Sobemovirus Coat Protein Gene Complements Long-Distance Movement of a Coat Protein-Null Dianthovirus

Home , Dianthovirus, Sobemovirus

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Virology 330 (2004) 186–195 www.elsevier.com/locate/yviro

A Sobemovirus coat protein gene complements long-distance movement of a coat protein-null Dianthovirus

Anton S. Callaway1, Carol G. George, Steven A. Lommel*

Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, United States

Received 16 August 2004; returned to author for revision 9 September 2004; accepted 28 September 2004

Abstract

Red clover necrotic mosaic virus (RCNMV; genus Dianthovirus) and Turnip rosette virus (TRoV; genus Sobemovirus) are taxonomically and ecologically distinct plant viruses. In addition, the two genera differ in the role of coat protein (CP) in cell-to-cell movement. However, both are small icosahedral viruses requiring CP for systemic movement in the host vasculature. Here, we show that the TRoV CP gene is capable of facilitating the vascular movement of a Dianthovirus. Substitution of the RCNMV CP gene with the TRoV CP gene permits movement of the resulting chimeric virus to non-inoculated leaves. RCNMV lacking a CP gene or containing a non-translatable TRoV CP gene do not move systemically. This report introduces the molecular characterization of TRoV and describes the unprecedented complementation of systemic movement function by intergenic complete substitution of a plant virus CP gene. D 2004 Elsevier Inc. All rights reserved.

Keywords: Long-distance movement; Complementation; Coat protein; Dianthovirus; Sobemovirus; Arabidopsis; Chimera

Introduction sal and encapsidation is not sufficient for long-distance movement of some viruses (Callaway et al., 2001; Sit et The mechanism(s) by which plant viruses move through al., 2001). It is thought that the viral CP interacts with the plant host vasculature system (long-distance or unknown host components, as well as with viral genomic systemic movement) is not well understood (Carrington RNA during vascular transport. Despite the common use et al., 1996). Likewise, the specific roles of viral gene of CP in long-distance movement, viral CPs vary widely in products in this process are largely unknown. In addition primary amino acid sequence among plant virus genera, to the viral gene products required for early infection possibly reflecting the highly divergent viral RNA events, that is, replication and cell-to-cell movement, coat sequences which they package (Koonin and Dolja, protein (CP) is required by most viruses for effective long- 1993). Perhaps partially as a consequence of their wide distance movement, although a slower, CP-independent sequence divergence, functional complementation among movement process appears to be an alternative mechanism viral CPs is rare. Members of the genus, Umbravirus, available to some viruses (Callaway et al., 2001). encode non-encapsidating proteins that can complement Encapsidation is necessary for most viruses to move the long-distance movement function of certain CP-null systemically. However, this requirement is far less univer- viruses from other genera (Ryabov et al., 1999a, 1999b). However, there are no reports, to our knowledge, of a plant virus CP complementing long-distance movement function * Corresponding author. Department of Plant Pathology, North of another virus from a different genus. Carolina State University, Box 7616, Raleigh, NC 27695-7616. Fax: +1 Members of the genera Dianthovirus and Sobemovirus are 919 515 7716. E-mail address: [email protected] (S.A. Lommel). both icosahedral viruses that require CP for movement in the 1 Current address: Department of Botany, North Carolina State vasculature of infected plants. However, they are taxonomi- University, Box 7612, Raleigh, NC 27695-7612, United States. cally distinct (van Regenmortel et al., 2000). Dianthoviruses

YVIRO-02783; No. of pages: 10; 4C: 6 A.S. Callaway et al. / Virology 330 (2004) 186–195 187 have a bipartite genome whereas Sobemovirus genomes are ecotypes of A. thaliana, sap from infected turnip plants monopartite. Dianthoviruses can move cell-to-cell without was passaged onto 65 Arabidopsis ecotypes. All ecotypes CP, whereas sobemoviruses require CP for cell-to-cell tested exhibited distinct vein clearing on non-inoculated movement. The two genera also differ in other important leaves and bhookingQ of the bolt near the terminal ways, including their genome strategy, particle size (Hiruki, inflorescence. The plants were also severely stunted. In 1987; Hollings and Stone, 1969), vector associations, and contrast to the distinct systemic symptoms, inoculated seed transmissibility characteristics (Matthews, 1991). Pre- leaves were usually indistinguishable from mock-inoculated dicted amino acid comparisons between ORFs from the two, controls, though occasionally local lesions formed, partic- otherwise distinct, genera suggest there is some phylogenetic ularly on ecotypes Can-0 and C24. Ecotypes tested are listed similarity between the CP genes (Tamm and Truve, 2000). in alphabetical order: Aa-0, Ag-0, Ala-1, An-1, Be-0, Bla-4, Turnip rosette virus (TRoV) is a species in the Bla-10, Bla-12, Bor-0, Br-0, Bs-5, Bu-4, Bur-0, C24, Can-0, Sobemovirus genus of positive-sense RNA viruses (van Co-4, Col-0, Ct-1, Cvi-0, Di-0, Di-1, Dy-0, Edi-0, Eil-0, En- Regenmortel et al., 2000) with a particle size of 24–26 nm 2, Est-0, Et-0, Fl-3, Fr-2, Ge-1, Gr-1, Gre-1, Ita-0, Kas-1, in diameter (Hollings and Stone, 1969). Typical of members La-0, Lc-0, Le-0, Ler, Li-5, Lip-0, Ll-0, Lou-2, Mh-0, Ms-0, of this genus, TRoV has a narrow host range, limited mainly Mt-0, Na-1, Nd-0, No-0, Np-0, Np-1, Nw-4, Oy-0, Pa-1, Pi- to infection of members of Cruciferae (Broadbent and 0, Pla-1, Po-1, RLD, Rsch-4, Se-0, Sei-0, Sf-1, Shadhara, Heathcote, 1958; Hollings and Stone, 1969). However, the Tsu-0, Wil-2, and Ws. Almost every plant inoculated earlier host range studies did not include the model plant, exhibited systemic symptoms and no consistent correlation Arabidopsis thaliana as a potential host. This study was was observed between ecotype and the appearance of rare initiated to characterize this virus on a molecular level, to individual plants that escaped obvious infection. determine if one or more Arabidopsis ecotypes are capable of supporting systemic infection by TRoV and to explore the TRoV genome arrangement possible functional conservation of the TRoV CP in movement. The size of the TRoV genome was determined to be 4037 nucleotides by sequencing of overlapping cloned and bulk cDNAs (GenBank accession number NC_004553). Results This is slightly smaller than the other species comprising the genus, whose genome sizes range between 4083 and 4451 TRoV infects Arabidopsis thaliana nucleotides. TRoV’s deduced genome is arranged similarly to previously described sobemoviruses (Fig. 1). Previous studies indicated that TRoV infects several The TRoV 5V untranslated region is 67 nucleotides in cruciferous species (Broadbent and Heathcote, 1958; Hol- length vs. 48–99 nts for previously described sobemovi- lings and Stone, 1969). However, those studies did not ruses. The primary and secondary structures of the 5Vend of include members of the genus, Arabidopsis. To determine the TRoV genomic RNA appear to be very much like whether TRoV was capable of infecting one or more several other sobemoviruses (Fig. 2). The CAAAAUA

