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.     

Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae.

psaA encodes a 37-kDa putative pneumococcal surface adhesin. Although its complete nucleotide sequence has been determined, its contribution to the pathogenicity of Streptococcus pneumoniae has not previously been assessed. In this study, we used a PCR-amplified internal fragment of the psaA gene from S. pneumoniae type 2 strain D39 cloned in pVA891, to direct the construction of D39 derivatives in which the psaA gene had been specifically interrupted, by insertion-duplication mutagenesis. Two independent D39 psaA mutants (PsaA-(1) and PsaA-(2)) were significantly less virulent (as judged by intranasal or intraperitoneal challenge of mice) than either the wild-type D39 strain or a derivative of PsaA-(1) in which the psaA gene had been reconstituted by back-transformation with an intact copy of the cloned gene. pVA891-directed mutagenesis of an open reading frame (designated ORF3) immediately 3' to psaA or insertion of pVA891 between psaA and ORF3 had no impact on intranasal virulence. However, a small but significant difference in virulence was observed between these two derivatives and the parental D39 strain in a low-dose intraperitoneal challenge model, suggesting that the ORF3 product may also contribute to pathogenesis. Adherence of PsaA-(1) to A549 cells (type II pneumocytes) was only 9% of that for D39, while the ORF3-negative strain exhibited intermediate adherence (23%). This is the first functional evidence that PsaA is an adhesin. Sequence analysis of the psaA gene from D39 indicated significant deviation from that previously published for the homolog from S. pneumoniae R36A. The deduced amino acid sequences of mature PsaA from the two strains had only 81% homology, with the bulk of the variation occurring in the amino-terminal portion.     

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.     

Complete nucleotide sequence of rose yellow leaf virus,a new member of the family Tombusviridae

The genome of the rose yellow leaf virus (RYLV) has been determined to be 3918 nucleotides long and to contain seven open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to encode an 87-kDa (p87) protein that has amino acid similarity to the RNA-dependent RNA polymerase (RdRp) of members of the family Tombusviridae. ORFs 3 and 4 have no significant amino acid similarity to known functional viral ORFs. ORF5 encodes a 6-kDa (p6) protein that has similarity to movement proteins of members of the Tombusviridae. ORF5A has no conventional start codon and overlaps with p6. A putative +1 frameshift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein that has high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CP) of members of the Tombusviridae. ORF7 has no significant amino acid similarity to known viral ORFs. Phylogenetic analysis of the RdRp amino acid sequences grouped RYLV together with the unclassified Rosa rugosa leaf distortion virus (RrLDV), pelargonium line pattern virus (PLPV), and pelargonium chlorotic ring pattern virus (PCRPV) in a distinct subgroup of the family Tombusviridae.     

Genome structure of tobacco necrosis virus strain A

An almost complete sequence of the RNA genome of tobacco necrosis virus (TNV) strain A has been determined. The genome organization is very similar to that of carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 5'-proximal open reading frame (ORF) encodes a 23-kDa protein and read-through of its amber codon into the second ORF is presumably used for the translation of a 82-kDa protein. The third large ORF encodes the 30-kDa coat protein. Two small ORFs are located upstream and one immediately downstream of this coat protein cistron. Extensive sequence similarity was found between the TNV 82-kDa protein and the putative polymerases of TCV, CarMV, cucumber necrosis virus (CNV), maize chlorotic mottle virus (MCMV), red clover necrotic mosaic virus (RCNMV), and barley yellow dwarf virus (BYDV). The TNV coat protein is very similar to southern bean mosaic virus (SBMV) capsid protein. Of the predicted small proteins only a 7.9-kDa protein shows some sequence similarity with a corresponding protein of MCMV, CarMV, and TCV. The others are unique to TNV. Except for the first four nucleotides at the 5' end no homology was found with the RNA of STNV (satellite of TNV).     

The genome of bovine ephemeral fever rhabdovirus contains two related glycoprotein genes.

