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.     

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 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     

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.     

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.     

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.     

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.     

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.     

Nucleotide sequence comparison of the 3′-terminal regions of severe,mild, and non-papaya infecting strains of papaya ringspot virus

Summary The 3-terminal 2,561 nucleotide residues of the severe HA strain of papaya ringspot virus (PRSV) was determined. Comparison with the published sequence of the mild strain PRSV HA 5-1 showed that they shared a 99.4% identity in their 3-terminal 2,235 residues. There were ten residues different at the NIb gene, resulting in five amino acid changes, and two residues different in the coat protein gene, resulting in two amino acid changes. The 3-untranslated regions were identical, but HA contained two more nucleotides (AG) at the 3 extreme. Comparison with the published non-papaya infecting type W strain PRSV-W revealed that they shared a 97.9% identity in their 3-terminal 2,235 residues. There were 40 nucleotides different in the coding region, which resulted in four amino acid changes in the NIb gene and six in the CP gene, and seven nucleotides different in the 3-untranslated region.     

Comparison of the entire nucleotide and deduced amino acid sequences of the attenuated hog cholera vaccine strain GPE− and the wild-type parental strain ALD

Summary We have determined the complete nucleotide sequences of a live attenuated hog cholera virus (HCV) and its progenitor strain. The viral RNA of each strain consisted of 12 298 nucleotides including untranslated regions of 373 and 228 bases at the 5 and 3 end, respectively. There was a single large open reading frame spanning 11 697 nucleotides which could encode a large protein of 3 899 amino acids with a calculated molecular weight of 438-kDa. We have found 225 nucleotide difference between the two strains, of which six were located in the untranslated region. Four-sixths of these differences resulted in amino acid substitutions.     

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     

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5′ trailer region

Summary. Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5′ trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.     

Determination of the 5′ and 3′ terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus

Summary Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5 ends (5RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5 end, whereas the 3 end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5 noncoding region compared to the 3 noncoding region (none on segment A and 1 on segment B).     

Molecular characterization of a Chinese isolate of potato virus A (PVA) and evidence of a genome recombination event between PVA variants at the 3′-proximal end of the genome

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84 % to 98 % and 93 % to 99 % sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99 %-sequence identity between PVA-Hunan and TamMV in the 3′-proximal end of the genome (~nt 9,160 to the 3′end) and a 50 %-94 % (average ~83 %) identity upstream of nt 9,160. In contrast, 98 % identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94 % for the 3′-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.     

Determination of the nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus genome

Summary.  The nucleotide sequences at the extreme 5′ and 3′ ends of swine hepatitis E virus (swine HEV) genome were determined, and genomic sequence of swine HEV is now complete. Sequence analysis revealed that the 3′ and 5′ non-coding regions (NCRs) of swine HEV are closely related to that of the US-1 and US-2 strains of human HEV. Like the two U.S. strains of human HEV, an extra G residue immediately proceeding the poly(A) tail was identified in swine HEV. The 5′ NCR of swine HEV also differed from many HEV strains: it lacks an A residue at its 5′ very end, and the extra 9 nucleotides in the US-2 strain. In the 3′ NCR, swine HEV shared 90–91% nucleotide sequence identities with the US-1 and US-2 strains but only about 58–65% identities with other HEV strains. This study further suggests that the US-1 and US-2 strains of human HEV may be of swine origin. The availability of the complete sequence of swine HEV should facilitate the construction of an infectious cDNA clone of swine HEV. Received April 20, 2001 Accepted July 10, 2001     

A conserved sequence motif at the 5′ terminus of the Southampton virus genome is characteristic of the Caliciviridae

We have determined the 5 terminal cDNA sequence for the genome of Southampton virus, a recently characterized, human, small round-structured virus (SRSV). Genomic RNA was extracted directly from a stool sample and amplified by RT-PCR by homopolymer tailing of the 3 terminus of the cDNA. The additional sequence increases the overall length of the Southampton virus genome by 12 nucleotides, resulting in a significant change to the genome organization by extending the first large open reading frame (ORF) by 51 amino acids. The 5 terminal bases pGpT and the presence of conserved genome and putative subgenomic RNA terminal motifs are now prominent features shared between the human SRSV Southampton virus and the animal caliciviruses rabbit hemorrhagic disease virus and feline calicivirus.     

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.     

Nucleotide sequence of the 3′ terminal region of lettuce mosaic potyvirus RNA shows a Gln/Val dipeptide at the cleavage site between the polymerase and the coat protein

Summary DNA complementary to the 3 terminal 1651 nucleotides of the genome of the common strain of lettuce mosaic virus (LMV-O) has been cloned and sequenced. Microsequencing of the N-terminus enabled localization of the coat protein gene in this sequence. It showed also that the LMV coat protein coding region is at the 3 end of the genome, and that the coat protein is processed from a larger protein by cleavage at an unusual Q/V dipeptide between the polymerase and the coat protein. This is the first report of such a site for cleavage of a potyvirus polyprotein, where only Q/A, Q/S, and Q/G cleavage sites have been reported. The LMV coat protein gene encodes a 278 amino acid polypeptide with a calculated M_r of 31,171 and is flanked by a region which has a high degree of homology with the putative polymerase and a 3 untranslated region of 211 nucleotides in length. Percentage of homology with the coat protein of other potyviruses confirms that LMV is a distinct member of this group. Moreover, amino acid homologies noticed with the coat protein of potexvirus, bymovirus, and carlavirus elongated plant viruses suggest a functional significance for the conserved domains.     

Sequence of the 3′ half of the Murray Valley encephalitis virus genome and mapping of the nonstructural proteins NS1, NS3, and NS5

We have determined the nucleotide sequence of the 3-terminal half of the RNA genome of Murray Valley encephalitis virus (MVE) using seven overlapping cDNA clones; an estimated 80–90 nucleotides at the extreme 3-end remain to be sequenced. In conjunction with previous sequence data for the 5 half (16), we can conclude that the MVE genome contains a long open reading frame of 10,302 nucleotides that encodes a polyprotein of 3434 residues. Comparison of the MVE deduced amino acid sequence with that of other flaviviruses shows that MVE is most closely related to Japanese encephalitis virus, consistent with serological studies. Using N-terminal amino acid sequence analysis, three nonstructural proteins (NS1, NS3, and NS5) have been identified and mapped on the MVE genome. MVE NS3 contains sequence motifs suggesting that its amino terminus may function as a serine protease. The central region of NS3 (in the linear amino acid sequence) has motifs that are found in NTP-binding proteins and helicases. MVE NS5 contains a conserved Gly-Asp-Asp sequence that is thought to be essential for RNA-dependent RNA polymerases.