Fig. 1. TRoV genome organization. The arrangement of open reading frames (ORFs) and other features are shown in comparison to other selected sobemoviruses. For each viral genome, ORFs are represented by open or shaded boxes. ORFs other than the coat protein (CP) gene are numbered according to their descriptions in the literature. The relative order of the putative protease, VPg and RdRP domains within the putative polyprotein composed of ORFs 2a and 2b within the TRoV genome are schematically represented by ovals. 188 A.S. Callaway et al. / Virology 330 (2004) 186–195

among the genomic and subgenomic RNAs of sobemoviruses, luteoviruses and Red clover necrotic mosaic virus (RCNMV-a Dianthovirus) RNA1 suggests that it or its minus-strand complement may be important in viral RNA synthesis (Hacker and Sivakumaran, 1997). The TRoV 3V untranslated region is somewhat shorter than previously described Sobemovirus 3VUTRs. It is 72 nts vs. 125–284 nts for other known Sobemovirus 3VUTRs. The 3V ends of sobemoviruses lack any discernible sequence similarity and it is unclear whether significant secondary structural similarities are present within Sobemovirus 3V UTRs (Hacker and Sivakumaran, 1997; Lokesh et al., 2001; Ryabov et al., 1996). The TRoV ORF1 encodes a predicted protein of 146 amino acid residues with a MW of 16.5 kDa. ORF1 proteins of other sequenced sobemoviruses range between 109 (Cocksfoot mottle virus;CfMV)and186(SCPMV) residues. Like all previously reported ORF1 Sobemovirus sequences, TRoV’s ORF1 has no significant primary deduced amino acid sequence homology with other Sobe- movirus ORF1 proteins or any other protein in the GenBank database. The major role of the Sobemovirus P1, appears to be in viral movement and/or host-specific interactions. The RYMV and SBMV P1’s suppress post-transcriptional gene silencing (PTGS) in Nicotiana benthamiana (Li and Ding, 2001; Voinnet et al., 1999). P1 may also exert some positive influence on RNA synthesis, though it is not essential for replication (Bonneau et al., 1998). A short intergenic region ranging from 34 to 143 nts is present between ORF1 and the next ORF in all sequenced sobemoviruses except SCPMV, SBMV and SeMV. This non-coding region is 56 nts in length in the TRoV genome, the same size as in the Nigerian RYMV isolate (Pinto and Baulcombe, 1995) and SeMV (Lokesh et al., 2001). This non-coding region is followed in the TRoV genome by a Fig. 2. Comparisons of the predicted 5Vterminal secondary structures from short ORF termed ORF1b, which encodes a putative protein selected Sobemovirus genomes. Nucleotides discussed in the text including of 78 amino acid residues or 8.4 kDa. Only LTSV may have those in the proposed conserved 5V-terminal sequence motif are boxed. a similarly positioned ORF, which is predicted to encode a somewhat smaller polypeptide of 61 amino acid residues sequence found at the 5V end of the TRoV genome is (Jeffries et al., 1995; Tamm and Truve, 2000). The possible identical to a sequence near the 5Vends of the genomes of role and existence of these small proteins are unknown. The Southern bean mosaic virus (SBMV), Southern cowpea two putative 1b polypeptides do not possess significant mosaic virus (SCPMV) and Sesbania mosaic virus (SeMV). sequence homology with each other (Table 1). However, the A similar sequence of CAAAUA is found near the 5Vends hydrophobicity plots (Kyte and Doolittle, 1982) of these of Lucerne transient streak virus (LTSV), and Ryegrass two putative polypeptides reveal possible secondary struc- mottle virus (RGMoV). This sequence typically starts at tural similarity (data not shown). position 2 or position 3 in other sobemoviruses, whereas it Sobemoviruses typically produce a serine protease, VPg constitutes the 5V terminus of TRoV as determined by and RNA-directed RNA polymerase (RdRp) as a single sequencing independent TRoV 5V-RACE clones. The 5V- polyprotein that is proteolytically processed to yield the terminal nucleotide of SBMV, SCPMV, LTSV, RGMoV, and mature functional proteins. Based on sequence alignments Rice yellow mottle virus (RYMV) is an A (Hacker and with other sobemoviruses, it is predicted that the full-length Sivakumaran, 1997; Jeffries et al., 1995; Ngon A Yassi et TRoV RdRp-containing polyprotein is translated via a -1 al., 1994; Zhang et al., 2001), thus making the consensus frameshift event (Jacks et al., 1988) at nt position 1784. ACAAA 5V-terminal motif described as important by Previously, this strategy for Sobemovirus polyprotein Hacker and Sivakumaran (1997). The biological signifi- production was only observed for CfMV (Ma¨kinen et al., cance of this motif is unclear, but its sequence conservation 1995) and Sweet clover mosaic virus (SCMoV) (Dwyer et A.S. Callaway et al. / Virology 330 (2004) 186–195 189