A 3789 nucleotide region of the bovine ephemeral fever virus (BEFV) genome, located 1.65 kb downstream of the N gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs) which are bounded by putative consensus (AACAGG) and polyadenylation (CATG[A]7) sequences and are separated by an intergenic region of 53 nucleotides. Discrete mRNAs corresponding to each ORF have been identified. The first ORF encodes a polypeptide comprising 623 residues which was identified by peptide sequencing as the virion G protein. The deduced amino acid sequence of the G protein includes putative signal and transmembrane domains and five potential glycosylation sites. The second ORF encodes a polypeptide of 586 amino acids which also has characteristics of a rhabdovirus glycoprotein, including putative signal and transmembrane domains and eight potential glycosylation sites, and appears to correspond to a 90-kDa nonstructural glycoprotein (GNS) identified in BEFV-infected cells (Walker et al. [1991] J. Gen. Virol. 72, 67-74). A database search indicated that both the G and GNS proteins share significant amino acid sequence homology with other rhabdovirus G proteins and with each other. Highest homology scores for each protein were with sigma virus and vesicular stomatitis virus serotypes.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus B

Sclerophthora macrospora Virus B (SmV B) found in S. macrospora, the pathogenic fungus responsible for downy mildew in gramineous plants, is a small icosahedral, monopartite virus containing a positive-strand ssRNA genome. In the present study, the complete nucleotide sequence of the SmV B genome was determined. The viral genome consists of 5533 nucleotides and has two large open reading frames (ORFs). ORF1 encodes a putative polyprotein containing the motifs of chymotrypsin-related serine protease and RNA-directed RNA polymerase. ORF2 encodes a capsid protein. The deduced amino acid sequence shows some similarity to those of certain positive-strand RNA viruses, but the genome organization is characteristic and distinct from those of other known fungal RNA viruses. These results suggest that SmV B should be classified into a new group of mycoviruses.     

Nucleotide sequence of the ononis yellow mosaic tymovirus genome

The nucleotide sequence of the genome of ononis yellow mosaic tymovirus (OYMV) has been determined. The genome is single-stranded RNA, 6211 nucleotides long, and has three main open reading frames (ORFs), two of them overlapping. The largest ORF (nucleotides 179-5509) encodes a polyprotein of 1776 amino acid residues that has sequence similarities with polymerases of other viruses with RNA genomes. The smaller overlapping ORF (nucleotides 172-1965) encodes a protein of 597 amino acids of unknown function. The third ORF located at the 3' end of the genome (nucleotides 5487-6065) is the virion protein gene, and it overlaps by 20 nucleotides the 3' terminus of the largest ORF. The organization of the OYMV genome, its sequence, and the sequences of the protein it encodes are clearly similar to those of two other tymoviruses, turnip yellow mosaic virus and eggplant mosaic virus. The 5' terminal noncoding region of the OYMV genome is much longer than the same region of other tymoviral genomes and includes a direct duplication of a sequence of 21-23 nucleotides.     

Molecular characterization and detection of plum bark necrosis stem pitting-associated virus

The complete RNA genome of plum bark necrosis stem pitting-associated virus (PBNSPaV) was cloned and sequenced and was determined to be 14, 214 nts long. The genome structure revealed seven major open reading frames (ORFs), and nontranslated regions at the 5' and 3' ends. PBNSPaV represents the simplest genome organization in the genus Ampelovirus, family Closteroviridae. The ORFs 1a and 1b encode, respectively, a large polyprotein with a molecular mass (Mr) of 259.6 kDa containing conserved domains characteristic of a papain-like protease, methyltransferase and helicase (ORF1a) and a 64.1-kDa protein of eight conserved motifs characteristic of viral RNA-dependent RNA polymerase (RdRp) (ORF1b). ORF1b is presumably expressed via a +1 ribosomal frameshift mechanism. ORF2 encodes a small 6.3-kDa hydrophobic protein of unknown function. ORF3 encodes a 57.4-kDa protein, a homologue of the HSP70 family of heat shock proteins. ORF4 encodes a 61.6-kDa protein with unknown function. ORF5 encodes a 35.9-kDa capsid protein (CP). Lastly, ORF6 encodes a 25.2-kDa minor capsid protein (CPm). Phylogenetic analyses performed on sequences of the HSP70h RdRp and CP support classification of the virus in the genus Ampelovirus. A real-time TaqMan RT-PCR assay and a one-step RT-PCR were developed for PBNSPaV detection and compared using three different sample preparation methods.     

Nucleotide sequence of the genome of an Australian isolate of turnip yellow mosaic tymovirus

The nucleotide sequence of the Club Lake isolate of turnip yellow mosaic virus (TYMV-CL) genomic RNA has been determined. The genome is 6319 nucleotide residues in length and has three major open reading frames (ORFs), two of which overlap. The smallest ORF is proximal to the 3' terminus and encodes the virion protein gene, which has 98% sequence similarity with the virion protein gene reported for the type strain of TYMV. The largest ORF is from nucleotide residues 96 to 5630, and encodes a protein some parts of which show sequence similarities to the possible RNA replicases and nucleotide binding proteins of other viruses. The third ORF is from nucleotide residues 89 to 1975 and overlaps the 5' end of the largest ORF in a manner similar to that found in several animal viral genomes. The function of the protein encoded by this ORF is unknown. The genomes of tymoviruses have, characteristically, an unusually large cytosine content and small guanosine content. This compositional bias is mirrored in the codon and dinucleotide frequencies of the TYMV-CL genome, but is only partially reflected in the amino acid sequences encoded by the genome.     

