Novel structure of the genome of Rice yellow stunt virus: identification of the gene 6-encoded virion protein

Yanwei Huang, Heng Zhao{dagger}, Zongli Luo{ddagger}, Xiaoying Chen and Rong-Xiang Fang

Laboratory of Plant Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China

Correspondence
Rong-Xiang Fang
fangrx{at}sun.im.ac.cn


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The genomic region encompassing the L protein gene and a small open reading frame (ORF 6) of Rice yellow stunt virus (RYSV) has been sequenced, thus completing the nucleotide sequence of the RYSV genome. The genome organization of RYSV is unique in the rhabdoviruses because it contains two additional genes when compared to the basic gene order of the family Rhabdoviridae. Phylogenetic analysis revealed that the amino acid sequence of the RYSV L protein is most closely related to that of the L protein of Sonchus yellows net virus, another nucleorhabdovirus. However, the RYSV L protein has a unique acidic N-terminal domain distinct from that of other rhabdoviruses. Moreover, the polypeptide encoded by the ORF 6 was detected by immunoblot analysis in purified RYSV virions. Thus RYSV provides the first example in the family Rhabdoviridae that a small ORF between the G and L genes encodes a virion protein.

The GenBank accession number for the complete sequence of the RYSV genome is AB011257.

{dagger}Present address: Cell Biology & Immunology, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands.

{ddagger}Present address: Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.


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Rice yellow stunt virus (RYSV), synonymous with Rice transitory yellowing virus, is a species in the genus Nucleorhabdovirus of the Rhabdoviridae (Walker et al., 2000). RYSV has a non-segmented, negative-sense single-stranded RNA genome. We have previously sequenced the RYSV genomic RNA from cDNA clones representing the 3' leader (Wang et al., 1999), the nucleocapsid protein (N) (Fang et al., 1994), the phosphoprotein (P, formerly designated non-structural protein) (Zhu et al., 1997), a protein (P3) of unknown function (Chen et al., 1998), the matrix protein (M) (Luo et al., 1998) and the glycoprotein (G) (Luo & Fang, 1998), as well as the 5' trailer region (Wang et al., 1999). In this paper, we report the sequence of the genomic region between the glycoprotein gene and the 5' trailer, thus completing the sequence of the entire RYSV genome (14 042 nucleotides). In this region, a large open reading frame (ORF) encoding the polymerase (L) and a small ORF (ORF 6) capable of encoding 93 amino acids (aa) were found on the viral complementary (vc) strand. Furthermore, we present evidence that the polypeptide encoded by ORF 6, named P6, is a virion-associated protein. Thus RYSV encodes seven genes and has, among the characterized rhabdoviruses, the unique genome organization 3' leader-N-P-3-M-G-6-L-5' trailer. In comparison to the basic gene order represented by the genome of Vesicular stomatitis virus (VSV), the RYSV genome has two additional genes: gene 3 and gene 6. Gene 3 is located between the P and M genes where additional ORFs were identified in the genomes of all plant rhabdoviruses examined so far [Sonchus yellows net virus (SYNV) (Scholthof et al., 1994), Lettuce necrotic yellows virus (LNYV) (Wetzel et al., 1994) and Northern cereal mosaic virus (NCMV) (Tanno et al., 2000)] and the insect rhabdovirus Sigma virus (SigmaV) (Teninges et al., 1993). However, in the position between the G and L genes where the RYSV gene 6 is interposed, the presence of the extra gene(s) has only been described for some animal rhabdoviruses [Adelaide River virus (ARV) (Wang et al., 1994), Bovine ephemeral fever virus (BEFV) (McWilliam et al., 1997), Rabies virus (RABV) (Tordo et al., 1986), Infectious haematopoietic necrosis virus (IHNV) (Schutze et al., 1995), Viral haemorrhagic septicaemia virus (VHSV) (Basurco & Benmansour, 1995) and Snakehead rhabdovirus (SHRV) (GenBank accession no. AF147498)]. The products of these extra viral genes are either non-structural or unidentified.

The nucleotide sequence of the RYSV genomic region spanning the untranscribed intergenic spacer 5' to the G gene to that 3' to the trailer was obtained by sequencing 10 overlapping cDNA clones isolated from a cDNA library of the RYSV genomic RNA (Fang et al., 1994) using a genome-walking strategy initiated with a probe from the G gene. This region consisted of 6560 nucleotides (nt) and each nucleotide was determined by sequencing both strands of at least two different clones. Sequence analysis using the DNASIS software (Hitachi Software Engineering) revealed two ORFs on the vc strand each of which is bordered by the stretch of nucleotides 5'-AAAUAAAACCCCAACA-3', similar to the gene junction sequences found between the other RYSV genes.

