Molecular characterization of a satellite RNA associated with blackcurrant reversion nepovirus

Summary.  A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M_r of 44 220. The coding region was bordered by a 5′ leader sequence of 25 nt and a 3′-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5′-terminal consensus sequence and a 40 nt motif (located at positions 264–303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs. Received March 22, 1999/Accepted August 30, 1999     

Size and sequence variability of the Arabis mosaic virus protein 2A

Summary.  The RNA 2 of the nepovirus Arabis mosaic virus (ArMV) encodes a polyprotein from which protein 2A is released by proteolytic cleavage at the N-terminus. The 2A gene of 19 ArMV isolates from different geographical origin and 9 distinct natural hosts was amplified by RT/PCR and subsequently cloned and sequenced. These 19 isolates and those from databanks were classified into four groups based on the size of the protein 2A which ranged from 233 to 280 amino acids, and sequence identities. Sequence variability was mainly located in the N-terminus of the proteins, whereas the core region and the C-terminus were conserved. Received January 14, 2002; accepted March 22, 2002 Published online June 21, 2002     

Complete nucleotide sequence of Cherry leaf roll virus (CLRV), a subgroup C nepovirus

The complete nucleotide sequence of both genomic (+)ss RNAs of a rhubarb isolate of Cherry leaf roll virus (CLRV) was determined. The larger RNA1 is 7918 nucleotides and the shorter RNA2 6360 nucleotides in size, each genome component comprising a single open reading frame (ORF). The RNA1-encoded polyprotein (P1) is 2112 amino acids long (235.6 kDa) containing domains characteristic for a proteinase-cofactor (PCo), nucleotide-binding helicase (Hel), genome-linked protein (VPg), proteinase (Pro), and an RNA-dependent RNA polymerase (Pol). The RNA2-encoded polyprotein (P2) has a molecular mass of 174.9 kDa (1589 aa) encoding the putative movement protein (MP) and the coat protein (CP) of CLRV. The genome region upstream of the MP has a coding capacity of 77 kDa, however processing of P2 by the putative virus-encoded proteinase and protein-function encoded by this region is unknown. Furthermore, it could be demonstrated that the 5′-termini including the N-terminal region (208 aa) of P1 and P2 of the rhubarb isolate of CLRV are nearly identical among the two genome segments.The taxonomic position of CLRV as member of the genus Nepovirus was confirmed by phylogenetic analyses employing the amino acid sequences of the conserved Pro-Pol region of RNA1, the complete P2, and the CP. However, clustering of Nepovirus-species according to allocated subgroups was inconsistent and depended on the compared genome fragment.     

Role of the RNA2 3' non-translated region of Blackcurrant reversion nepovirus in translational regulation

The 3' non-translated regions (NTRs) of mRNAs of eukaryotes and their viruses often contain translational enhancers (TEs). Blackcurrant reversion nepovirus (BRV) has a genome composed of two uncapped polyadenylated RNAs with very long 3' NTRs, nucleotide sequences of which are very conserved between different BRV isolates. In this work, we studied a role of the RNA2 3' NTR in translation, using mutagenesis of the firefly luciferase reporter mRNA, in protoplasts of Nicotiana benthamiana. The RNA2 3' NTR was found to contain a cap-independent TE (3' CITE), which must base pair with the 5' NTR to facilitate translation. The BRV 3' CITE and poly(A) tail provided a major contribution to translational efficiency, with less input from other 3' NTR parts. The BRV 3' CITE does not share similarity in nucleotide sequence and secondary structure with other viruses and thus represents a new class of 3' CITE.     

Nucleotide sequence analysis of the coat protein genes of two Korean isolates of sweet potato feathery mottle potyvirus

Summary.  The coat protein (CP) genes of the genomic RNA of two Korean isolates of sweet potato feathery mottle potyvirus (SPFMV), SPFMV-K1 and SPFMV-K2, were cloned and their complete nucleotide sequences were determined. Sequence comparisons of the two Korean isolates showed 97.8% amino acid identity in the CP cistron, and 79.9% to 99.0% identity with those of 6 other known SPFMV strains. Of 74 amino acid changes totally among the SPFMV strains, 39 changes were located at the N-terminal region. Pairwise amino acid sequence comparison revealed sequence similarities of 48.6 to 70.2% between SPFMV and 20 other potyviruses, indicating SPFMV to be a quite distinct species. Multiple alignment of the CP cistrons from other potyviruses showed that most of the conserved amino acid residues of the genus Potyvirus are well preserved in the corresponding locations. Accepted November 13, 1997 Received September 1, 1997     

The complete genome sequences of two isolates of potato black ringspot virus and their relationship to other isolates and nepoviruses

The complete nucleotide sequences of RNA 1 and RNA 2 of the nepovirus potato black ringspot virus (PBRSV) from two different isolates were determined, as well as partial sequences from two additional isolates. RNA1 is 7,579-7,598 nucleotides long and contains one single open reading frame (ORF), which is translated into a large polyprotein with 2,325 amino acids and a molecular weight of 257 kDa. The complete sequence of RNA2 ranges from 3857 to 3918 nt between the different isolates. It encodes a polyprotein of 1079-1082 amino acids with a molecular weight of 120 kDa. Sequence comparison using the Pro-Pol region and CP showed that all four isolates formed two distinct groups, corresponding to potato and arracacha, that were closely related to each other and also to tobacco ringspot virus (TRSV). Comparing our data to those obtained with other nepoviruses, our results confirm that PBRSV belongs to a distinct species and is a member of subgroup A in the genus Nepovirus based on its RNA2 size, genome organization, and nucleotide sequence.     

