Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs

Summary The complete sequences of genomic and defective interfering (DI) RNAs of carnation Italian ringspot tombusvirus (CIRV) were determined. The genome (4760 nt) has an organization identical to that reported for other tombusviruses except that the pre-readthrough domain of the viral replicase encoded by the 5-proximal open reading frame (ORF) is larger. In particular, the N-terminal region of this protein differs from the corresponding region of the other members of the genusTombusvirus andCarmovirus. Two DI RNAs were found in infected tissues whose sequences were completely derived from genomic RNA. The smaller molecule (474 nt) is contained completely within the larger molecule (656 nt), which suggests that it is derived from the larger one.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Database under the accession number X85215.     

Molecular cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA

Summary.  The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3 803 nucleotides and has five open reading frames (ORFs). The 5′ ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria. Accepted August 25, 1997 Received June 18, 1997     

The complete sequence of the genomic RNAs of spinach latent virus

Summary.  We describe the sequence for the complete genome of spinach latent virus (SpLV). Comparisons of this genome with that of the only other complete genome described for a species within the genus Ilarvirus (citrus leaf rugose virus – CiLRV) indicate that while there are marked differences between the RNA 3 of the two viruses, their respective RNAs 1 and 2 share many similarities. However, the putative 2a protein of SpLV contains a C₂H₂ type “zinc finger”-like motif located towards the carboxy terminal of the protein which is absent in CiLRV and has not been reported for other members of the family Bromoviridae. A second open reading frame (2b), located at a similar position to that described for the cucumoviruses, occurs in the RNA 2 of both SpLV and CiLRV. The putative coat protein of SpLV is similar to that of citrus variegation virus (CVV) and asparagus virus 2 (AV-2), both members of subgroup 2 of the ilarviruses. We have subsequently demonstrated a serological relationship between SpLV and other viruses in subgroup 2 and suggest that SpLV should be included in this subgroup rather than remain in a separate group (subgroup 6). However, while the putative movement protein of SpLV is remarkably similar to that of AV-2, it shows little relationship with the corresponding protein of CVV and the lack of similarity suggests that a recombination event may have occurred in the past. The relationship between the genera Alfamovirus and Ilarvirus is discussed in the light of the data for the genome of SpLV and recently published information for other members of the genus Ilarvirus. Received October 21, 1996 Accepted December 3, 1996     

The complete nucleotide sequence of plum pox potyvirus RNA

The complete nucleotide sequence of the plum pox virus (PPV) RNA genome has been determined. The RNA sequence is 9786 nucleotides in length, excluding the 3'-terminal poly(A) tail. An AUG triplet at position 147-149 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3140 amino acid residues. The nucleotide sequence of the non-coding regions and the predicted amino acid sequence of the polyprotein of PPV were compared with those previously reported for two other potyviruses (tobacco etch virus, TEV, and tobacco vein mottling virus, TVMV), with nucleotide and amino acid sequences of other viruses, as well as with sequences from data banks. The potyvirus genomic expression is discussed in relation to the homologies observed, in particular the predicted protease recognition sequences in related viruses.     

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus

The complete nucleotide sequence of the genomic RNA of the tobamovirus tobacco mild green mosaic virus (TM-GMV) was determined. It shows 64.4% sequence homology with the genomic RNA of tobacco mosaic virus (TMV) and 66.0% with that of tomato mosaic virus (ToMV). Its genomic organization is similar to that of TMV and ToMV. The 5' proximal open reading frame (ORF) encodes a 126K polypeptide and a 183K readthrough product in which nucleotide-binding and polymerase-sequence motifs are found. The third ORF encodes a 28.5K protein homologous to TMV and ToMV movement proteins. A conserved core is found with four other tobamoviruses and two tobraviruses suggesting a common mechanism of cell-to-cell movement for tobamo- and tobraviruses. The fourth ORF encodes the 17.5K coat protein. Homology between the RNAs of TMGMV and its satellite virus STMV is limited to their 3' termini, and structural comparisons suggest that this region may determine the nature of the satellite/helper virus interaction.     

The complete nucleotide sequence of Pepper mottle virus-Florida RNA

Summary.  The Pepper mottle virus-Florida (PepMoV-FL) RNA genome was cloned and sequenced, and shown to consist of 9,717 nucleotides (nt) excluding the poly (A) tail. A single open reading frame was identified beginning at nucleotide position 169 encoding a polyprotein of 3068 amino acids. Phylogenetic sequence analysis revealed that of 44 full-length viral RNA genomes analyzed within the family Potyviridae, PepMoV-FL was most closely related to PepMoV-California (PepMoV-CA), Potato virus Y-H (PVY-H), PVY-N, PVY^o and Potato virus V-DV42 (PVV-DV42). Using the PepMoV-FL sequence as a basis for comparison, the overall nucleotide sequence identity was highest between PepMoV-FL and PepMoV-CA at 93%, while the relationship was more distant with PVV-DV42 at 64% and for the PVY strains at 61%. A unique direct repeat sequence of 76 nucleotides was identified in the PepMoV-FL 3′-untranslated region (UTR), and this repeat sequence was confirmed not to occur in the PepMoV-CA sequence. Since the Florida isolate was among the first of the PepMoV isolates described, extensive biological and serological data on this isolate are available, and it has now been cloned and sequenced, we recommend that PepMoV-FL be recognized as the PepMoV type strain. Received March 25, accepted July 16, 2002     

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     

Identification of sequence elements of tombusvirus-associated defective interfering RNAs required for symptom modulation

Summary. Defective interfering (DI) RNAs of tombusviruses are short, non-coding, symptom-modulating RNAs originating from the viral genome. The presence of homologous DI RNA in virus infection attenuates the otherwise lethal viral symptoms. Nicotiana benthamiana plants infected with tomato bushy stunt tombusvirus pepper isolate (TBSV-P) show severe symptoms, which culminate in the death of the plant. In contrast, plants co-inoculated with TBSV-P and TBSV-P-derived DI RNA display attenuated symptoms. However, co-inoculation of TBSV-P with heterologous DI RNA, originating from Carnation Italian ringspot tombusvirus results in development of apical necrotic symptoms. To localize the symptom-determining factors on DI RNA genome, chimeras of protective and non-protective DI RNAs have been constructed. All chimeras were biologically active and accumulated to a high level in the presence of helper virus. We identified a 5′ proximal sequence element of the DI RNA as the most important symptom determinant region. However, our results demonstrated that the symptom modulating ability of this region is also influenced by the sequence composition of whole DI RNAs.     

