Complete nucleotide sequence of an isolate of the nepovirus raspberry ringspot virus from grapevine

The complete nucleotide sequence of the RNAs 1 and 2 of the nepovirus Raspberry ringspot virus cherry isolate (RpRSV-ch) from grapevine was determined. The RNA 1 is 7935 nucleotides (nt) long excluding the poly(A) tail, and contains one long open reading frame (ORF) encoding a polypeptide of 2367 amino acids. This ORF is preceeded by a 136nt 5' non-coding region, and followed by a 695nt 3' non-coding region. Conserved amino acid motifs, characteristic of the viral protease cofactor, the NTP-binding protein, proteinase and polymerase, were found in the sequence of the RNA 1-encoded polyprotein. The RNA 2 is 3915nt long excluding the poly(A) tail, and contains one long ORF encoding a polypeptide of 1106 amino acids. This ORF is preceeded by a 203nt 5' non-coding region, and followed by a 390nt 3' non-coding region. When compared to the corresponding sequences of other nepoviruses, a maximum level of 34% identity was found between the RNA 1-encoded polypetides of RpRSV-ch and other nepoviruses. For the RNA 2-encoded polypeptide, 88% identity was found between RpRSV-ch and RpRSV-S, a Scottish isolate of RpRSV from raspberry, and a maximum 29% identity between RpRSV-ch and other nepoviruses.     

Complete nucleotide sequence and construction of an infectious clone of Chinese yam necrotic mosaic virus suggest that macluraviruses have the smallest genome among members of the family Potyviridae

The complete nucleotide sequence of Chinese yam necrotic mosaic virus (CYNMV) was determined from cloned virus cDNA. The CYNMV genomic RNA is 8224 nucleotides in length, excluding the poly(A) tail, and contains one long open reading frame encoding a large polyprotein of 2620 amino acids. CYNMV has no counterpart to the P1 cistron and a short HC-Pro cistron located at the 5?? side of the potyvirus genome. A full-length cDNA clone, pCYNMV, was assembled under the control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. Biolistic inoculation of Nagaimo plants with cDNA resulted in systemic necrotic mosaic symptoms typical of CYNMV infection. To our knowledge, this is the first report of the complete nucleotide sequence and construction of an infectious cDNA clone of a member of the genus Macluravirus.     

Co-infection by two distinct totivirus-like double-stranded RNA elements in Chalara elegans (Thielaviopsis basicola)

A full-length cDNA clone was developed from a 5.3 kb double-stranded (ds) RNA element present in strain CKP of the plant pathogenic fungus Chalara elegans. The complete nucleotide sequence was 5310 bp in length and sequence analysis revealed that it contained three large putative open reading frames (ORFs). ORF1 was initiated at nucleotide position 329 and encoded a putative coat protein, which shared some homology (35-45% amino acid identity) to other dsRNAs in the family Totiviridae. Both ORF2 and ORF3 were initiated at nucleotide positions 2619 and 4071, respectively, and encoded a putative RNA-dependent RNA polymerase (RdRp). Sequence comparison using deduced amino acid sequences of both ORF2 and ORF3 revealed that all RdRp conserved motifs shared highest homology (41% identity) to that of SsRNA1 of Totiviridae. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 1 (CeRV1). During the development of the full-length cDNA clone of CeRV1, several partial cDNA clones from an additional dsRNA fragment in strain CKP were obtained, which when aligned with each other, produced one linear fragment which was 2336 bp long. Northern blot and sequence analysis of this second clone showed it differed in sequence composition from CeRV1. This dsRNA in C. elegans was designated Chalara elegans RNA Virus 2 (CeRV2). Sequence analysis of CeRV2 showed it contained all conserved motifs and shared some homology (45% amino acid identity) to RdRp regions of Totiviridae. The nucleotide and amino acid sequences of the conserved motifs of the RdRp regions between CeRV1 and CeRV2 showed an identity of 56% and 50%, respectively. These findings suggest that co-infection of two distinct totivirus-like dsRNAs (CeRV1 and CeRV2) in C. elegans, a first report in this fungus. Transmission electron microscopy of strain CKP of C. elegans revealed the presence of putative virus-like particles in the cytoplasm, which were similar both in shape and size to viruses in the Totiviridae.     