The nucleotide sequence and genome organization of Sclerophthora macrospora virus A

Sclerophthora macrospora virus A (SmV A) found in S. macrospora, the pathogenic fungus responsible for downy mildew of gramineous plants, is a small icosahedral virus containing three segments (RNAs 1, 2, and 3) of the positive-strand ssRNA genome. In the present study we report the complete nucleotide sequence of the SmV A genome. The viral genome RNA 1 consists of 2928 nucleotides (nt) and has two open reading frames (ORFs 1a and 1b). ORF 1a contains the motifs of RNA-directed RNA polymerase (RdRp). The function of ORF 1b is unknown. RNA 2 consists of 1981 nt and single ORF (ORF 2). ORF 2 encodes a capsid protein. RNA 3 consists of 977 nt but not any ORFs, suggesting it as a satellite RNA. The deduced amino acid sequence of ORF 1a shows some similarity to those of RdRp of certain positive-strand RNA viruses, especially to the members of the family Nodaviridae, and that of ORF 2 to CP of the members in the family Tombusviridae. The nucleotide sequence of RNA 3 shows a 40-nucleotide length of partial similarity to S. macrospora virus B (SmV B) RNA. The capsid of SmV A is composed of two capsid proteins, CP 1 (p43) and CP 2 (p39), both encoded in ORF 2. CP 2 is apparently derived from CP 1 via proteolytic cleavage at the N-terminus. The genome organization of SmV A is characteristic and distinct from those of other known fungal RNA viruses, including SmV B. These results suggest that SmV A should be classified into a new group of mycoviruses.     

Localization of a r-protein gene within the chloroplast DNA replication origin of Chlamydomonas

Summary In our previous study of chloroplast (Cp) DNA replication in Chlamydomonas reinhardtii, one D-loop site with its flanking regions was cloned and sequenced. The D-loop site mapped by electron mircroscopy (EM) overlaps with an open reading frame (ORF) potentially coding for a polypeptide of 136 amino acids. In this report, the corresponding D-loop isolated from another species of Chlamydomonas was sequenced. An ORF was also detected. Sequence comparison indicated that most conserved sequences between these two cloned origins are located within the ORE Amino acid sequences of these two ORFs are highly conserved. The corresponding sequence for this ORF in the tobacco Cp genome was located by a Southern blotting analysis. Since the complete sequence data of Cp DNAs from a liverwort and from tobacco have been determined in 2 Japanese laboratories recently, it has been possible for us to show that this ORF encodes a protein homologous to the Cp ribosomal protein (r-protein) L16, by sequence comparison.     

Nucleotide sequence, genome organization and phylogenetic analysis of Strawberry pallidosis associated virus, a new member of the genus Crinivirus

Summary. The complete nucleotide sequence of Strawberry pallidosis associated virus (SPaV), a newly identified member of the genus Crinivirus, family Closteroviridae has been determined. RNA 1 is 8067 nucleotides long and encodes at least three open reading frames (ORFs). The first ORF (ORF 1a) specifies a multifunctional protein that has papain-like proteinase, methyltransferase and RNA helicase domains. The RNA-dependent-RNA polymerase is encoded in ORF 1b and is probably expressed by a +1 ribosomal frameshift. The 3 ORF of RNA 1 encodes a small protein with two potential transmembrane helices. RNA 2 is 7979 nucleotides long and encodes 8 ORFs, similar in amino acid sequence and arrangement with those of other criniviruses. SPaV encodes the largest structural protein of closteroviruses sequenced to date as the minor coat protein of the virus has molecular mass of approximately 80kDa. The 3 non-translated regions share nucleotide sequence identities of about 56% and the predicted folding of the non-translated regions is similar. Phylogenetic analyses reveal that SPaV is related most closely to Abutilon yellows virus and Beet pseudo-yellows virus, another virus that has been identified recently to cause identical symptoms on strawberry indicator plants as SPaV.     

