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At least 1,369 records · Page 76Linked to original sources
PubMed2006

The genomic sequence of Wisteria vein mosaic virus and its similarities with other potyviruses.

The complete nucleotide sequence of a Beijing isolate of Wisteria vein mosaic virus was determined to be 9695 nucleotides in length excluding the poly(A) tail. Sequence analysis predicted a single large open reading frame of 9279 nucleotides potentially encodes a polyprotein of 3092 amino acids. Phylogenetic analysis based on the genomic and deduced amino acid sequences support the current status of Wisteria vein mosaic virus (WVMV) as a distinct virus of the genus Potyvirus and a member of the Bean common mosaic virus (BCMV) subgroup. Sequence comparisons of WVMV and other members of the BCMV subgroup showed that WVMV is most closely related to both soybean mosaic virus and watermelon mosaic virus.

Amino Acid Sequence↗
PubMed2007

Identification and characterisation of tomato torrado virus, a new plant picorna-like virus from tomato.

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of 'torrado disease'; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.

Amino Acid Motifs↗
PubMed1999

Comparison of the complete sequences of five different isolates of Potato virus A (PVA), genus Potyvirus.

The complete nucleotide (nt) and deduced amino acid (aa) sequences of isolates Ali, U, Her (from potato, Solanum tuberosum) and TamMV (from tamarillo, Solanum betacea) of Potato virus A (PVA, genus Potyvirus) were determined and compared with the previously reported sequence of PVA isolate B11. Most parts (proteins) of the polyprotein showed over 95% aa sequence similarity. The cylindrical inclusion (CI) protein and the 6K 1 protein were the most conserved proteins among the five isolates. TamMV was the most different isolate. Sequence similarity between TamMV and the other isolates was the lowest in regions close to the 5'-end [5'-non-translated region (NTR) and P1 region] and 3'-end (N-terminus of coat protein) of the genome. However, the termini of the genome (the first 60 nt of the 5'-NTR and the entire 3'-NTR) were highly similar in all five isolates. A frameshift region in the replicase (NIb) was identified the PVA isolates Ali, B11, Her and U, as compared to TamMV and other potyviruses.

5' Untranslated Regions↗
PubMed2006

Genome comparison of a novel classical swine fever virus isolated in China in 2004 with other CSFV strains.

The genome of a novel classical swine fever virus (CSFV), SWH/CA/2004, isolated from a hog pen in Henan Province, central China, is 12,296 nucleotides (nt) in length. It is composed of a 373-nt 5' terminal non-translated region (NTR), a 11,697-nt open reading frame (ORF) encoding a polyprotein of 3,898 amino acids (aa), and a 226-nt 3'-NTR. Genome comparison of the SWH/CA/2004 isolate (GenBank Accession: DQ127910) with other known CSFV isolates was performed and analyzed. Corresponding segments from SWH/CA/2004 and other reported strains shared 80.4-99.8% identity at the nucleotide level and 89.5-99.8% identity at the amino acid level. From an evolutionary point of view, isolate SWH/CA/2004 is closely related to the highly virulent isolate cF114/CA/2001, with a pairwise distance of 0.013; and distantly related to the moderately virulent isolate GXWZ02/CA/2003, with pairwise distance 0.170. The phylogenetic trees of the full-length genome and the following region E(rns), E1, E2, and NS5B-based neighbor-joining (NJ) method were constructed and approximately divided into different genetic groups according to avirulence, moderate virulence and high virulence, while other region-based NJ trees demonstrated sequence conservation between these groups. The four genomic regions may constitute important criteria for genetic typing of diverse CSFV isolates. Based on these analyses, isolate SWH/CA/2004 was deduced to belong to the highly virulent isolate group. However, SWH/CA/2004 also contains a 14-U deletion in the 3'-NTR that is characteristic of avirulent isolates. These analyses constitute a comprehensive study of the phylogenetics of CSF based on distinct regions of the genome and may provide the basis for future molecular epidemiology research to identify virulent strain outbreaks and trigger implementation of appropriate control measures.

Animals↗
PubMed2005

Complete nucleotide sequence of the RNA-2 of grapevine deformation and Grapevine Anatolian ringspot viruses.

