Nucleotide sequence of the 3′-terminal region of citrus tatter leaf virus RNA

The 3-terminal sequence of citrus tatter leaf virus lily isolate (CTLV-L) was determined from cloned cDNA. The sequence contains two open reading frames (ORFs). ORF1 encodes a protein that contains consensus sequences associated with the RNA-dependent RNA polymerase. ORF2, which is in a different reading frame within ORF1, can encode a 36 kD protein, putatively identified as a movement protein. CTLV-L coat protein (CP) was found to be located in the C-terminal region of the polyprotein encoded by ORF1. Evolutionary relationships and classification of capilloviruses is discussed.The nucleotide sequence data reported in this paper have been submitted to GenBank nucleotide sequence database and have been assigned the accession number D14455.     

Molecular cloning and sequence analysis of the 3′-terminal region of iris severe mosaic virus RNA

Summary Sequence analysis of iris severe mosaic potyvirus genomic RNA revealed an unusual E/G cleavage site between the deduced large nuclear-inclusion protein and coat protein sequences. The latter showed an N-terminus of only 15 amino acids. The 3 non-translated region of the viral RNA was 340 nucleotides long.     

Genetic diversity in the 3′-terminal region of papaya ringspot virus (PRSV-W) isolates from watermelon in Oklahoma

The 3′-terminal region (1191 nt) containing part of the NIb gene, complete coat protein (CP) and poly-A tail of 64 papaya ringspot virus (PRSV-W) isolates collected during 2008-2009 from watermelon in commercial fields of four different counties of Oklahoma were cloned and sequenced. Nucleotide and amino acid sequence identities ranged from 95.2-100% and 97.1-100%, respectively, among the Oklahoman PRSV-W isolates. Phylogenetic analysis showed that PRSW-W isolates clustered according to the locations where they were collected within Oklahoma, and each cluster contained two subgroups. All subgroups of Oklahoman PRSV-W isolates were on separate branches when compared to 35 known isolates originating from other parts of the world, including the one reported previously from the USA. This study helps in our understanding about the genetic diversity of PRSV-W isolates infecting cucurbits in Oklahoma.     

The nucleotide sequence of the coat protein gene and 3′ untranslated region of papaya ringspot virus type W (Aust)

Summary The nucleotide sequence of the 3 terminal region of the Australian isolate of papaya ringspot virus type W [PRSV-W (Aust)] was determined. An open reading frame (864 bp), encoding the putative coat protein gene, occurs upstream of the putative 3 untranslated region (206 bp) and poly(A) tail. A 23 amino acid sequence was obtained from N-terminal analysis of the coat protein from purified virions. This sequence has 100% homology with a region of the amino acid sequence inferred from the nucleic acid sequence of the coat protein gene. However, this region is 13 amino acids downstream from the N terminus predicted for two American isolates of PRSV. The coat protein gene of PRSV-W (Aust) was shown to have 96.8% and 96.4% nucleotide sequence similarity with American isolates of PRSV-W and PRSV-P, respectively.     

Cloning of the 3′-terminal region encoding movement and coat proteins of a Korean isolate of odontoglossum ringspot virus

Summary The 3-terminal 1855 nucleotides (nts) of a Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) were cloned and sequenced. The sequence contained two open reading frames, which encode the cell-to-cell movement protein (MP) and coat protein genes, and are 912 nts and 477 nts long, respectively. The MP gene contained a conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA locating between 1117 nts and 1292 nts from the 3-end. The 3 untranslated region (UTR) of the virus comprises 414 nts, includes nine pseudoknots and a tRNA-like structure domain containing aminoacyl acceptor arm and the anticodon hairpin loop coding for histidine.The nucleotide sequence data for ORSV-Cy reported in this paper will appear in the EMBL, GenBANK and DDBJ nucleotide databases under the accession numbers X78966 and X80053.     

Nucleotide sequence of the 3′-terminal region of the genome confirms that pea mosaic virus is a strain of bean yellow mosaic potyvirus

Summary The 1035 nucleotides at the 3end of the I strain of pea mosaic potyvirus (PMV-I) genomic RNA, encoding the coat protein, have been cloned and sequenced. A comparison of the derived coat protein sequence with those of the bean yellow mosaic virus (BYMV) strains, CS, S, D and GDD, indicates that PMV-I is a strain of BYMV. Sequence comparisons and hybridisation studies using the 3-noncoding region support this classification. The nucleotide and protein sequence data also suggest that PMV-I and BYMV-CS form one subset of BYMV strains while the other three strains form another.     

