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.     

Nucleotide sequence of the putative replicase gene of the sour cherry strain of plum pox potyvirus

Summary.  The complete nucleotide sequence of the NIb coding region of the sour cherry strain of plum pox potyvirus (PPV-SoC) has been determined. It consists of 1554 nucleotides and encodes a putative replicase protein of 518 amino acids. Sequence identity scores between NIb of PPV-SoC and other isolates of PPV are significantly low (c. 78%). Many of the nucleotide substitutions, however, are silent. PPV-SoC differs from isolates of PPV-D, PPV-M and PPV-El Amar at multiple amino acid positions that are conserved between the other isolates. The NIb sequence extends the PPV-SoC sequence presently available to 2781 nt from the 3′ end (∼ 28% of the genome). Received March 16, 1998 Accepted{June 10, 1998     

Composition of the sequence downstream of the dengue virus 5' cyclization sequence (dCS) affects viral RNA replication

RNA replication of dengue virus (DENV) requires an RNA-RNA mediated circularization of the viral genome, which includes at least three sets of complementary RNA sequences on both ends of the genome. The 5′ and the 3′ untranslated regions form several additional RNA elements that are involved in regulation of translation and required for RNA replication. Communication between the genomic termini results in a structural reorganization of the RNA elements, forming a functional RNA panhandle structure. Here we report that the sequence composition downstream of the 5′ CS element in the capsid gene, designated as downstream CS (dCS) sequence - but not the capsid protein - also influences the ability of the viral genome to circularize and hence replicate by modulating the topology of the 5′ end. These results provide insights for the design of reporter sub-genomic and genomic mosquito-borne flavivirus constructs and contribute to the understanding of viral RNA replication.     

Determination of polyprotein processing sites by amino terminal sequencing of nonstructural proteins encoded by plum pox potyvirus

Nonstructural proteins of plum pox potyvirus were partially purified following a procedure described for the isolation of tobacco etch virus nuclear inclusion proteins. Plum pox virus proteins with electrophoretic mobilities corresponding to 49, 59 and 68 kDa reacted with antibodies against the 49 kDa and 54 kDa components of the nuclear inclusions and the 70 kDa component of the cylindrical inclusions of tobacco etch virus, respectively. Further purification by size exclusion high performance liquid chromatography or SDS-polyacrylamide gel electrophoresis, and amino terminal amino acid sequencing permitted the location in the plum pox virus polyprotein of the cleavage sites from which the 49 kDa (NIa-type, protease), 59 kDa (NIb-type, putative RNA replicase), and 68 kDa (CI-type) proteins originate. A 110 kDa protein which copurified with the plum pox virus inclusion proteins reacted with both anti-NIa and anti-NIb sera and had the same amino terminus as the plum pox virus 49 kDa protein, indicating that it is a non-processed 49-59 kDa polypeptide.     

Proteolytic activity of the plum pox potyvirus NIa-like protein in Escherichia coli

The nucleotide sequence of the small nuclear inclusion protein (NIa)-like cistron of plum pox potyvirus (PPV) has been determined. Viral proteolytic activity was expressed in Escherichia coli cells harboring plasmids with a PPV cDNA insert approximately 7000 nt long. Free PPV capsid protein was detected in these cells, but it was not produced when a mutation was introduced in the PPV cDNA insert which induced a Gln to Pro substitution at the large nuclear inclusion protein (NIb)-capsid protein junction. By mutational analysis, the NIa-like protein was determined to be responsible for the proteolytic activity. A Gln to Ser substitution at the presumed NIa-NIb junction, which inhibited proteolytic processing at the carboxyl end of the protease, had no effect on proteolytic cleavage at the NIb-capsid protein junction. In contrast with the high efficiency of proteolytic processing at the NIb-capsid protein cleavage site, processing at the ends of the PPV protease was not complete, suggesting that the PPV polyprotein, like that of other potyviruses, contains cleavage sites with different properties.     

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication

We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.     

Replacement of the coat protein gene of plum pox potyvirus with that of zucchini yellow mosaic potyvirus: characterization of the hybrid potyvirus

Infectious hybrid virus was generated by replacing part of the coat protein gene of plum pox potyvirus with that of the zucchini yellow mosaic potyvirus. This viable hybrid contains 84.5% of zucchini yellow mosaic potyvirus coat protein gene while the rest of the sequence was derived from plum pox potyvirus. Changing the coat protein gene between these two viruses had no effect on the experimental host range. Pathogenicity, stability and replication capacity of the hybrid virus were nearly identical to the parent viruses.     