The nucleotide sequence of RNA-2 of Grapevine Anatolian ringspot virus (GARSV) and Grapevine deformation virus (GDefV), two recently described nepoviruses, has been determined. These RNAs are 3753 nt (GDefV) and 4607 nt (GARSV) in size and contain a single open reading frame encoding a polyprotein of 122 kDa (GDefV) and 150 kDa (GARSV). Full-length nucleotide sequence comparison disclosed 71-73% homology between GDefV RNA-2 and that of Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV), and 62-64% homology between GARSV RNA-2 and that of Grapevine chrome mosaic virus (GCMV) and Tomato black ring virus (TBRV). As previously observed in other nepoviruses, the 5' non-coding regions of both RNAs are capable of forming stem-loop structures. Phylogenetic analysis of the three proteins encoded by RNA-2 (i.e. protein 2A, movement protein and coat protein) confirmed that GDefV and GARSV are distinct viruses which can be assigned as definitive species in subgroup A and subgroup B of the genus Nepovirus, respectively.

5' Untranslated Regions↗
PubMed2006

Novel recognition sequence of coxsackievirus 2A proteinase.

Coxsackievirus B1 (CVB1) 2A proteinase (2A(pro)) is a cysteine proteinase that cleaves the viral monocistronic polyprotein between the C-terminus of the VP1 region and the N-terminus of the 2A region, and also shuts off translational initiation in host cells by cleavage of eukaryotic initiation factor 4G (eIF4G) isoforms. We expressed in Escherichia coli a series of fusions in which various C-terminal fragments of VP1 were linked to the N-terminus of 2A(pro), and we also employed site-directed mutagenesis to introduce mutations of several amino acid residues. Our results showed that the presence of the C-terminal three amino acid residues of VP1 at the N-terminus of 2A(pro) is sufficient for specific self-cleavage between VP1 and 2A(pro) to generate mature 2A(pro), but the P4 amino acid also plays an important role. We further found that 2A(pro) cleaves the amino acid sequence Leu-Val-Pro-Arg-( *)Gly-Ser (LVPRGS motif), which is the target sequence of thrombin.

Amino Acid Motifs↗
PubMed2004

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotransposon.

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansoni. Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRn1 and kabuki, as well as to the conserved integrase core domain, GenBank rve (). Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.

Amino Acid Sequence↗
PubMed2004

Functional relationship between the matrix proteins of feline and simian immunodeficiency viruses.

To investigate the functional relationship between the matrix (MA) proteins of feline and simian immunodeficiency viruses (FIV and SIV, respectively), we generated chimeric proviruses in which the MA-coding region of an SIV infectious molecular clone was partially or fully replaced by its FIV counterpart. Chimeric SIV proviruses containing the amino-terminal 36 residues or the central and carboxy-terminal regions of the FIV MA assembled into virions as efficiently as wild-type SIV. However, the resulting virions were noninfectious in single-cycle infectivity assays. Furthermore, a chimeric SIV provirus containing the entire FIV MA was found to be severely impaired in virion production due to inefficient membrane binding of the chimeric Gag polyprotein. Interestingly, the assembly defective phenotype of this chimeric Gag precursor could be reversed either by introducing the G31K/G33K double amino acid substitution in the FIV-derived MA domain or by coexpression with wild-type SIV Gag. Of note, a chimeric FIV provirus expressing the SIV MA not only assembled into particles as efficiently as wild-type FIV, but also replicated in feline T cells with wild-type kinetics. Our results thus provide novel information about the functional homology between the MA proteins of distantly related lentiviruses.

Amino Acid Sequence↗
PubMed2003

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.

3' Untranslated Regions↗
PubMed2004

New Calicivirus isolated from walrus.

The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.

Amino Acid Sequence↗
PubMed2005

Nucleotide sequence analysis of Plum pox virus isolate W3174: evidence of a new strain.