The large ORF located at the 3' region of the vc strand could encode a protein of 1967 aa with a deduced molecular mass of 223·6 kDa, and probably encodes the L protein. The transcription initiation sequence of the L gene has been determined to be 5'-AACA-3' by 5'RACE analysis performed on poly(A+) RNA from RYSV-infected rice plants (Luo & Fang, 1998). This sequence motif is identical to the 5' end sequence of other RYSV genes (N, P, M and G) and similar to that of gene 3 (5'-AACU-3') or gene 6 (see below). As in the case of genes G, M and 6 of RYSV, non-viral nucleotides were found at the 5' terminus of the mRNA for the L gene preceding the initiation sequence (Luo & Fang, 1998), suggesting the possibility that the initiation of transcription of RYSV genes proceeds via a cap snatching mechanism as originally demonstrated for influenza virus (Krug, 1981). To determine the exact 3' end of the L gene, 3'RACE was performed as described by Fang et al. (1994). The termination sequence of the L gene was defined as 5'-AAAUAAAAA-3', which is consistent with the conserved termination sequence of other RYSV genes. We thus conclude that the L gene contains a 39 nt 5'-untranslated sequence and a 52 nt 3'-untranslated sequence, and in total is composed of 5988 nt extending from positions 7860 to 13847 relative to the 3' end of the RYSV genomic RNA.

The RYSV L protein is the smallest of all characterized non-segmented negative-strand RNA viruses (NNSV) except for the 1608 aa L protein of Borna disease virus (Briese et al., 1994). Nevertheless, the RYSV L protein harbours multiple functional domains typical of the RNA polymerases of NNSV, e.g. the catalytic domain, the RNA template-binding site and a metal-binding motif (data not shown). A phylogenetic tree was generated by comparison of the amino acid sequence of the RYSV L protein with sequences of the L protein of 32 NNSV (Fig. 1). It is clear that the RYSV L protein is most closely related to the L protein of SYNV, also a nucleorhabdovirus (Choi et al., 1992), with a sequence similarity of 36·9 %. However, the RYSV L protein is distinct from the L proteins of other rhabdoviruses and most members of other families of the Mononegavirales in that it is an acidic protein with a calculated isoelectric point of 6·22 (Fig. 1). Inspection of the RYSV L protein sequence revealed an overall Asp+Glu composition of 12·1 % and a Lys+Arg content of 10·9 %. More significantly, the N-terminal 110 amino acids contained 30 % Asp+Glu and 7·3 % Lys+Arg. This acidic domain is not present in the L proteins of the order Mononegavirales, and its function is unknown.



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Fig. 1. Phylogenetic tree constructed by the neighbour-joining method using entire amino acid sequences of the L proteins of 33 NNSV. Amino acid sequence alignments using ClustalW between the L proteins of RYSV (NP_620502), Australian bat lyssavirus (ABLV; NP_478343), Avian paramyxovirus 6 (APMV-6; NP_150063), Borna disease virus (BDV; NP_042024), BEFV (NP_065409), Bovine parainfluenza virus 3 (BPIV-3; NP_037646), Bovine respiratory syncytial virus (BRSV; NP_048058), Canine distemper virus (CDV; NP_047207), Hendra virus (HeV; NP_047113), Human metapneumovirus (HMPV; NP_690071), Human parainfluenza virus 1 (HPIV-1, NP_604442), Human parainfluenza virus 2 (HPIV-2; NP_598406), Human parainfluenza virus 3 (HPIV-3; NP_067153), Human respiratory syncytial virus (HRSV; NP_056866), IHNV (NP_042681), Marburg virus (MARV; NP_042031), Measles virus (MeV; NP_056924), Mumps virus (MuV; NP_054714), Newcastle disease virus (NDV; NP_071471), Nipah virus (NiV; NP_112028), NCMV (NP_597914), RABV (NP_056797), Respiratory syncytial virus (RSV; NP_044598), Reston Ebola virus (REBOV; NP_690587), Sendai virus (SeV; NP_056879), Snakehead rhabdovirus (SHRV; NP_050585), SYNV (NP_042286), Spring viraemia of carp virus (SVCV; NP_116748), Tioman virus (TiV; NP_665871), Tupaia paramyxovirus (TPMV; NP_054697), Vesicular stomatitis Indianavirus (VSIV; NP_041716), VHSV (NP_049550) and Zaire Ebola virus (ZEBOV; NP_066251). Numbers at each node represent the percentage bootstrap support (1000 replicates). Calculated isoelectric points higher than 7 are underlined. The isoelectric point of the RYSV L protein is indicated in bold. Branch lengths are proportional to genetic distances. The scale bar corresponds to substitutions per amino acid site.

