Sequence of the PV2 gene of rice hoja blanca tenuivirus RNA-2

Comparison of a partial sequence of rice hoja blanca tenuivirus RNA-2 with 40% similarity to rice stripe tenuivirus RNA-2 revealed regions of high local sequence homology at the 5 terminus, within the coding region (the pv2 gene), and in the intergenic region separating this gene from the other protein (pc2) encoded by this ambisense RNA. Analysis of the conserved regions of the pv2 protein identified two motifs found principally in viral membrane glycoproteins and six motifs found each in a wide variety of proteins. The possible significance of these results is discussed.The GenBank accession number of the sequence reported in this paper is L39989.     

Maize stripe tenuivirus RNA2 transcripts in plant and insect hosts and analysis of pvc2, a protein similar to the Phlebovirus virion membrane glycoproteins

The complete sequence of the maize stripe tenuivirus (MstV) RNA2 was determined (3337 nucleotides). RNA2 contains two large open reading frames (ORFs) arranged in an ambisense orientation and specific RNAs of ca. 700 and 2600 nucleotides corresponding to the ORFs were detected in MStV-infected plants and planthoppers. The deduced amino acid sequence of the 23,500 MW protein (pv2) encoded by viral RNA2 (vRNA2) was similar to proteins encoded by the rice stripe (RStV) and rice hoja blanca tenuiviruses vRNA2. Sequence analysis suggested that pv2 is membrane associated. The 93,900 MW protein (pvc2) encoded by viral complementary MStV RNA2 (vcRNA2) was similar to the 94,000 MW protein of RStV RNA2 and to the virion membrane glycoproteins for Phlebovirus members of the Bunyaviridae. The phlebovirus glycoprotein cleavage sitewas similar to a region in the MStV and RStV proteins suggesting that the tenuivirus pvc2 may be processed analogous to the phlebovirus glycoproteins.     

The M RNA of impatiens necrotic spot Tospovirus (Bunyaviridae) has an ambisense genomic organization.

The nucleotide sequence of Impatiens necrotic spot virus (INSV) M RNA was determined from cDNA clones. The INSV M RNA was 4972 nucleotides in length with two open reading frames (ORFs) in an ambisense genomic organization. The larger ORF near the 3' end of the viral RNA, coding for a protein with a predicted molecular weight of 124.9 kDa, was in the viral complementary sense and produced the G2 and G1 proteins. A smaller ORF in the viral sense was capable of coding for a 34.1-kDa polypeptide, designated the NSm protein. Two subgenomic RNA species were detected in INSV-infected tissue that corresponded to the predicted sizes (3.3 and 1.0 kb) of the G2-G1 and NSm mRNAs. The ORFs were separated by a 478 nucleotide A-U-rich intergenic region similar to the regions found in other viral RNAs with ambisense ORFs. The intergenic region was predicted to form a stable stem-loop structure (-81.2 kcal/mole). The ambisense genomic organization is characteristic of the S RNA for members of the Phlebovirus, Uukuvirus, and Tospovirus genera in the Bunyaviridae family. This is the first report of an ambisense Bunyaviridae M RNA.     

Sequence of Echinochloa hoja blanca tenuivirus RNA-5

The sequence is presented of RNA-5 of Echinochloa hoja blanca tenuivirus, a second tenuivirus associated with rice cultivation in Latin America (after rice hoja blanca virus). The RNA is 1334 nucleotides long and contains in the complementary sense RNA a single long open reading frame. The deduced amino acid sequence of this open reading frame shows that it encodes a highly basic and hydrophilic 44 kD protein (pc5) with about 50% similarity to the pc5 protein of maize stripe virus (MStV). This and other features of the RNA are discussed.The GenBank accession number of the sequence reported in this paper is L47430.     

Sequence of Echinochloa hoja blanca tenuivirus RNA-4

The sequence is presented of RNA-4 of Echinochloa hoja blanca tenuivirus (EHBV), one of two tenuiviruses associated with rice cultivation in Latin America (together with rice hoja blanca virus; RHBV). Analysis of the sequence shows that the coding regions of EHBV RNA-4 are closely related to those of RHBV RNA-4. However, the intergenic region separating the two ambisense open reading frames, are highly distinct for the two viruses. The features of the RNA and the comparisons with the sequences of RNA-4 of RHBV, rice stripe virus (RStV) and maize stripe virus (MStV) are discussed.The GenBank accession number of the sequence reported in this paper is L48441     

