Letteratura scientifica selezionata sul tema "ORF2 protein"
Cita una fonte nei formati APA, MLA, Chicago, Harvard e in molti altri stili
Consulta la lista di attuali articoli, libri, tesi, atti di convegni e altre fonti scientifiche attinenti al tema "ORF2 protein".
Accanto a ogni fonte nell'elenco di riferimenti c'è un pulsante "Aggiungi alla bibliografia". Premilo e genereremo automaticamente la citazione bibliografica dell'opera scelta nello stile citazionale di cui hai bisogno: APA, MLA, Harvard, Chicago, Vancouver ecc.
Puoi anche scaricare il testo completo della pubblicazione scientifica nel formato .pdf e leggere online l'abstract (il sommario) dell'opera se è presente nei metadati.
Articoli di riviste sul tema "ORF2 protein":
1
Tsai, Chi-Wei, Margaret G. Redinbaugh, Kristen J. Willie, Sharon Reed, Michael Goodin e Saskia A. Hogenhout. "Complete Genome Sequence and In Planta Subcellular Localization of Maize Fine Streak Virus Proteins". Journal of Virology 79, n. 9 (1 maggio 2005): 5304–14. http://dx.doi.org/10.1128/jvi.79.9.5304-5314.2005.
Abstract (sommario):
ABSTRACT The genome of the nucleorhabdovirus maize fine streak virus (MFSV) consists of 13,782 nucleotides of nonsegmented, negative-sense, single-stranded RNA. The antigenomic strand consisted of seven open reading frames (ORFs), and transcripts of all ORFs were detected in infected plants. ORF1, ORF6, and ORF7 had significant similarities to the nucleocapsid protein (N), glycoprotein (G), and polymerase (L) genes of other rhabdoviruses, respectively, whereas the ORF2, ORF3, ORF4, and ORF5 proteins had no significant similarities. The N (ORF1), ORF4, and ORF5 proteins localized to nuclei, consistent with the presence of nuclear localization signals (NLSs) in these proteins. ORF5 likely encodes the matrix protein (M), based on its size, the position of its NLS, and the localization of fluorescent protein fusions to the nucleus. ORF2 probably encodes the phosphoprotein (P) because, like the P protein of Sonchus yellow net virus (SYNV), it was spread throughout the cell when expressed alone but was relocalized to a subnuclear locus when coexpressed with the MFSV N protein. Unexpectedly, coexpression of the MFSV N and P proteins, but not the orthologous proteins of SYNV, resulted in accumulations of both proteins in the nucleolus. The N and P protein relocalization was specific to cognate proteins of each virus. The subcellular localizations of the MFSV ORF3 and ORF4 proteins were distinct from that of the SYNV sc4 protein, suggesting different functions. To our knowledge, this is the first comparative study of the cellular localizations of plant rhabdoviral proteins. This study indicated that plant rhabdoviruses are diverse in genome sequence and viral protein interactions.
2
Kondo, Hideki, Takanori Maeda, Yukio Shirako e Tetsuo Tamada. "Orchid fleck virus is a rhabdovirus with an unusual bipartite genome". Journal of General Virology 87, n. 8 (1 agosto 2006): 2413–21. http://dx.doi.org/10.1099/vir.0.81811-0.
Abstract (sommario):
Orchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21–38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3′-UGUGUC---GACACA-5′), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.
3
Melzer, M. J., A. V. Karasev, D. M. Sether e J. S. Hu. "Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2". Journal of General Virology 82, n. 1 (1 gennaio 2001): 1–7. http://dx.doi.org/10.1099/0022-1317-82-1-1.
Abstract (sommario):
The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3′-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5′ to 3′, potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3′ terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.
4
Li, Jiapeng, Xiaoyin Wu, Hui Liu, Xiaomei Wang, Shaokui Yi, Xueting Zhong, Yaqin Wang e Zhanqi Wang. "Identification and Molecular Characterization of a Novel Carlavirus Infecting Chrysanthemum morifolium in China". Viruses 15, n. 4 (21 aprile 2023): 1029. http://dx.doi.org/10.3390/v15041029.
Abstract (sommario):
Chrysanthemum (Chrysanthemum morifolium) is an important ornamental and medicinal plant suffering from many viruses and viroids worldwide. In this study, a new carlavirus, tentatively named Chinese isolate of Carya illinoinensis carlavirus 1 (CiCV1-CN), was identified from chrysanthemum plants in Zhejiang Province, China. The genome sequence of CiCV1-CN was 8795 nucleotides (nt) in length, with a 68-nt 5′-untranslated region (UTR) and a 76-nt 3′-UTR, which contained six predicted open reading frames (ORFs) that encode six corresponding proteins of various sizes. Phylogenetic analyses based on full-length genome and coat protein sequences revealed that CiCV1-CN is in an evolutionary branch with chrysanthemum virus R (CVR) in the Carlavirus genus. Pairwise sequence identity analysis showed that, except for CiCV1, CiCV1-CN has the highest whole-genome sequence identity of 71.3% to CVR-X6. At the amino acid level, the highest identities of predicted proteins encoded by the ORF1, ORF2, ORF3, ORF4, ORF5, and ORF6 of CiCV1-CN were 77.1% in the CVR-X21 ORF1, 80.3% in the CVR-X13 ORF2, 74.8% in the CVR-X21 ORF3, 60.9% in the CVR-BJ ORF4, 90.2% in the CVR-X6 and CVR-TX ORF5s, and 79.4% in the CVR-X21 ORF6. Furthermore, we also found a transient expression of the cysteine-rich protein (CRP) encoded by the ORF6 of CiCV1-CN in Nicotiana benthamiana plants using a potato virus X-based vector, which can result in a downward leaf curl and hypersensitive cell death over the time course. These results demonstrated that CiCV1-CN is a pathogenic virus and C. morifolium is a natural host of CiCV1.
5
Shafat, Zoya. "Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus". Bioinformation 18, n. 1 (31 gennaio 2022): 19–25. http://dx.doi.org/10.6026/97320630018019.
Abstract (sommario):
Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein’s function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/β structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.
6
Shafat, Zoya. "Intrinsically disordered regions in the rodent hepevirus proteome". Bioinformation 18, n. 2 (28 febbraio 2022): 111–18. http://dx.doi.org/10.6026/97320630018111.
Abstract (sommario):
Hepatitis E virus (HEV) is the causative agent of Hepatitis E infections across the world. Intrinsically disordered protein regions (IDPRs) or intrinsically disordered proteins (IDPs) are regions or proteins that are characterized by lack of definite structure. These IDPRs or IDPs play significant roles in a wide range of biological processes, such as cell cycle regulation, control of signaling pathways, etc. IDPR/IDP in proteins is associated with the virus’s pathogenicity and infectivity. The prevalence of IDPR/IDP in rat HEV proteome remains undetermined. Hence, we examined the unstructured/disordered regions of the open reading frame (ORF) encoded proteins of rat HEV by analyzing the prevalence of intrinsic disorder. The intrinsic disorder propensity analysis showed that the different ORF proteins consisted of varying fraction of intrinsic disorder. The protein ORF3 was identified with maximum propensity for intrinsic disorder while the ORF6 protein had the least fraction of intrinsic disorder. The analysis revealed ORF6 as a structured protein (ORDP); ORF1 and ORF4 as moderately disordered proteins (IDPRs); and ORF3 and ORF5 as highly disordered proteins (IDPs). The protein ORF2 was found to be moderately as well as highly disordered using different predictors, thus, was categorized into both IDPR and IDP. Such disordered regions have important roles in pathogenesis and replication of viruses.
7
Garcia, Miguel, Madalena Pimentel e José Moniz-Pereira. "Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA". Journal of Bacteriology 184, n. 11 (1 giugno 2002): 3034–43. http://dx.doi.org/10.1128/jb.184.11.3034-3043.2002.
Abstract (sommario):
ABSTRACT A mycobacteriophage Ms6 strong promoter region (Plys ) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region Plys drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.
8
Kiatpapan, Pornpimon, Yoshiteru Hashimoto, Hisako Nakamura, Yong-Zhe Piao, Hisayo Ono, Mitsuo Yamashita e Yoshikatsu Murooka. "Characterization of pRGO1, a Plasmid from Propionibacterium acidipropionici, and Its Use for Development of a Host-Vector System in Propionibacteria". Applied and Environmental Microbiology 66, n. 11 (1 novembre 2000): 4688–95. http://dx.doi.org/10.1128/aem.66.11.4688-4695.2000.
