Virology 295, 328–336 (2002) doi:10.1006/viro.2001.1349, available online at http://www.idealibrary.com on CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Characterization of Two Kinds of Subgenomic RNAs Produced by Citrus Leaf Blotch Virus Marı´a C. Vives, Luis Galipienso, Luis Navarro, Pedro Moreno, and Jose´Guerri1 Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain Received October 26, 2001; returned to author for revision November 21, 2001; accepted January 7, 2002 Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RP), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3Ј-coterminal and two 5Ј-coterminal subgenomic RNAs (sgRNAs). The 3Ј-coterminal sgRNAs contain the MP (3ЈMP sgRNA) and CP (3ЈCP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5Ј end. These sgRNAs start with a hexanucleotide which is also present at the 5Ј terminus of the gRNA. The 5Ј-coterminal sgRNAs have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5ЈRPMP sgRNA) and most ORF1 (5ЈRP sgRNA), respectively. Their 3Ј termini map 35 and 40 nt upstream of the transcription initiation of the 3ЈCP and 3ЈMP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3Ј-coterminal sgRNAs transcribed from the minus gRNA strand. The 5Ј-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis. © 2002 Elsevier Science (USA) INTRODUCTION resembles that of members of genus Trichovirus, but considering biological, structural, and molecular differ- Citrus leaf blotch virus (CLBV) was first detected in a ences with known trichoviruses, it has been proposed Nagami kumquat (Fortunella margarita (Lour) Swing), that CLBV should be included in a new virus genus clone SRA-153 from Corsica (France), which showed bud (Vives et al., 2001). union crease when propagated on Troyer citrange (Cit- Synthesis of 3Ј-coterminal subgenomic RNAs (sgRNAs) rus sinensis (L.) Osb. ϫ Poncirus trifoliata (L.) Raf.) (Na- is used by many positive-stranded RNA viruses to express varro et al., 1984; Galipienso et al., 2000). Later, it was internal ORFs (Miller and Koev, 2000). In most cases the also detected in several citrus cultivars from Australia, mechanism by which sgRNAs are produced has not Japan, USA, and Spain, usually associated with bud been experimentally established, but it is believed to union crease on citrange or citrumelo (C. paradisi involve internal initiation of RNA synthesis by the viral (Macf.) ϫ P. trifoliata), which indicated that CLBV may be replicase on the minus gRNA strand (Miller et al., 1985; widespread (Galipienso et al., 2001). Gargouri et al., 1989; Adkins et al., 1997; Wang and CLBV virions are filamentous particles about 960 ϫ 14 Simon, 1997). However, for some viruses it has been nm in size, containing a single-stranded (ss), positive- sense, genomicRNA (gRNA), encapsidatedby a 42-kDa suggested that the negative-stranded sgRNAs could be Ј coat protein (Galipienso et al., 2001). The gRNA has 8747 initiated at the 3 terminus and internally terminated, and nt organized in three open reading frames (ORFs) and then they would serve as templates for the positive- untranslated regions (UTR) of 73 and 541 nt at the 5Ј and strand replication (Sit et al., 1998; Choi et al., 2001; 3Ј termini, respectively. ORF1 potentially encodes a Sawicki et al., 2001). Viruses of different families also Ј 227.4-kDa polyprotein containing methyltransferase, pa- produce 5 -coterminal sgRNAs (German et al., 1992; pain-like protease, helicase, and RNA-dependent RNA MacBeth and Patterson, 1995; Mawassi et al., 1995; Wiel- polymerase motifs (RP). ORF2 encodes a 40.2-kDa gosz and Huang, 1997; Rubio et al., 2000; Che et al., 2001; polypeptide containing a motif characteristic of the 30K Gowda et al., 2001), but the role of these sgRNAs in viral superfamily of cell-to-cell movement proteins (MP). The replication is still unknown. 40.7-kDa polypeptide encoded by ORF3 was identified as CLBV-infected tissue contains a set of double-stranded the coat protein (CP). The genome organization of CLBV RNAs (dsRNAs), which include a full-genome-sized dsRNA (ϳ8.5 kb) and two less-than-full-genome-size dsRNAs of ϳ6.5 and ϳ5.5 kb, the ϳ6.5 kb species being the most Ј 1 abundant. A cDNA probe located in the 5 half of gRNA To whom correspondence and reprint requests should be ad- hybridized in Northern blot with the three dsRNAs, suggest- dressed at Instituto Valenciano de Investigaciones Agrarias, Ctra. ϳ ϳ Ј Moncada-Na´quera Km.4.5, 46113 Moncada, Valencia, Spain. Fax: 34- ing that the 6.5 and the 5.5 kb dsRNAs are 5 coterminal 96-1390240. E-mail: [email protected]. with the gRNA (Galipienso et al., 2001). 