical (Bacher, 1993). This duplicated region consists of 1390 nucleotides, with differences occurring at only seven positions. Highly conserved 3’ termini have also been found on all the other nepoviruses sequenced (Grief et al., 1986; Brault et al., 1989; Serghini et al., 1990; Bertioli et al., 1991; Rott et al., 1991; Scott et al., 1992). In fact, conservation of the 3’ terminus is a common feature for most multipartite viruses (Matthews, 1991). How this duplication originated, what function or functions it is involved in, and how it is maintained is not known. However, conservation of the 3’ terminus could reflect important functions related to this region. The 3’ termini of genomic viral RNAS are essential for the replication of the minus sense Strand (Matthews, 1991). In some cases, a specific secondary Structure of the 3’ noncoding region is required for it’s proper functioning (Dreher & Hall, 1988; Takamotso et al., 1990). Possible roles for the 3’ noncoding region suggested for other viruses include: promotor activity (Dreher & Hall, 1988), viral 65 66 assembly through interactions with the coat protein, and Signals for the polyadenylation of the 3’ terminus. High mutation rates in RNA viruses means strong selection pressure must constantly counteract these changes if a specific sequence is to be conserved. The rate of point mutations for RNA viruses has been estimated to average approximately 10'3 to 10'5 per nucleotide per round Of replication (Steinhauer & Holland, 1987). As a consequence of the high mutation rates of RNA polymerases, all RNA virus populations examined have been found to exist as heterogeneous mixtures of related genomes that Share a consensus sequence but differ from each other by one or more mutations (Holland et al., 1982; Domingo et al., 1985). The highly heterogenous nature of viral populations is illustrated by phage OB studies, which Show that individual clones isolated after multiple passages differed from the consensus sequence of the original parent virus by an average of one to two nucleotides (Domingo et al., 1978). Domingo proposed that, a 0,8 phage population is in dynamic equilibrium, with mutants arising at a high frequency, but being strongly selected against. It is possible that Other mechanisms are working in conjunction with selection to help maintain identity in the 3' termini of BBLMV. Rott et al. (1991) found that the 3’ terminal 1533 nt of tomato ringspot nepovirus (TomRSV) RNA1 and RNA2 were identical and noncoding and hypothesized that the similar sequences were a result of recombination between the two genomic RNA components. It was proposed that, "in TomRSV, replication begins in cis with RNA1 and that trans replication or RNA2 occurs only 67 following disassociation and reassociation of the initial negative-strand transcript with the corresponding region in RNA2" (i.e., the 3’ terminal region of only one of the genomic RNAS serves as a template for both 3’ noncoding regions, thereby maintaining identity). Scott et al. (1992) found a 1.5 kb sequence homology in the 3' terminal regions of RNA1 and RNA2 of a birch isolate of cherry leaf roll nepovirus. The authors questioned why "the RNA1 and RNA2 duplication is retained during countless cycles of replication in natural and glasshouse hosts when mutation rates are so high in RNA viruses" and suggested that "this duplicated sequence is the site for high levels of recombination between RNA1 and RNA2". What role recombination plays in the conservation of the 3’ terminus of BBLMV is unknown, but the presence of a duplicated region suggests that recombination has occurred at least once in the evolution of this virus. In this study we search for evidence Of recombination between RNA1 and RNA2 as a means of determining the relative importance of recombination vs. selection in maintenance the duplicated region. Materials and Methods To determine if recombination was occurring in BBLMV, we examined cDNA clones at the seven positions where nucleotide differences were found between the 3’ noncoding regions of RNA1 and RNA2 (Bacher, 1993). If recombination had occurred in a particular viral RNA, then a recombinant clone would contain sequences unique to RNA1 on one Side of the crossover point, 68 and sequences unique to RNA2 on the other side. The ability to use these differences between RNA1 and RNA2 as "markers"'in identifying recombinants, depended upon their conservation and uniqueness to either RNA1 or RNA2. A series of 3’ cDNA clones, from the same library that the original RNA1 and RNA2 sequences were obtained, were sequenced around the marker sites. Consensus sequences for both RNA1 and RNA2 were developed and deviations from these sequences were examined to determine if they were due to Single base mutations or recombinational events. Virus propagation and purification. The viral population from which the cDNA library was made is likely to be a heterogeneous mixture Of genotypes. The BBLMV isolate was maintained by repeated passage in Chenopodium quinoa plants over a period of many years. The initial inoculation source of BBLMV was infected shoot terminals of Vaccinium corymbosum L. cv. Rubel collected in southern Michigan. The virus population used to generate the cDNA library was propagated in Chenopodium quinoa (approx. 