Table 1 Krsinich and Hull, 1981). A DXXD motif characteristic of Amino acid sequence relatedness of TRoV to SBMV Sobemovirus Sobemovirus, Carmovirus, and Tombusvirus CPs is thought Sequence Identity (%) Similarity (%) to be a Ca2+-binding site (Lokesh et al., 2001). The TRoV Genomic RNA 49 N.A. CP is predicted to have two of these motifs (nt positions ORF1 9 47 3586–3597 and 3928–3939), though it is not known if both ORF 1b* 17 52 of these sites are functional. A phylogenetic analysis of Polyprotein 33 56 ORF3 35 66 predicted CP amino acid sequences suggested that the CP 31 70 Sobemovirus CP is most closely related to CPs from * vs. LTSV because SBMV lacks an ORF1b. Luteoviridae and Tombusviridae (Tamm and Truve, 2000), of which RCNMV is a member. al., 1999). The shifty heptanucleotide, UUUAAAC, starts at TRoV CP gene complements systemic movement function in position 1750 in TRoV and is followed by a similar stem– a CP-null RCNMV loop structure (Fig. 3) observed in several other viruses employing a -1 ribosomal frameshift including other To investigate whether the TRoV CP could function in a sobemoviruses (Ma¨kinen et al., 1995; Zhang et al., 2001), chimeric context, a cDNA copy of the deduced TRoV CP Human immunodeficiency virus (Parkin et al., 1992), and ORF was used to replace the RCNMV CP ORF in the Red clover necrotic mosaic virus (RCNMV) (Kim and plasmidpRC169(modifiedfromXiong and Lommel, Lommel, 1994). In addition, the six nucleotides between the 1991). Even though RCNMV is a member of a different shifty heptanucleotide and the stem–loop are identical to viral genus, Dianthovirus, limited phylogenetic similarity those in CfMV and SCMoV. A feature unique to TRoV is has been suggested between the Sobemovirus and Diantho- that the initiation codon for this polyprotein appears to be a virus CPs at the amino acid level (Dolja and Koonin, 1991; CUG, as there are no AUG codons suitably positioned (Fig. Tamm and Truve, 2000). As controls, an antisense TRoV 3). The resulting predicted 867 amino acid, 97 kDa CP cDNA copy or a plus-sense CP gene with the ATG polyprotein contains the viral protease, VPg, ORF3 and initiation codon deleted was cloned into the same site in the RdRp domains, respectively from the N-to C-terminus of RCNMV genome. Infectious transcripts from each of these the polyprotein. This order within the polyprotein is vectors were synthesized in vitro with T7 RNA polymerase, identical to the order in CfMV (Ma¨kinen et al., 2000b). mixed with transcripts made from a wild-type cDNA copy Several other sobemoviruses follow this order in the of RCNMV RNA2 (Xiong and Lommel, 1991)and nucleotide sequence, but ORF3 is translated separately as inoculated onto N. benthamiana plants. To test for the a -1 frameshift product (Lokesh et al., 2001; Sivakumaran et ability of wild-type TRoV to infect N. benthamiana, al., 1998; Zhang et al., 2001). Most other VPg-encoding infectious extract from TRoV-infected Arabidopsis plants RNA viruses are arranged as VPg-proteinase-polymerase was inoculated onto additional plants. Local lesions and (Koonin and Dolja, 1993). Several motifs within the RdRp are highly conserved among the sobemoviruses, including the GDD motif (2895–2903 in TRoV) that is a hallmark of RdRp enzymes. The 5V-terminal region of TRoV ORF2b, the RdRp, encodes a putative protein domain that is similar to the ORF3 described for other sobemoviruses (Table 1). This same arrangement is found in CfMV (Ma¨kinen et al., 1995; Ryabov et al., 1996) and SCMoV (Dwyer et al., 1999). In all other sobemoviruses, ORF3 is nested within the replicase- containing polyprotein also, but is in a different reading frame. The function of the ORF3 protein or domain is not known, though a translatable SCPMV ORF3 is required for cell-to-cell movement (Sivakumaran et al., 1998). Like other sobemoviruses, the TRoV ORF nearest the 3V end of the genome is most likely translated from a subgenomic RNA and encodes the CP (Morris-Krsinich and Hull, 1981). The TRoV CP is 256 amino acid residues with a predicted MW of 27.5 kDa, very similar to the 195– Fig. 3. Predicted secondary TRoV RNA structure proposed to be involved b 280 residues in other Sobemovirus CPs (Nigerian RYMV in translation of the -1 frameshift polyprotein. The conserved shifty heptanucleotidesQ (boxed) at the beginning of the selected RNA sequence isolate; Pinto and Baulcombe, 1995 and SCPMV; Wu et al., are followed by stem-loops characteristic of -1 frameshifting structures 1987, respectively) and to the 30-kDa apparent molecular reported in other viruses. The uracil residue at which the-1 frameshift is weight of TRoV CP on polyacrylamide gels (Morris- proposed to occur is shown in bold italic. 190 A.S. Callaway et al. / Virology 330 (2004) 186–195 ringspots typical of RCNMV infections appeared 4–5 days recovered in the plants inoculated with the sense CP post-inoculation (dpi) on N. benthamiana leaves inoculated chimera or the antisense CP chimera could have been lost with wild-type RCNMV RNA1 + RNA2. Local symptoms somehow during passaging. To test these possibilities, on plants inoculated with the three chimeric RCNMV/TRoV additional N. benthamiana plants were inoculated with (Fsense and DATG) CP ORF transcripts were significantly transcripts generated in vitro of either sense or antisense CP- less apparent, but appeared within the same 4–5 dpi time chimeric RNA1 mixed with wild-type RCNMV RNA2. period. Ringspots were not observed on plants inoculated Viral RNA was present in inoculated leaves from plants with the chimeras. No symptoms were observed on N. inoculated with either of the CP chimeras (Table 2) benthamiana inoculated with wild-type TRoV. suggesting that both chimeric viruses were able to replicate Surprisingly, a mild mosaic formed approximately 15 dpi and perhaps move locally within inoculated tissue. Viral on non-inoculated leaves of plants inoculated with the plus- RNA was also readily detectable in non-inoculated leaves of sense CP chimera. A similar mosaic was produced 15 dpi plants inoculated with the sense-CP chimera (Table 2). when sap from the leaves showing this mild mosaic was However, despite numerous attempts, no viral RNA was passaged onto new N. benthamiana plants. Non-inoculated detectable from systemic leaves of plants inoculated with leaves of plants inoculated with the antisense CP chimera the antisense-CP chimera. Furthermore, viral RNA was not and the DATG chimera were indistinguishable from mock- detectable in virion preparations from inoculated leaves of inoculated controls. To determine if the antisense and DATG plants inoculated with the antisense-CP chimera, whereas CP chimeras were also moving into the non-inoculated parallel virion extracts from sense-CP inoculated plants leaves and to verify that infection had occurred, RT-PCR yielded easily detectable amounts of viral RNA (Table 2). with various primer combinations was performed. Attempts to detect a relatively abundant control phytoene Initial experiments using virion RNA from N. benthami- desaturase (PDS) RNA in virion preparations from the host ana plants inoculated with the second passage of the CP were unsuccessful, whereas parallel total RNA preparations sense chimera indicated that genomic RNA1 was present in from the same plants consistently amplified PDS RNA non-inoculated tissue (data not shown). Additional virion (Table 2), suggesting that unprotected RNAs are destroyed RNA was extracted from non-inoculated leaves of plants during the virion preparation. To test whether the inability to inoculated with the 2nd passage sense or antisense CP detect the antisense CP-chimera was due to changes in RNA chimeras and tested by RT-PCR for the presence of viral structure as a consequence of inversion of the CP-gene, RNA. Viral RNA was detectable only in plants inoculated additional N. benthamiana plants were inoculated with the with the sense CP chimera (Fig. 4). These results suggest DATG chimera, also a sense-CP chimera, but lacking the that it is important for the TRoV CP gene to be in the sense first TRoV CP gene initiation codon, the original CP-sense orientation for the chimeric virus to infect N. benthamiana chimera. Wild-type RCNMV RNAs 1 + 2 or only with plants. Alternatively, adaptive changes could have occurred RCNMV RNA2. RCNMV RNA2 cannot replicate as it during the first two passages that by chance were only encodes no replicase proteins, but this mock inoculum provides a measure of whether inoculum RNA persisted to significant levels under our conditions on the inoculated leaves. As in the previous experiments, only the CP-sense chimera and wild-type RCNMV were detectable in non- inoculated parts of the plants (Table 2). These results strongly suggest that the presence of a translatable TRoV CP gene in the chimeric RCNMV RNA1 was able to protect the viral RNA from degradation during the virion preparation procedures and facilitate movement to non-inoculated regions of N. benthamiana plants. Furthermore, these data suggest that the previous results were not artifacts due to passaging the virus. However, because selection for adaptive changes could occur shortly after inoculation with the sense chimeric virus, symptom timing and reproducibility of movement patterns are not sufficient to determine whether or not the observed Fig. 4. RT-PCR detection of viral RNA from N. benthamiana plants successful infections were due to rapid evolution of the inoculated with RCNMV chimeras bearing either a plus- or anti-sense sense chimeric virus. To further test for the presence of TRoV CP gene. All template RNAs represented were extracted from non- adaptive changes within the viral genome, cDNA copies of inoculated tissues after subjecting the material to a typical virion chimeric viral RNA from infected plants were cloned using preparation procedure. Viral RNA was only detected in plus-sense CP plants. Each sample was divided equally just prior to cDNA synthesis. RT-PCR. Eight independent clones derived from the sense Reverse transcriptase was added to one half (+) and the other half lacking chimeric RNA1 and 10 independent clones derived from the reverse transcriptase (À) served as a control for DNA contamination. co-inoculated RCNMV RNA2 were sequenced. A sequence A.S. Callaway et al. / Virology 330 (2004) 186–195 191