0042-6822/02 $35.00 © 2002 Elsevier Science (USA) 328 All rights reserved. SUBGENOMIC RNAs OF CITRUS LEAF BLOTCH VIRUS 329 FIG. 1. Northern blot hybridization of denatured dsRNAs obtained from 5 g CLBV-infected (right lane of each membrane) or healthy (left lane) plant tissue, with digoxigenin-labeled cDNA probes 5ЈT, rp, mp, cp, and 3ЈT, corresponding to the genomic regions marked with horizontal lines. An outline of the CLBV genomic RNA is shown at the top: boxes indicate the three ORFs and the size and potential function of the protein products encoded by each ORF (replicase, RP; movement protein, MP; and coat protein, CP) are given. Arrowheads show the positions of the genomic RNA (gRNA), 5Ј-coterminal subgenomic RNAs (5ЈRPMP and 5ЈRP), 3Ј-coterminal subgenomic RNAs (3ЈMP and 3ЈCP), defective RNA (D-RNA), and unidentified RNAs (?), reacting with each probe. Sizes (bp) of marker RNAs are at the left. Here we have identified and characterized the pected for CLBV gRNA and sgRNAs, as estimated from sgRNAs produced by CLBV in total RNA extracts, in the gRNA sequence (Vives et al., 2001). Five major partially purified virions and in dsRNA-rich preparations dsRNAs of ϳ8.5, ϳ6.5, ϳ5.5, ϳ3.0, and ϳ2.0 kb were using probes derived from different regions of CLBV consistently detected (gRNA, 5ЈRPMP,5ЈRP,3ЈMP, and gRNA. We have found that, in addition to the gRNA, 3ЈCP in Fig. 1). The ϳ8.5-kb dsRNA hybridized with the CLBV-infected plants contain four smaller RNAs species five probes and its size was equal to that of the RNA of ϳ6.5, ϳ5.5, ϳ3, and ϳ2 kb. The ϳ3- and ϳ2-kb RNAs extracted from partially purified virions (see Fig. 3); there- were not detected previously and have been identified as fore, this dsRNA is likely the replicative form of the gRNA. 3Ј-coterminal sgRNAs which contain at their 5Ј end the The sizes of the ϳ3- and ϳ2-kb dsRNAs correspond to MP and CP gene, respectively. These sgRNAs may be sg messenger RNAs involved in expression of the MP encapsidated. The ϳ6.5- and ϳ5.5-kb RNAs have been and CP genes, respectively. The ϳ3-kb dsRNA hybrid- identified as 5Ј-coterminal sgRNAs. In an effort to under- ized with the probes mp, cp, and 3ЈT, and the ϳ2-kb stand the CLBV transcriptional strategy, the 5Ј terminus dsRNA with probes cp and 3ЈT. These two RNAs did not of the 3Ј-coterminal sgRNAs, and the 3Ј terminus of the hybridize with the probes 5ЈT or rp. Therefore, these 5Ј-coterminal sgRNAs, were cloned and sequenced. dsRNAs are likely double-stranded forms of 3Ј-cotermi- nal sgRNAs synthesized to express the MP (3ЈMP RESULTS sgRNA) and the CP (3ЈCP sgRNA) genes (Fig. 1). The ϳ6.5-kb dsRNA hybridized with the probes 5ЈT, rp, and Characterization of CLBV subgenomic RNAs mp, but not with the probes cp and 3ЈT, and the ϳ5.5-kb To characterize the different dsRNA molecules de- dsRNA only reacted with the probes 5ЈT and rp. These tected in infected tissues, denatured dsRNA was hybrid- dsRNAs could be double-stranded forms of potential ized in a Northern blot format with five cDNA probes from 5Ј-coterminal sgRNAs, one of them comprising the RP different regions of the CLBV gRNA (Fig. 1). The size of and at least part of the MP ORFs (5ЈRPMP sgRNA), and these RNAs was estimated by regression analysis of the the other, at least part of the RP ORF (5ЈRP sgRNA) (Fig. electrophoretic mobility measured in Northern-blotted 1). In addition to the five major dsRNAs, an ϳ1-kb dsRNA membranes in comparison with known RNA size mark- was found which hybridized with the probes 3ЈT and 5ЈT, ers (Fig. 1). Sizes were then compared with those ex- but not with the probes of the central region, suggesting 330 VIVES ET AL. FIG. 2. Northern blot hybridization analysis of dsRNA (from 5 g CLBV-infected tissue) and total RNA (from 50 mg infected tissue), using digoxigenin-labeled riboprobes genome-sense (ϩ) or complementary (Ϫ) to the 5ЈT (A) or to the 3ЈT (B) terminal regions of the CLBV gRNA. Arrowheads indicate the gRNA and sgRNAs reacting with each probe (see Fig. 1). that it could be a defective RNA (D-RNA). Other minor gRNA, whereas the (Ϫ) mp riboprobe additionally hybrid- bands hybridizing with the probes cp and 3ЈT, whose size ized with the 3ЈMP sgRNA, and the (Ϫ)3ЈT riboprobe did not correspond to any predictable sgRNA, were additionally hybridized with both the 3ЈMP and the 3ЈCP sometimes observed in Northern blots.