 
Gene 6 is located between the G and L genes. The junction sequence between the G gene and gene 6 is 5'-UAAUAAAAACCCAAUA-3' on the vc strand, where UAAUAAAAA represents the termination sequence of the G gene, and AAUA the initiation sequence of gene 6 as determined by 5'RACE (Luo & Fang, 1998).

The 3' end of gene 6 was determined by 3'RACE. Gene 6 terminates with the sequence 5'-AAAUAAAA-3' followed by a tetranucleotide CCCC as an untranscribed intergenic spacer before the L gene (Fig. 2). Thus gene 6 is flanked by two junction sequences which are homologous to the RYSV intergenic consensus sequence. Since 5' and 3'RACE have provided evidence for the presence of the mRNA for gene 6 in RYSV-infected rice plants, this confirms that RYSV encodes an extra gene located between the G and L genes that is unique to plant rhabdoviruses.



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Fig. 2. Nucleotide and amino acid sequences of RYSV gene 6. The nucleotide sequence is presented in the vc sense with the first nucleotide of the transcription initiation sequence as 1. The intergenic spacers between the G gene and gene 6 and between gene 6 and the L gene, and the start and stop codons of ORF 6 are indicated in bold. The transcription termination sequence of the G gene and the transcriptional start site of the L gene are indicated with bent arrows. Potential phosphorylation sites of P6 are underlined. The putative aspartic protease motif is boxed.

 
These analyses indicated that gene 6 consists of 568 nt extending from positions 7288 to 7855 relative to the 3' end of the RYSV genomic RNA. Gene 6 contains a 38 nt 5'-untranslated leader sequence, a 251 nt 3'-untranslated region and a small ORF (ORF 6) of 279 nt that is capable of encoding a polypeptide (P6) of 93 aa with a calculated molecular mass of 10·5 kDa (Fig. 2). Comparison of the nucleotide sequence of the RYSV gene 6 with that of the large non-coding region preceding the L gene found in the genomes of Hendra virus (Wang et al., 2000) and lyssaviruses (Le Mercier et al., 1997; Tordo et al., 1986) revealed sequence similarity of 38·3 % to Hendra virus and 36·6 % to RABV. The cytorhabdovirus NCMV also has a small ORF (52 aa) between the G and L genes (Tanno et al., 2000). However, little sequence similarity was found when compared this small putative peptide with the RYSV P6.

RYSV P6 is an acidic protein with an isoelectric point of 3·49, and has five potential phosphorylation sites (consensus pattern S/T-X-X-D/E) and one possible aspartic protease motif (D-T-G). Homology analysis and pair-wise comparison of the P6 amino acid sequence was conducted against GenBank/EMBL and SWISSPROT entries, but no clear similarity was found with any protein from these databases. Small non-virion (NV) genes preceding the L gene are present in all characterized novirhabdoviruses (Basurco & Benmansour, 1995; Schutze et al., 1995; Johnson et al., 2000). The RYSV P6 has very limited sequence similarities with the NV proteins: 22·6 % to IHNV and 25·4 % to VHNV – only slightly higher than those generated from random sequences (about 20 %).

To elucidate whether RYSV P6 is a viral protein, SDS-PAGE analysis of purified RYSV virions was performed. Since the P6 protein was not visible in the SDS-PAGE profile of the RYSV virion proteins using Coomassie blue R-250 staining (Fang et al., 1994), an antiserum against glutathione S-transferase (GST)–P6 fusion protein was raised and used in immunoblot analysis. ORF 6 was inserted into the BamHI site of the pGEX-3X vector (Amersham Pharmacia) in-frame with the GST gene. Following transformation of E. coli BL21 with the recombinant clone pGEX-3X-6 and induction by IPTG, the GST–P6 fusion protein was expressed and purified with the Bulk GST Purification Module kit (Amersham Pharmacia) (Fig. 3a). Rabbit anti-GST–P6 antiserum was prepared as previously described (Luo et al., 1998). The total proteins from purified RYSV, healthy leafhoppers and viruliferous leafhoppers were extracted, separated on a 16 % Tris/Tricine gel (Schagger & von Jagow, 1987), and electro-transferred onto ImmobilonTM-P PVDF membrane (Millipore). Immunoblots were done as described by Fang et al. (1994) using rabbit anti-GST–P6 antiserum diluted 1 : 3000. P6 protein (10·5 kDa) was detected in purified virions and also in viruliferous leafhoppers, which transmit RYSV among rice plants (Fig. 3b). Such a protein band was not detected in the total protein extracted from RYSV-infected rice plants in a similar immunoblot assay (data not shown), probably due to the very low P6 content in infected rice plants. Thus, unlike the NV proteins of novirhabdoviruses and as the first case in the family Rhabdoviridae, the protein encoded by the small ORF between the G and L genes in the RYSV genome is associated with purified virions and appears to be a viral structural protein.