Nucleotide sequence of the 3′ terminal region of the genome of four Lettuce mosaic virus isolates from Greece and Yemen

Summary.  Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1 ¹ and mo1 ² and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR. Received July 1, 1998 Accepted March 25, 1999     

Nucleotide and deduced amino acid sequence of the 3′ end of the BYMV-MI genome

Summary We have cloned and sequenced cDNA transcribed from the 3 1239 nucleotides of the genomic RNA of a Western Australian isolate (MI) of bean yellow mosaic potyvirus (BYMV). This sequence contains 246 nucleotides of the NIb (replicase) gene and 819 nucleotides representing the entire coding region of the viral coat protein gene, followed by a 3 non-coding region of 174 nucleotides. The coding region of the coat protein gene is identical in length (273 amino acids) to that already reported for other isolates of this virus. The sequence identities obtained for BYMV-MI and published sequences of BYMV isolates range between 85% and 92% for the coding region of the coat protein and 90% to 98% for the 3 non-coding region. Likewise, the region of the NIb gene sequenced shows 99% and 97% sequence identity in the deduced amino acid and the nucleotide sequences, respectively.     

Nucleotide sequence comparison of the 3′ terminal region of the genome of pepper vein mottle virusisolates from Tunisia and Ivory Coast

Summary.  Three Tunisian PVMV isolates identified in pepper and tomato fields and one isolate from Ivory Coast were submitted to biological and molecular analysis. Phenotypically, Tunisian isolates induced mild symptoms while the Ivory Coast one is more aggressive on tobacco. As no PVMV sequence data are available, detailed sequence comparisons of coat protein gene (CP) were made. No nucleotide or amino acid changes in this region could be related to the pathogenicity of the isolates analysed. With the aim to increase our molecular understanding of the biological properties, we have sequenced the 3′-non translated region (3′NTR). Results suggest that this region of the RNA genome may be involved in the modulation of disease symptoms. Received February 28, 2000 Accepted August 3, 2000     

Rice tungro spherical virus: Nucleotide sequence of the 3′ genomic half and studies on the two small 3′ open reading frames

Rice tungro spherical virus (RTSV) consists of a single-stranded RNA genome of about 12 kilobases that contains one large open reading frame, ORF 1 and two small ORFs 2 and 3 at its 3 end (Shen et al., 1993, Virology 193:621–630); it was suggested that ORF 2 was expressed via a frameshift. To study the genomic information of RTSV and the variation between different RTSV isolates, the 3 half of a Philippine isolate and parts of a Thai and an Indian isolate were cloned and sequenced. Significant sequence differences were found in ORF 2 and in the 3 non-translated region. Additional stop codons have been revealed in the previously described ORF 2 in several independent clones from the three different virus isolates, the most conserved stop codon in the middle of ORF 2 being confirmed by direct RNA sequencing. These results suggest that ORF 2 could only express a peptide of about 5 kDa instead of 12 kDa as proposed earlier. Polyclonal antisera were raised against ORF 2 and 3 proteins as fusions with glutathione-S-transferase. Using these antisera we failed to detect any virus-specific peptides in extracts from infected rice plants and in virus preparations. The nucleotide sequence of the 3 end of our RTSV isolates contains several small ORFs and does not contain a repeat of 256 nucleotides found in the published sequence. These results indicate that RTSV could contain an unusually long 3 non-coding region of 1240 nucleotides in length.     

The nucleotide sequence of the 5′ non-coding and capsid coding genome regions of two bovine enterovirus strains

Summary The sequence of cDNA clones representing the 5 non-coding regions (NCR) and capsid regions of two bovine enteroviruses (strains PS-87 and RM-2; serotype two viruses) have been determined and compared with that obtained from a serotype one strain (VG-5-27). All three strains showed a longer 5 NCR compared to human enteroviruses and rhinoviruses due in part to a hundred residue insertion approximately at a hundred residues in from the 5 end. However, another domain occurring at nucleotide 187–222 in poliovirus is absent in each bovine enterovirus. Comparisons of the predicted structural protein amino acid sequences indicate that PS-87 shares most sequence identity with RM-2 and then with VG-5-27 in that order. The VP1 protein of PS-87 and RM-2 are shorter than the equivalent VP1 of VG-5-27 due in part to a truncation at their C-terminii. VP3 is only slightly smaller than VP2 in each virus.The nucleotide sequences have been submitted to the EMBL database under the accession numbers X79368 and X79369 for PS-87 and RM-2 respectively     