Nucleotide sequence at the 3' terminus of pepper mottle virus genomic RNA: evidence for an alternative mode of potyvirus capsid protein gene organization

The sequence of the 3'-terminal 1481 nucleotides of the pepper mottle virus (PeMV) genome has been determined. The sequence was determined by dideoxy nucleotide sequencing of complementary DNA which had been inserted into M13 bacteriophage cloning vectors and was confirmed by sequencing selected regions of PeMV RNA. A discrete open reading frame of 993 nucleotides, ending 333 nucleotides from the 3'-terminal polyadenylate tract, was identified that potentially encoded a 37,669-MW protein. The amino acids predicted at positions 64 through 84 of this putative polypeptide were identical to the amino-terminal 21 amino acids of the PeMV capsid protein ascertained by chemical sequencing. These combined nucleotide and amino acid sequence data suggest that the PeMV capsid protein is encoded by the 3'-most cistron on the genomic RNA and that it may be expressed as a precursor that is proteolytically processed to produce the mature capsid protein.     

Nucleotide sequence of the 3'-terminal region of potato virus A RNA.

The sequence of the 3'-terminal region of the genome of the potato virus A (PVA) was obtained from two independent cDNA clones. This sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon TAA and followed by untranslated region of 205 nucleotides. Since the N-terminal amino acid of the coat protein of PVA was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus sequences and by the analogy to other potyviruses. The resulting coat protein is 269 amino acids long and has a calculated MW of 30257. Two independently sequenced cDNA clones show sequence heterogeneity at four nucleotide positions: C422/A422, G432/A432, G446/A446 and T706/C706. Three first nucleotide differences are located at the PVA coat protein N-terminal region and led to the change of the amino acid. The coat protein of PVA displayed significant (73-78%) sequence homology to the coat proteins of six other potyviruses: papaya ringspot virus (PRV), pepper mottle virus (PeMV), plum pox virus (PPV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and tobacco vein mottling virus (TVMV). Even higher sequence homology (82%) was detected with a coat protein of a seventh potyvirus, tobacco etch virus (TEV). Major differences among the coat protein of PVA and of other potyviruses are located at the N-terminal region of the coat protein.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

Summary The sequence of the 3 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M_r of 30 210. The 3 non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3 non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.     

Nucleotide sequence analysis of the apolipoprotein B 3' VNTR

Variable number of tandem repeat (VNTR) loci typically exhibithigh rates of germline mutations that alter allele length andthus are ideal models for examining processes governing repeatsequence instability. We have characterized by nucleotide sequencingthe internal structure of the apolipoprotein B (Apo B) 3' VNTRin a sample of same- and different-sized alleles previouslyassociated with flanking marker haplotypes. Significant linkagedisequilibrium between flanking polymorphisms and minisatellitealleles excludes unequal recombinatlon as the predominant mechanismof mutation at the Apo B VNTR and is consistent with intra-allelicmutational processes such as replication slippage and/or unequalsister chromatid exchange. Diversity among different lengthalleles was distinctly polar and was usually atiributable tochanges in copy number at one particular repeat sequence. Analysisof predicted secondary structures for the dimeric repeats demonstrateda relationship between variability and the potential to formself-complementary intermediates. Preferential instability ofthe variable repeat: (i) was a function of its location withinthe tandem array; (ii) was not solely dependent on copy number;and (iii) may be related to the base composition of the VNTRand the degree of self-complementarity between the dimeric repeatsequences. The data suggest that polarized variability may beindependent of the mutational process(es) generating lengthvariation at minisatellite loci and suggest a possible alternativemechanism of mutation that involves the formation of secondarystructures.     

