Nucleotide sequence of the genome of an Australian isolate of turnip yellow mosaic tymovirus

The nucleotide sequence of the Club Lake isolate of turnip yellow mosaic virus (TYMV-CL) genomic RNA has been determined. The genome is 6319 nucleotide residues in length and has three major open reading frames (ORFs), two of which overlap. The smallest ORF is proximal to the 3' terminus and encodes the virion protein gene, which has 98% sequence similarity with the virion protein gene reported for the type strain of TYMV. The largest ORF is from nucleotide residues 96 to 5630, and encodes a protein some parts of which show sequence similarities to the possible RNA replicases and nucleotide binding proteins of other viruses. The third ORF is from nucleotide residues 89 to 1975 and overlaps the 5' end of the largest ORF in a manner similar to that found in several animal viral genomes. The function of the protein encoded by this ORF is unknown. The genomes of tymoviruses have, characteristically, an unusually large cytosine content and small guanosine content. This compositional bias is mirrored in the codon and dinucleotide frequencies of the TYMV-CL genome, but is only partially reflected in the amino acid sequences encoded by the genome.     

Complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV). Brief report

The complete nucleotide sequence of the genomic RNA of a French isolate of Pepino mosaic virus (PepMV) was determined. With a length of 6,425 nucleotides excluding the poly(A) tail at the 3' terminus, the PepMV genome contains five major open reading frames encoding a 164 kDa replicase, triple gene block proteins of 26 kDa, 14 kDa and 9 kDa, and a 25 kDa coat protein. This genome organization, as well as characteristic consensus motifs and high degrees of similarity of PepMV proteins with those of other potexviruses confirmed that PepMV belongs to the Potexvirus genus. Phylogenetic analyses carried out on replicase, triple gene block protein 1 and coat protein amino acid sequences revealed that PepMV is closely related to species Narcissus mosaic virus (NMV), Scallion virus X (SVX), Cymbidium mosaic virus (CymMV), and Potato aucuba mosaic virus (PAMV).     

The coat protein genes of squash mosaic virus: cloning,sequence analysis,and expression in tobacco protoplasts

Summary Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3 end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.     

Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3

Summary.  The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic α-helical structure preceding the conserved motif of the ‘30K superfamily’ proposed by Mushegian and Koonin [26] for MP’s. Within this ‘30K’ motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP’s of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses. Received June 4, 1996 September 25, 1996     

Nucleotide sequence of the ononis yellow mosaic tymovirus genome

The nucleotide sequence of the genome of ononis yellow mosaic tymovirus (OYMV) has been determined. The genome is single-stranded RNA, 6211 nucleotides long, and has three main open reading frames (ORFs), two of them overlapping. The largest ORF (nucleotides 179-5509) encodes a polyprotein of 1776 amino acid residues that has sequence similarities with polymerases of other viruses with RNA genomes. The smaller overlapping ORF (nucleotides 172-1965) encodes a protein of 597 amino acids of unknown function. The third ORF located at the 3' end of the genome (nucleotides 5487-6065) is the virion protein gene, and it overlaps by 20 nucleotides the 3' terminus of the largest ORF. The organization of the OYMV genome, its sequence, and the sequences of the protein it encodes are clearly similar to those of two other tymoviruses, turnip yellow mosaic virus and eggplant mosaic virus. The 5' terminal noncoding region of the OYMV genome is much longer than the same region of other tymoviral genomes and includes a direct duplication of a sequence of 21-23 nucleotides.     

The complete nucleotide sequenceof prune dwarf ilarvirus RNA-1

Summary.  The sequence of prune dwarf ilarvirus (PDV) RNA-1 has been determined; it consists of 3 374 nucleotides and contains a single open reading frame of 3 168 nucleotides. The putative translation product is 1 055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to the corresponding RNA-1 and ORF-1 of apple mosaic ilarvirus and alfalfa mosaic alfamovirus than to spinach latent mosaic ilarvirus or citrus leaf rugose ilarvirus. These findings are consistent with the inclusion of alfalfa mosaic virus in the ilarvirus genus. The reported sequence of PDV RNA-1 and its single ORF conform to the genomic organization typical of the Bromoviridae family. Received September 4, 1997 Accepted March 27, 1997     