The complete nucleotide sequence of bean yellow mosaic potyvirus RNA

Summary The complete nucleotide sequence of an Australian strain of bean yellow mosaic virus (BYMV-S) has been determined from cloned viral cDNAs. The BYMV-S genome is 9 547 nucleotides in length excluding a poly(A) tail. Computer analysis of the sequence revealed a single long open reading frame (ORF) of 9 168 nucleotides, commencing at position 206 and terminating with UAG at position 9 374–6. The ORF potentially encodes a polyprotein of 3 056 amino acids with a deduced Mr of 347 409. The 5 and 3 untranslated regions are 205 and 174 nucleotides in length respectively. Alignment of the amino acid sequence of the BYMV-S polyprotein with those of other potyviruses identified nine putative proteolytic cleavage sites. The predicted consensus cleavage site of the BYMV NIa protease was found to differ from that described for other potyviruses. Processing of the BYMV polyprotein at the designated proteolytic cleavage sites would result in a typical potyviral genome arrangement. The amino acid sequences of the putative BYMV encoded proteins were compared to the homologous gene products of twelve individual potyviruses to identify overall and specific regions of amino acid sequence homology.The nucleotide sequence data reported in this paper has been submitted to GenBank nucleotide sequence database and has been assigned the accession number U47033.     

Molecular cloning and complete nucleotide sequence of genomic RNA of the AIK-C strain of attenuated measles virus

Twelve cDNA clones covering the entire genome of the AIK-C strain of a seed for live measles vaccine were obtained, and the nucleotide sequences were determined. The full viral genomic RNA consists of 15,894 nucleotides. Comparisons of the nucleotide sequence and the deduced amino acid sequence between the AIK-C and other Edmonston strains revealed the following changes: 56 nucleotide differences and one C residue insertion, 31 amino acid changes, and 19 silent mutations.     

The complete nucleotide sequence of RNA2 of blackcurrant reversion nepovirus

The complete nucleotide sequence of blackcurrant reversion nepovirus (BRV) RNA2 was determined from cDNA clones. RNA2 was 6400 nucleotides (nt) in length excluding the 3' poly(A)-tail. It contained a single open reading frame of 4878 nts encoding a polypeptide of 1626 amino acids with a calculated M(r) of 178? omitted?860. The genome organization of BRV RNA2 was similar to that of other nepoviruses, especially those with a large RNA2. The coat protein (CP) was located in the C-terminal region of the large polyprotein and contained amino acid motifs conserved among nepovirus CPs. Sequence comparisons revealed a proline (P) residue surrounded by hydrophobic amino acid residues located upstream of the CP. This P motif is conserved among the putative movement proteins of nepo-, como-, caulimo- and capilloviruses. An N-terminal domain of 350 amino acids of RNA2-encoded polyprotein shared 34 and 35% sequence identity with the N-terminal domains of tomato ringspot nepovirus RNA1- and RNA2-encoded polyproteins, respectively. Sequence identities between the N-terminal domains of BRV RNA2 and other nepoviral RNA2s were less than 20%; no common N-terminal motif was found.     

The 5' terminal nucleotide of RNA from vesicular stomatitis virus defective interfering particles.

The RNAs of three well-characterized and vastly dissimilar VSV defective interfering particles (DI_LT, DI_T, and DI₀₁₁) have the same 5′ terminus, pppA, as the infectious particle.     

The complete nucleotide sequence and genome organization of odontoglossum ringspot tobamovirus RNA

Summary The complete nucleotide sequence of the genomic RNA of odontoglossum ringspot tobamovirus (ORSV) was determined. The RNA genome of ORSV is 6618 nucleotides long and contains five open reading frames (ORFs 1 to 5) coding for proteins of Mr 126 K, 181 K, 34 K, 18 K and 52 K, respectively. This is the longest RNA of the known viruses of theTobamovirus genus. The sequences of the ORSV RNA encoded proteins exhibit high homology to the proteins of the members of theTobamovirus genus. The genomic organization and sequence analysis showed that ORSV is more closely related to tobacco mild green mosaic virus (TMGMV), pepper mild mottle virus (PMMV), tomato mosaic virus (ToMV) and TMV than to cucumber green mottle mosaic virus (CGMMV) and sunn-hemp mosaic virus (SHMV).The nucleotide sequence reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession number X82130.     

The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain

Summary The complete nucleotide sequence of the RNA genome of turnip mosaic virus Japanese strain (TuMV-J) has been determined from five overlapping cDNA clones and by direct sequencing of viral RNA. The RNA sequence was 9833 nucleotides in length, excluding a 3 terminal poly(A) tail. An AUG triplet at position 130–132 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3164 amino acid residues. Interestingly, a different amino acid sequence (continuous twenty amino acids) within the cytoplasmic inclusion protein between TuMV-J and Canadian strain of TuMV was observed, caused by an insertion and a deletion of nucleotides.DDBJ/EMBL/GenBank accession number D83184.     

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.