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     

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 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     

Investigation of the 5′ terminal structures of genomic and subgenomic RNAs of potato virus S

Hybrid arrest translation involving an antisense RNA, generated from a cloned cDNA close to the 5 region of potato virus S genomic RNA, blocked the synthesis of genomic encoded products but had little effect on subgenomic RNA encoded products. Similarly, the synthesis of PVS genomic RNA-directed peptides was inhibited by the cap analogue m⁷G⁵ ppp⁵G, suggesting the presence of a cap structure at the 5 terminus whilst subgenomic RNA encoded products remained unaffected, suggesting an uncapped structure. This was confirmed by artificially produced uncapped subgenomic RNAs translating as efficiently in in vitro translation systems as authentic viral subgenomic RNAs.     

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.     

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.     

Determination of 5′-leader sequences from radically disparate strains of porcine reproductive and respiratory syndrome virus reveals the presence of highly conserved sequence motifs

Summary.  We determined the untranslated 5′-leader sequence for three different isolates of porcine reproductive and respiratory syndrome virus (PRRSV): pathogenic European- and American-types, as well as an American-type vaccine strain. 5′-leader from European- and American-type PRRSV differed in length (220 and 190 nt, respectively), and exhibited only approximately 50% nucleotide homology. Nevertheless, highly conserved areas were identified in the leader of all 3 PRRSV isolates, which constitute candidate motifs for binding of protein(s) involved in viral replication. These comparative data provide a priori knowledge for mutational identification of virulence determinants in the 5′ nontranslated part of the PRRSV genome. Received October 12, 1998 Accepted January 8, 1999     

Long,nearly identical untranslated sequences at the 3′ terminal regions of the genomic RNAs of cherry leafroll virus (walnut strain)

Hybridization analyses of cDNA clones derived from the two genomic RNAs, RNA1 and RNA2, of the walnut strain of the nepovirus cherry leafroll nepovirus (wCLRV) demonstrated a long region of high homology between the two viral RNAs. Subsequent mapping and nucleotide sequencing revealed a long, noncoding, presumably untranslated, region (3 UTR) immediately 5 of the terminal polyadenylate, a region that is almost identical in the two RNAs. This 3 UTR is 1567 nucleotide residues long in RNA1. Homologies of about 80% were found with corresponding regions of genomic RNAs from other strains of CLRV, but not with the corresponding regions of other nepovirus genomic RNAs.The nucleotide sequence reported in this paper has been submitted to the EMBL data library and assigned the accession number Z34265.     

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.     

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     

The 3′ terminal region is strictly required for clover yellow vein virus genome replication

Summary.  To identify the cis-element in the 3′ terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3′ terminal non-coding region (3′-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3′ terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3′-NCR were not infectious. Various mutants with duplicated 3′ terminal sequences were infective only when the authentic 3′ terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3′-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication. Received June 11, 2002; accepted October 25, 2002     

The capsid protein of infectious bursal disease virus contains a functional α4β1 integrin ligand motif

Infectious bursal disease virus (IBDV), a member of the dsRNA Birnaviridae family, is an important immunosuppressive avian pathogen. We have identified a strictly conserved amino acid triplet matching the consensus sequence used by fibronectin to bind the α4β1 integrin within the protruding domain of the IBDV capsid polypeptide. We show that a single point mutation on this triplet abolishes the cell-binding activity of IBDV-derived subviral particles (SVP), and abrogates the recovering of infectious IBDV by reverse genetics without affecting the overall SVP architecture. Additionally, we demonstrate that the presence of the α4β1 heterodimer is a critical determinant for the susceptibility of murine BALB/c 3T3 cells to IBDV binding and infectivity. Our data suggests that the IBDV might also use the α4β1 integrin as a specific binding receptor in avian cells.     

The viral RNA 3′- and 5′-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.     

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.     

Roles of the polypyrimidine tract and 3′ noncoding region of hepatitis C virus RNA in the internal ribosome entry site-mediated translation

Summary. Hepatitis C virus (HCV) genome contains a 3noncoding region (3NCR) consisting of a variable region, a polypyrimidine tract (polyU/UC) and the X region. To examine the roles of 3NCR and polyU/UC tract in the internal ribosome entry site (IRES)-mediated translation process, a variety of 3NCRs containing different lengths of polyU/UC tract were obtained from HCV infected patients and cloned respectively to the downstream of the firefly luciferase coding gene linked to HCV 5NCR and 30 nucleotides of core gene (containing IRES element). The results of in vitro translation in rabbit reticulocyte lysate (RRL) and cell transfection assay in mammalian cells showed that the IRES-mediated translation efficiency could be enhanced by the full-length of 3NCR of HCV RNA. However, contradictory results were observed when the role of polyU/UC tract in the IRES-mediated translation was studied. While the IRES-mediated translation efficiency was inhibited by the presence of polyU/UC tract in in vitro translation experiments, transfection of these expression cassettes into hepatic cell line showed that polyU/UC tract enhanced IRES-mediated translation efficiency in vivo. Cellular-fraction complement experiments showed that cellular factors were required for the enhancement by the polyU/UC tract. Further antibody blocking assay and UV cross-linking assay suggested the correlation of IRES-mediated translation with host factors, including the La protein. The data above also indicated that the modulations of the IRES-mediated translation by the HCV 3NCR and the polyU/UC tract were in a length-independent manner.These authors contributed equally to this work.     

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.     

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.     

Determination of the 5′- and 3′-Terminal Sequences Completes the Sequences of the Two Double-stranded RNAs of Penicillium stoloniferum Virus S

The two genomic segments of Penicillium Stoloniferum virus S (PsV-S), a member of the Partitiviridae, were recently sequenced and published. We independantly sequenced PsV-S and showed that the original sequence was missing nucleotides at both the 5′ and 3′ termini of both segments. We determined the correct sequence in three independent experiments and found the segments to be 1753 bp (encoding the RNA-dependant RNA polymerase) and 1581 bp (encoding the Capsid Protein). Homology was shown between the 5′ and 3′ ends of PsV-S and other members of the Partitiviridae. The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers AM040148 and AM040149.