Nucleotide sequence andE. coli expression of the coat protein gene of the yellowing strain of soybean dwarf luteovirus

Summary The nucleotide sequence of the coat protein gene of the yellowing (Y) strain of soybean dwarf virus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated M_r of 22 200. The identity of the open reading frame (ORF) encoding the coat protein was confirmed by expression of anEscherichia coli -galactosidase fusion protein and detection by a dot blot immunoassay. Sequence comparisons of the deduced coat protein amino acids to several luteoviruses demonstrated that the SbDV-Y coat protein ORF shares greatest similarity with bean leaf roll virus (BLRV) (65%).     

Molecular characterization of two prunus necrotic ringspot virus isolates from Canada

We determined the entire RNA1, 2 and 3 sequences of two prunus necrotic ringspot virus (PNRSV) isolates, Chr3 from cherry and Pch12 from peach, obtained from an orchard in the Niagara Fruit Belt, Canada. The RNA1, 2 and 3 of the two isolates share nucleotide sequence identities of 98.6%, 98.4% and 94.5%, respectively. Their RNA1- and 2-encoded amino acid sequences are about 98% identical to the corresponding sequences of a cherry isolate, CH57, the only other PNRSV isolate with complete RNA1 and 2 sequences available. Phylogenetic analysis of the coat protein and movement protein encoded by RNA3 of Pch12 and Chr3 and published PNRSV isolates indicated that Chr3 belongs to the PV96 group and Pch12 belongs to the PV32 group.     

The complete genome sequence of a Brazilian isolate of yam mild mosaic virus

In this study, the complete genome of an isolate of yam mild mosaic virus (YMMV) from Brazil was sequenced, and the predicted amino acid sequence was analyzed. The YMMV RNA genome consists of 9538 nt without the poly(A) tail, encoding a putative typical potyvirus polyprotein of 3084 amino acids. Furthermore, the small overlapping ORF (PIPO) in the P3 gene was also deduced, and the cleavage sites of the polyprotein were predicted. Multiple alignment with other potyviruses showed a maximum nucleotide sequence identity of 64 % to wild tomato mosaic virus. A phylogenetic tree showed that YMMV clustered with Asian potyviruses that mainly infect solanaceous plants.     

The complete genome sequence of a new necrovirus isolated from <Emphasis Type="Italic">Olea europaea</Emphasis> L.

Summary. The complete nucleotide sequence of a virus isolated from Olea europaea L. (GP isolate), previously identified as an isolate of Tobacco necrosis virus D (TNV-D) based on its coat protein sequence, was determined. The viral RNA genome consists of 3683 nucleotides and contains five open reading frames. The putative RNA-dependent RNA polymerase shows 91.2% amino acid identity with that of an isolate of Olive latent virus 1 (OLV-1) and the coat protein reveals highest sequence identity with that of TNV-D. Based on the deduced genome organization and phylogenetic analysis of predicted functional translation products with that of other necroviruses, the GP isolate genome appears to represent an example of a new virus arisen by gene exchange and is proposed to be a new necrovirus, provisionally named Olive mild mosaic virus.     

Complete nucleotide sequence of the tick-borne, orthomyxo-like Dhori/Indian/1313/61 virus nucleoprotein gene

The complete nucleotide sequence of the fifth largest segment of single-stranded RNA of the tick-borne, orthomyxo-like Dhori/Ind/1313/61 virus was determined by using cloned cDNA derived from infected cell mRNA and dideoxynucleotide sequencing of viral RNA. The fifth RNA contains 1479 nucleotides and can code for a protein of 477 amino acids with a molecular weight of 53,679 Da. The RNA 5 protein of the Dhori/Ind/1313/61 virus possesses five short regions (16-26 amino acids) which share a high degree (50-59%) of amino acid sequence homology with a computer-aligned consensus sequence of the influenza nucleoprotein gene family. These and other structural features of the RNA 5 protein suggest that RNA 5 of Dhori viruses codes for the nucleoprotein. The data also suggest that Dhori viruses are orthomyxoviruses, but that they are more distantly related to the influenza viruses than type A, B, and C viruses are to each other.     