Nucleotide sequence analysis of the 3′ terminal region of a wasabi strain of crucifer tobamovirus genomic RNA: subgrouping of crucifer tobamoviruses

Summary.  The 3′ terminal 2378 nucleotides of a wasabi strain of crucifer tobamovirus (CTMV-W) infectious to crucifer plants was determined. This includes the 3′ non-coding region of 235 nucleotides, coat protein(CP) gene (468 nucleotides), movement protein (MP) gene (798 nucleotides) and C-terminal partial readthrough portion of 180 K protein gene (940 nucleotides). Comparison of the sequence with homologous regions of thirteen other tobamovirus genomes showed that it had much higher identity to those of four other crucifer tobamoviruses, 85.2% to cr-TMV and turnip vein-clearing virus (TVCV), 87.4% to oilseed rape mosaic virus (ORMV) and 87.1% to TMV-Cg, than to those of other tobamoviruses. Thus CTMV-W was most similar to ORMV and TMV-Cg in sequence, but only marginally so, whereas the location and size of its MP gene was the same as cr-TMV amd TVCV. These results, together with other analyses, show that CTMV-W is a new crucifer tobamovirus, that the five crucifer tobamoviruses can be classified into two subgroups based on MP gene organization, and that the rate of sequence change is not the same in all lineages. Received November 24, 1998 Accepted April 29, 1998     

Nucleotide sequence of RNA 3 of the British type isolate (Blencowe strain) of tomato aspermy virus

The complete sequence of RNA 3 of the Blencowe, British (B) isolate of tomato aspermy virus (TAV) is presented. The RNA 3 of TAV-B is dicistronic, encoding the putative movement protein 3a and the capsid protein.     

Nucleotide sequence of the coat protein cistron and the 3' noncoding region of cucumber green mottle mosaic virus (watermelon strain) RNA

Double-stranded cDNA copies of cucumber green mottle mosaic virus (watermelon strain, CGMMV-W) RNA polyadenylated in vitro were cloned into the pBR322 at the PstI site. The sequence of 1071 nucleotides from the Tend of the genomic RNA was determined using two recombinant plasmids and the genomic RNA. The coat protein cistron was located in residues 176-661 from the 3' end. The coat protein was composed of 160 amino acid residues with the molecular weight of 17,261. The 3' noncoding region of the CGMMVW genome was 175 nucleotides long and highly homologous to that of the common strain of TMV. The assembly origin of reconstitution is positioned within the coat protein cistron as predicted previously. In the 5' flanking region of the coat protein cistron a long open frame, probably of 30K protein, was found. The predicted 30K and the coat protein cistron would overlap each other as is the case of the cowpea strain of TMV.     

Proteolytic processing of the plum pox potyvirus polyprotein by the NIa protease at a novel cleavage site.

The expression of potyvirus genomic RNA takes place through translation of its unique long and functional open reading frame into a large polyprotein that undergoes extensive proteolytic processing. Most of the cleavages are performed by the virus-encoded NIa protease, which cuts the polyprotein at defined sites that are characterized by conserved heptapeptide sequences. We have demonstrated in vitro cleavage activity by the plum pox potyvirus (PPV) NIa protease at a novel site, previously identified by sequence analysis, thus allowing a further refinement of the potyviral genetic map. This novel site is located 52 amino acids upstream from the site corresponding to the N-terminus of the CI protein (the NIa cleavage site previously considered the closest to the beginning of the polyprotein). The specificity of the processing was demonstrated by its abolishment when the Gln at position -1 of the cleavage site was changed to His. This novel NIa cleavage site was only partially processed, a characteristic that was not altered when its heptapeptide sequence was modified to become that of the efficiently cleaved NIb-CP junction. On the contrary, substitutions at the nonconserved position +3 had notable effects, positive or negative, on the efficiency of processing. These results show the relevance of sequence and/or conformational context outside the conserved heptapeptide for modulating the cleavage reaction catalyzed by the NIa protease.     

Expression of the plum pox virus coat protein region in Escherichia coli

A cDNA complementary to the 3 end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.     

Nucleotide sequence of pea seed-borne mosaic potyvirus coat protein gene

cDNA of pea seed-borne mosaic potyvirus (PSbMV) RNA was synthesized and cloned in E. coli. Four overlapping clones that cover the complete PSbMV genome, except the extreme 5 terminus, were identified by restriction enzyme mapping, hybridization analysis, and partial sequencing. Overlapping cDNA clones covering 1386 nucleotides of the 3 terminus were sequenced. The nucleotide sequence contains one open reading frame (ORF), followed by an untranslated region of 163 nucleotides and a poly(A)-tract. The deduced amino acid sequence was found to include the C-terminus of the predicted RNA-dependent RNA polymerase and the coat protein. A glutamine-alanine dipeptide was identified as a putative 49-kD proteinase cleavage site at the N-terminus of the coat protein.