Sequence of Echinochloa hoja blanca tenuivirus RNA-3

Analysis of the sequence of the 2336 nucleotide RNA-3 of Echinochloa hoja blanca tenuvirus shows that it is closely related to RNA-3 of rice hoja blanca tenuivirus, the principal virus disease of rice in Latin America. This is especially true for the coding regions, where the viruses are almost 90% similar. However, the non-coding regions of RNA-3 of these viruses, principally the intergenic region separating the two ambisense open reading frames, are only about 50% similar, suggesting that these are distinct viruses. The results closely resemble those obtained for the analysis of RNA-4 of these viruses, both in the absolute and relative percentage similarities of the coding and non-coding regions. This implies a coordinated evolution of the different tenuivirus RNA segments. The features of the RNA and the comparisons with the sequences of RNA-3 of RHBV, rice stripe virus (RStV) and maize stripe virus (MStV) are discussed.The GenBank accession number of the sequence reported in this paper is L75930     

Nucleotide sequence of the S RNA of Lassa virus (Nigerian strain) and comparative analysis of arenavirus gene products

The nucleotide sequence of the small (S) genomic RNA of Lassa virus (strain GA391, of Nigerian origin) has been determined. The RNA has features which conform to those seen in most other arenavirus S RNAs which have been characterised, including conserved terminal sequences, an ambisense arrangement of the coding regions for the precursor glycoprotein (GPC) and nucleocapsid (N) proteins and an intergenic region capable of forming a base-paired "hairpin" structure. Comparison of the nucleotide sequence with that of the Josiah strain of Lassa virus (from Sierra Leone) reveals considerable nucleotide divergence in the third base of codons in the reading frames of all three proteins, although the resulting protein sequences are highly conserved, with 92, 94 and 91% identical residues for the mature glycoproteins G1 and G2 and the N protein, respectively. Sequence alignments of the available arenavirus structural proteins and dendrograms summarising the relationships between the viral proteins are presented.     

The S RNA segment of Sandfly Fever Sicilian virus: evidence for an ambisense genome

The complete nucleotide sequence of the S RNA segment of Sandfly Fever Sicilian (SFS) virus (Phlebovirus, Bunyaviridae) was determined from overlapping cDNA clones and by primer extension. The RNA is 1746 nucleotides in length and has two large open reading frames (ORF), one of which (24.8 kDa) is viral-complementary in sense, and the other (30.4 kDa) is in the viral sense. This ambisense genome arrangement has been seen in another member of the Phlebovirus genus, Punta Toro (PT) virus (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306), but not in representatives of either the Bunyavirus or Hantavirus genera of the Bunyaviridae. Comparison of the predicted amino acid sequences for SFS virus with the recognized products of PT S RNA (T. Ihara, Y. Matsuura, and D. H. L. Bishop, 1985, Virology 147, 317-325; H. A. Overton, T. Ihara, and D. H. L. Bishop, 1987, Virology 157, 338-350) indicated that the 24.8-kDa ORF encodes the nucleoprotein (N) of SFS virus, and the 30.4-kDa ORF codes for a nonstructural protein (NSs). Subgenomic messenger RNAs, from which these two proteins are presumably translated, were detected in virus-infected cells.     

Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA

Two overlapping cDNA clones corresponding to the 5' region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an ambisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5' terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3' termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression.     

Comparison of Sequences of RNAs 3 and 4 of Rice Stripe Virus from China with those of Japanese Isolates

The sequences were determined of RNAs 3 and 4 of a Chinese isolate (Y) of rice stripe tenuivirus (RStV) and were compared with those of two RStV isolates (M and T) from Japan. Both RNAs of the Y isolate were longer than those of the M and T isolates. There was almost complete conservation in the 5′ and 3′ non-coding regions for each RNA between the isolates. The analogous ambisense coding regions for each isolate were exactly the same size and the sequences were highly conserved. The major differences were in the intergenic regions, the sizes of which accounted for the differences in size of each RNA of the three isolates. There were no obvious patterns of differences in comparisons of the two RNA over the three isolates. The significance of the similarities and differences in sequences of isolates of RStV separated by more than 3500 km is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analyses of the mRNA transcription processes of Punta Toro phlebovirus (Bunyaviridae)