Table 2 Infectivity of RCNMV/TRoV chimeras in Nicotiana benthamiana Inoculum Inoculated leaves Systemica leaves Total RNA virion prep Total RNA virion prep Systemic symptomsb CP-sense + + + + 15 DATG + N.D. À N.D. À CP-antisense + ÀÀÀÀ RNA2 only ÀÀÀÀÀ RCNMV wt + + + + 7 PDSc + À + À N.A. Infectivity was determined by RT-PCR and symptom appearance. + = amplification of RNA1 and/or RNA2 sequences, À = no detectable viral RNA, N.D. = Not Determined, N.A. = Not Applicable. a Systemic = non-inoculated leaves. b Number of days post-inoculation visible symptoms appeared on non-inoculated leaves. c PDS = Phytoene desaturase, an endogenous N. benthamiana RNA used as a measure of RNA quality and stability in all the above inocula. Thus, PDS RNA was not an inoculum, but an internal control. change was considered to be influenced by selection if it Attempts to generate an infectious wild-type TRoV clone was found in more than two clones. Three such nucleotide were not successful. 5V- and 3V-RACE were used to ensure substitutions, all resulting in changes in deduced protein that the respective nucleotide ends of the viral genome were sequences, were found in the chimeric RNA1. A C N A represented. Because the 3Vuntranslated region is somewhat transversion was found in 3/8 chimeric virus clones at shorter than those of other sobemoviruses, an additional nucleotide 619 in the p27/p88 RCNMV ORFs that is measure was taken to determine the 3V end sequence. predicted to result in a threonine to proline substitution. A Sequencing was conducted directly on random hexamer- second mutation was found in the p88 RCNMV replicase primed TRoV cDNA. As is evident from Fig. 6, the gene in 6/8 chimeric clones. It was a C N T transition at sequencing trace file shows a dramatic loss of signal after nucleotide 1945 that results in a predicted alanine to valine the last nucleotide in the reported TRoV sequence, indicat- substitution. The third substitution at nucleotide 3295 of the ing that sequences downstream to those reported were not chimeric RNA1 genomic sequence was observed in 4/8 represented in the cDNA mixture. Neither capped nor clones. This change within the TRoV CP gene was a T N C uncapped RNAs generated in vitro from either T7 or SP6 transition which would be predicted to result in a lysine to promoters were infectious on Arabidopsis or turnip plants. serine substitution. Two of the eight clones accumulated all three of the described nucleotide substitutions. No adaptive sequence changes were found in cDNAs derived from Discussion RCNMV RNA2. To test for the ability of the chimeric viruses to produce CP is dispensable for replication and cell-to-cell move- TRoV CP, total protein from inoculated leaves harvested 17 ment of the Dianthovirus, RCNMV (Xiong et al., 1993), thus dpi was subjected to SDS-PAGE and Western blotting using it is not surprising that viral RNA from both sense and a polyclonal antibody raised against TRoV virion prepara- antisense CP-chimeric RCNMV constructs was detected in tions. Only the plus-sense CP chimera produced detectable total RNA from inoculated leaves. However, Sobemovirus TRoV CP in N. benthamiana (Fig. 5). CP is an early gene product (Bonneau et al., 1998) required for cell-to-cell movement of SCPMV (Sivakumaran et al., 1998) and possibly RYMV (Brugidou et al., 1995). Both dianthoviruses and sobemoviruses require CP for efficient long-distance movement. It is possible that the substitution of