Abstract (sommario):
ABSTRACT The complete nucleotide sequence of pRGO1, a cryptic plasmid fromPropionibacterium acidipropionici E214, was determined. pRGO1 is 6,868 bp long, and its G+C content is 65.0%. Frame analysis of the sequence revealed six open reading frames, which were designated Orf1 to Orf6. The deduced amino acid sequences of Orf1 and Orf2 showed extensive similarities to an initiator of plasmid replication, the Rep protein, of various plasmids of gram-positive bacteria. The amino acid sequence of the putative translation product of orf3 exhibited a high degree of similarity to the amino acid sequences of DNA invertase in several bacteria. For the putative translation products of orf4,orf5, and orf6, on the other hand, no homologous sequences were found. The function of these open reading frames was studied by deletion analysis. A shuttle vector, pPK705, was constructed for shuttling between Escherichia coli and a Propionibacterium strain containingorf1 (repA), orf2(repB), orf5, and orf6 from pRGO1, pUC18, and the hygromycin B-resistant gene as a drug marker. Shuttle vector pPK705 successfully transformed Propionibacterium freudenreichii subsp. shermanii IFO12426 by electroporation at an efficiency of 8 × 106 CFU/μg of DNA under optimized conditions. Transformation of various species of propionibacteria with pPK705 was also performed at efficiencies of about 104 to 107 CFU/μg of DNA. The vector was stably maintained in strains of P. freudenreichiisubsp. shermanii, P. freudenreichii, P. pentosaceum, and P. freudenreichii subsp.freudenreichii grown under nonselective conditions. Successful manipulation of a host-vector system in propionibacteria should facilitate genetic studies and lead to creation of genes that are useful industrially.
9
Li, Tong, Massoud Kheir Khah, Snjezana Slavnic, Ingegerd Johansson e Nicklas Strömberg. "Different Type 1 Fimbrial Genes and Tropisms of Commensal and Potentially Pathogenic Actinomyces spp. with Different Salivary Acidic Proline-Rich Protein and Statherin Ligand Specificities". Infection and Immunity 69, n. 12 (1 dicembre 2001): 7224–33. http://dx.doi.org/10.1128/iai.69.12.7224-7233.2001.
Abstract (sommario):
ABSTRACT Actinomyces spp. exhibit type 1 fimbria-mediated adhesion to salivary acidic proline-rich proteins (PRPs) and statherin ligands. Actinomyces spp. with different animal and tissue origins belong to three major adhesion types as relates to ligand specificity and type 1 fimbria genes. (i) In preferential acidic-PRP binding, strains of Actinomyces naeslundiigenospecies 1 and 2 from human and monkey mouths displayed at least three ligand specificities characterized by preferential acidic-PRP binding. Slot blot DNA hybridization showed seven highly conserved type 1 fimbria genes (orf1- to -6 andfimP) in genospecies 1 and 2 strains, except thatorf5 and orf3 were divergent in genospecies 1. (ii) In preferential statherin binding, oralActinomyces viscosus strains of rat and hamster origin (and strain 19246 from a human case of actinomycosis) bound statherin preferentially. DNA hybridization and characterization of the type 1 fimbria genes from strain 19246 revealed a homologous gene cluster of four open reading frames (orfA to -C andfimP). Bioinformatics suggested sortase (orfB, orf4, and part oforf5), prepilin peptidase (orfC andorf6), fimbria subunit (fimP), and usher- and autotransporter-like (orfA and orf1to -3) functions. Those gene regions corresponding toorf3 and orf5 were divergent, those corresponding to orf2, orf1, andfimP were moderately conserved, and those corresponding to orf4 and orf6 were highly conserved. Restriction fragment length polymorphism analyses using a fimP probe separated human and monkey and rat and hamster strains into phylogenetically different groups. (iii) In statherin-specific binding, strains of A. naeslundii genospecies 1 from septic and other human infections displayed a low-avidity binding to statherin. Only theorf4 and orf6 gene regions were highly conserved. Finally, rat saliva devoid of statherin bound bacterial strains avidly irrespective of ligand specificity, and specific antisera detected either type 1, type 2, or both types of fimbria on the investigated Actinomyces strains.
10
Shimizu, Takeo, Hiroshi Kinoshita e Takuya Nihira. "Identification and In Vivo Functional Analysis by Gene Disruption of ctnA, an Activator Gene Involved in Citrinin Biosynthesis in Monascus purpureus". Applied and Environmental Microbiology 73, n. 16 (22 giugno 2007): 5097–103. http://dx.doi.org/10.1128/aem.01979-06.
Abstract (sommario):
ABSTRACT Citrinin, a secondary fungal metabolite of polyketide origin, is moderately nephrotoxic to vertebrates, including humans. From the red-pigment producer Monascus purpureus, a 21-kbp region flanking pksCT, which encodes citrinin polyketide synthase, was cloned. Four open reading frames (ORFs) (orf1, orf2, orf3, and orf4) in the 5′-flanking region and one ORF (orf5) in the 3′-flanking region were identified in the vicinity of pksCT. orf1 to orf5 encode a homolog of a dehydrogenase (similarity, 46%), a regulator (similarity, 38%), an oxygenase (similarity, 41%), an oxidoreductase (similarity, 26%), and a transporter (similarity, 58%), respectively. orf2 (2,006 bp with four introns) encodes a 576-amino-acid protein containing a typical Zn(II)2Cys6 DNA binding motif at the N terminus and was designated ctnA. Although reverse transcriptase PCR analysis revealed that all of these ORFs, except for orf1, were transcribed with pksCT under citrinin production conditions, the disruption of ctnA caused large decreases in the transcription of pksCT and orf5, together with reduction of citrinin production to barely detectable levels, suggesting that these two genes are under control of the ctnA product. Complementation of the ctnA disruptant with intact ctnA on an autonomously replicating plasmid restored both transcription and citrinin production, indicating that CtnA is a major activator of citrinin biosynthesis.
Tesi sul tema "ORF2 protein":
1
Guo, Hailong. "Antigenic epitope composition and protectivity of avian hepatitis E virus (avian HEV) ORF2 protein and vertical transmission of avian HEV". [Ames, Iowa : Iowa State University], 2006.
2
Ankavay, Maliki. "Étude des modifications post-traductionnelles de la protéine de capside ORF2 du virus de l'hépatite E (HEV)". Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S013.
Abstract (sommario):
L’infection par le virus de l’hépatite E (HEV) est un problème majeur de santé publiquequi touche plus de 20 millions de personnes et tue environ 70 000 chaque année à travers lemonde. Le HEV est la cause majeure d’hépatite virale aiguë dans le monde. En France, laséroprévalence du HEV est de 22,4% dans la population générale. Ce virus se transmet parvoie féco-orale ou par consommation de viande contaminée mal cuite. Très récemment, nousavons décrit un système de culture cellulaire permettant d’amplifier efficacement le HEV. Cesystème représente un outil unique pour caractériser les différentes étapes du cycle infectieuxdu HEV qui sont très mal connues à ce jour. Dans le cadre de ma thèse, je me suis intéresséplus particulièrement à la protéine de capside ORF2 qui est l’unité structurale des particulesvirales et est donc un acteur central du cycle infectieux du HEV. La protéine ORF2 est uneprotéine de 660 acides aminés (aa) qui possède un peptide signal (PS) et trois sites potentielsde N-glycosylation (N1, N2 et N3). J’ai donc étudié le rôle de la N-glycosylation de cetteprotéine ORF2 dans le cycle infectieux du HEV. Pour atteindre cet objectif, la soucheinfectieuse HEV-p6 génotype 3 a été utilisée pour infecter les cellules de la lignéehépatocytaire PLC3, sous clone de la lignée PLC/PRF/5. Les résultats obtenus ont montré quela protéine ORF2 est N-glycosylée uniquement au niveau des sites N1 et N3 ; le site N2 estdéfavorable à la N-glycosylation. Nos résultats ont montré que la N-glycosylation de laprotéine ORF2 n’est pas importante pour sa stabilité, son oligomérisation, sa reconnaissancepar des anticorps, l’assemblage et l’infectiosité des particules virales. Nous avons aussimontré que la protéine ORF2 est importée dans le noyau des cellules hôtes au cours du cycleinfectieux du HEV de manière indépendante de la N-glycosylation. Nous avons identifié pourla première fois un signal de localisation nucléaire (NLS) fonctionnel en position N-terminalede la protéine ORF2 lui permettant d'interagir probablement avec l’importine alpha1. Demanière surprenante, ce NLS régule non seulement l’import nucléaire de cette protéine virale,mais aussi sa translocation réticulaire. Nous avons également démontré que la protéine ORF2est exportée du noyau des cellules hôtes en interagissant avec l’exportine1 et possède 3signaux d’export nucléaire (NES) en position C-terminale. Ces résultats améliorent laconnaissance du trafic intracellulaire de la protéine ORF2 et pourraient orienter les stratégiesantivirales dirigées contre le HEVHepatitis E virus (HEV) infection is a major public health problem that affects more than20 million people and kills approximately 70,000 worldwide each year. HEV is the leadingcause of acute viral hepatitis in the world. In France, the seroprevalance of HEV is 22.4% inthe general population. This virus is transmitted by fecal-oral route or by consumption ofundercooked contaminated meat. Very recently, we have described an efficient cell culturesystem to amplify HEV. This system represents a unique tool for characterizing the variousstages of the infectious HEV lifecycle that are very poorly understood to date. As part of mythesis, I focused on the ORF2 capsid protein which is the structural unit of viral particles andis therefore a central player in the HEV lifecycle. ORF2 is a 660 amino acids protein thatcontains a signal peptide and three potential N-glycosylation sites (N1, N2 and N3). My workaimed to identify the role of the ORF2 N-glycosylation in the HEV lifecycle. Our resultsrevealed that the ORF2 protein is N-glycosylated on the N1 and N3 sites, whereas the N2 siteis not N-glycosylated. Also, the N-glycosylation has no significant relevance neither for thestability, oligomerization, antibody recognition of the ORF2 protein nor for viral assemblyand viral infectivity. We also revealed that, the ORF2 protein is translocated into the nucleusof infected cells, independently of N-glycosylation. We identified for the first time afunctional nuclear localization signal (NLS) located at the N-terminus of the ORF2 proteinthat interacts probably with the importin alpha1. Surprisingly, this NLS regulates both nuclearimport and endoplasmic reticulum translocation of the ORF2 protein. Finally, wedemonstrated that, the ORF2 protein possesses three nuclear export signals (NES) located atits C-terminus. The ORF2 protein interacts with the exportin1 and is exported from the cellnuclei. This interaction drives the ORF2 protein from the nucleus to the cytoplasm of theinfected cells. Hence, this study led to new insights into the molecular mechanisms of theORF2 protein and could help to the design of novel antiviral strategies against HEV
3
Ferrié, Martin. "Étude de la maturation protéolytique et du trafic intracellulaire de la protéine de capside ORF2 du virus de l'hépatite E (HEV)". Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS067.
