The complete nucleotide sequence and genome organization of tomato infectious chlorosis virus: a distinct crinivirus most closely related to lettuce infectious yellows virus

The complete nucleotide sequence of tomato infectious chlorosis virus (TICV) was determined and compared with those of other members of the genus Crinivirus. RNA 1 is 8,271 nucleotides long with three open reading frames and encodes proteins involved in replication. RNA 2 is 7,913 nucleotides long and encodes eight proteins common within the genus Crinivirus that are involved in genome protection, movement and other functions yet to be identified. Similarity between TICV and other criniviruses varies throughout the genome but TICV is related more closely to lettuce infectious yellows virus than to any other crinivirus, thus identifying a third group within the genus. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (FJ815440 and FJ815441).     

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3′ non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.     

Complete nucleotide sequence of the RNA2 of the crinivirus tomato chlorosis virus

Summary. The complete sequence of genomic RNA2 of Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae), isolate AT80/99 from Spain, was determined and compared with those from the other members of the genus sequenced to date. RNA2 is 8244 nucleotides (nt) long and putatively encodes nine ORFs that encompass the hallmark gene array of the family Closteroviridae, which includes a heat shock protein 70 family homologue, a 59 kDa protein, the coat protein, and a diverged coat protein. Phylogenetic analysis confirmed assignment of ToCV in the genus Crinivirus, being most similar to sweet potato chlorotic stunt virus and cucurbit yellow stunting disorder virus.     

Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus

Viruses express proteins with silencing suppression activity to counteract the RNA silencing-mediated defense response of the host. In the family Closteroviridae, examples of multiple-component RNA silencing suppression systems have been reported. To ascertain if this is a general strategy in this group of viruses, we have explored the bipartite genome of Tomato chlorosis virus (ToCV, genus Crinivirus). We have identified the RNA1-encoded p22 protein as an effective silencing suppressor by using a Agrobacterium co-infiltration assay. p22 suppressed local RNA silencing induced either by sense RNA or dsRNA very efficiently, but did not interfere with short or long-distance systemic spread of silencing. We have also demonstrated by using the heterologous vector PVX the silencing suppression activity of the RNA-2 encoded coat protein (CP) and minor coat protein (CPm). In this study, we demonstrate an even greater complexity of silencing suppressor activity for a plant virus, and for the first time we show the presence of RNA silencing suppressor genes encoded by both genomic RNA molecules of a bipartite genome in the complex family Closteroviridae.     

Identification and characterisation of tomato torrado virus,a new plant picorna-like virus from tomato

Summary. A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of ‘torrado disease’; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.     

Functional analysis of the grapevine virus A genome

Grapevine virus A (GVA) carries five open reading frames (ORFs). Only the coat protein ORF has been experimentally identified as such; the roles of some of the other ORFs have been deduced by sequence homology to known genes (Minafra et al., 1997). The construction of a full-length, infectious clone of GVA has been previously reported. In an attempt to experimentally define the role of the various genes of GVA, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. Mutations in ORF 1 abolished RNA replication. Mutations in ORF 2 did not affect any of the aforementioned parameters. Mutations in ORFs 3 and 4 restricted viral movement. Mutations in ORF 5 rendered the virus asymptomatic, and partially restricted its movement.     

Genetic diversity and silencing suppression activity of the p22 protein of <Emphasis Type="Italic">Tomato chlorosis virus</Emphasis> isolates from tomato and sweet pepper

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3′-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a “strong” silencing suppressor is a conserved feature of ToCV isolates.     

Heterologous minor coat proteins of Citrus tristeza virus strains affect encapsidation, but the coexpression of HSP70h and p61 restores encapsidation to wild-type levels