Fig. 5. Western analysis of total protein from plants inoculated with RCNMV/TRoV chimeras and controls. All plants were N. benthamiana unless otherwise stated. Film was exposed for 30 s. Loading order (left to right): mock inoculated, RCNMV/TRoV CP chimera with full-length CP gene in antisense orientation, chimera with full-length CP gene in the sense orientation, chimera with a truncated CP gene corresponding to the first 135 amino acid residues (407 nts) in the sense orientation, chimera with CP Fig. 6. Sequence chromatogram trace from direct sequencing of random- gene in sense orientation, but lacking initial ATG start codon, sense chimera hexamer primed TRoV cDNAs. A primer specific to TRoV (5V- lacking start codon and truncated to first 407 nts of the CP gene, RCNMV CTCTAAACGTTGACGCTTCGCTGGG-3V) was used for the sequencing virions and TRoV-infected A. thaliana. reaction. 192 A.S. Callaway et al. / Virology 330 (2004) 186–195 a translatable TRoV CP ORF for RCNMV’s CP gene possibility that the presence of these changes within some facilitated long-distance movement per se by functional viral genomes of the infecting population were necessary for substitution. Even so, it is doubtful that the TRoV CP fully systemic infection by the CP-sense chimera, the fact that the substitutes for all functions of the RCNMV CP as symptoms few changes we found were either represented by a minority from the resultant virus occurred significantly later and of clones or an apparently conservative alteration suggests attempts to detect virus particles from chimera-infected plants that little, if any, selection is necessary for the chimera to by electron microscopy were not successful. Alternatively, move into non-inoculated leaves. the TRoV CP may have enhanced cell-to-cell movement such In addition to demonstrating the ability of the TRoV CP that the sense-CP chimeric virus was able to spread to non- gene to complement movement of a Dianthovirus, we also inoculated leaves. RCNMV CP-null mutants can spread out demonstrate the ability of wild-type TRoV to infect the of inoculated N. benthamiana leaves when plants are grown widely used model plant, Arabidopsis thaliana, and report at 15 8C, albeit at a much slower rate, but cannot move at 25 the genomic nucleotide sequence of TRoV. ClustalW 8C(Xiong et al., 1993). In this study, all plants were grown at alignments (Thompson et al., 1994) of the nucleotide and 18 8C. Because no systemic virus movement was observed in deduced amino acid TRoV sequences with those of other plants inoculated with chimeras bearing non-translatable CP sobemoviruses and the presence of similar structural genes, it is considered unlikely that the ability of the characteristics suggest that the sequence is complete. The translatable plus-sense CP chimera to move to non-inoculated reasons for the lack of infectivity of our clones are unclear. leaves can be entirely explained by the growth temperature. Nevertheless, until an infectious clone is generated, an Interestingly, neither chimeric virus in this study was able to incomplete sequence cannot be ruled out. infect N. clevelandii systemically, a host of wild-type Alternatively, the Sobemovirus VPg protein may be RCNMV that was previously shown to be incapable of essential for full infectivity. The VPg protein is a protease supporting the CP-independent form of RCNMV systemic which is covalently linked to native genomic RNA in movement (Xiong et al., 1993). Distinguishing between true infected plants. The SBMV VPg has been described as vascular systemic movement and slower alternate mecha- essential for measurable infectivity (Veerisetty and Sehgal, nisms awaits localization of the virus to vascular or non- 1980). In vitro-generated SCPMV transcripts can initiate vascular tissue. On the other hand, the previously described infection without VPg, but infectivity is reduced 5- to 28- differences in movement capabilities may be more due to fold compared to native SCPMV RNA (Sivakumaran et al., differences in ability of the viral variants to suppress or avoid 1998). It is quite possible that the dependency on the post-transcriptional gene silencing (PTGS) than a direct presence of the attached VPg varies among Sobemovirus effect on movement per se. Recent work has shown that isolates and species. Among the sequenced sobemoviruses, PTGS is effectively suppressed by 15 8C temperatures infectious in vitro-generated transcripts have been reported (Szittya et al., 2003), but remains active at 25 8C. Addition- for SCPMV (Sivakumaran et al., 1998), CfMV (Ma¨kinen et ally, Turnip crinkle virus CP can effectively suppress PTGS al., 2000a), and RYMV (Brugidou et al., 1995), but not for (Qu et al., 2003; Thomas et al., 2003). Thus, it is quite RGMoV, SeMV, LTSV, SBMV, SCMoV, or TRoV. possible that the RCNMV and TRoV CPs suppress PTGS. It is interesting to speculate that a reduced binding strength of TRoV CP to the RCNMV genomic RNAs, relative to the CP- Materials and methods RNA binding strengths of either parent virus, could even enhance PTGS suppression. The CP-RNA pair in the chimera Cloning, assembly and sequencing the TRoV genome had little time to co-evolve. Recent findings suggest that reduced binding affinity of the TCV CP can increase Superscript II reverse transcriptase (Gibco-BRL, Gaithers- symptom severity, possibly through enhanced PTGS sup- burg, MD) was used to synthesize cDNAs from virion RNA pression (Zhang and Simon, 2003). Alternatively, the from TRoV-infected turnip and Arabidopsis plants. Initial alteration of the genomic RNA1 structure itself made in the clones were made from random hexamer reverse tran- deletion or inversion of the CP gene may predispose the RNA scription. Subsequent clones used TRoV-specific primers to degradation by PTGS, though this hypothesis may not for cDNA synthesis except for the 3Vand 5Vtermini. Clones explain the lack of infectivity of the chimera lacking only the representing the TRoV 5V terminus were generated with CP initiation codon. 5VRACE using Superscript II reverse transcriptase, terminal Selection for adaptive sequence changes within the viral transferase and an inosinylated primer according to the RNA is another possible influence on the observed ability of manufacturer’s instructions (Gibco-BRL). The bulk of the 3V the CP-sense chimera to systemically infect plants. How- end was generated by a type of 3VRACE. First-strand cDNA ever, only three nucleotide changes were found in more than synthesis from TRoV virion RNA was primed with tailed 1/4 of clones from recovered chimera’s genomic RNA. Only random hexamers, bRandecQ (5V-CGCCNNNNNN-3V). The one of these three alterations was present in the majority of 5Vtail of the primer constitutes a partial AscI site. Reverse clones, a C N T nucleotide substitution resulting in a transcription with these primers was performed at 25 8C for conservative Ala N Val change. While we cannot rule out the 10 min followed by 50 min at 42 8C. Two microliters of this A.S. Callaway et al. / Virology 330 (2004) 186–195 193 reverse transcription reaction was then added to a 100 Al PCR amplification of TRoV virion RNA with the primers 5V- reaction and amplified with Taq polymerase and 2 Â 107 Mof ATGGAGAAAGGAAACAAGAAGCTCAC-3V and 5V- a TRoV-specific forward primer (5V-AACTCAAGAAT- CTATACGTTTAAGGACGAGGCGATC-3V into PmlI- CAGGGTAGCAATGGC-3V)and1Â 106 M of a primer digested pRCDCP. The plasmid pRCDCP was made by that extends the Randec products in the 5Vdirection to include site-directed, long-inverse PCR mutagenesis (Callaway, a complete AscI site and additional restriction sites for EcoRI 1998) of a cDNA clone of RCNMV RNA1 (Xiong and and NcoI(5V-GGAATTCGGCCATGGCGCGCC-3V). Lommel, 1991) with the primers 5V-CACGTGTTGGTTC- Finally, 2 Al of this amplification reaction was re-amplified TTTTAAGCGTAGCCTCC-3Vand 5V-TTTATCGGGCTTT- with Pfu polymerase (Stratagene, La Jolla, CA), a nested GATTAGATCTTTGTGGATTC-3V. To remove the TRoV TRoV-specific primer (5V-GAGCTCGTTGGAAGAATCC- CP gene initiation codon, the gene was amplified with the GAGCAAAAC-3V) and another extension primer (5V- primers 5V-ATCGAGAAAGGAAACAAGAAGCTCAC-3V GGAATTCGGCCATGGCGCGCC-3V) to allow greater and 5V-CTATACGTTTAAGGACGAGGCGATC-3V and stringency during amplification. Products from this reaction cloned into the PmlI site of pRCDCP resulting in plasmid N 700 bp were digested with AscI and cloned into pNEB193 pCPDATG. All preparative amplifications were done using a (New England Biolabs, Beverly, MA) which had been mixture of Taq (Promega, Madison, WI) and Pfu (Stra- cleaved with AscIandSmaI. These inserts were then tagene) DNA polymerases. sequenced. To extend this sequence to include any additional nucleotides that may have been missed by the 3VRACE, Detection of viral RNA sequencing was also performed directly on random hexamer- primed TRoV cDNA. A TRoV-specific forward primer (5V- Virion preparations of TRoV and RCNMV/TRoV CTCTAAACGTTGACGCTTCGCTGGG-3V) was used for chimeras were done as described (Hull, 1977). RNA the sequencing reaction. extractions from plant tissue or virion preparations were done using the RNeasy extraction kit according the Plant growth and inoculation conditions manufacturer’s instructions (Qiagen, Valencia, CA). Re- verse transcription used Stratascript reverse transcriptase N. benthamiana and turnip (Brassica campestris cam- (Stratagene) according to manufacturer’s recommendations pestris dNaboT) plants were grown under standard green- except that the reactions were supplemented with DTT to house conditions and transferred to a chamber held at 18 8C 10 mM. Diagnostic amplifications were done using Red- just before inoculation. Arabidopsis plants were sown in pre- Taq polymerase (Sigma-Aldrich, St. Louis, MO) with moistened Metro-Mix 100 plus Osmocote and held at 4 8C for primers specific to the N. benthamiana phytoene desatur- 1 week prior to germination and subsequent growth in an 18 ase gene (PDS), RNA1 or RNA2 as appropriate. PDS 8C chamber with approximately 200 AE lighting. Inocula- primers: CTGACGAGCTTTCGATGCAGTGC-3V and 5V- tions were performed essentially as previously described ATATATGGACATTTATCACAGGAACTCC-3V.RCNMV (Vaewhongs and Lommel, 1995) on 6–8 true-leaf stage RNA 1 and TRoV primers to detect the various genomic Nicotiana plants except that 10 mM sodium phosphate, pH RNA 1’s, which include: 5V-AAGCTGCAGTTAGAG- 7.0 was used as diluent. Turnip and Arabidopsis inoculations CAACTGCCA-3V,5V-CGGGTACCCTACGACGGCATCC- were done at the 4–6 true-leaf stage. TRoV was obtained from TTCGGG-3V,5V-ATGGAGAAAGGAAACAAGAAGCTC- the American Type Culture Collection and was inoculated AC-3V, and 5V-CTATACGTTTAAGGACGAGGCGATC-3V. onto turnip and Arabidopsis plants by passage of infected sap RNA2 primers included: 5V-ATGTGGAAAATTTAAGT- ground in 10 mM sodium phosphate buffer pH 7.0. In vitro GATTTGGCAAAGAC-3V, and 5V-GAGTCTTTCCGGAT- transcription reactions were performed essentially as TTGGGTCGG-3V. described (Pokrovskaya and Gurevich, 1994)usingT7 RNA polymerase (New England Biolabs). Detection of TRoV coat protein