Comparative sequence analysis of the 5′ noncoding region of classical swine fever virus strains from Europe,Asia, and America

Summary Polymerase chain reaction was utilized to determine the sequence of a 280 base pair fragment from cDNAs of the 5 noncoding region of 29 isolates of classical swine fever virus. Phylogenetic analysis of the sequences revealed low level genomic variation that correlated with the geographic origins of the isolates.     

Nucleotide sequence of the 3′ terminal region of the RNA of two filamentous grapevine viruses

Summary The 3 terminal region of grapevine virus A (GVA) and grapevine virus B (GVB), encompassing 1883 and 2136 nucleotides, respectively, was sequenced by the deoxynucleotide chain termination method. Three putative open reading frames (ORF) were identified in both genomic viral RNAs, denoted 1 to 3 in the 5 to 3 direction. ORF 1 encoded a polypeptide with estimated M_r of 31 kDa (GVA) and 36.5 kDa (GVB), possessing the G/D motif of the 30 K superfamily movement proteins, and showing good alignments with putative movement proteins of trichoviruses and capilloviruses. ORF 2 was identified as the coat protein (CP) cistron, coding for polypeptides with an estimated M_r of 21.5 kDa (GVA) and 21.6 kDa (GVB). These CPs showed substantial sequence homology with one another and with CPs of tricho- and capilloviruses, but not of closteroviruses. ORF 3 potentially coded for two small polypeptides with estimated M_r of 10 kDa (GVA) and 14 kDa (GVB). The ORF 3 product of GVB (14 K), but not that of GVA, shared some homology with the 3 terminal polypeptides of different plant viruses that exhibit the zinc finger domain of proteins with nucleic acid-binding properties. GVA and GVB have many properties in common with trichoviruses but possess an extra open reading frame (ORF 3). Whether this finding may have a bearing on the classification of these viruses is unclear. However, until the taxonomic significance of this difference in genome structure is established, it seems plausible to include GVA and GVB as tentative species in theTrichovirus genus.     

Cloning of the genome of cricket paralysis virus: sequence of the 3′ end

DNA complementary to the single-stranded RNA genome of the insect picornavirus, cricket paralysis virus (CrPV), was cloned into the plasmid pBR322 by a hybrid RNA-cDNA cloning strategy. Positive CrPV-specific clones were selected by colony hybridization and characterised by restriction enzyme mapping. Overlapping clones spanning 7.5 kb of the estimated 8.5 kb genome were obtained, the largest being 7.0 kb. Comparison of the restriction enzyme map with those of mammalian picornaviruses revealed no conserved pattern of cleavage sites. The CrPV-cDNA was sequenced using the M13-dideoxy chain-terminating method although the chemical method of Maxam and Gilbert was employed to complete gaps in the sequence. The sequence of the 3′-terminal 1600 nucleotides is presented and is compared with those of mammalian picornaviruses. Computer comparisons of the CrPV sequence and those of mammalian picornaviruses revealed no significant homology between either the nucleotide or the predicted amino acid sequence of this region.     

On the variability of the 3′ terminal sequence of the turnip mosaic virus genome

Summary The sequence of the 3-terminal 1223 nucleotides (nts) of a Japanese isolate of turnip mosaic virus (TuMV-Jap) RNA has been determined. The sequence reveals a single open reading frame (ORF) which terminates at a position 212 nts upstream of the 3 poly(A)-tract. Determination of the N-terminal amino acids of TuMV-Jap coat protein (CP) mapped the CP cistron within this ORF and revealed a Glu-Ala dipeptide sequence as the putative cleavage site by which the CP is released from the viral polyprotein. The predicted amino acid sequence of the TuMV-Jap CP shows 97.2% identity with that of a Canadian isolate of TuMV (TuMV-Can) and 99% with a second, Chinese, isolate (TuMV-Chi). However, the 3-terminal non-translated region (NTR) of TuMV-Jap RNA is significantly shorter (212 nts) than the 3-NTR of TuMV-Can RNA (668 nts), but of equal length as the 3-NTR of the TuMV-Chi isolate which also measures 212 nts. The 3-NTRs of both the TuMV-Jap and TuMV-Chi RNAs show homology with the first 201 nucleotides of the TuMV-Can RNA 3-NTR. A search in the EMBL nucleotide sequence database revealed that the 467 nt-long unique extension of the 3-NTR of TuMV-Can RNA has 89.8% homology to a part of the chloroplast ribosomal protein 12 gene (rsp 12-gene). Irrespective of the origin of this extra sequence in the reported TuMV-Can sequence, which may have been introduced by a genuine RNA recombination event, it is concluded that the standard TuMV genome has a CP gene of 864 nts and an conserved 3-NTR of approximately 212 nucleotides in length.     