200 plants) after rub inoculation with leaf extract (in 0.05 M Na phosphate buffer pH 7.0) from infected stock plants. Subsequent virus purification was as described by Ramsdell & Stace-Smith (1981). cDNA synthesis andc/oning. Total viral RNA was extracted from purified virus particles according to Ramsdell and Stace—Smith (1981) and fractionated on a linear log sucrose density gradient made with RNase-free sucrose in 1X SSC buffer, pH7, containing 6ug/ml purified bentonite (Fraenkel-Conrat et al., 1961). A mixture containing both RNA1 and RNA2 molecules was used as 69 templates for oligo(dT) primed cDNA synthesis essentially as described by Gubler & Hoffman (1983). Size fractionated cDNA was blunt end ligated into Bluescript KS + vector cut with Smal, then cloned in XL1-Blue Escherichia coli cells (Sambrook et al., 1991). Nucleotide sequencing and analysis. One hundred and sixty cDNA clones were sized and those longer than 1 kb (about 30%) were selected for sequencing by the dideoxynucleotide chain termination method (Sanger, 1981) using single stranded templates prepared according to Viera and Messing (1987). In order to avoid sequencing the entire length of each clone, subclones were made by restriction enzyme digestion that removed intervening sequences that were not informative (i.e., the same on both RNAS). The particular restriction enzyme combinations varied depending upon the direction of the cDNA insert, its size, and the marker selected for sequencing. The desired fragment was separated on 1.2% low melting point agarose, blunt ended with klenow enzyme when appropriate, and re-ligated with T4 DNA Iigase (Sambrook et al., 1991). Two to four hundred nucleotides Of sequence from one end Of each clone was determined initially. Next, similar Sized regions of sequence containing the most distant flanking markers were determined. Clones exceeding 1.4 kb (the length of the noncoding region) contained coding sequence, depending upon direction of cDNA insert. Sequence data were analyzed using the Genetics Computer Group Sequence Analysis Software Package for the VAX, Version 7.1 (Devereux et al, 1984). 70 Results and Discussion Identification of differences in consensus sequence. The marker genotypes of 43 cDNA clones were determined and aligned with the sequences from the 3’ noncoding region of RNA1 and RNA2 of BBLMV (Figure 1). Consensus sequences were determined for both RNAS. Three of the seven nucleotide differences found between the original RNA1 and RNA2 sequences (positions 2,3, and 4) were not present in any other clones. However, the remaining four differences (positions 1, 5, 6 and 7) did consistently distinguish between RNA1 and RNA2. Positive identification Of RNA1 and RNA2 was possible in clones longer that 1.4 kb, since they contained coding regions which were not homologous. No evidence of recombination was found in any of the cDNA clones sequenced, that is, there were no clones that contained unique markers from both RNAS (Figure 1). All differences from the consensus sequences of either RNA1 or RNA2 were at Single Sites and did not occur at any of the four marker positions used to distinguish the RNAS. This indicates that identity in the 3’ noncoding regions of RNA1 and RNA2 of BBLMV is being maintained without high levels of recombination. Mutations found in the viral population. Identity was conserved even though a high frequency of mutations were found (Figure 1). A total of 11 mutations were identified out of about 30,000 nucleotides sequenced and appeared to be randomly distributed throughout the 3’ terminus. The origin of these mutations can not be clearly identified Since they may have occurred 71 1 2 3 4 5 6 CON1 T G c — 'r — T A — — poly(A) CON2 C G C — T — C G —— — poly(A) (24) [111* T‘ c — T — T A — T —— poly(A) (34) [21c G T‘— c‘ — c G — — poly(A) (1) — / ................. /—- G — — poly(A) (3) G —*- - P01Y(A) (8) [21¢ / ......................... /— c — — poly(A) (9) /- G — — P01Y(A) (11) [21c G —/ ................. /— G — —- poly(A) (12) l— G —- — P°1Y(A) (14) [1]/ ............................. /— A — —- poly(A) (17) — T — C ./— G — — poly(A) (19) [1]/ ............................. /— A — — poly(A) (20) G —*—— /..../- C ./— G —— — poly(A) (23) G c — //— c ./— G — — poly(A) (25) [2]/ ............................. /— G —— — poly(A) (26) G c — //— T ./— A — — poly(A) (28) [1]/ ............................. /— A —— — poly(A) Figure 1. Consensus sequences for the 3’ noncoding region of RNA1 and RNA2 of BBLMV for the seven positions where differences were found between clones 24 and 34. Clones 24 and 34 were used originally to determine the sequence of the 3’ noncoding region of RNA1 and RNA2 (Bacher, 1993). The marker genotypes of cDNA clones of BBLMV are aligned with consensus sequences. RNA1 =[1] or RNA2 =[2]; (#l = cDNA clone number; * cDNA sequence; and ..... = area not sequenced. Bracketed [] numbers indicate coding sequence from either mutation; 1 2 CON1 T — G CON2 C — G (29) G (30) — (31) (32) (33) (38) [1]T — / ......................... (45) (49) (54) [1]T — G ——— / ................... (55) [1]T — / ......................... (66) *[21/ ............................. (67) *[11/ ............................. (68) [1]T — / ......................... (74) — G ———— (80) — G ———— (83) (87) (99) [1]/ ............................. (103) (104) — (107) G at» I T 72 HUI C C T C /.... /.... /... 