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.     

Translation mechanisms involving long-distance base pairing interactions between the 5' and 3' non-translated regions and internal ribosomal entry are conserved for both genomic RNAs of Blackcurrant reversion nepovirus

One of the mechanisms of functioning for viral cap-independent translational enhancers (CITEs), located in 3' non-translated regions (NTRs), is 3' NTR-5' leader long-distance base pairing. Previously, we have demonstrated that the RNA2 3' NTR of Blackcurrant reversion nepovirus (BRV) contains a CITE, which must base pair with the 5' NTR to facilitate translation. Here we compared translation strategies employed by BRV RNA1 and RNA2, by using mutagenesis of the BRV NTRs in firefly luciferase reporter mRNA, in plant protoplasts. Translation mechanisms, based on 3' CITEs, 5' NTR-3' NTR base pairing and poly(A) tail-stimulation, were found conserved between RNA1 and RNA2. The 40S ribosomal subunit entry at the RNA1 leader occurred, at least partly, via an internal ribosomal entry site (IRES). Two RNA1 leader segments complementary to plant 18S rRNA enhanced translation. A model for BRV RNAs translation, involving IRES-dependent 40S subunit recruitment and long-distance 5' NTR-3' NTR base pairing, is discussed.     

Structure of the mite-transmitted Blackcurrant reversion nepovirus using electron cryo-microscopy

Blackcurrant reversion nepovirus (BRV; genus Nepovirus) has a single-stranded, bipartite RNA genome surrounded by 60 copies of a single capsid protein (CP). BRV is the most important mite-transmitted viral pathogen of the Ribes species. It is the causal agent of blackcurrant reversion disease. We determined the structure of BRV to 1.7 nm resolution using electron cryo- microscopy (cryoEM) and image reconstruction. The reconstruction reveals a pseudo T=3 viral capsid similar to that of tobacco ringspot virus (TRSV). We modelled the BRV capsid protein to that of TRSV and fitted it into the cryoEM reconstruction. The fit indicated that the extended C-terminus of BRV-CP is located on the capsid surface and the N-terminus on the interior. We generated peptide antibodies to two putatively exposed C-terminal sequences and these reacted with the virus. Hence homology modelling may be useful for defining epitopes for antibody generation for diagnostic testing of BRV in commercial crops.     

Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1.

Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.     

Complete nucleotide sequence of a Spanish isolate of <Emphasis Type="Italic">Parietaria mottle virus</Emphasis> infecting tomato

The genome of a Spanish isolate of Parietaria mottle virus (PMoV) obtained from tomato (strain PMoV-T) was completely sequenced. Protein motifs conserved for RNA viruses were identified: the p1 protein contained a metyltransferase domain in its N-terminal half and a triphosphatase/helicase domain in its C-terminal half, the p2 protein contained a RNA polymerase domain; the 3a protein contained a RNA-binding domain with α-helix and β-sheet secondary structures. In addition, stem-loop structures with potential capacity of protein interactions were predicted on the untranslated terminal regions. Comparison with the other sequenced PMoV isolate showed nucleotide identities of 93, 90, and 93% for genomic RNAs 1, 2 and 3, respectively, and amino acid identities ranging from 88 to 97% for the different proteins. A cytosine deletion was detected at position 1,366 of RNA 3, involving a start codon for the coat protein (CP) gene different from the other PMoV isolate, resulting in a CP 16 amino acids shorter. Comparison of synonymous and nonsynonymous mutations revealed different selective constraints along the genome.     

Characterization of proteinase cleavage sites in the N-terminal region of the RNA1-encoded polyprotein from Arabis mosaic virus (subgroup A nepovirus)

Arabis mosaic virus is a subgroup A nepovirus. The RNA1-encoded polyprotein (P1) contains the domains for the NTP-binding protein (NTB), VPg, proteinase (Pro) and polymerase at its C-terminus. Putative cleavage sites delineating these domains have been proposed. However, the number and location of cleavage sites upstream of the NTB domain are not known. Using in vitro processing assays, we have confirmed proteolytic cleavage at the NTB-VPg and VPg-Pro sites. In addition, we have identified two cleavage sites in the N-terminal region of P1. Site-directed mutagenesis and immunoprecipitation experiments using inserted peptide tags confirmed that the position of these cleavage sites corresponds to that of cleavage sites delineating the X1 and X2 domains in Tomato ringspot virus (subgroup C nepovirus). Amino acid alignments implied the presence of similar cleavage sites in the P1 polyprotein of other nepoviruses. Our results suggest that the presence of two protein domains upstream of NTB is a common feature of nepoviruses.     