The time course of the syntheses of Punta Toro (PT) phlebovirus (Bunyaviridae) small (S)-size viral RNA (S vRNA), viral complementary RNA (S vcRNA), and messenger RNA (S mRNA) species has been analyzed using single-stranded DNA probes representing the two S-coded gene products. The data obtained support the conclusion that PT S RNA has an ambisense coding strategy (T. Ihara, H. Akashi, and D. H. L. Bishop, Virology 136, 293-306, 1984) with the viral nucleocapsid protein, N, encoded in a viral-complementary, subgenomic, mRNA species and a putative nonstructural protein, NSs, encoded in a viral-sense, subgenomic, second S mRNA species. In the absence of puromycin (or cycloheximide) full-length S vRNA, S vcRNA, and subgenomic N mRNA and putative NSs mRNA species were identified in PT virus-infected cell extracts. In the presence of inhibitors of protein synthesis (puromycin or cycloheximide) newly synthesized N mRNA species were detected, but not full-length S vcRNA, nor S vRNA, nor the S coded NSs mRNA species. The mRNA species recovered from drug-treated cells have been translated in vitro to synthesize viral N protein. Analyses of the 5' ends of the N and NSs mRNA species have shown them to be heterogeneous in sequence and some 11-18 bases longer than the ends of the genomic RNA species, indicating that they represent nonviral primer sequences like those identified for bunyavirus mRNA species (D. H. L. Bishop, M. E. Gay, and Y. Matsuoka, Nucleic Acids Res. 11, 6409-6418, 1983). The presence of such additional sequences on mRNA derived from representatives of two Bunyaviridae genera appears by these analyses to be a more conserved feature than the S RNA coding arrangement of the respective viruses.     

The glycoprotein gene of Chrysanthemum stem necrosis virus and <Emphasis Type="Italic">Zucchini lethal chlorosis virus</Emphasis> and molecular relationship with other tospoviruses

Two tospoviruses, Chrysanthemum stem necrosis virus (CSNV) and Zucchini lethal chlorosis virus (ZLCV), cause economical losses in several ornamental and vegetable crops in Brazil. The nucleocapsid gene and movement protein sequences had already been reported for both viruses, though the glycoprotein precursor gene sequence was not available. In this study, cDNA fragments (ca. 4 kb) of the M RNA 3' portion of CSNV (isolate Chry-1) and ZLCV (isolate 1038), including the complete glycoprotein precursor gene, partial NSm gene, and the entire intergenic and 3' untranslated regions, were cloned and sequenced. The sequences were assembled with the corresponding 5' region sequence (NSm gene and 5'UTR) of the same isolates to build up the complete sequence of the M RNA segment of both species. The M RNA of CSNV was 4,828 nucleotide-long, while of ZLCV 4,836 nucleotides. Both M RNA molecules comprised two ORFs in an ambisense arrangement. The vcRNA coded for the viral glycoprotein (Gn/Gc) precursor gene of CSNV and ZLCV (both with 127.5 kDa). Comparison of deduced amino acids of the CSNV and ZLCV glycoprotein precursor genes with those of other tospoviruses showed the highest identity with that of Tomato spotted wilt virus (86%) and with that of CSNV (82%), respectively. However, the nucleotide sequence of the intergenic and 3' untranslated regions of CSNV and ZLCV shared lower identities with other tospoviruses. The glycoprotein precursor gene is thought to be a good candidate as additional classification parameter for Tospovirus taxonomy. The presence of the RGD motif in both Gc proteins indicated that they are typical American tospoviruses, which was confirmed by phylogenetic analysis. The membrane topology of both glycoproteins is discussed.     

Cell-to-cell movement of plant viruses

Summary Cell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function is secured by specific virus-encoded proteins. Amino acid sequence comparisons of these proteins revealed a vast superfamily containing a conserved sequence motif that may comprise a hydrophobic interaction domain. This superfamily combines proteins of viruses belonging to all principal groups of positive-strand RNA viruses, as well as single-stranded DNA containing geminiviruses, double-stranded DNA-containing pararetroviruses (caulimoviruses and badnaviruses), and tospoviruses that have negative-strand RNA genomes with two ambisense segments. In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. A distinct type of movement proteins with very high content of proline is found in tymoviruses. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Recombination between unrelated or distantly related viruses could have played a major role in the evolution of the movement function. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. It is hypothesized that all movement proteins of plant viruses may mediate hydrophobic interactions between viral and cellular macromolecules.     

Nucleotide sequence of the Lassa virus (Josiah strain) S genome RNA and amino acid sequence comparison of the N and GPC proteins to other arenaviruses

The complete nucleotide sequence of the S genome RNA of the Josiah strain of Lassa virus was determined from cloned cDNA. The S RNA is 3402 nucleotides long with a calculated molecular weight of 1.09 x 10(6) Da. The nucleotide base composition is 26.84% adenine, 21.40% guanine, 22.75% cytosine, and 29.01% uridine. The 5' and 3' terminal nucleotide sequences are conserved and complimentary for 19 nucleotides, the nucleoprotein and glycoprotein genes are arranged in ambisense coding strategy, and the intergenic region contains an inverted complimentary sequence, as do all other arenavirus S RNAs characterized to date. Amino acid sequence comparisons between the nucleoproteins and glycoproteins of the Josiah and Nigerian (N sequences only) strains of Lassa virus, the WE and ARM strains of lymphocytic choriomeningitis virus (LCMV), Tacaribe, and Pichinde viruses are presented. These findings reveal that the G2 envelope glycoprotein is more conserved among different arenaviruses than the internal nucleoprotein.     