The nucleotide sequence of Plum pox virus (PPV) isolate W3174 was determined. The virus genome consists of 9788 nucleotides (nt), excluding the poly(A) tail at the 3'-terminus, with 5'- and 3'-untranslated regions (UTRs) of 146 and 219 nt, respectively. The deduced polyprotein consists of 3141 amino acid (aa) residues, with the coat protein region consisting of 993 nt (331 aa). In comparisons with PPV-D, -M, -EA and -C isolates, nucleotide identity levels ranged from 79 to 80% for the entire genome and from 78 to 79%, 78 to 81%, and 92 to 95% for the NIb, CP, and 3'-UTR regions, respectively. The majority of nucleotide substitutions in the NIb region of W3174 are silent, while substitutions in the CP region are not silent, giving aa identities of 89-91% and 79-81%, respectively. The HC-Pro region contains the KITC and PTK motifs, and the DAG motif is located at positions 12-14 of the deduced CP aa sequence, all associated with aphid transmission. Phylogenetic analysis based on the complete genome sequence, the NIb, CP, and 3'-UTR region were performed. PPV-W3174 consistently formed a distinct clade or group, when compared to members of all four recognized strains of PPV, indicating that it is genetically distinct. These results are consistent with serological and nucleic acid-based strain typing data and justify recognition of this isolate as representative of a new strain identified as PPV-W.

3' Untranslated Regions↗
PubMed2005

Simultaneous production of two foreign proteins from a polyvirus-based vector.

With the aim of developing a biotechnological tool for the production of foreign proteins in plants, we first engineered an infectious turnip mosaic virus (TuMV) cDNA that contained the jellyfish green fluorescent protein (GFP) gene or the bacterial beta-glucuronidase (GUS) gene (uidA). Two insertion sites were assessed, either between P1 and HCPro cistrons or Pol and CP cistrons. In each construct, the junctions flanking the inserted gene coded for P1 and/or VPg-Pro cleavage recognition site sequences, to produce free GUS or GFP. After transfection by particle bombardment on Brassica perviridis, characteristic symptoms for TuMV infection appeared and Western blot analyses showed that GFP and GUS had been excised from the viral polyprotein. No significant differences in expression level were noticed between the two insertion sites. By RT-PCR, gfp was found to be stable over 30 days post-transfection (dpt) while uidA was gradually lost at 15 dpt. We also created two constructs containing either gene at each insertion sites on the same molecule. Attenuated systemic symptoms were observed after particle bombardment on B. perviridis and Western blot analyses showed that both foreign proteins were produced. Also, the same stability/instability as for the single-gene constructs were observed. These results indicate that it is possible to produce at least two foreign proteins simultaneously in a TuMV-based vector.

Amino Acid Sequence↗
PubMed2002

The mechanical hierarchies of fibronectin observed with single-molecule AFM.

Mechanically induced conformational changes in proteins such as fibronectin are thought to regulate the assembly of the extracellular matrix and underlie its elasticity and extensibility. Fibronectin contains a region of tandem repeats of up to 15 type III domains that play critical roles in cell binding and self-assembly. Here, we use single-molecule force spectroscopy to examine the mechanical properties of fibronectin (FN) and its individual FNIII domains. We found that fibronectin is highly extensible due to the unfolding of its FNIII domains. We found that the native FNIII region displays strong mechanical unfolding hierarchies requiring 80 pN of force to unfold the weakest domain and 200 pN for the most stable domain. In an effort to determine the identity of the weakest/strongest domain, we engineered polyproteins composed of an individual domain and measured their mechanical stability by single-protein atomic force microscopy (AFM) techniques. In contrast to chemical and thermal measurements of stability, we found that the tenth FNIII domain is mechanically the weakest and that the first and second FNIII domains are the strongest. Moreover, we found that the first FNIII domain can acquire multiple, partially folded conformations, and that their incidence is modulated strongly by its neighbor FNIII domain. The mechanical hierarchies of fibronectin demonstrated here may be important for the activation of fibrillogenesis and matrix assembly.

Amino Acid Sequence↗
PubMed2003

Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system.

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.

3C Viral Proteases↗
PubMed2001

Expression and partial purification of recombinant tomato ringspot nepovirus 3C-like proteinase: comparison of the activity of the mature proteinase and the VPg-proteinase precursor.