RNA polymerase (L) gene and genome terminal sequences of ephemeroviruses bovine ephemeral fever virus and Adelaide River virus indicate a close relationship to vesiculoviruses

The sequence of the RNA genome of bovine ephemeral fever virus (BEFV) was determined from the start of the L (polymerase) gene to the end of the untranslated 5′ trailer sequence, completing the sequence of the 14 900 nucleotide (nt) genome. The 6470 nt L gene encodes a single long ORF of 2144 amino acids with a deduced molecular weight of 249 766 Da. The 70 nt BEFV 5′ trailer region displays partial terminal complementarity with the 3′ leader sequence and contains a 26 nt direct repeat of the U-rich domain of the 3′ leader region. The 47 nt 5′ trailer region of Adelaide River virus (ARV) displays terminal sequence similarity to the BEFV trailer and partial terminal complementarity with the ARV 3′ leader sequence, but does not contain the direct repeat sequence. The BEFV L protein contains all characteristic sequence motifs of amino acid blocks I–VI, conserved among RNA polymerase proteins of single-stranded (−) RNA viruses, separated by regions of lower homology. Phylogenetic analysis using the complete BEFV L protein sequence indicated a closer relationship to vesicular stomatitis virus than to rabies virus. Sequence comparison of two conserved central domains encompassing blocks II and III and block VI of the BEFV and ARV L proteins indicated they are closely related. An extended phylogenetic analysis using the block III sequence, confirmed the relationship of these ephemeroviruses to vesiculo- and lyssaviruses and to other single-stranded (−) RNA viruses.     

Analysis of the genome of satellite panicum mosaic virus

The relatedness of the genomes of satellite panicum mosaic virus (SPMV) and its helper virus, panicum mosaic virus (PMV), were investigated by nucleic acid hybridization. The results show that the satellite and helper virus RNAs have no appreciable homology or complementarity as assessed by hybridization with cDNA probes derived from the genomes of PMV and SPMV and with a probe complementary to the 3' terminus of SPMV RNA. The complete nucleotide sequence of SPMV RNA reveals that the genome is 826 nucleotides (nt) long. The ability to label SPMV RNA with polynucleotide kinase only after phosphatase treatment suggests that the 5' terminus is phosphorylated, but the extent of phosphorylation was not determined. The first open reading frame (ORF), encountered after an 88-nt 5'-untranslated region, encodes a 17,000 mol wt protein of a size and amino acid composition that are consistent with analysis of SPMV coat protein. An additional short ORF, located near the 3' end of the RNA, could encode a 6300 mol wt polypeptide. The minus strand also contains two ORFs that could potentially encode polypeptides of 7100 and 11,000 mol wt. No evidence is available to determine whether the second positive-strand ORF or the two minus-strand ORFs are expressed. The data presented here clearly show the SPMV RNA is distinct from the RNAs of other satellite viruses, in both size and nucleotide sequence. However, the 5'-untranslated portions of SPMV and satellite tobacco mosaic virus RNAs share some structural features that may be important in initiation of translation.     

Nucleotide sequence,serology and symptomatology suggest that vanilla necrosis potyvirus is a strain of watermelon mosaic virus II

Summary Vanilla necrosis potyvirus (VNV) is the cause of significant losses to the South Pacific islands vanilla crop. The gene for the coat protein of VNV has been cloned and sequenced. Comparison of this gene with other potyviral coat protein sequences revealed 97% nucleotide sequence homology (98% amino acid homology) to a US isolate of watermelon mosaic virus II (WMV-II), 93% nucleotide sequence homology (96% amino acid homology) to an Australian isolate of WMV-II and 81% nucleotide sequence homology (88% amino acid homology) to soybean mosaic virus-N (SMV-N). Serological analysis, by Western blot and ELISA, confirmed the close relationship between VNV and WMV-II. Furthermore, a limited host range determination found VNV and WMV-II able to infect the same series of test plants. However, symptoms differed significantly on three test species demonstrating that VNV and WMV-II are not identical in biological properties. We suggest that VNV be renamed WMV-II (Tonga).     