A molecular switch in the capsid protein controls the particle polymorphism in an icosahedral virus

The recombinant coat protein (CP) of Sesbania mosaic virus (SeMV; genus Sobemovirus) was found to self-assemble into capsids encapsidating 23S rRNA and CP mRNA in Escherichia coli. The CP lacking 22 amino acids from the N-terminus assembled into stable T = 3 capsids that appeared similar to SeMV, indicating that the N-terminal 22 amino acid residues are dispensable for T = 3 assembly. Two distinct capsids, T = 1 and pseudo T = 2, were observed when the N-terminal 36 amino acids encompassing the arginine-rich motif (N-ARM) were removed. Only T = 1 particles were observed upon deletion of 65 amino acids from the N-terminus, which also included the sequence element for the beta-annulus. These results reveal that N-ARM acts as a molecular switch in regulating T = 3 assembly. Formation of stable pseudo T = 2 particles shows that pentamers of AB dimers could nucleate assembly at icosahedral-5-folds. Capsids assembled from the N-terminally truncated proteins also encapsidated 23S rRNA and CP mRNA, suggesting the presence of sites outside the N-terminal 65 residues that may be involved in RNA--protein interactions.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

汉坦病毒A9株全长L片段cDNA克隆和核苷酸序列测定

目的克隆我国分离的汉坦病毒A9株L片段全长cDNA,并测定其核苷酸序列.方法用逆转录-聚合酶链反应(RT-PCR)技术分段扩增汉坦病毒A9株全部L片段,用T-A克隆方法进行PCR产物克隆,测定PCR产物的核苷酸序列.通过亚克隆将分段的L片段连接成全长cDNA克隆.结果A9株的基因组L片段长度为6533个核苷酸,腺嘌呤核苷酸和尿嘧啶核苷酸丰富(%A+U=62.47).包含有一个单一的开放读码框架(ORF),编码一个标准的质量为2.46×105的蛋白,含有2151个氨基酸.A9株与76-118、C1-1和C1-2株的同源性最高,达到83.8%.与TULA病毒的关系较远,其核酸序列的同源性为65.8%.将推导的A9株编码的氨基酸序列与其他21种负链RNA病毒的依赖RNA的RNA聚合酶的氨基酸序列以及汉坦病毒几个代表株的L片段氨基酸序列进行比较,显示A9编码的RNA聚合酶也有6个比较保守的区域以及几个极端保守的氨基酸残基.结论汉坦病毒A9株L片段具有和其他汉坦病毒RNA聚合酶相似的核苷酸一级结构,通过对推导的氨基酸分析,该片段具有一些在RNA病毒聚合酶中都存在的保守区域.     

Molecular cloning and sequence analysis of the fusion glycoprotein gene of human parainfluenza virus type 2

A cDNA clone containing a 2.0-kb insert was identified as the human parainfluenza virus type 2 (PI2) fusion glycoprotein gene by hybridizing with a viral RNA probe and a synthetic oligonucleotide derived from a conserved sequence found in other paramyxovirus fusion protein genes. The complete nucleotide sequence of the glycoprotein gene was determined by the dideoxynucleotide sequencing procedure and found to contain a single, large open reading frame encoding a protein of 551 amino acids with a calculated molecular weight of 59,664. Comparison of the P12 fusion protein with those of other paramyxoviruses indicated similarities in overall length, N-terminal signal peptide sequence (amino acids 7 to 25), C-terminal membrane-spanning region (amino acids 486 to 513), and a highly conserved fusion sequence region at the N-terminus of the F1 subunit (amino acids 107 to 132).     

Complete cDNA sequence of a South American isolate of potato virus X

The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.     

The interchain disulfide bond between TCR alpha beta heterodimers on human T cells is not required for TCR-CD3 membrane expression and signal transduction

In the present paper, it was attempted to define the amino acids or regions on TCR beta molecules that determine the TCR alpha-TCR beta interaction. Sequence studies on HBP-ALL variant cells with an intrinsic deficiency in TCR alpha beta dimer formation elucidated a conserved amino acid motif in the TCR-C beta beta-strand E, = Y(C)(L)(S)SRLR(V)(S)(A); this motif seems to represent one interaction area for the TCR alpha-TCR beta interaction. In addition, amino acids in the connecting peptide may be shaped in a precise structure (by the interactions with CD3 molecules?) involved in TCR alpha-TCR beta dimerization. This result was supported by the finding that the interchain disulfide bond between TCR alpha and beta chains is not required for membrane expression or transmembrane signal transduction of TCR alpha beta-CD3 complexes. Finally, comparative results from two membrane TCR-CD3-negative Jurkat variants R4.9 and E6.E12 suggest that TCR-C beta exon 1- and 2-encoded amino acids are important for the TCR beta-CD3 gamma epsilon association.