The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5′ 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this study, we found encapsidation of CTV CPm in the absence of other assembly-related proteins is highly specific in contrast to most plant viruses that allow virion assembly by a range of heterologous coat proteins. Heterologous CPms with 95-96% amino acid identity from related strains in CTV-CPm, a replicon with CPm as the only assembly-related ORF, either failed to initiate encapsidation or reduced encapsidation substantially. Substitution of subsets of amino acids revealed that the amino acids that differ between positions 121 and 180 of the VT strain, and 61 and 120 of the T3 strain were involved in specific encapsidation. We further mapped the specific encapsidation to a single amino acid: mutation of methionine₁₆₅ to threonine (VT type) or serine₁₀₅ to proline (T3 type) in CTV-CPm failed to form nucleocapsids. However, the heterologous CPm in combination with both HSP70h and p61 proteins, but not HSP70h or p61 alone, encapsidated at wild-type levels, suggesting that specific encapsidation by CPm was mitigated by the combination of HSP70h and p61. Thus, in addition to the previously described functions of HSP70h and p61 of greatly enhanced virion formation and restriction of CPm encapsidation to the 5′ 630 nts of the genomic RNA, these proteins facilitate encapsidation by heterologous CPms.     

Foveavirus,a new plant virus genus

Summary.  Foveavirus is a novel genus of plant viruses with helically constructed filamentous particles ca. 800 nm long, typified by apple stem pitting virus (ASPV). Virions do not contain lipids or carbohydrates, have a positive sense, single-stranded, polyadenylated RNA genome 8.4 to 9.3 kb in size, and a single type of coat protein with a size of 28 to 44 kDa. The genome of definitive viral species is made up of five ORFs encoding respectively, the replication-related proteins (ORF 1), the putative movement proteins (ORF 2 to 4, constituting the triple block gene), and the coat protein (ORF 5). Virions accumulate in the cytoplasm, where replication is likely to occur with a strategy comparable to that of potexviruses, based on direct expression of the 5'-proximal ORF, and expression of downstream ORFs through subgenomic RNAs. No vector is known. Virus transmission is by grafting, and dispersal is through infected propagating material. The genome structure and organization (i.e. number and order of genes) closely resembles that of the genera Potexvirus, Carlavirus and Allexvirus, but ORF 1 and the coat protein cistron (ASPV only) are significantly larger.     

Tomato marchitez virus, a new plant picorna-like virus from tomato related to tomato torrado virus

Summary A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as ‘marchitez disease’: severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898 nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24 kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed. Correspondence: Martin Verbeek, Plant Research International BV, P.O. Box 16, NL-6700 AA Wageningen, The Netherlands     

Genome characterization of a flexuous rod-shaped mycovirus, Botrytis virus X, reveals high amino acid identity to genes from plant ‘potex-like’ viruses

Summary. This study reports the molecular characterization of a flexuous rod-shaped mycovirus, Botrytis virus X (BVX), infecting the plant-pathogenic fungus, Botrytis cinerea. BVX contains a ssRNA genome of 6966 nucleotides, and a poly(A) tract at or very near the 3′ terminus. Computer analysis of the genomic cDNA sequence of BVX revealed five potential open reading frames (ORFs). ORF1 showed significant amino acid sequence identity to the replicase proteins of plant ‘potex-like’ viruses, including 73% identity to the RNA-dependent RNA polymerase (RdRp) region of the allexivirus, garlic virus A (GarV-A). The C-terminal region of ORF3 shared amino acid homology with plant ‘potex-like’ coat proteins. The remaining ORFs did not reveal significant homology with known protein sequences. BVX differs substantially from Botrytis virus F (BVF), another flexuous rod-shaped mycovirus characterized from the same B. Cinerea isolate. It is proposed that the mycovirus BVX belongs to a new, as yet unassigned genus in the plant ‘potex-like’ virus group, distinct from BVF.     

Genetic analysis of tomato golden mosaic virus: ORF AL2 is required for coat protein accumulation while ORF AL3 is necessary for efficient DNA replication

Tomato golden mosaic virus (TGMV) is a geminivirus whose genome is divided between two DNA components, designated A and B. The TGMV genome contains six open reading frames (ORFs) which can encode proteins of greater than 10 kDa. We have used a protoplast transfection system to determine the effects of viral proteins, as defined by these ORFs, on the accumulation of viral DNA in infected cells. The accumulation of cost protein was also examined in leaf discs. Our results indicate that mutations in ORFs AR1 and AL2 do not affect viral double-stranded DNA (dsDNA) levels, although AR1 and AL2 mutants accumulate only small amounts of single-stranded viral DNA (ssDNA). In contrast, a large reduction in both ss- and dsDNA levels is observed when a mutation is introduced into ORF AL3. Mutations within either of the two DNA B ORFs do not affect DNA replication. The AL3, BR1, and BL1 mutants are capable of synthesizing coat protein; however, coat protein is not detected in leaf discs inoculated with AR1 or AL2 mutants. Testable models are proposed to explain the influence of AL2 protein on coat protein accumulation and to account for the stimulation of viral DNA synthesis mediated by the AL3 gene product.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

