Two types of defective RNAs arising from the tomato black ring virus genome

Short defective RNAs (D-RNAs) associated with tomato black ring virus (TBRV) were isolated, cloned and sequenced. As a result, two types of D-RNAs associated with different TBRV isolates were identified. Both types were derived from RNA1. The first one contained sequences from the 5′ and 3′ untranslated regions (UTR) and from the 5′ region of a single large open reading frame. The second one included a portion of the coding region for the RNA-dependent RNA polymerase flanked by a short fragment of the 5′ UTR and the entire 3′ UTR. The possible nature and origin of these RNA species is discussed.     

Synthesis of genomic and subgenomic RNAs by a membrane-bound RNA-dependent RNA polymerase isolated from oat plants infected with cereal yellow dwarf virus

Summary. A membrane-bound RNA-dependent RNA polymerase (RdRp) complex was isolated by differential sedimentation from oat plants infected with cereal yellow dwarf virus (CYDV). When incubated with ³²P-labelled UTP, unlabelled ATP, CTP and GTP, and Mg²⁺ ions, the RdRp preparation catalysed the synthesis of double-stranded (ds) RNAs corresponding in size to the virus genomic RNA (5.7 kbp) and two putative subgenomic RNAs (2.8 and 0.7 kbp). Hybridisation using strand-specific hybridization targets showed that the 5.7-kbp dsRNA was labelled mainly in the plus strand, whereas the 2.8- and 0.7-kbp dsRNAs were labelled only in the minus strand. Genomic-length single-stranded, plus-strand RNA of 5.7 kb and single-stranded, plus-strand subgenomic RNAs of 2.8 and 0.7 kbp were detected in RNA isolated from oat plants infected with CYDV. Mapping experiments were consistent with the genomic and subgenomic RNAs having common 3′ ends, but different 5′ ends, whether produced in vitro or in vivo. The RdRp-encoding region of the CYDV genome was cloned and expressed in Escherichia coli, and the purified protein was used to raise antibodies in a rabbit. In immunoblots, the antibodies detected a protein of about 68 kDa in RdRp preparations from CYDV-infected oat plants, but not from equivalent preparations from healthy oats. As far as we are aware, this is the first report of an in vitro RNA synthesis system for a phloem-limited virus.     

The RNA 5 of Prunus necrotic ringspot virus is a biologically inactive copy of the 3′-UTR of the genomic RNA 3

Summary.  In addition to the four RNAs known to be encapsidated by Prunus necrotic ringspot virus (PNRSV) and Apple mosaic virus (ApMV), an additional small RNA (RNA 5) was present in purified preparations of several isolates of both viruses. RNA 5 was always produced following infection of a susceptible host by an artificial mixture of RNAs 1, 2, 3, and 4 indicating that it was a product of viral replication. RNA 5 does not activate the infectivity of mixtures that contain the three genomic RNAs (RNA 1 + RNA 2 + RNA 3) nor does it appear to modify symptom expression. Results from hybridization studies suggested that RNA 5 had partial sequence homology with RNAs 1, 2, 3, and 4. Cloning and sequencing the RNA 5 of isolate CH 57/1-M of PNRSV, and the 3′ termini of the RNA 1, RNA 2 and RNA 3 of this isolate indicated that it was a copy of the 3′ untranslated terminal region (3′-UTR) of the genomic RNA 3. Received March 15, 2000 Accepted September 13, 2000     

Characterization of the large (L) RNA of peanut bud necrosis tospovirus

Summary.  The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing ‘core polymerase region’ were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 5′ and 3′ termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 nt complementary sequence with one mismatch. Comparisons of the predicted amino acid sequence of the L protein of PBNV with other members of Bunyaviridae suggest that the L protein of PBNV is a viral polymerase. The L protein had highest identity in the ‘core-polymerase domain’ with the corresponding regions of other tospoviruses, tomato spotted wilt virus and impatiens necrotic spot virus. Received October 17, 1997 Accepted June 16, 1998     

Characterization and in vitro translation analysis of pelargonium flower break virus

Summary.  Pelargonium flower break virus (PFBV) is one of the common viruses in the glasshouses of Western Europe and has been assigned to the genus Carmovirus. A Spanish isolate obtained from nursery-grown Pelargonium zonale plants (PFBV-m) has been characterized. The molecular weight of genomic RNA and coat protein of PFBV-m were determined to be 1.36 × 10⁶ (corresponding to approximately 4 kb) and 36,000, respectively. Only genomic-size RNA was encapsidated in PFBV virions; making necessary to purify double-stranded RNA from infected tissue in order to detect putative PFBV subgenomic RNAs. PFBV RNA directed the synthesis of a major polypeptide of 34 kDa and three other relevant polypeptides of estimated sizes 88–90 kDa, 42 kDa and 35–36 kDa. Antisera specific to PFBV immunoprecipitated the in vitro translated 35–36 kDa polypeptide indicating that this polypeptide is the PFBV coat protein. The PFBV in vitro translation pattern was very similar to that of CarMV although the relative levels of translated coat protein differed dramatically between the two viruses, most probably due to the lack of encapsidation of subgenomic PFBV. In vitro translation studies with a different biological clone obtained from the same PFBV-m isolate revealed a prominent additional polypeptide which is postulated to be a truncation of the 5′ proximal ORF. Received November 27, 1998 Accepted March 24, 1999     