Nucleotide sequence of black currant reversion associated nepovirus RNA1

The RNA1 of black currant reversion associated nepovirus (BRAV) is 7711 nucleotides (nt) long, excluding the poly-A tail, and contains one long open reading frame (ORF) which is translated into a polyprotein of 2094 amino acids. The 5' NTR of BRAV RNA1 is 66 nt long and 78% identical with RNA2 5' NTR only over the first 57 nucleotides. The 3' non-translated region (3'NTR) is 1360 nucleotides long, and after the first 24 nucleotides 95% identical with the 3'NTR of RNA2. RNA1 3'NTR contains several stretches, 694-24 nucleotides in length, which are 60-80% similar to corresponding areas of the other viruses of the subgroup c of nepoviruses (BLMV, CLRV, PRMV or TomRSV). The 2094 amino acids-long polypeptide encoded by BRAV RNA1 is 33% identical with that of PRMV between amino acids 9 and 2057, and has significant similarity also to those of other nepoviruses and comoviruses. Conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cysteine protease and the RdRp core domains, known to occur in the polyproteins of different viruses of the picornavirus-like supergroup, are all detected in the amino acid sequences encoded by BRAV RNA1.     

Analysis of the complete genome sequence of acute bee paralysis virus shows that it belongs to the novel group of insect-infecting RNA viruses

The complete genome sequence of acute bee paralysis virus (ABPV) was determined. The 9470 nucleotide, polyadenylated RNA genome encoded two open reading frames (ORF1 and ORF2), which were separated by 184 nucleotides. The deduced amino acid sequence of the 5' ORF1 (nucleotides 605 to 6325) showed significant similarity to the RNA-dependent RNA polymerase, helicase, and protease domains of viruses from the picornavirus, comovirus, calicivirus, and sequivirus families, as well as to a novel group of insect-infecting RNA viruses. The 3' ORF2 (nucleotides 6509-9253) was proposed as encoding a capsid polyprotein with three major structural proteins (35, 33, and 24 kDa) and a minor protein (9.4 kDa). This was confirmed by N-terminal sequence analysis of two of these proteins. The overall genome structure of ABPV showed similarities to those of Drosophila C virus, Plautia stali intestine virus, Rhopalosiphum padi virus, and Himetobi P virus, which have been classified into a novel group of picorna-like insect-infecting RNA viruses called cricket paralysis-like viruses. It is suggested that ABPV belongs to the cricket paralysis-like viruses.     

Cloning and Nucleotide Analysis of Segment A Gene of Infectious Bursal Disease Virus Detected in Korea

A strain of infectious bursal disease virus (IBDV) was detected from bursal tissues of chicks which suffered from infectious bursal disease (IBD) in Chinju, Korea and provisionally named as Chinju strain. A full-length cDNA clone for segment A gene of the virus was constructed, and complete nucleotide sequence of the gene including noncoding region was determined and analyzed by comparison with that of other IBDV strains. The segment A gene of Chinju strain consisted of 3,269 nucleotides including 862 adenine (26.4%), 917 cytosine (28.0%), 854 guanine (26.1%) and 636 thymine (19.5%). There were regions for two open reading frames (ORFs), ORF1 encoding the VP5 with ATG codon at nucleotides 98–100 and ORF2 encoding the polyprotein of VP2, VP4 and VP3 in the nucleotides 132–3,170. In deduced translation the ORF2 encoded 1,012 amino acids. The full nucleotide sequence of segment A gene and amino acid sequence of ORF2 of the Chinju strain showed 98–99% homology with those of the very virulent IBDVs (vvIBDVs) such as HK46, OKYM, UK661, UPM97/61, D6948 and BD3/99. In phylogenetic analysis of nucleotide and amino acid sequences, the Chinju strain was also related closely to the vvIBDVs. Hence, it was suggested that the Chinju strain is a vvIBDV. The nucleotide and amino acid sequences of the Chinju strain with pertinent information can be useful for the development of genetically engineered vaccines and diagnostic reagents against vvIBDV.     

The nucleotide sequence of apple stem grooving capillovirus genome.

The complete nucleotide sequence of apple stem grooving virus (ASGV) genome has been determined. The genome is 6496 nucleotides in length excluding a 3'-terminal poly(A) tail and contains two overlapping open reading frames (ORFs). ORF1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at position 4788 and can encode a 36-kDa protein. The 241-kDa protein contains two consensus sequences associated with the RNA-dependent RNA polymerase and the NTP-binding helicase. Comparisons of amino acid sequences around these conserved motifs with other RNA viruses revealed that ASGV has extensive similarities with apple chlorotic leaf spot, tymo-, carla-, and potexviruses, and is a member of the sindbis-like supergroup. ASGV coat protein is found to be located in the C-terminal region of the 241-kDa polyprotein. The 36-kDa protein encoded by ORF2 contains the consensus sequence Gly-Asp-Ser-Gly found in the active site of several cellular and viral serine proteases.     