Nucleotide Sequence and Phylogenetic Analysis of Cucurbit Yellow Stunting Disorder Virus RNA 2

The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA 2, a whitefly (Bemisia tabaci)-transmitted closterovirus with a bi-partite genome, is reported. CYSDV RNA 2 is 7,281 nucleotides long and contains the closterovirus hallmark gene array with a similar arrangement to the prototype member of the genus Crinivirus, Lettuce infectious yellows virus (LIYV). CYSDV RNA 2 contains open reading frames (ORFs) potentially encoding in a 5 to 3 direction for proteins of 5 kDa (ORF 1; hydrophobic protein), 62 kDa (ORF 2; heat shock protein 70 homolog, HSP70h), 59 kDa (ORF 3; protein of unknown function), 9 kDa (ORF 4; protein of unknown function), 28.5 kDa (ORF 5; coat protein, CP), 53 kDa (ORF 6; coat protein minor, CPm), and 26.5 kDa (ORF 7; protein of unknown function). Pairwise comparisons of CYSDV RNA 2-encoded proteins (HSP70h, p59 and CPm) among the closteroviruses showed that CYSDV is closely related to LIYV. Phylogenetic analysis based on the amino acid sequence of the HSP70h, indicated that CYSDV clusters with other members of the genus Crinivirus, and it is related to Little cherry virus-1 (LChV-1), but is distinct from the aphid- or mealybug-transmitted closteroviruses.     

Nucleotide sequence and genome organization of <Emphasis Type="Italic">Cucurbit yellow stunting disorder virus</Emphasis> RNA1

Summary. The complete nucleotide sequence of Cucurbit yellow stunting disorder virus (CYSDV) RNA1, a member of the Crinivirus genus in the Closteroviridae, was determined. CYSDV RNA1 is 9126 nucleotides long and contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyl transferase, helicase, and polymerase domains, a small 5kDa hydrophobic protein and two further downstream ORFs potentially encoding proteins respectively 25 and 22kDa in size. The genomic position and homology of the four domains comprising the replication module appear to be similar for all sequenced criniviruses but there is divergence in the downstream ORFs, in terms of number, size, position and sequence homology.Received January 24, 2003; accepted May 14, 2003 Published online July 17, 2003     

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.     

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     

Alfalfa mosaic virus temperature-sensitive mutants. I. Mutants defective in viral RNA and protein synthesis

The replication in cowpea protoplasts of temperature-sensitive (ts) mutants of alfalfa mosaic virus (AIMV) was studied at the permissive (25 degrees) and the restrictive (30 degrees) temperature. Using the Northern blot hybridization technique, it was shown that at the restrictive temperature two RNA 1 mutants, Bts 03 and Bts 04, and two RNA 2 mutants, Mts 03 and Mts 04, were all defective in the synthesis of viral minus-strand RNA, whereas the synthesis of the plus-strand genomic RNAs 1, 2, and 3 and the subgenomic coat protein messenger, RNA 4, was relatively unimpaired. In Bts 04 inoculated protoplasts the RNA 4 produced at 30 degrees was translated into coat protein and viral RNA was encapsidated to give infectious virus. RNA 4 in Bts 03 and Mts 04 infected protoplasts was not translated into coat protein at 30 and consequently there was no assembly of infectious virus. Protein synthesis by Mts 03 was not investigated. A1MV RNAs 1 and 2 encoded proteins are both involved in the synthesis of viral minus-strand RNA and the translation of RNA 4 and possibly other viral messengers. The results with Bts 03 and Bts 04 show that the two functions of the RNA 1 encoded protein can be mutated separately.