Abstract (sommario):
L'infection par le virus de l'Hépatite E (HEV) est un problème majeur de santé publique qui toucherait 100 millions de personnes et tuerait 100 000 personnes chaque année dans le monde. Le HEV est la première cause d'hépatite aigüe dans le monde. En France, la séroprévalence s'élève à 22,4%. Ce virus se transmet par voie féco-orale ou par la consommation de viande contaminée mal cuite. Au cours de ma thèse, je me suis intéressé plus particulièrement à la protéine de capside ORF2, qui est l'unité structurale des particules virales et un acteur central du cycle infectieux du HEV. La protéine ORF2 est une protéine de 660 acides aminés qui possède un peptide signal N-terminal et trois sites potentiels de N-glycosylation. Au cours de son cycle infectieux, le HEV produit au moins trois formes de sa protéine de capside : (i) la forme ORF2g (pour glycosylée) et (ii) la forme ORF2c (pour clivée), abréviées ORF2g/c, qui sont des formes glycosylées et massivement sécrétées dans les surnageants de culture ou le sérum des patients infectés, et (iii) la forme ORF2i (pour infectieuse), qui n'est pas glycosylée et qui est associée aux particules virales.Dans le cadre de mes travaux de thèse, j'ai étudié les mécanismes de maturation protéolytique et de trafic intracellulaire de la protéine ORF2. Plus précisément, j'ai d'abord montré que la protéine ORF2 était importée dans le noyau des cellules infectées par le biais d'un mécanisme dépendant de l'Importine-alpha1, ceci grâce à un motif riche en résidus arginine situé à l'extrémité N-terminale de l'ORF2 et nommé ARM. La protéine ORF2 est ensuite exportée vers le cytoplasme par un mécanisme dépendant de l'exportine CRM1, ceci grâce à trois sites d'export nucléaire (NES9, NES10 et NES12) que nous avons identifiés dans la séquence de l'ORF2. J'ai également montré que le trafic nucléo-cytoplasmique de l'ORF2 régule probablement l'expression de certains gènes de l'immunité antivirale aux temps précoces de l'infection.Dans un second temps, j'ai participé à l'identification et à la caractérisation des usines virales du HEV. En ce sens, nous avons montré que les protéine virales ORF1, ORF2 et ORF3, ainsi que l'ARN viral sont enrichis dans des structures vésiculaires et tubulaires localisées dans les régions périnucléaires des cellules infectées. Nous avons montré que ces structures sont également enrichies en marqueurs du compartiment endosomal de recyclage (ERC), comme Rab11 et CD71, indiquant que les usines virales du HEV dérivent probablement de l'ERC. En réalisant des expériences d'extinction de Rab11 avec des ARN interférents, nous avons confirmé l'importance de l'ERC dans la production des particules virales du HEV.Dans un troisième temps, j'ai démontré que la protéine ORF2i, qui est associée aux membranes de la voie de sécrétion, est adressée aux usines virales par un mécanisme faisant intervenir le complexe adapteur AP-1.Dans un quatrième temps, j'ai caractérisé les mécanismes de maturation protéolytique des formes ORF2g/c et ORF2i. J'ai montré que la furine, une proproprotéine convertase de la voie de sécrétion, est impliquée dans la maturation protéolytique des glycoprotéines ORF2g/c. J'ai également montré que la préséniline, qui est la sous-unité catalytique du macro-complexe Gamma-sécrétase, est impliquée dans la maturation protéolytique de la forme infectieuse ORF2i. De manière intéressante, j'ai montré que l'inhibition pharmacologique de la préséniline réduit drastiquement l'infectiosité virale dans des lignées d'hépatocarcinome humain et dans des hépatocytes primaires humains. Ces données suggèrent que l'inhibition pharmacologique de la préséniline représente une stratégie antivirale prometteuse.En conclusion, les résultats obtenus dans le cadre de ma thèse permettent de mieux comprendre les mécanismes d'adressage subcellulaire et de maturation protéolytique de la protéine de capside ORF2, et ouvrent la voie au développement de nouvelles thérapies pour lutter contre le HEVHepatitis E virus (HEV) infection is a major public health problem, affecting an estimated 100 million people and killing 100,000 every year worldwide. HEV is the leading cause of acute hepatitis worldwide. In France, seroprevalence is 22.4%. This virus is transmitted via the feco-oral route, or by eating contaminated undercooked meat. During my thesis, I focused on the ORF2 capsid protein, which is the structural unit of viral particles and a central player in the HEV lifecycle. ORF2 is a 660 amino acid protein with an N-terminal signal peptide and three potential N-glycosylation sites. During its lifecycle, HEV produces at least three forms of its capsid protein: (i) the ORF2g (for glycosylated) form and (ii) the ORF2c (for cleaved) form, abbreviated ORF2g/c, which are glycosylated forms and massively secreted in culture supernatants or serum from infected patients, and (iii) the ORF2i (for infectious) form, which is not glycosylated and is associated with viral particles.As part of my thesis work, I studied the mechanisms of proteolytic maturation and intracellular trafficking of the ORF2 protein. More specifically, I first showed that ORF2 is imported into the nucleus of infected cells via an Importin-alpha1-dependent mechanism, thanks to an arginine-rich motif located at the N-terminus of ORF2 and named ARM. The ORF2 protein is then exported to the cytoplasm via a CRM1 exportin-dependent mechanism, thanks to three nuclear export sites (NES9, NES10 and NES12) that we have identified in the ORF2 sequence. I have also shown that nucleocytoplasmic trafficking of ORF2 probably regulates the expression of certain antiviral immunity genes in the early stages of infection.Secondly, I participated in the identification and characterization of HEV viral factories. We have shown that the viral proteins ORF1, ORF2 and ORF3, as well as viral RNA, are enriched in vesicular and tubular structures located in the perinuclear regions of infected cells. We have shown that these structures are also enriched in markers of the endosomal recycling compartment (ERC), such as Rab11 and CD71, indicating that HEV viral factories probably derive from the ERC. By performing Rab11 silencing experiments with siRNAs, we confirmed the importance of the ERC in the production of HEV viral particles. Thirdly, I demonstrated that the ORF2i protein, which is associated with the membranes of the secretion pathway, is addressed to viral factories by a mechanism involving the AP-1 adaptor complex.In a fourth step, I characterized the proteolytic maturation mechanisms of the ORF2g/c and ORF2i forms. I showed that furin, a proproprotein convertase of the secretory pathway, is involved in the proteolytic maturation of ORF2g/c glycoproteins. I have also shown that presenilin, which is the catalytic subunit of the macro-complex Gamma-secretase, is involved in the proteolytic maturation of the ORF2i form. Interestingly, I have shown that pharmacological inhibition of presenilin drastically reduces viral infectivity in human hepatocarcinoma lines and in primary human hepatocytes. These data suggest that pharmacological inhibition of presenilin represents a promising antiviral strategy. In conclusion, the results obtained during my thesis provide a better understanding of the mechanisms of subcellular addressing and proteolytic maturation of the ORF2 capsid protein, and pave the way for the development of new therapies to combat HEV
4
Horridge, Jackie J. "RNA-protein interactions of the adenovirus proteins E1B 55K and E4 Orf6". Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322435.
5
Baghbadorani, G. Ahmadian. "Analysis of the utilisation of second ORFs in pneumovirus mRNA". Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323416.
6
Chand, Puran. "Molecular and immunological characterisation of a major envelope protein of capripoxvirus". Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/2774/.