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA:the large coding region of the P1 gene

Summary. The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3 untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus. Received October 28, 1996 Accepted April 3, 1997     

Complete sequence of RNA1 of grapevine Anatolian ringspot virus

The nucleotide sequence of RNA1 of grapevine Anatolian ringspot virus (GARSV), a nepovirus of subgroup B, was determined from cDNA clones. It is 7,288 nucleotides in length excluding the 3′ terminal poly(A) tail and contains a large open reading frame (ORF), extending from nucleotides 272 to 7001, encoding a polypeptide of 2,243 amino acids with a predicted molecular mass of 250 kDa. The primary structure of the polyprotein, compared with that of other viral polyproteins, revealed the presence of all the characteristic domains of members of the order Picornavirales, i.e., the NTP-binding protein (1B^Hel), the viral genome-linked protein (1C^VPg), the proteinase (1D^Prot), the RNA-dependent RNA polymerase (1E^Pol), and of the protease cofactor (1A^Pro-cof) shared by members of the subfamily Comovirinae within the family Secoviridae. The cleavage sites predicted within the polyprotein were found to be in agreement with those previously reported for nepoviruses of subgroup B, processing from 1A to 1E proteins of 67, 64, 3, 23 and 92 kDa, respectively. The RNA1-encoded polyprotein (p1) shared the highest amino acid sequence identity (66 %) with tomato black ring virus (TBRV) and beet ringspot virus (BRSV). The 5′- and 3′-noncoding regions (NCRs) of GARSV-RNA1 shared 89 % and 95 % nucleotide sequence identity respectively with the corresponding regions in RNA2. Phylogenetic analysis confirmed the close relationship of GARSV to members of subgroup B of the genus Nepovirus.     

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 and coding capacity of the large (L) genomic RNA segment of Seoul 80-39 virus, a member of the hantavirus genus.

The nucleotide sequence of the large (L) genomic RNA segment of Seoul 80-39 virus was determined from overlapping cDNA clones. The virion L RNA segment is 6530 nucleotides long. The 3' and 5' terminal sequences are inversely complementary for 15 bases. The viral complementary-sense RNA contains a single open reading frame from an AUG codon at nucleotide position 37-39 to a UAA stop codon at nucleotide position 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 kDa) which likely corresponds to the L protein detected in purified viral particles (Elliott et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). Comparison of the L protein of the Seoul 80-39 virus with the polymerase proteins encoded by other negative-stranded RNA viruses revealed 44% similarity only with the part of the Bunyamwera virus L protein (Elliott, 1989) and a very weak homology with the PB1 protein of influenza virus.     

The complete nucleotide sequence of RNA2 of blackcurrant reversion nepovirus

The complete nucleotide sequence of blackcurrant reversion nepovirus (BRV) RNA2 was determined from cDNA clones. RNA2 was 6400 nucleotides (nt) in length excluding the 3' poly(A)-tail. It contained a single open reading frame of 4878 nts encoding a polypeptide of 1626 amino acids with a calculated M(r) of 178? omitted?860. The genome organization of BRV RNA2 was similar to that of other nepoviruses, especially those with a large RNA2. The coat protein (CP) was located in the C-terminal region of the large polyprotein and contained amino acid motifs conserved among nepovirus CPs. Sequence comparisons revealed a proline (P) residue surrounded by hydrophobic amino acid residues located upstream of the CP. This P motif is conserved among the putative movement proteins of nepo-, como-, caulimo- and capilloviruses. An N-terminal domain of 350 amino acids of RNA2-encoded polyprotein shared 34 and 35% sequence identity with the N-terminal domains of tomato ringspot nepovirus RNA1- and RNA2-encoded polyproteins, respectively. Sequence identities between the N-terminal domains of BRV RNA2 and other nepoviral RNA2s were less than 20%; no common N-terminal motif was found.     

The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein

The complete nucleotide sequence of the tobacco etch virus (TEV) RNA genome has been determined excepting only the nucleotide(s) present at the extreme 5' terminus. The assembled TEV genomic sequence is 9496 nucleotides in length followed by a polyadenylated tract ranging from 20 to 140 residues. A computer search of the sequence reveals the following. A 5' untranslated region, rich in adenosine and uridine, is present between nucleotides 1 and 144. A putative initiation codon, at nucleotides 145-147, marks the beginning of a large open-reading frame (ORF) which ends with an opal (UGA) termination codon at positions 9307-9309. A 186-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3' polyadenylated region. The predicted translation product of this ORF is a 3054 amino acid polyprotein with a mol wt of 345,943. A function for the large (54,000 Mr) nuclear inclusion protein is suggested by a comparison of the deduced amino acid sequence with a protein data bank. This protein displays biochemical similarities to other viral RNA-dependent, RNA polymerases.     

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.