6 A_ G— /—*A* /—G— /—A— ./— A ——— /—A— G_ G— /—A— /—A— /—G— /—A— /—A— A— A_ A— G_ /—A— /—G— /—A— ./— G ——— O *3 l-3 H O a '6 O O #3 8 #3 O H 0 Pi poly(A) poly(A) P°1Y(A) p01Y(A) P01Y(A) poly(A) poly(A) polY(A) P01Y(A) P°1Y(A) p01Y(A) p01Y(A) p01Y(A) poly(A) poly(A) poly(A) polY(A) P01Y(A) p01Y(A) poly(A) p01Y(A) poly(A) P01Y(A) ngre1.(confinued) 1 2 3 4 5 CON1 T — G c — T — T CON2 c — G c — T — c (122) * ——— (129) — T — c ——— /.../— (130) c — / ......................... /— (131) [2]/ ............................. /— (139) * ——— (146) [1]/ ............................. /— (149) [2]/ ............................. /— G (152) [1]/ ............................. /— .......... poly(A) p°1Y(A) poly(A) poly(A) p01Y(A) poly(A) poly(A) poly(A) poly(A) poly(A) Hgtne 1.(Confinued) 74 during viral replication or during the cDNA synthesis by the reverse transcriptase enzyme. Both the viral RNA polymerase and the reverse transcriptase are thought to lack error correction mechanism and, therefore, have high error rates. The heterogeneity found in the BBLMV population was expected as a consequence of the proposed high error rates of RNA polymerases and is consistent with the heterogeneous nature reported for other viral populations (Holland et al., 1982; Domingo et al., 1985). It is possible that the differences between the 3’ noncoding regions of RNA1 and RNA2 represent neutral mutations. However, the fact that only two of the four nucleotides were found in these four positions indicates selection was still limiting sequence divergence or our sample sizes were too small to uncover the other mutations. Alternately, the conserved differences may be critical and reflect some important variation in function or secondary structure between the two RNAs. Estimation of recombination frequencies. There were a total of 91 intervals between markers where recombination could have been detected, since all clones contained multiple markers. These markers span a distance of about 1360 bases. Based on recombination studies in other RNA viruses, this distance should be sufficient for recombination to occur. For example, in poliovirus, recombination was detected between markers only 190 bases apart (Kirkegaard & Baltimore, 1986); and in brome mosaic virus, recombination occurs in a 3’ noncoding region about 300 bases long (Bujarski & Kaesberg, 1986). We found no recombination in the 3’ noncoding region of BBLMV, and 75 even if a recombinant was found in the next clone sequenced, rates would be below 2.3% (1\44) in the overall population sampled and below 1.1% (1/91) between markers within in the 3’ noncoding region. These estimates compare to recombination frequencies found in other RNA viruses. In closely related strains of poliovirus the frequency of recombination between genetic markers 190 bases apart, was estimated at 0.13% (Kirkegaard & Baltimore, 1986). If this frequency of recombination per nucleotide was extended over 1390 nt (the length of the 3’ noncoding region of BBLMV) it would be close to 1%. Recombinants between isogeneic strains of another picornavirus, foot—and-mouth disease virus, were detected at a frequency of 0.92% in vitro (McCohon et al., 1977). Recombination frequencies in plant RNA viruses have not been determined. In most studies of recombination in plant RNA viruses, modified viral RNAs were placed under strong selection for rescue of functional recombinants. For example, in brome mosaic virus, a 20 nt deletion was made in the tRNA-like structure of the 3’ noncoding region which is involved in initiation of BMV RNA replication (Bujarski & Kaesberg, 1986); and in cowpea chlorotic mottle virus, deletions were made in either the 3a (putative movement gene) or the coat protein gene of the RNA3 component, both of which are required for systemic infection (Allison et al., 1990). In both cases, co— infection of the mutant RNA componentls) with the remaining wild type components resulted in the restoration of wild type sequences of the mutant RNA component. This research does indicate that under conditions of strong 76 selection pressure, recombination can play an important role in restoring optimal (wild type) sequences in defective or poorly adapted genotypes. However, rescue of wild type genome could have resulted from extremely low levels of recombination, due to the high rates of replication of viruses. Role of recombination in maintenance of identity in the 3’ noncoding region. The conservation of duplicated regions on the 3’ terminus of BBLMV and other nepoviruses has caused speculation that recombination may be involved in preserving this identity. The exchange of RNA sequences between genomic RNAS would transfer unique mutations from one RNA to the other, equalizing any changes that have occurred during replication and preventing the accumulation of neutral mutations. However, in order for recombination to significantly contribute to the maintenance of identity it would have to occur at relatively high frequencies. Rott et al. (1991) has suggested that only the 3’ terminal region of one of the genomic RNAs may serve as a template for both 3’ noncoding regions in TomRSV. 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