Abstract (sommario):
Analysis of the proteins of capripoxvirus (KS-1) revealed a 32kd protein that is one of the major structural proteins of the virus and is localised in the virus envelope. Monospecific serum prepared against the 32kd envelope protein neutralised the virus indicating that this protein contains neutralising epitopes. Lymphocyte proliferation studies, using the 32kd protein and peripheral blood mononuclear cells from capripoxvirus (KS-i) vaccinated sheep, showed that this protein strongly induced cellmediated immune responses. The 32kd protein is capripoxvirus specific and induced antibodies in early stages of capripoxvirus infections. Immunoblot analysis of antibody responses against this protein has provided a basis for the differential diagnosis of capripoxvirus and orf virus infections. The 32kd protein bound to the surface of cultured lamb testis cells. The binding of the 32kd protein was completely inhibited by prior incubation of cells with purified capripoxvirus (KS-1) but not by bovine serum albumin. Trypsin treatment of capripoxvirus (KS-1) degraded the majority of the 32kd protein with a minimal effect on a few other virus proteins. Trypsin removed an external 10kd fragment from the 32kd protein, leaving a 22kd fragment associated with the virus. In addition, the trypsin treatment reduced the virus infectivity by at least ten fold, suggesting that the cell surface binding domain of the 32kd protein is located within the external 10kd fragment. The monospecific serum to the 32kd protein had no effect of the infectivity titre of the trypsin treated virus further supporting the concept that the external 10kd fragment of the 32kd protein is involved in binding of the virus particle to the cell surface. A degenerate oligonucleotide probe, based on an internal amino acid sequence obtained from V8 protease cleavage products of the 32kd protein, was used to identify the gene encoding the 32kd protein. The gene encoding the 32kd protein was identified within the 2.8kb HindI1l Q1 fragment of the capripoxvirus (KS-1) genome. The nucleotide sequence analysis of the Hindu Q1 fragment revealed five open reading frames (Q11L, Q12R, Q13L, Q14R and Q15L), one of these open reading frames, Q13L, is capable of encoding a 30.6kd protein and contains the complete internal amino acid sequence obtained from the V8 protease cleavage products of the 32kd protein, indicating that the Q13L encodes the 32kd envelope protein of capripoxvirus (KS-1). The deduced amino acid sequence of the Q13L shows a 34.1% identity and 61.3% similarity with that of H3L open reading frame of vaccinia virus.
7
Koyuncu, Emre. "Expression, Purification, And Functional Analysis Of Adenovirus Type 5 E4 Orf3 Protein". Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/12605415/index.pdf.
Abstract (sommario):
In this study, structural and functional aspects of adenovirus type 5 (Ad5) E4orf3 protein were analyzed by biophysical and biochemical methods. Ad5 is one of the mostly used gene therapy vectors to date. However, some of its proteins possess oncogenic potential and their presence comprises safety risks. E4orf3 is one of the oncoproteins of Ad5. It also takes important roles in viral infection, and is beneficial for therapy vectors. Therefore, understanding the functions of E4orf3 is very important for developing efficient and safe adenovirus vectors.
Most of the present knowledge about the functions of E4orf3 comes from its mutational analysis. It has never been expressed or purified successfully due to its extreme insolubility. Therefore, this study focused on the optimization of expression of E4orf3 protein. As a result, full-length E4orf3 was obtained in soluble form as a Glutathione-S-transferase (GST) fusion protein and purified by GST affinity chromatography for the first time. Subsequently, the interaction of E4orf3 with four different proteins, DNA-PK, Aup1, E1B-55 kDa and E4orf6 was analyzed in detail by GST-pulldown technique. In these experiments, E4orf3 was shown to associate with Aup1, E1B-55 kDa and E4orf6 in vitro, and the C-terminal of E4orf3 was determined to be responsible for these interactions. Finally, basic structural information about E4orf3 protein was also obtained for the first time by the direct analysis of the fusion protein in glutathione beads with Fourier Transform Infrared (FTIR) spectroscopy. Since the purified E4orf3 protein could not be separated from the glutathione beads due to its hydrophobic regions, the secondary structures in this protein were determined after subtracting glutathione and H2O absorption bands, and the GST moiety.
8
Bowers, Laura Yvonne. "Orf protein modulates phage and bacterial pathways of genetic recombination". Thesis, Durham University, 2008. http://etheses.dur.ac.uk/2345/.
Abstract (sommario):
The emergence of novel pathogenic organisms due to the acquisition of virulence determinants from bacteriophages has generated significant interest in the pathways responsible for genomic rearrangements. Phageλ encodes its own recombination system, the Red system, comprising Exo, β and γ proteins. In addition,λ encodes another recombinase, Orf, which participates in the initial stages of genetic exchange and supplies a frmction equivalent to that of the Escherichia coli RecFOR proteins. This thesis focuses on determining the function of Orf in phage and bacterial recombination pathways by analysing its impact on recombinases encoded by λ and E. coli. Experiments revealed that Orf interacts with bacterial and phage recombination proteins in the initial exchange step of recombination, modulating the activities of both Exo and RecA. Orf, along with β, attenuates the 5'-3' exonuclease activity of Exo, a feature that depends largely on the ability of Orf to bind DNA. Orf also facilitates loading of RecA onto ssDNA pre-coat SSB but only if a ssDNA:dsDNA intersection is incorporated in the substrate. A motif similar to that found at the BRCA2-Rad51 interface may be responsible for Orf mimicking a RECA monomer to initiate nucleoprotein filament formation. Significantly, this would direct recombination down the bacterial RecA pathway of break restoration rather than the phage Red pathway with potentially important consequences for the outcome of the exchange reaction.
9
Liedeman, Kerwin. "Transient transgene expression of human Coronavirus nl63 orf3 protein in a baculovirus system". University of the Western Cape, 2020. http://hdl.handle.net/11394/8042.
Abstract (sommario):
>Magister Scientiae - MScInsect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.
10
Yadav, Kush Kumar. "Genotype 1 hepatitis E virus (HEV) ORF4 protein enhances genotype 3 HEV replication". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574781581580768.
Libri sul tema "ORF2 protein":
1
Horridge, Jackie J. RNA-protein interactions of the adenovirus proteins E1B 55K and E4 Orf6. [s.l.]: typescript, 1998.
2
Manfred, Schlapp, PEN-Club Liechtenstein e ORF Landesstudio Vorarlberg (Austria), a cura di. Das verrantene Gewissen: Unser Tun und Lassen wider besseres Wissen : ein öffentliches Symposion des PEN-Club Lichtenstein in Zusammenarbeit mit dem ORF Landesstudio Vorarlberg. Vaduz: PEN-Club Liechtenstein, 1989.
3
Meng, X. J. Hepatitis E virus. Oxford University Press, 2011. http://dx.doi.org/10.1093/med/9780198570028.003.0048.
Abstract (sommario):
Hepatitis E virus (HEV) is a small, non-enveloped, single-strand, positive-sense RNA virus of approximately 7.2 kb in size. HEV is classified in the family Hepeviridae consisting of four recognized major genotypes that infect humans and other animals. Genotypes 1 and 2 HEV are restricted to humans and often associated with large outbreaks and epidemics in developing countries with poor sanitation conditions, whereas genotypes 3 and 4 HEV infect humans, pigs and other animal species and are responsible for sporadic cases of hepatitis E in both developing and industrialized countries. The avian HEV associated with Hepatitis-Splenomegaly syndrome in chickens is genetically and antigenically related to mammalian HEV, and likely represents a new genus in the family. There exist three open reading frames in HEV genome: ORF1 encodes non-structural proteins, ORF2 encodes the capsid protein, and the ORF3 encodes a small phosphoprotein. ORF2 and ORF3 are translated from a single bicistronic mRNA, and overlap each other but neither overlaps ORF1. Due to the lack of an efficient cell culture system and a practical animal model for HEV, the mechanisms of HEV replication and pathogenesis are poorly understood. The recent identification and characterization of animal strains of HEV from pigs and chickens and the demonstrated ability of cross-species infection by these animal strains raise potential public health concerns for zoonotic HEV transmission. It has been shown that the genotypes 3 and 4 HEV strains from pigs can infect humans, and vice versa. Accumulating evidence indicated that hepatitis E is a zoonotic disease, and swine and perhaps other animal species are reservoirs for HEV. A vaccine against HEV is not yet available.
Capitoli di libri sul tema "ORF2 protein":
1
Cernooka, Elina, Janis Rumnieks e Andris Kazaks. "Structural Characterization of a Single-Stranded DNA-Binding Protein: A Case Study of the ORF6 Protein from Bacteriophage Enc34". In Methods in Molecular Biology, 343–73. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1290-3_23.
2
Yamakawa, Hisashi. "High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins". In Methods in Molecular Biology, 25–39. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60761-232-2_3.
3
Flint, S. J., e R. A. Gonzalez. "Regulation of mRNA Production by the Adenoviral E1B 55-kDa and E4 Orf6 Proteins". In Current Topics in Microbiology and Immunology, 287–330. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/978-3-662-05597-7_10.