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Fig. 3. Detection and in vitro phosphorylation of RYSV P6. (a) Expression and purification of the GST–P6 fusion protein. Total proteins from E. coli harbouring pGEX-3X before (lane 1) or after (lane 2) IPTG induction and from E. coli harbouring pGEX-3X-6 before (lane 4) or after (lane 5) IPTG induction were resolved by 12 % SDS-PAGE followed by Coomassie blue staining. GST (lane 3) and the GST–P6 fusion protein (lane 6) purified from E. coli extracts with the GST–glutathione affinity system were run in parallel in the gel. (b) Immunoblot detection of RYSV P6 with anti-GST–P6 antiserum. Protein samples were resolved by 16 % Tris/Tricine-PAGE followed by Coomassie blue staining (lanes 1–3) or by immunoblot analysis (lanes 4–6). Healthy leafhoppers (lanes 1 and 4); viruliferous leafhoppers (lanes 2 and 5); purified RYSV virion (lanes 3 and 6). (c) In vitro phosphorylation of GST–P6 fusion protein. Samples were resolved by 12 % SDS-PAGE followed by Coomassie blue staining (lanes 1 and 2) or autoradiography (lanes 3 and 4). Lanes 1 and 3, GST–P6 fusion protein; lanes 2 and 4, GST. (d) TLC analysis of phosphoamino acids. Phosphoamino acids makers were visualized by spraying 0·25 % ninhydrin in acetone (lane 1). 32P-labelled phosphoamino acids of GST–P6 fusion protein phosphorylated by CKII were identified by autoradiography (lane 2).

 
As described above, the RYSV P6 protein contains five putative phosphorylation motifs. To determine if these sites are candidate targets of phosphorylation, in vitro phosphorylation analysis was performed. Purified GST–P6 fusion protein was phosphorylated in vitro, along with purified GST protein as a control, in a 10 µl assay mixture containing the reaction buffer, 1 µCi [{alpha}-32P]ATP (3000 Ci mmol-1, 10 µCi µl-1) and 1 unit casein kinase II (CKII, Promega). After incubation for 15–20 min at 37 °C the reactions were stopped, and resolved on a 15 % SDS-PAGE gel followed by autoradiography (Chigaev et al., 2001). The results showed that the GST–P6 fusion protein was phosphorylated by CKII, but the GST protein was not (Fig. 3c). To determine the phosphorylated amino acid residues, gel pieces containing 32P-labelled bands were excised, and the proteins were recovered from gel and precipitated with trichloroacetic acid. The recovered proteins were partially hydrolysed in 6 M HCl at 110 °C for 1 h. The released amino acids were mixed with phosphoamino acid markers and resolved on a thin-layer chromatography plate (Merck). Markers were visualized by spraying the plate with ninhydrin and compared with positions of the 32P-labelled spots (Fig. 3d). Both Thr and Ser were found to be 32P-labelled. In vitro phosphorylation and phosphoamino acid analysis showed that P6 can be phosphorylated by CKII in vitro and the phosphorylated amino acid residues are Thr and Ser.

Although P6 appears to be a virion structural protein and can be phosphorylated, its function remains unknown. P6 has a limited (24 %) sequence similarity with the N-terminal 110 aa of the SYNV L protein. Moreover, P6 contains 32 Asp+Glu (34·4 %) and 4 Lys+Arg (4·3 %) with a large net negative charge, similar to the N-terminal acidic domain of the RYSV L protein (see above), although they share only 18 % sequence similarity. This suggests that P6 may have a close evolutionary relationship with the L protein. Reverse genetic studies have demonstrated that although the NV protein of IHNV was not required for virus replication in cell cultures, it greatly improved virus growth (Biacchesi et al., 2000). Therefore, the small gene preceding the L gene may play an important role in rhabdovirus replication.


   ACKNOWLEDGEMENTS
 
The authors thank Dr Ralf Dietzgen (University of Queensland, Australia) for critical reading of the manuscript. Yanwei Huang and Heng Zhao contributed equally to this work. This investigation was supported by a grant from the National Natural Science Foundation of China (No. 30070036).


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Received 3 March 2003; accepted 17 April 2003.