Evidence for a segmented genome and partial nucleotide sequences of maize yellow stripe virus,a proposed new tenuivirus

Summary Attempts at molecular characterization of a maize yellow stripe virus (MYSV) isolate from Egypt revealed that it has a tenuivirus-like segmented genome consisting of five RNA segments (>9.5, 2.4, 2.1, 1.6 and 1.6 kb). Whereas the complete sequence of RNA-5 consists of 1562 nts, only 1152, 1085, 1213, and 808 nts of RNA-1, -2, -3, and -4, respectively, were determined from the MYSV genome, estimated to be 18 kb. Four of the MYSV segments had complementary and conserved 5′ and 3′ termini similar to those of tenuiviruses and phleboviruses. No cross hybridization was observed between MYSV and maize stripe virus (MSpV), a definite member of the genus Tenuivirus. Also, no nucleotide or peptide sequence similarities were detected between the five sequenced stretches of the MYSV genome and any virus sequences, including those of tenuiviruses, available in the databases.     

The nucleotide sequence of the 3′-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production

Summary.  A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA. The sequence of the 3′-terminal 3 158 nucleotides, which consisted of the 3′-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57–68% similar at the nucleotide level and 72–82% similar at the amino acid level when compared with other potyviruses. Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus. A recombinant DsMV-Ch CP (∼39 kDa) expressed in E. coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA. Received March 19 Accepted June 26, 1998     

The genome organization of the broad bean necrosis virus (BBNV)

Summary.  The genome of the broad bean necrosis virus Oita-isolate (BBNV-O) [RNA1 (6.0 kb), RNA2 (2.8 kb) and RNA3 (2.4 kb)] was cloned and sequenced. Computer analysis indicates that methyltransferase, helicase and RNA-dependent RNA polymerase (RdRp) motifs are present in RNA1. The viral capsid protein (CP) cistron is located at the 5′ terminal end of RNA2 and the Mr of CP (20 K) is close to that determined by SDS-PAGE analysis. An ochre codon (UAA) in the CP cistron is thought to be partially suppressed to produce a large readthrough protein. RNA3 possesses typical motifs of triple gene block proteins, which are also reported in several other plant viruses. The furovirus genome organization and phylogenetic analysis using RdRp and CP amino acid sequences suggest that BBNV is closely related to potato mop-top virus (PMTV), but is relatively distantly related to other furoviruses. The data also suggest that the genus Furovirus should be separated into several genera: the prototypical genus Furovirus, which excludes the following viruses: the PMTV group including BBNV; the beet necro- tic yellow vein virus (BNYVV) group; and the peanut clump virus (PCV) group. Received November 14, 1997 Accepted Febuary 12,1998     

The complete sequence of <Emphasis Type="BoldItalic">Oat mosaic virus</Emphasis> and evidence for deletion and duplication in RNA2

Summary.  The complete nucleotide sequence of a UK isolate of oat mosaic virus (OMV) was determined. RNA1 was 7550 nt long with one large open reading frame potentially encoding a polyprotein of 262.8 kDa with features typical of bymoviruses. RNA2 was 2284 nt in length, substantially smaller than those of other bymoviruses sequenced. It appeared that most of the P2 region had been deleted during repeated mechanical transmission of the isolate. The 3′-UTR of RNA2 was very long (>1.25 kb) and proved to have a 532 nt slightly overlapping repeat. Phylogenetic analysis confirmed that OMV is an independent member of the genus Bymovirus. Received June 27, 2001 Accepted October 18, 2001     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus

Summary.  The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5′-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5′-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3′-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF. JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5′ end of the viral RNA is uncapped. The 3′ end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae. Accepted September 23, 1999     

Molecular characterization of a satellite RNA associated with blackcurrant reversion nepovirus

Summary.  A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M_r of 44 220. The coding region was bordered by a 5′ leader sequence of 25 nt and a 3′-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5′-terminal consensus sequence and a 40 nt motif (located at positions 264–303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs. Received March 22, 1999/Accepted August 30, 1999     

Effects of structural modifications upon the accumulation in planta of replicons derived from beet necrotic yellow vein virus RNA 3

Summary. Beet necrotic yellow vein virus (BNYVV) RNA 3 from which all but the 3′ and 5′‘core’ replication origins (promotors) have been deleted replicates when coinoculated to Chenopodium quinoa with viral RNAs 1 and 2. The resulting ‘replicon’ can be used to express inserted heterologous sequences in planta. The effects of alterations of replicon structure on its efficiency of accumulation in planta were examined. Inclusion of up to ∼240 nucleotides of sequence from the region immediately upstream of the core 3′-promoter sequence increased replicon accumulation, suggesting that this region contains specific replication enhancer elements. Insertion of non-viral ‘spacer’ sequences between the core promoters also increased replicon accumulation, provided that no strong secondary structure was present. The highly homologous 3′-terminal core promoters of BNYVV RNAs 1, 2 and 4 could substitute for the RNA 3 core promoter but were generally somewhat less effective. Co-inoculation of full-length RNA 3 but not RNA 4 interfered with accumulation of the RNA 3-based replicons. Received December 7, 1998 Accepted February 11, 1999     