Sequence analysis Inoculated N. benthamiana leaves were harvested at 17 dpi. A. thaliana Col-0 leaves from plants infected with wild- RNA secondary structure predictions were accomplished type TRoV were harvested at 5 dpi. Protein extracts were using the MFOLD algorithms (Mathews et al., 1999; Zuker made from 0.5 g leaf tissue that was ground in 1 ml of et al., 1999). Pairwise sequence comparisons were done 10 mM Tris HCl buffer pH 6.5. The leaf extract was mixed using BLAST (Altschul et al., 1990), multiple sequence in a ratio of 4:1 with 8% SDS, 0.2% h-mercaptoethanol and comparisons used ClustalW (Thompson et al., 1994). 0.2–0.4% bromophenol blue in glycerol. The samples were boiled at 100 8C for 10 min, placed on ice for 5 min and Construction of RCNMV/TRoV chimeras then centrifuged at 5000 rpm for 45 s. To separate the proteins, 20 Al of each sample was run on a 12% Chimeric RNA1 cDNA clones pRCPTR and pRCPTRap polyacrylamide Tris–HCl Ready Gel (BioRad, Hercules, were constructed by insertion of RT-PCR products from CA) with a 1Â Laemmli Buffer at 200V for approximately 194 A.S. Callaway et al. / Virology 330 (2004) 186–195