4
Nigam, Mohit, Ruchi Yadav e Garima Awasthi. "In-Silico Construction of Hybrid ORF Protein to Enhance Algal Oil Content for Biofuel". In Advances in Biomedical Engineering and Technology, 67–89. Singapore: Springer Singapore, 2020. http://dx.doi.org/10.1007/978-981-15-6329-4_8.
5
Czerny, Claus-Peter, Michaela Alex, Jana Pricelius e Christiane Zeller-Lue. "Development of Quantitative PCR Tests for the Detection of the Orthopox Virus Adsorption Protein Gene (ORF D8L) on the LightCycler". In Rapid Cycle Real-Time PCR, 371–79. Berlin, Heidelberg: Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/978-3-642-59524-0_40.
6
Ferro-Novick, S. "BET2". In Secretory Pathway, 131. Oxford University PressOxford, 1994. http://dx.doi.org/10.1093/oso/9780198599425.003.0081.
Abstract (sommario):
Abstract ORF2, an essential yeast gene of unknown function. The DNA sequences of BET2 and ORF2 are identical with the exception of one nucleotide alteration that results in a conserved amino acid change in the protein (asparagine in Orf2 and lysine in Bet2 at position 22)213. This change may be attributable to differences in strain backgrounds.
7
Ferro-Novick, Susan. "Bet2p". In Guidebook to the Sinall GTPases, 61–62. Oxford University PressOxford, 1995. http://dx.doi.org/10.1093/oso/9780198599456.003.0015.
Abstract (sommario):
Abstract The DNA sequence of BET2 predicts a hydrophilic protein of-36 500 daltons (Peterson-Bjorn et al. 1990; Rossi et al. 1991). Antibody raised to this protein has confirmed this prediction (Jiang et al. 1993). Bet2 shares 56.6% similarity and 34% identity with Dpr1 (Ram1), an essential component of a protein prenyltransferase that adds farnesyl (a 15 carbon lipid) to the COCH-terminus of Ras and the yeast mating pheromone afactor (Schafer et al. 1990; Goodman et al. 1990; He et al. 1991).8ET2wasalsofoundto be the same as ORF2, an essential yeast gene of unknown function (Peterson-Bjorn et al. 1990; Rossi et al. 1991). The DNA sequences of BET2 andORF2 are identical with one exception that results in a conserved amino acid change (asparagine in Orf2 and lysine in Bet2 at position 22) in the protein. This change may be attributable to differences in strain backgrounds.
8
Sriram, Anitha, Ravindra Vasave, Indrani Maji, Rahul Kumar, Dharmendra Kumar Khatri, Shashi Bala Singh, Neelesh K. Mehra e Pankaj Kumar Singh. "Virology of SARS-CoV2". In An Update on SARS-CoV-2: Damage-response Framework, Potential Therapeutic Avenues and the Impact of Nanotechnology on COVID-19 Therapy Volume 1, 16–42. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9789815039863122010005.
Abstract (sommario):
The 2019-nCoV RNA genome is highly protected by its unique structure equipped with mechanistic spike proteins on its surface. Its RNA genome contains almost all 14 Orfs encoding for at least 27 proteins. An Orf is a part of genetic material that can undergo translation and produce proteins. Four structural proteins (SPs) likely spike (S), envelope (E), membrane (M), and nucleocapsid (N) are present in 2019- nCoV and afford its structure. In this chapter, we have discussed in detail the virology of 2019-nCoV including structural proteins (SPs), accessory proteins, non-structural proteins (NSPs), genomic structure, and its components. The role of SPs, accessory proteins, NSPs of 2019-nCoV is discussed and this review also explains, how the interaction of 2019-nCoV occurs with that of hACE2. Additionally, topics such as stabilization of virus-binding hotspots on hACE2 by 2019-nCoV, the role of thioldisulfide interchanges in the interplay between 'S' protein and hACE2, and the similarity (in terms of amino acid sequence homology) (%) of 2019-nCoV with SARS-COV are discussed.
9
Skolnick, Jeffrey, e Yang Zhang. "Protein Structure Prediction". In Systems Biology, 187–218. Oxford University PressNew York, NY, 2006. http://dx.doi.org/10.1093/oso/9780195300819.003.0007.
Abstract (sommario):
Abstract Over the past decade, the success of genome sequence efforts has brought about a paradigm shift in biology [1]. There is increasing emphasis on the large-scale, high-throughput examination of all genes and gene products of an organism, with the aim of assigning their functions [2]. Of course, biological function is multifaceted, ranging from molecular/biochemical to cellular or physiological to phenotypical [3]. In practice, knowledge of the DNA sequence of an organism and the identification of its open reading frames (ORFs) does not directly provide functional insight. Here, the focus is on the proteins in a genome, namely, the proteome, but recognizes that proteins are only a subset of all biologically important molecules and addresses aspects of molecular/biochemical function and protein–protein interactions.
10
Kondegowda, Nagesha Guthalu, Sheela Joshi Gokhale, George Harb, Katoura Williams, Xiaoying Zhang, Karen K. Takane, Pili Zhang et al. "Parathyroid Hormone Related Protein (PTHrP) Enhances Human β-Cell Proliferation and Function with Simultaneous Induction of Cyclin-Dependent Kinase 2 (cdk2) and Cyclin E Expression." In The Endocrine Society's 92nd Annual Meeting, June 19–22, 2010 - San Diego, OR32–1—OR32–1. Endocrine Society, 2010. http://dx.doi.org/10.1210/endo-meetings.2010.part3.or2.or32-1.
Atti di convegni sul tema "ORF2 protein":
1
Stoicescu, Ramona, Razvan-Alexandru Stoicescu, Codrin Gheorghe, Adina Honcea e Iulian Bratu. "CONSIDERATIONS ON SARS-COV-2 DIAGNOSIS IN THE LABORATORY OF UNIVERSITY EMERGENCY CLINICAL HOSPITAL OF CONSTANTA". In GEOLINKS Conference Proceedings. Saima Consult Ltd, 2021. http://dx.doi.org/10.32008/geolinks2021/b1/v3/07.
Abstract (sommario):
Coronaviruses are members of the Coronaviridae family. They are enveloped, non-segmented, positive-sense, single-stranded RNA viruses. Their genome size is about 30 kilobases (kb) which consist, at the 5’ end, of non-structural open reading frames (ORFs: ORF1a, ORF 1b) which code for 16 non structural proteins, and at the 3’ end the genes which code for four structural proteins including membrane (M), envelope (E), spike (S), and nucleocapsid (N) proteins. Due to the rapid spread of COVID-19, a reliable detection method is needed for patient diagnosis especially in the early stages of the disease. WHO has recommended nucleic acid amplification tests such as real-time reverse transcription-polymerase chain reaction (RT-PCR). The assay detects three SARS-CoV-2 RNA targets: the envelope (E) gene, the nucleocapsid (N) gene and a region of the open reading frame (ORF1) of the RNA-dependent RNA polymerase (RdRp) gene from SARS-CoV-2 virus isolate Wuhan-Hu-1. Our study was made in the first 3 months of the year 2021 using the real-time RT PCR results obtained in the Cellular Biology ward of the University Emergency Clinical Hospital. In our lab we are testing the inpatients from the hospital wards (Neurology, Pediatrics, Surgery, Internal medicine, ICU, Cardiology, etc.); we are also testing the outpatients from Dialysis and Oncology, 2 days prior to their therapy; we also test the health care personnel. The number of tests we performed was: in January 1456, with 399 positive results (27.4%), 33 deaths; in February 1273 tests, 221 positive (17.36%), 16 deaths; in March 1471 tests, 373 positive (25.36%), 37 deceased.
2
Yang, Fan, Hong-Liang Liu, Xiao-Wan Liu, Bo Guo, Song Li, Ping Li e Ling Zhug. "Development of a TTSuV2-ORF1 Protein-based Indirect Blocking ELISA for Serological Testing". In 2015 International Conference on Medicine and Biopharmaceutical. WORLD SCIENTIFIC, 2016. http://dx.doi.org/10.1142/9789814719810_0023.
3
Shan, Si, Nie Peng, Xiaojun Yan e Caifeng Ba. "Study on the secondary structure and B-cell epitopes of Mycoplasma suis ORF9 protein by Bioinformatics". In 2015 International Power, Electronics and Materials Engineering Conference. Paris, France: Atlantis Press, 2015. http://dx.doi.org/10.2991/ipemec-15.2015.194.
4
"Lettuce Big Vein associated Virus ORF3 encodes a 30K-like protein that facilitates cell-to-cell movement". In IS-MPMI Congress. IS-MPMI, 2023. http://dx.doi.org/10.1094/ismpmi-2023-50.