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression inEscherichia coli

Total RNA from infectedPhysalis floridana was isolated to generate complementary DNA corresponding to the coat protein (CP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1–3) for fusion protein expression inE. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97–99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRV_cub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.     

Genome sequence and phylogenetic analysis of a novel comovirus from tabasco pepper (Capsicum frutescens)

A virus isolate from tabasco pepper (Capsicum frutescens) has been reported as a strain of the comovirus Andean potato mottle virus (APMoV). Using the replicative intermediate viral dsRNA, the pepper virus strain was sequenced by Illumina MiSeq. The viral genome was de novo assembled resulting in two RNAs with lengths of 6028 and 3646 nt. Nucleotide sequence analysis indicated that they corresponded to the RNA-1 and RNA-2 of a novel comovirus which we tentatively named pepper mild mosaic virus (PepMMV). Predictions of the open reading frame (ORF) of RNA-1 resulted in a single ORF of 5871 nt with five cistrons typical of comoviruses, cofactor proteinase, helicase, viral protein genome-linked, 3C-like proteinase (Pro), and RNA-dependent RNA polymerase (RdRP). Similarly, sequence analysis of RNA-2 resulted in a single ORF of 3009 nt with two cistrons typical of comoviruses: movement protein and coat protein (large coat protein and small coat proteins). In pairwise amino acid sequence alignments using the Pro-Pol protein, PepMMV shared the closest identities with broad bean true mosaic virus and cowpea mosaic virus, 56% and 53.9% respectively. In contrast, in alignments of the amino acid sequence of the coat protein (small and large coat proteins) PepMMV shared the closest identities to APMoV and red clover mottle virus, 54% and 40.9% respectively. A phylogenetic tree constructed using the conserved domains for the Pro-Pol from all members of the family Secoviridae confirmed the comovirus nature of the virus. Phylogenetic and sequence analyses supports proposing PepMMV as a new species of the genus Comovirus.

     

The Genome-linked Protein (VPg) of Southern Bean Mosaic Virus is Encoded by the ORF2

The sequence of the 20 N-terminal amino acids of the viral protein (VPg) which is covalently attached to the genomic RNA of the bean strain of Southern bean mosaic virus (SBMV-B) has been determined. The obtained VPg sequence mapped to position 327 to 346 of the SBMV-B ORF2 product, downstream of the putative protease domain and in front of the RNA-dependent RNA polymerase. Thus indicating that the sobemovirus genomic arrangement is similar to that of subgroup II luteoviruses. Comparison with other viral sequences revealed a high similarity with the sequence of the ORF2-product of the cowpea strain of SBMV (SBMV-C). No significant similarities were detected with amino acid sequences derived of other sobemoviruses or non-related viruses. This revised version was published online in July 2006 with corrections to the Cover Date.     

Molecular characterization of a dsRNA totivirus infecting the sclerotial parasite Coniothyrium minitans

The complete nucleotide sequence, 4975 bp, of the double-stranded RNA (dsRNA) mycovirus infecting the sclerotial parasite Coniothyrium minitans (CmRV) was determined. Sequence analysis revealed the occurrence of two overlapping open reading frames (ORFs): the 5'-proximal large ORF (ORF1; nucleotide positions 62-2389) encodes a putative coat protein (CP) with a predicted molecular mass of 80344 Da, and the 3'-proximal ORF (ORF2, nucleotide positions 2386-4875) encodes a putative RNA dependent RNA Polymerase (RdRp) with a predicted molecular mass of 82551 Da. The tetranucleotide AUGA at nucleotide positions 2386-2389 includes the predicted start codon of ORF2, which overlaps with the stop codon for ORF1. Based on genome organization and sequence analysis encoded proteins, the virus infecting C. minitans strain Chy-1, designated C. minitans RNA virus (CmRV), belongs to the family Totiviridae. Pairwise sequence comparisons of the deduced amino acid sequences encoded by CmRV as well as phylogentic analysis indicated that it is more closely related to the totiviruses that infect filamentous fungi than to those infecting protozoa, yeast and smut fungi. The role of CmRV in the abnormal phenotype associated with a variant of C. minitans is discussed.