40 min. Resolved proteins were transferred to a nitro- Hollings, M., Stone, O.M., 1969. Purification and serological reactions of cellulose filter using the Towbin buffer with 20% methanol four polyhedral viruses from Cruciferae. Zentralbl. Bakteriol. Para- sitenkd. 123, 237–248. according to the manufacturer’s instructions using a Trans- Hull, R., 1977. The banding behaviour of the viruses of Southern bean Blot SD Semi-Dry Transfer Cell (BioRad). The membrane mosaic virus group in gradients of caesium sulphate. Virology 79, was blocked overnight at 4 8C with 50 ml of 1Â TBST (20 50–57. mM Tris–HCl, 500 mM NaCl, 0.05% Tween-20 pH 7.5 and Jacks, T., Madhani, H.D., Masiarz, F.R., Varmus, H.E., 1988. Signals for 3% non-fat dry milk). The blocking buffer was poured off ribosomal frameshifting in the Rous sarcoma virus Gag-pol region. Cell 55, 447–458. and the filter was incubated for 2 h at room temperature with Jeffries, A.C., Rathjen, J.P., Symons, R.H., 1995. Lucerne transient streak 30 ml of rabbit anti-TRoV polyclonal antibody diluted virus complete genome. GenBank Accession No. U31286. 1:1000 in 1Â TBST + 3% non-fat dry milk. The filter was Kim, K.H., Lommel, S.A., 1994. Identification and analysis of the site of -1 washed 5Â with 30 ml 1Â TBST at room temperature for 5 ribosomal frameshifting in Red clover necrotic mosaic virus. Virology min. The membrane was next incubated for 1 h at room 200, 574–582. Koonin, E.V., Dolja, V.V., 1993. Evolution and taxonomy of positive-strand temperature with a 1:10,000 dilution of goat anti-rabbit IgG- RNA viruses: implications of comparative analysis of amino acid horseradish peroxidase conjugate (Sigma). This solution sequences. Crit. Rev. Biochem. Mol. Biol. 28, 375–430. was then poured off and the filter was again washed as Kyte, J., Doolittle, R.F., 1982. A simple method for displaying the above. The antibody was detected using the ECL system hydropathic character of a protein. J. Mol. Biol. 157, 105–132. (Amersham, Piscataway, NJ) and Kodak BioMax Light Li, W.X., Ding, S.-W., 2001. Viral suppressors of RNA silencing. Curr. Opinion Biotech. 12, 150–154. Film (Eastman Kodak, Rochester, NY). Lokesh, G.L., Gopinath, K., Satheshkumar, P.S., Savithri, H.S., 2001. Complete nucleotide sequence of Sesbania mosaic virus: a new species of the genus Sobemovirus. Arch. Virol. 146, 209–223. Acknowledgment M7kinen, K., N&ss, V., Tamm, T., Truve, E., Aaspfllu, A., Saarma, M., 1995. The putative replicase of the Cocksfoot mottle Sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift. We would like to extend special thanks to Dr. J.M. Virology 207, 566–571. Thornsberry for assistance in sequencing the 3Vend of the M7kinen, K., Generozov, E., Arshava, N., Kaloshin, A., Morozov, S., TRoV genome. Zavriev, S., 2000a. Detection and characterization of defective interfering RNAs associated with the Cocksfoot mottle Sobemovirus. Mol. Biol. 34, 291–296. M7kinen, K., M7kel7inen, K., Arshava, N., Tamm, T., Merits, A., Truve, E., References Zavriev, S., Saarma, M., 2000b. Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus). Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. J. Gen. Virol. 81, 2783–2789. Basic local alignment search tool. J. Mol. Biol. 215, 403–410. Matthews, R.E.F., 1991. Plant Virology. Academic Press, San Diego. Bonneau, C., Brigidou, C., Chen, L., Beachy, R., Fauquet, C., 1998. Mathews, D.H., Sabina, J., Zuker, M., Turner, D.H., 1999. Expanded Expression of the Rice yellow mottle virus P1 protein in vitro and in sequence dependence of thermodynamic parameters improves predic- vivo and its involvement in virus spread. Virology 244, 79–86. tion of RNA secondary structure. J. Mol. Biol. 288, 911–940. Broadbent, L., Heathcote, G.D., 1958. Properties and host range of Morris-Krsinich, B.A.M., Hull, R., 1981. Translation of Turnip rosette turnip crinkle, rosette and yellow mosaic viruses. Ann. Appl. Biol. virus RNA in rabbit reticulocyte lysates. Virology 114, 98–112. 46, 585–592. Ngon A Yassi, M., Ritzenthaler, C., Brugidou, C., Fauquet, C., Beachy, R., Brugidou, C., Holt, C., Yassi, M.N.A., Zhang, S., Beachy, R., Fauquet, C., 1994. Nucleotide sequence and genome characterization of Rice yellow 1995. Synthesis of an infectious full-length cDNA clone of Rice yellow mottle virus RNA. J. Gen. Virol. 72, 2633–2638. mottle virus and mutagenesis of the coat protein. Virology 206, 108–115. Parkin, N.T., Chamorro, M., Varmus, H.E., 1992. Human immunodefi- Callaway, A.S., 1998. A genetic approach to study host factors of ciency virus type 1 gag-pol frameshifting is dependent on downstream Arabidopsis (Arabidopsis thaliana) that influence susceptibility to mRNA secondary structure: demonstration by expression in vivo. Cauliflower mosaic virus. Ph.D. Dissertation. Cornell University, J. Virol. 66, 5147–5151. Ithaca, NY. Pinto, Y.M., Baulcombe, D.C., 1995. Rice yellow mottle virus from Nigeria, Callaway, A., Giesman-Cookmeyer, D., Gillock, E.T., Sit, T.L., Lommel, complete genome. The Sainsbury Laboratory, John Innes Institute. S.A., 2001. The multifunctional capsid proteins of plant RNA viruses. Accession U23142. Annu. Rev. Phytopathol. 39, 419–460. Pokrovskaya, I.D., Gurevich, V.V., 1994. In vitro transcription: prepara- Carrington, J.C., Kasschau, K.D., Mahajan, S.K., Schaad, M.C., 1996. Cell- tive RNA yields in analytical scale reactions. Anal. Biochem. 220, to-cell and long-distance transport of viruses in plants. Plant Cell 8, 420–423. 1669–1681. Qu, F., Ren, T., Morris, T.J., 2003. The coat protein of Turnip crinkle virus Dolja, V.V., Koonin, E.V., 1991. Phylogeny of capsid proteins of small suppresses posttranscriptional gene silencing at an early initiation step. icosahedral RNA plant viruses. J. Gen. Virol. 72, 1481–1486. J. Virol. 77, 511–522. Dwyer, G.I., Williamson, S., Njeru, R., Lai, Y., Hopkins, R., Jones, R.A.C., Ryabov, E.V., Krutov, A.A., Novikov, V.K., Zheleznikova, O.V., Morozov, Waterhouse, P.M., Jones, M.G.K., 1999. The complete nucleotide S.Y., Zavriev, S.K., 1996. Nucleotide sequence of RNA from the sequence of Subterrenean clover mottle virus (SCMoV). GenBank Sobemovirus found in infected cocksfoot shows a Luteovirus-like Accession number NC004346. arrangement of the putative replicase and protease genes. Phytopatho- Hacker, D.L., Sivakumaran, K., 1997. Mapping and expression of southern logy 86, 391–397. bean mosaic virus genomic and subgenomic RNAs. Virology 234, Ryabov, E.V., Roberts, I.M., Palukaitis, P., Taliansky, M., 1999a. Host- 317–327. specific cell-to-cell and long-distance movements of Cucumber mosaic Hiruki, C., 1987. The dianthoviruses: a distinct group of isometric plant virus are facilitated by the movement protein of Groundnut rosette viruses with a bipartite genome. Adv. Virus Res. 33, 257–300. virus. Virology 260, 98–108. A.S. Callaway et al. / Virology 330 (2004) 186–195 195