5
Saksaganskaia, Alla S., Victoria S. Muntyan, Alexey N. Muntyan, Boris V. Simarov e Marina L. Roumiantseva. "ABUNDANCE OF PHAGE-RELATED SEQUENCES ON NON-SYMBIOTIC PLASMIDS OF SINORHIZOBIUM MELILOTI FROM CENTERS OF LEGUME PLANTS DIVERSITY". In 22nd SGEM International Multidisciplinary Scientific GeoConference 2022. STEF92 Technology, 2022. http://dx.doi.org/10.5593/sgem2022/6.1/s25.06.
Abstract (sommario):
Genomes of alfalfa root nodule bacteria, Sinorhizobium meliloti, symbionts of alfalfa are enriched in non-symbiotic (cryptic) plasmids, which gene pool is remained weakly studied. S. meliloti strains are significantly varied in number and size of these plasmids. The goal of the study was to assess the occurrence of phage-related sequences (PRS) on cryptic plasmids. Whole genome sequences of 12 S. meliloti strains native to Caucasian and Kazakhstan centers of alfalfa diversity (NCG and PAG, correspondingly) were studied and 20 cryptic plasmids, which sizes varied from 17.2 to 453.8 kb, were assembled. In total 55 PRS were identified on cryptic plasmids, and these sequences were represented by intact, questionable and incomplete sequences according to PHASTER. Significant differences in the occurrence of above-mentioned types of PRS on cryptic plasmids was detected between strains native to NCG and PAG (X2 = 6.73, p = 0.03). The sizes of the desired PRS varied from 5.1 to 33 kb, and their number was from 1 to 11 per replicon in tested strains. It was revealed that PRS on plasmids of strains from NCG were predominantly related to Siphoviridae family (p smaller than 0.05), while PRS homologous to phages of Siphoviridae and Podoviridae families prevailed with equal frequencies on plasmids of strains from PAG. For 40% of tested PRS the attL/attR sequences were detected and that is proving their site-specific integration type. ORFs of PRS as it was revealed are encoded integrases, fiber protein and tail shaft, and nearly all PRS are contained ORFs encoded transposases. Summarizing, S. meliloti strains native to origins of alfalfa diversity are enriched in cryptic plasmids, and the latest are attractive for soil bacteriophages, that is strongly evident the participation of small size plasmids in horizontal gene transfer process.
6
Nogueira, Raniery Augusto dos Santos Beserra, Ana Beatriz Silva Barbosa, Francisco Das Chagas Diassis Jácome Valentim, Sâmya Pires Batista De Azevêdo e Jamile Rodrigues Cosme De Holanda. "MECANISMOS DE MANUTENÇÃO DA LATÊNCIA DO VÍRUS VARICELA ZÓSTER: UMA ADAPTAÇÃO NECESSÁRIA". In I Congresso Nacional de Microbiologia Clínica On-Line. Revista Multidisciplinar em Saúde, 2021. http://dx.doi.org/10.51161/rems/1182.
Abstract (sommario):
Introdução: O Vírus Varicela Zóster pertence à subfamília α-herpesviridae e é um DNA-vírus de fita dupla, possuindo 68 Fases de Leitura Aberta (ORF) únicas. Sendo responsável por varicela, em um primeiro contato, o vírus mantém-se latente por um longo período em gânglios das raízes dorsais e de nervos cranianos, disseminando-se por essas estruturas após décadas, quando reativado, configurando Herpes Zóster, cujas complicações prejudicam a qualidade de vida dos pacientes. Posteriormente à infecção primária, o vírus classifica-se como neurotrópico e seus meios de sobrevida em neurônios, ainda não são claros. Objetivo: Este estudo busca elucidar os mecanismos de latência desse patógeno, visando ao desenvolvimento do conhecimento científico no assunto. Material e Métodos: Utilizando-se da plataforma online PubMed, foram pesquisados os descritores “varicella zoster virus”, em adição a “apoptosis”, e “herpes zoster infection”, com o operador booleano “AND” tendo como filtro a delimitação temporal entre 01/01/2018 e 01/05/2021. Resultados: Ao longo do período de latência, o DNA viral é encontrado em células neuronais, estando em forma não-integrada (cita-se epissoma infinito ou comprimento concateméro) e sua transcrição é fortemente restrita. Em meio ao genoma viral, o ORF63 mostrou-se de grande relevância na proteção de neuronal em humanos. Durante monitorização, foi-se percebido a migração da proteína OFR63 quando se induz apoptose na célula, tornando-se mais citoplasmática. A partir disso, percebeu-se interação inibitória entre a proteína ORF63 e a estaurosporina, molécula indutora da apoptose. O que até então se mostrava como hipótese, foi melhor embasado pela redução dos níveis de caspase-3, marcador apoptótico celular, em neurônios infectados pelo VVZ quando comparados a outras células (infectadas ou não). Conclusão: Diante de todas as questões, ainda cientificamente obscuras, acerca da capacidade desse vírus de sobreviver durante décadas nos gânglios nervosos, ressalta-se a necessidade de mais pesquisas na área, para que se tenha melhor manejo de pacientes infectados com o Vírus Varicela Zóster. Ademais, não se pode negar que a inibição da apoptose é uma evolução adaptativa muito favorável ao microrganismo, já que as células neuronais são hospedeiras senescentes e seu aumento de vida significa aumento de vida do patógeno.
7
Silva, Victoria Regina Da, Gabriela Rodrigues De Aguiar, Jackson Nascimento De Souza e Rhayanny Kethylly Pereira Santos. "CORRELAÇÃO DOS RETROVÍRUS ENDÓGENOS HUMANOS (HERVS) COM A SEVERIDADE DOS CASOS DE COVID-19 ABRE A POSSIBILIDADE DE SEREM USADOS COMO MARCADORES DE PROGNÓSTICO NA DOENÇA". In I Congresso Brasileiro de Imunologia On-line. Revista Multidisciplinar em Saúde, 2021. http://dx.doi.org/10.51161/rems/1150.
Abstract (sommario):
Introdução: A COVID-19 é uma doença pandêmica com graves consequências socioeconômicas causada pelo vírus SARS-COV-2. Ela é caracterizada por sintomas respiratórios leves a graves, como pneumonia e falência de órgãos. Comorbidades e idade avançada são fatores de risco para a doença, todavia, a intensidade da doença não é explicada somente por esses fatores, o que evidencia que mecanismos ainda desconhecidos contribuem no seu prognóstico. HERVs são fragmentos virais incorporados no genoma humano que agravam danos teciduais quando superexpressos em algumas condições patológicas, como neurodegeneração. Estudos recentes investigaram sua correlação com os quadros de COVID-19, tendo relevância para definir biomarcadores de prognóstico na doença. Objetivos: Investigar se os dados da literatura sustentam a hipótese de que HERVs estão associados com o agravamento do quadro da COVID-19, sendo potenciais biomarcadores de prognóstico. Material e métodos: Artigos foram buscados nos bancos de dados Portal Capes, ScienceDirect e Scholar Google utilizando os descritores “HERV” e “COVID-19”. Os critérios de inclusão foram: ser um artigo experimental, correlacionar HERVs com a infecção pelo SARS-COV-2 e ter sido publicado entre 2020-2021. 7 artigos atenderam aos requisitos e foram revisados. Resultados: HERVs foram associados à intervenção leucocitária em pacientes com COVID-19, assim como a marcadores de senescência CD57, mediadores pró-inflamatórios (IL-6, IL-10, TNF) e quimiocinas (CCL-2, CXCL6). Através da produção proteica e de miRNA modulam o padrão de ORFs COVID-19 e também alteram a ribose do RNA viral, facilitando a introdução na célula hospedeira. Está ligado ainda à gravidade da doença, pois há achados semelhantes ao imunofenótipo I. O aumento da transcrição do mRNA correlaciona-se com a severidade da pneumonia, impactando a necessidade de oxigenação. Pacientes saudáveis apresentaram expressão normal de HERVs. Conclusão: A superexpressão dos HERVs associa-se a casos graves de COVID-19 pela influência na expressão de citocinas e quimiocinas, já consideradas marcadores de mau prognóstico.
Rapporti di organizzazioni sul tema "ORF2 protein":
1
Gafny, Ron, A. L. N. Rao e Edna Tanne. Etiology of the Rugose Wood Disease of Grapevine and Molecular Study of the Associated Trichoviruses. United States Department of Agriculture, settembre 2000. http://dx.doi.org/10.32747/2000.7575269.bard.