The complete nucleotide sequence of RNA 3 of a peach isolate of Prunus necrotic ringspot virus

The complete nucleotide sequence of RNA 3 of the PE-5 peach isolate of Prunus necrotic ringspot ilarvirus (PNRSV) was obtained from cloned cDNA. The RNA sequence is 1941 nucleotides and contains two open reading frames (ORFs). ORF 1 consisted of 284 amino acids with a calculated molecular weight of 31,729 Da and ORF 2 contained 224 amino acids with a calculated molecular weight of 25,018 Da. ORF 2 corresponds to the coat protein gene. Expression of ORF 2 engineered into a pTrcHis vector in Escherichia coli results in a fusion polypeptide of approximately 28 kDa which cross-reacts with PNRSV polyclonal antiserum. Analysis of the coat protein amino acid sequence reveals a putative "zinc-finger" domain at the amino-terminal portion of the protein. Two tetranucleotide AUGC motifs occur in the 3'-UTR of the RNA and may function in coat protein binding and genome activation. ORF 1 homologies to other ilarviruses and alfalfa mosaic virus are confined to limited regions of conserved amino acids. The translated amino acid sequence of the coat protein gene shows 92% similarity to one isolate of apple mosaic virus, a closely related member of the ilarvirus group of plant viruses, but only 66% similarity to the amino acid sequence of the coat protein gene of a second isolate. These relationships are also reflected at the nucleotide sequence level. These results in one instance confirm the close similarities observed at the biophysical and serological levels between these two viruses, but on the other hand call into question the nomenclature used to describe these viruses.     

Complete nucleotide sequence of Rosa rugosa leaf distortion virus, a new member of the family Tombusviridae

This report describes the complete nucleotide sequence and genome organization of Rosa rugosa leaf distortion virus (RrLDV), the causal agent of a previously undescribed virus disease of Rosa rugosa. The RrLDV genome is a positive-sense ssRNA, 3971 nucleotides in length, containing five open reading frames (ORFs). ORF1 encodes a 27-kDa peptide (p27). ORF2 shares a common start codon with ORF1 and continues through the amber stop codon of p27 to produce an 87-kDa protein (p87) with amino acid sequence similarity to the RNA-dependent RNA polymerases (RdRp) of members of the family Tombusviridae. ORF3 encodes a protein of 8 kDa with no significant similarity to known viral sequences. ORF4 encodes a 6-kDa protein (p6) with similarity to the p13 movement proteins of members of the family Tombusviridae. ORF5 has no conventional start codon and overlaps with p6. A putative +1 frame shift mechanism allows p6 translation to continue through the stop codon and results in a 12-kDa protein with high homology to the carmovirus p13 movement protein. The 37-kDa protein encoded by ORF6 has amino acid sequence similarity to coat proteins (CPs) of members of the family Tombusviridae. Phylogenetic analyses of the RdRp and CP amino acid sequences placed RrLDV in a subgroup close to members of the genus Carmovirus of the family Tombusviridae.     

The complete nucleotide sequence of Beet black scorch virus (BBSV), a new member of the genus <Emphasis Type="Italic">Necrovirus</Emphasis>

The complete nucleotide sequence of Beet black scorch virus (BBSV) was determined. The BBSV genome is composed of 3641 nucleotides and has similar organization with Tobacco necrosis virus D of 61% nucleotide identity. The 5'-proximal open reading frame (ORF) encodes a putative 23 kDa protein and a 82 kDa protein by reading-through of an amber termination codon. Three small ORFs located in the center of the genome may encode for a 4.2 kDa protein and two 7 kDa proteins. The 3'-proximal ORF encodes a 24.5 kDa protein equivalent in mass to the viral coat protein. Considering biological and molecular similarities with TNV, it is concluded that BBSV is a new member of the genus Necrovirus.     

Molecular characterization of the genome of Maize rayado fino virus, the type member of the genus Marafivirus

The complete nucleotide sequence of the single-stranded RNA genome of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus, is 6305 nucleotides (nts) in length and contains two putative open reading frames (ORFs). The largest ORF (nt 97-6180) encodes a polyprotein of 224 kDa with sequence similarities at its N-terminus to the replication-associated proteins of other viruses with positive-strand RNA genomes and to the papainlike protease domain found in tymoviruses. The C-terminus of the 224-kDa ORF also encodes the MRFV capsid protein. A smaller, overlapping ORF (nt 302-1561) encodes a putative protein of 43 kDa with unknown function but with limited sequence similarities to putative movement proteins of tymoviruses. The nucleotide sequence and proposed genome expression strategy of MRFV is most closely related to that of oat blue dwarf virus (OBDV). Unlike OBDV, MRFV RNA does not appear to contain a poly(A) tail, and it encodes a putative second overlapping open reading frame.