Sequence analysis of the S RNA of the African Arenavirus Mopeia: An unusual secondary structure feature in the intergenic region

Mopeia virus is an apparently nonpathogenic African arenavirus which can protect animals from subsequent challenge by the closely related Lassa virus. As a step toward understanding these differences in pathogenicity and the means by which Mopeia virus infection can protect against subsequent Lassa virus infection, cDNA clones corresponding to 3419 nucleotides of Mopeia virus S RNA were isolated and sequenced. Two open reading frames, encoding the glycoprotein precursor (GPC) and nucleocapsid (N) proteins, were located in the ambisense arrangement characteristic of the arenaviruses. Comparison of the amino acid sequences of the translation products with those of two Lassa virus strains showed considerable conservation, with 74 and 80% identity for the two glycoproteins G1 and G2, and 74% identity for the N protein. The putative dibasic site of GPC cleavage (R-R) was conserved, as were the potential N-linked glycosylation sites. A striking difference between Mopeia virus and Lassa virus was identified in the noncoding intergenic region. Instead of the single hairpin structure formed by base-pairing of complementary sequences which is usually found, the Mopeia virus S RNA has the potential to form two hairpins. These hairpins were similar in sequence and may have been formed in a duplication event during RNA replication. The possible contribution of this secondary structure feature to differences in pathogenicity between Mopeia and Lassa viruses is discussed.     

Genetic organisation of <Emphasis Type="Italic">Iris yellow spot virus</Emphasis> M RNA: indications for functional homology between the G<Subscript>(C)</Subscript> glycoproteins of tospoviruses and animal-infecting bunyaviruses

Summary.  The complete nucleotide sequence (4838 nucleotides) of Iris yellow spot virus (IYSV) M RNA indicates, typical for tospoviruses, the presence of two genes in ambisense arrangement. The vRNA ORF codes for the potential cell-to-cell movement (NSm) protein (34.8 kDa) and the vcRNA ORF for the viral glycoprotein (G1/G2) precursor (128.6 kDa). Multiple sequence alignment of the NSm and G1/G2 precursor proteins of IYSV with those of other tospoviruses, showed highest homologies to Peanut bud necrosis virus (PBNV) and Watermelon silver mottle virus (WSMV). The potential cell-to-cell movement protein of tospoviruses is highly conserved (40–70% identity), with the exception of the first 60 N terminal amino acids, a domain that clearly diverged. For the G1 and G2 viral glycoproteins, blast searches revealed a significant homology between the C-terminally located tospoviral G1 (G_(C)) protein with the counterpart of the animal-infecting bunyaviruses, suggesting a functional homology for these proteins. Received January 15, 2002; accepted July 10, 2002     

Dugbe nairovirus M RNA: Nucleotide sequence and coding strategy

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.     

Peanut bud necrosis tospovirus S RNA: complete nucleotide sequence,genome organization and homology to other tospoviruses

Summary The complete nucleotide sequence of the S RNA of peanut bud necrosis virus (PBNV) has been determined. The RNA is 3 057 nucleotides in length, contains inverted repeats and two open reading frames (ORFs) with an ambisense coding strategy that are separated by an A+U-rich intergenic region. One ORF (1 320 nucleotides in the viral sense strand) encodes a Mr 49.5 kDa protein, identified as the nonstructural (NSs) protein based on similarity to published tospovirus sequences. The second ORF (831 nucleotides in virus complementary strand) encodes a Mr 30.6 kDa protein. This protein was identified as the nucleocapsid (N) protein based on sequence similarities. Amino acid sequence comparison of N and NSs proteins revealed identities of 22–34% with the reported tospovirus isolates of serogroups I, II, and III, whereas it had 82–86% identity with viruses in serogroup IV, watermelon silver mottle virus (WSMV) and tomato isolate of peanut bud necrosis virus (PBNV-To). Two subgenomic RNA species detected in PBNV infected tissue corresponded to the predicted sizes (1.65 and 1.4 kb) of the NSs and N mRNAs. The data presented show conclusively that PBNV should be included in serogroup IV, along with WSMV and PBNV-To.