Ryabov, E.V., Robinson, D.J., Taliansky, M.E., 1999b. A plant virus- Pringle, C.R., Wickner, R.B., 2000. Virus Taxonomy. Academic Press, encoded protein facilitates long-distance movement of heterologous New York. viral RNA. Proc. Natl. Acad. Sci. U.S.A. 96, 1212–1217. Veerisetty, V., Sehgal, O.P., 1980. Proteinase K-sensitive factor essential for Sit, T.L., Haikal, P.R., Callaway, A.S., Lommel, S.A., 2001. A single amino the infectivity of Southern bean mosaic virus ribonucleic acid. acid mutation in the Carnation ringspot virus capsid protein allows vi- Phytopathology 70, 282–284. rion formation but prevents systemic infection. J. Virol. 75, 9538–9542. Voinnet, O., Pinto, Y.M., Baulcombe, D.C., 1999. Suppression of gene Sivakumaran, K., Fowler, B.C., Hacker, D.L., 1998. Identification of viral silencing: a general strategy used by diverse DNA and RNA viruses of genes required for cell-to-cell movement of Southern bean mosaic plants. Proc. Natl. Acad. Sci. U.S.A. 96, 14147–14152. virus. Virology 252, 376–386. Wu, S., Rinehart, C., Kaesberg, P., 1987. Sequence and organization of Szittya, G., Silhavy, D., Molna´r, A., Havelda, Z., Lovas, A., Lakatos, L., Southern bean mosaic virus genomic RNA. Virology 161, 73–80. Ba´nfalvi, Z., Burgya´n, J., 2003. Low temperature inhibits RNA Xiong, Z., Lommel, S.A., 1991. Red clover necrotic mosaic virus infectious silencing-mediated defence by the control of siRNA generation. EMBO transcripts synthesized in vitro. Virology 182, 388–392. J. 22, 633–640. Xiong, Z., Kim, K.H., Giesman-Cookmeyer, D., Lommel, S.A., 1993. The Tamm, T., Truve, E., 2000. Sobemoviruses. J. Virol. 74, 6231–6241. roles of the Red clover necrotic mosaic virus capsid and cell-to-cell Thomas, C.L., Leh, V., Lederer, C., Maule, A.J., 2003. Turnip crinkle virus movement proteins in systemic infection. Virology 192, 27–32. coat protein mediates suppression of RNA silencing in Nicotiana Zhang, F., Simon, A.E., 2003. Enhanced viral pathogenesis associated with benthamina. Virology 306, 33–41. a virulent mutant virus or a virulent satellite RNA correlates with Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: reduced virion accumulation and abundance of free coat protein. improving the sensitivity of progressive multiple sequence alignment Virology 312, 8–13. through sequence weighting, position-specific gap penalties and weight Zhang, F., Toriyama, S., Takahashi, M., 2001. Complete nucleotide matrix choice. Nucleic Acids Res. 22, 4673–4680. sequence of Ryegrass mottle virus: a new species of the genus Vaewhongs, A.A., Lommel, S.A., 1995. Virion formation is required for the Sobemovirus. J. Gen. Plant Pathol. 67, 63–68. long-distance movement of Red clover necrotic mosaic virus in Zuker, M., Mathews, D.H., Turner, D.H., 1999. Algorithms and thermody- movement protein transgenic plants. Virology 212, 607–613. namics for RNA secondary structure prediction: a practical guide. In: van Regenmortel, M.H.V., Fauquet, C.M., Vishop, D.H.L., Carstens, E.B., Barciszewski, J., Clark, B.F.C. (Eds.), RNA Biochemistry and Estes, M.K., Lemon, S.M., Maniloff, J., Mayo, M.A., McGeoch, D.J., Biotechnology. Kluwer Academic Publishers, pp. 11–43.