A new virus related to <Emphasis Type="Italic">Satsuma dwarf virus</Emphasis>: the nucleotide sequence of the 3′-terminal regions of Hyuganatsu virus RNAs 1 and 2

Summary. The sequences of the 3-terminal 1306 and 2160 nucleotides of RNAs 1 and 2 of a virus serologically related to Satsuma dwarf virus (SDV) from Hyuganatsu (Citrus tamurana Hort. ex Tan.) were determined, respectively. We found that the partial RNA-dependent RNA polymerase region in RNA1 and the coat proteins (CPs) region in RNA2 of the virus tentatively named Hyuganatsu virus (HV) have 78.3–84.0% and 76.9–80.7% amino acid sequence identities to those of known SDV-related viruses (SDV-RVs), i.e., SDV, Citrus mosaic virus, and Navel orange infectious mottling virus. Sequence analyses show that HV is classifiable as a new SDV-RV species.     

Comparison of the nucleotide sequences of the 3′-terminal regions of one aphid and two non-aphid transmissible isolates of potato A potyvirus

Summary The sequences of the 3-terminal 1145 nucleotides of two non-aphid transmissible (NAT) isolates (Ali and Juliniere) and one aphid transmissible (AT) isolate (Rouge) of potato virus A (PVA) RNA were determined. Those sequences contained the complete coding region of the coat protein (CP) followed by a 3-nontranslated region (3-NTR) of 225 (Ali and Juliniere) and 227 (Rouge) nucleotides. The obtained sequences were compared to those of the 3'-regions of four published PVA isolates and a virus described as tamarillo mosaic virus (TamMV) which on the basis of sequence data is a strain of PVA. The analysis of the 3-terminal region of PVA isolates indicated that the CP N-terminal variable domain (32 residues) divides PVA isolates into two subgroups, where only tripeptide DAG correlates with aphid transmissibility. In addition to DAG/DAS sequence we found four other amino acids at the N-terminus of PVA CP, which are conserved in two subgroups. The central region (core part and C-termini) of CP is highly conserved among all PVA isolates (96.6 to 99.6%). 3-NTR, separates PVA isolates into two subgroups on the basis of its length and homology.     

Zucchini tigré mosaic virus is a distinct potyvirus in the papaya ringspot virus cluster: molecular and biological insights

In recent years, three new potyviruses have been described in the papaya ringspot virus (PRSV) cluster. In addition, two types of PRSV are recognized, type W, infecting cucurbit plants, and type P, infecting papaya and also cucurbits. A third type, PRSV-T, was also partially described in Guadeloupe. Complete genome sequencing of four PRSV-T isolates showed that this virus is a related virus that is distinct from PRSV, and the name zucchini tigré mosaic virus (ZTMV) is proposed, in reference to the typical symptoms observed in zucchini squash. Eleven other viral isolates from different geographic origins were confirmed as ZTMV isolates using the complete sequence of the cylindrical inclusion (CI) coding region, whereas pairwise sequence similarities in the coat protein (CP) coding region did not unambiguously distinguish ZTMV isolates from PRSV isolates. The use of the CI coding region for species demarcation appears more suitable than the CP coding region for closely related viruses. Principal coordinates analysis based on the biological behavior of the viral isolates studied clustered PRSV-P, PRSV-W and ZTMV isolates into three different groups. Therefore, ZTMV is different from PRSV in its molecular and biological properties.     

Nucleotide sequence of the 3′ terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins

Summary The 3 terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3 terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110–111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.