Abstract (sommario):
Rugose wood is a complex disease of grapevines, characterized by modification of the woody cylinder of affected vines. The control of rugose wood is based on the production of healthy propagation material. Detection of rugose wood in grapevines is difficult and expensive: budwood from tested plants is grafted onto sensitive Vitis indicators and the appearance of symptoms is monitored for 3 years. The etiology of rugose wood is complex and has not yet been elucidated. Several elongated clostero-like viruses are consistently found in affected vines; one of them, grapevine virus A (GVA), is closely associated with Kober stem grooving, a component of the rugose wood complex. GVA has a single-stranded RNA genome of 7349 nucleotides, excluding a polyA tail at the 3' terminus. The GVA genome includes five open reading frames (ORFs 1-5). ORF 4, which encodes for the coat protein of GVA, is the only ORF for which the function was determined experimentally. The original objectives of this research were: 1- To produce antisera to the structural and non-structural proteins of GVA and GVB and to use these antibodies to establish an effective detection method. 2- Develop full length infectious cDNA clones of GVA and GVB. 3- Study the roll of GVA and GVB in the etiology of the grapevine rugose wood disease. 4- Determine the function of Trichovirus (now called Vitivirus) encoded genes in the virus life cycle. Each of the ORFs 2, 3, 4 and 5 genes of GVA were cloned and expressed in E. coli and used to produce antisera. Both the CP (ORF 4) and the putative MP (ORF 3) were detected with their corresponding antisera in-GVA infected N. benthamiana and grapevine. The MP was first detected at an early stage of the infection, 6-12 h after inoculation, and the CP 2-3 days after inoculation. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available ELISA kit. Antisera to ORF 2 and 5 encoded proteins could react with the recombinant proteins but failed to detect both proteins in GVA infected plants. A full-length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro transcribed RNA was infectious in N. benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. The full-length clone was modified to include a gus reporter gene and gus activity was detected in inoculated and systemic leaves of infected plants. Studies of GVA mutants suggests that the coat protein (ORF 4) is essential for cell to cell movement, the putative movement protein (ORF 3) indeed functions as a movement protein and that ORF 2 is not required for virus replication, cell to cell or systemic movement. Attempts to infect grapevines by in-vitro transcripts, by inoculation of cDNA construct in which the virus is derived by the CaMV 35S promoter or by approach grafting with infected N. benthamiana, have so far failed. Studies of the subcellular distribution of GFP fusion with each of ORF 2, 3 and 4 encoded protein showed that the CP fusion protein accumulated as a soluble cytoplasmatic protein. The ORF 2 fusion protein accumulated in cytoplasmatic aggregates. The MP-GFP fusion protein accumulated in a large number of small aggregates in the cytoplasm and could not move from cell to cell. However, in conditions that allowed movement of the fusion protein from cell to cell (expression by a PVX vector or in young immature leaves) the protein did not form cytoplasmatic aggregates but accumulated in the plasmodesmata.
2
Coplin, David, Isaac Barash e Shulamit Manulis. Role of Proteins Secreted by the Hrp-Pathways of Erwinia stewartii and E. herbicola pv. gypsophilae in Eliciting Water-Soaking Symptoms and Initiating Galls. United States Department of Agriculture, giugno 2001. http://dx.doi.org/10.32747/2001.7580675.bard.
Abstract (sommario):
Many bacterial pathogens of plants can inject pathogenicity proteins into host cells using a specialized type III secretion system encoded by hrpgenes. This system deliver effector proteins, into plant cells that function in both susceptible and resistant interactions. We have found that the virulence of Erwinia stewartii(Es; syn. Pantoea stewartii) and Erwinia herbicola pv. gypsophilae (Ehg, syn. Pantoea agglomerans), which cause Stewart's wilt of corn and galls on Gypsophila, respectively, depends on hrpgenes. The major objectives of this project were: To increase expression of hrpgenes in order to identify secreted proteins; to identify genes for proteins secreted by the type-III systems and determine if they are required for pathogenicity; and to determine if the secreted proteins can function within eukaryotic cells. We found that transcription of the hrp and effector genes in Es and Ehg is controlled by at least four genes that constitute a regulatory cascade. Environmental and/or physiological signaling appears to be mediated by the HrpX/HrpY two component system, with HrpX functioning as a sensor-kinase and HrpY as a response regulator. HrpYupregulateshrpS, which encodes a transcriptional enhancer. HrpS then activates hrpL, which encodes an alternate sigma factor that recognizes "hrp boxes". All of the regulatory genes are essential for pathogenicity, except HrpX, which appears only to be required for induction of the HR in tobacco by Es. In elucidating this regulatory pathway in both species, we made a number of significant new discoveries. HrpX is unusual for a sensor-kinase because it is cytoplasmic and contains PAS domains, which may sense the redox state of the bacterium. In Es, a novel methyl-accepting protein may function upstream of hrpY and repress hrp gene expression in planta. The esaIR quorum sensing system in Es represses hrp gene expression in Es in response to cell-density. We have discovered six new type III effector proteins in these species, one of which (DspE in Ehg and WtsE in Es) is common to both pathogens. In addition, Es wtsG, which is a homolog of an avrPpiB from P. syringae pv. pisi, and an Ehg ORF, which is a homolog of P. syringae pv. phaseolicola AvrPphD, were both demonstrated to encode virulence proteins. Two plasmidborne, Ehg Hop proteins, HsvG and PthG, are required for infection of gypsophilia, but interestingly, PthG also acts as an Avr elicitor in beets. Using a calmodulin-dependent adenylate cyclase (cyaA) reporter gene, we were successful in demonstrating that an HsvG-CyaA fusion protein can be transferred into human HeLa cells by the type-III system of enteropathogenic E. coli. This is a highly significant accomplishment because it is the first direct demonstration that an effector protein from a plant pathogenic bacterium is capable of being translocated into a eukaryotic cell by a type-III secretion system. Ehg is considered a limiting factor in Gypsophila production in Israel and Stewart’s Wilt is a serious disease in the Eastern and North Central USA, especially on sweet corn in epidemic years. We believe that our basic research on the characterization of type III virulence effectors should enable future identification of their receptors in plant cells. This may lead to novel approaches for genetically engineering resistant plants by modifying their receptors or inactivating effectors and thus blocking the induction of the susceptible response. Alternatively, hrp gene regulation might also provide a target for plant produced compounds that interfere with recognition of the host by the pathogen. Such strategies would be broadly applicable to a wide range of serious bacterial diseases on many crops throughout the USA and Israel.
3
Barefoot, Susan F., Bonita A. Glatz, Nathan Gollop e Thomas A. Hughes. Bacteriocin Markers for Propionibacteria Gene Transfer Systems. United States Department of Agriculture, giugno 2000. http://dx.doi.org/10.32747/2000.7573993.bard.
Abstract (sommario):
The antibotulinal baceriocins, propionicin PLG-1 and jenseniin G., were the first to be identified, purified and characterized for the dairy propionibaceria and are produced by Propionibacterium thoenii P127 and P. thoenii/jensenii P126, respectively. Objectives of this project were to (a) produce polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1; (b) identify, clone and characterize the propionicin PLG-1 (plg-1) and jenseniin G (jnG) genes; and (3) develop gene transfer systems for dairy propionibacteria using them as models. Polyclonal antibodies for detection, comparison and monitoring of propionicin PLG-1 were produced in rabbits. Anti-PLG-1 antiserum had high titers (256,000 to 512,000), neutralized PLG-1 activity, and detected purified PLG-1 at 0.10 mg/ml (indirect ELISA) and 0.033 mg/ml (competitive indirect ELISA). Thirty-nine of 158 strains (most P. thoenii or P. jensenii) yielded cross-reacting material; four strains of P. thoenii, including two previously unidentified bacteriocin producers, showed biological activity. Eight propionicin-negative P127 mutants produced neither ELISA response nor biological activity. Western blot analyses of supernates detected a PLG-1 band at 9.1 kDa and two additional protein bands with apparent molecular weights of 16.2 and 27.5 kDa. PLG-1 polyclonal antibodies were used for detection of jenseniin G. PLG-1 antibodies neutralized jenseniin G activity and detected a jenseniin G-sized, 3.5 kDa peptide. Preliminary immunoprecipitation of crude preparations with PLG-1 antibodies yielded three proteins including an active 3-4 kDa band. Propionicin PLG-1 antibodies were used to screen a P. jensenii/thoenii P126 genomic expression library. Complete sequencing of a cloned insert identified by PLG-1 antibodies revealed a putative response regulator, transport protein, transmembrane protein and an open reading frame (ORF) potentially encoding jenseniin G. PCR cloning of the putative plg-1 gene yielded a 1,100 bp fragment with a 355 bp ORF encoding 118 amino acids; the deduced N-terminus was similar to the known PLG-1 N-terminus. The 118 amino acid sequence deduced from the putative plg-1 gene was larger than PLG-1 possibly due to post-translational processing. The product of the putative plg-1 gene had a calculated molecular weight of 12.8 kDa, a pI of 11.7, 14 negatively charged residues (Asp+Glu) and 24 positively charged residues (Arg+Lys). The putative plg-1 gene was expressed as an inducible fusion protein with a six-histidine residue tag. Metal affinity chromatography of the fused protein yielded a homogeneous product. The fused purified protein sequence matched the deduced putative plg-1 gene sequence. The data preliminarily suggest that both the plg-1 and jnG genes have been identified and cloned. Demonstrating that antibodies can be produced for propionicin PLG-1 and that those antibodies can be used to detect, monitor and compare activity throughout growth and purification was an important step towards monitoring PLG-1 concentrations in food systems. The unexpected but fortunate cross-reactivity of PLG-1 antibodies with jenseniin G led to selective recovery of jenseniin G by immunoprecipitation. Further refinement of this separation technique could lead to powerful affinity methods for rapid, specific separation of the two bacteriocins and thus facilitate their availability for industrial or pharmaceutical uses. Preliminary identification of genes encoding the two dairy propionibacteria bacteriocins must be confirmed; further analysis will provide means for understanding how they work, for increasing their production and for manipulating the peptides to increase their target species. Further development of these systems would contribute to basic knowledge about dairy propionibacteria and has potential for improving other industrially significant characteristics.