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors

Summary.  Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adh I intron and 68-bp TMV Ω RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3′ of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV Ω RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems. Received September 4, 1998 Accepted March 4, 1999     

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants

Summary.  A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUU potential shifty-heptanucleotide at the end of the 5′-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient − 1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5′-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3′-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known. Received May 15, 2000 Accepted August 24, 2000     

The 3′ terminal region is strictly required for clover yellow vein virus genome replication

Summary.  To identify the cis-element in the 3′ terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3′ terminal non-coding region (3′-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3′ terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3′-NCR were not infectious. Various mutants with duplicated 3′ terminal sequences were infective only when the authentic 3′ terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3′-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication. Received June 11, 2002; accepted October 25, 2002     

Complete nucleotide sequences and genome characterization of double-stranded RNA 1 and RNA 2 in the Raphanus sativus-root cv. Yipinghong

Summary. Four distinct double-stranded (ds) RNA bands were extracted from leaves of Raphanus sativus-root cv. Yipinghong with yellowing at the leaf edge in China. Purified viral particles of 28–30 nm in diameter contained dsRNA segments with the same number and mobility as these extracted directly from radish leaves. The two major dsRNA segments, namely RasR 1 and RasR 2, were 1866 and 1791 bp in length, respectively. Computer analysis predicted that they both contained a single open reading frame (ORF) on their plus-stranded RNA, putatively encoding a RNA dependent RNA polymerase and a capsid protein similar to that encoded by members of the family Partitiviridae. In addition, both RasR 1 and RasR 2 were highly conserved at the 5′ untranslated regions (UTR) and had an adenosine-uracil rich stretch at the 3′ UTR, with an identical terminal motif (5′-AAAAUAAAACC-3′). Taken together, these results suggest that the two major dsRNA segments constitute the genome of a partitivirus infecting radish.     

A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family

Summary.  A universal primer (Sprimer: 5^′-GGX AAY AAY AGY GGX CAZ CC-3^′, X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province). Received June 30, 2000 Accepted September 28, 2000     

Complete nucleotide sequence of wheat yellow mosaic bymovirus genomic RNAs

Summary.  The complete sequences of wheat yellow mosaic bymovirus (WYMV) RNA1 and RNA2 were determined. RNA1 is 7 636 nucleotides long [excluding the 3′-poly(A)], and codes for a 269 kDa polyprotein of 2 404 amino acids which contains the capsid protein (CP) at the C terminus and seven putative non-structural proteins. RNA2 is 3 659 nucleotides long and codes for a polyprotein of 904 amino acids which contains a 28 kDa putative proteinase and a 73 kDa polypeptide. These functional proteins are arranged as in RNA1 and RNA2 of barley yellow mosaic bymovirus (BaYMV). Comparisons with the sequence reported for the 3′ half of RNA1 of wheat spindle streak mosaic bymovirus (WSSMV) from Southern France show that WYMV and WSSMV have a similar genetic organization. However, WYMV and WSSMV share only 77% amino acid sequence identity in their deduced CPs in spite of their close serological relationship, and 74% nucleotide sequence identity in their 3′ non-coding regions. Thus, the sequence data indicate that WYMV and WSSMV are not strains of the same virus, which has long been suggested, but are distinct virus species within the genus Bymovirus of the family Potyviridae. Accepted December 19, 1997 Received November 14, 1997     

<Emphasis Type="Italic">Melandrium yellow fleck bromovirus</Emphasis> infects <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and has genomic RNA sequence characteristics that are unique among bromoviruses

Melandrium yellow fleck bromovirus (MYFV) systemically infected Arabidopsis thaliana, although the susceptibility of several A. thaliana accessions to MYFV differed from their susceptibility to the other two bromoviruses infecting A. thaliana. We constructed full-length cDNA clones of MYFV genomic RNAs 1, 2, and 3 and determined their complete nucleotide sequences. Similar to Broad bean mottle bromovirus, (1) the 5′-terminal nucleotide of the MYFV genomic RNAs was adenine, and (2) the “D-arm” was absent from the tRNA-like structure in the 3′ untranslated regions (UTRs) of MYFV RNAs. As unique characteristics, MYFV RNA3 lacked the poly(A) tract in the intercistronic region and contained a directly repeated sequence of about 200 nucleotides and polypyrimidine tracts of heterogeneous lengths in the 5′ UTR. Co-infection experiments using RNA3 clones with or without the duplicated sequence demonstrated that the duplication contributed to the competitive fitness of the virus in Nicotiana benthamiana.