The 3C-like proteinase (Pro) from Tomato ringspot virus (genus Nepovirus) is responsible for the processing of the RNA1-encoded (P1) and RNA2-encoded (P2) polyproteins. Cleavage between the VPg and Pro domains is inefficient in vitro and in E. coli, resulting in the accumulation of the VPg-Pro. In this study, we have compared the trans-activity of the Pro and VPg-Pro on various P1- and P2-derived precursors. Recombinant Pro and VPg-Pro were partially purified using an E. coli expression system. A mutation of the VPg-Pro cleavage site was introduced into the VPg-Pro to prevent slow release of the Pro. The Pro was five to ten times more active than the VPg-Pro on two P2 cleavage sites (at the N- and C-termini of the movement protein domain) and was approximately two times more active than the VPg-Pro on the third P2 cleavage site (between the X3 and X4 domains). Neither the Pro nor the VPg-Pro could cleave in trans P1-derived substrates containing the three cleavage sites delineating the X1, X2, putative NTP-binding protein and VPg domains. These results are discussed in light of the possible regulation of the proteinase activity during virus replication.

3C Viral Proteases↗
PubMed2006

Protein structure by mechanical triangulation.

Knowledge of protein structure is essential to understand protein function. High-resolution protein structure has so far been the domain of ensemble methods. Here, we develop a simple single-molecule technique to measure spatial position of selected residues within a folded and functional protein structure in solution. Construction and mechanical unfolding of cysteine-engineered polyproteins with controlled linkage topology allows measuring intramolecular distance with angstrom precision. We demonstrate the potential of this technique by determining the position of three residues in the structure of green fluorescent protein (GFP). Our results perfectly agree with the GFP crystal structure. Mechanical triangulation can find many applications where current bulk structural methods fail.

Calibration↗
PubMed2004

The isolation and characterisation of astroviruses from chickens.

The isolation, cultivation and characterization of three chicken astroviruses (CAstV) isolates are described. They are antigenically related to each other but unrelated to avian nephritis virus (ANV) and duck hepatitis virus type 2 (DVH2) in neutralization, immunofluorescence and gel diffusion tests. CAstV, ANV and DVH2 all grew well in the LMH cell line, which was used for assay and serological tests. Serological surveys in 1982 and 2001 showed that antibody to CAstV virus was widespread in broiler and broiler breeder flocks and present in some turkey flocks. Infection of 1-day-old specific pathogen free chicks with one isolate in the laboratory resulted in mild diarrhoea and some distention of the small intestine. The virus could be isolated in high titres from all parts of the small intestine but rarely from other organs. Electron microscopic examination of purified particles of this agent revealed the presence of clusters of small round viruses with a diameter ranging from 25 to 30 nm. The amino acid sequence derived from the relatively conserved non-structural polyprotein region of this virus shows 62% identity with the corresponding region of turkey astrovirus 2, 58% identity with turkey astrovirus 1, 55% identity with avian nephritis virus and 33% identity with sheep astroviruses. Taken together, the results indicate that the agent is a new chicken astrovirus belonging to the family Astroviridae.

Amino Acid Sequence↗
PubMed2004

A programmed -1 ribosomal frameshift signal can function as a cis-acting mRNA destabilizing element.

Nonsense-mediated mRNA decay (NMD) directs rapid degradation of premature termination codon (PTC)-containing mRNAs, e.g. those containing frameshift mutations. Many viral mRNAs encode polycistronic messages where programmed -1 ribosomal frameshift (-1 PRF) signals direct ribosomes to synthesize polyproteins. A previous study, which identified consensus -1 PRF signals in the yeast genome, found that, in contrast to viruses, the majority of predicted -1 PRF events would direct translating ribosomes to PTCs. Here we tested the hypothesis that a -1 PRF signal can function as a cis-acting mRNA destabilizing element by inserting an L-A viral -1 PRF signal into a PGK1 reporter construct in the 'genomic' orientation. The results show that even low levels of -1 PRF are sufficient to target the reporter mRNA for degradation via the NMD pathway, with half-lives similar to messages containing in-frame PTCs. The demonstration of an inverse correlation between frameshift efficiency and mRNA half-lives suggests that modulation of -1 PRF frequencies can be used to post-transcriptionally regulate gene expression. Analysis of the mRNA decay profiles of the frameshift-signal- containing reporter mRNAs also supports the notion that NMD remains active on mRNAs beyond the 'pioneer round' of translation in yeast.

Codon, Nonsense↗