4
Ostersetzer-Biran, Oren, e Alice Barkan. Nuclear Encoded RNA Splicing Factors in Plant Mitochondria. United States Department of Agriculture, febbraio 2009. http://dx.doi.org/10.32747/2009.7592111.bard.
Abstract (sommario):
Mitochondria are the site of respiration and numerous other metabolic processes required for plant growth and development. Increased demands for metabolic energy are observed during different stages in the plants life cycle, but are particularly ample during germination and reproductive organ development. These activities are dependent upon the tight regulation of the expression and accumulation of various organellar proteins. Plant mitochondria contain their own genomes (mtDNA), which encode for a small number of genes required in organellar genome expression and respiration. Yet, the vast majority of the organellar proteins are encoded by nuclear genes, thus necessitating complex mechanisms to coordinate the expression and accumulation of proteins encoded by the two remote genomes. Many organellar genes are interrupted by intervening sequences (introns), which are removed from the primary presequences via splicing. According to conserved features of their sequences these introns are all classified as “group-II”. Their splicing is necessary for organellar activity and is dependent upon nuclear-encoded RNA-binding cofactors. However, to-date, only a tiny fraction of the proteins expected to be involved in these activities have been identified. Accordingly, this project aimed to identify nuclear-encoded proteins required for mitochondrial RNA splicing in plants, and to analyze their specific roles in the splicing of group-II intron RNAs. In non-plant systems, group-II intron splicing is mediated by proteins encoded within the introns themselves, known as maturases, which act specifically in the splicing of the introns in which they are encoded. Only one mitochondrial intron in plants has retained its maturaseORF (matR), but its roles in organellar intron splicing are unknown. Clues to other proteins required for organellar intron splicing are scarce, but these are likely encoded in the nucleus as there are no other obvious candidates among the remaining ORFs within the mtDNA. Through genetic screens in maize, the Barkan lab identified numerous nuclear genes that are required for the splicing of many of the introns within the plastid genome. Several of these genes are related to one another (i.e. crs1, caf1, caf2, and cfm2) in that they share a previously uncharacterized domain of archaeal origin, the CRM domain. The Arabidopsis genome contains 16 CRM-related genes, which contain between one and four repeats of the domain. Several of these are predicted to the mitochondria and are thus postulated to act in the splicing of group-II introns in the organelle(s) to which they are localized. In addition, plant genomes also harbor several genes that are closely related to group-II intron-encoded maturases (nMats), which exist in the nucleus as 'self-standing' ORFs, out of the context of their cognate "host" group-II introns and are predicted to reside within the mitochondria. The similarity with known group-II intron splicing factors identified in other systems and their predicted localization to mitochondria in plants suggest that nuclear-encoded CRM and nMat related proteins may function in the splicing of mitochondrial-encoded introns. In this proposal we proposed to (i) establish the intracellular locations of several CRM and nMat proteins; (ii) to test whether mutations in their genes impairs the splicing of mitochondrial introns; and to (iii) determine whether these proteins are bound to the mitochondrial introns in vivo.
5
Dawson, William O., Moshe Bar-Joseph, Charles L. Niblett, Ron Gafny, Richard F. Lee e Munir Mawassi. Citrus Tristeza Virus: Molecular Approaches to Cross Protection. United States Department of Agriculture, gennaio 1994. http://dx.doi.org/10.32747/1994.7570551.bard.
Abstract (sommario):
Citrus tristeza virus (CTV) has the largest genomes among RNA viruses of plants. The 19,296-nt CTV genome codes for eleven open reading frames (ORFs) and can produce at least 19 protein products ranging in size from 6 to 401 kDa. The complex biology of CTV results in an unusual composition of CTV-specific RNAs in infected plants which includes multiple defective RNAs and mixed infections. The complex structure of CTV populations poses special problems for diagnosis, strain differentiation, and studies of pathogenesis. A manipulatable genetic system with the full-length cDNA copy of the CTV genome has been created which allows direct studies of various aspects of the CTV biology and pathology. This genetic system is being used to identify determinants of the decline and stem-pitting disease syndromes, as well as determinants responsible for aphid transmission.
6
Antignus, Yehezkiel, Ernest Hiebert, Shlomo Cohen e Susan Webb. Approaches for Studying the Interaction of Geminiviruses with Their Whitefly Vector Bemisia tabaci. United States Department of Agriculture, luglio 1995. http://dx.doi.org/10.32747/1995.7604928.bard.
Abstract (sommario):
The DNA of tomato yellow leaf curl virus (TYLCB) was detected in its whitefly vector, Bemisia tabaci, by dot spot hybridization as early as 1 h after acquisition access. The retention of the virus nucleic acid in the vector was at least 23 days after a 48 h acquisition access. However, the retention of TYLCV coat protein did not exceed 10 days. No replicative forms of TYLCV could be detected in B. tabaci, indicating a non-propagative relationship with the vector. Whiteflies were not able to accumulate naked virion ssDNA, virus cloned dsDNA, or virions with impaired coat protein. Deletion, frameshift, and single amino acid mutations were inserted into open reading frames (ORFs) V1 and V2 (Coat protein) of TYLCV. The ability of these mutants to replicate, to spread and to induce symptoms was tested both in leaf disks and in intact plants. No replication was found in tissues that were infected with a deletion mutant that lacked the carboxy half of the coat protein gene. Residual amounts of ssDNA and dsDNA were detected i tissues infected with a frameshift mutant in which an early termination at the extreme part of the protein. Two other mutants in which a single amino acid was changed in the overlapping part of V1 and V2 were able to spread systemically but infections remained symptomless and the production of ssDNA and dsDNA were significantly lower. These mutants were acquired and transmitted by Bemisia tabaci. Procedures for the the dissection, fixation and embedding of whiteflies were developed. The anatomy and ultrastructure of the salivary gland and the midgut of Bemisia tabaci and Trialeurodes vaporariorum (a vector and non-vector of geminiviruses respectively) was studied and described. Monoclonal antibodies against bean golden mosaic virus (BGMV) with narrow and broad spectrum were prepared. Transmission studies of tomato mottle geminivirus (TMoV) by B. tabaci were carried out. These studies were essential for a further work aimed to understand the interaction of geminiviruses with the insect and their localization in its tissues. To enable the production of transgenic plants procedures were developed for tomato transformation with both Agrobacterium and microparticle bombardment.
7
Ostersetzer-Biran, Oren, e Jeffrey Mower. Novel strategies to induce male sterility and restore fertility in Brassicaceae crops. United States Department of Agriculture, gennaio 2016. http://dx.doi.org/10.32747/2016.7604267.bard.
Abstract (sommario):
Abstract Mitochondria are the site of respiration and numerous other metabolic processes required for plant growth and development. Increased demands for metabolic energy are observed during different stages in the plants life cycle, but are particularly ample during germination and reproductive organ development. These activities are dependent upon the tight regulation of the expression and accumulation of various organellar proteins. Plant mitochondria contain their own genomes (mtDNA), which encode for rRNAs, tRNAs and some mitochondrial proteins. Although all mitochondria have probably evolved from a common alpha-proteobacterial ancestor, notable genomic reorganizations have occurred in the mtDNAs of different eukaryotic lineages. Plant mtDNAs are notably larger and more variable in size (ranging from 70~11,000 kbp in size) than the mrDNAs in higher animals (16~19 kbp). Another unique feature of plant mitochondria includes the presence of both circular and linear DNA fragments, which undergo intra- and intermolecular recombination. DNA-seq data indicate that such recombination events result with diverged mitochondrial genome configurations, even within a single plant species. One common plant phenotype that emerges as a consequence of altered mtDNA configuration is cytoplasmic male sterility CMS (i.e. reduced production of functional pollen). The maternally-inherited male sterility phenotype is highly valuable agriculturally. CMS forces the production of F1 hybrids, particularly in predominantly self-pollinating crops, resulting in enhanced crop growth and productivity through heterosis (i.e. hybrid vigor or outbreeding enhancement). CMS lines have been implemented in some cereal and vegetables, but most crops still lack a CMS system. This work focuses on the analysis of the molecular basis of CMS. We also aim to induce nuclear or organellar induced male-sterility in plants, and to develop a novel approach for fertility restoration. Our work focuses on Brassicaceae, a large family of flowering plants that includes Arabidopsis thaliana, a key model organism in plant sciences, as well as many crops of major economic importance (e.g., broccoli, cauliflower, cabbage, and various seeds for oil production). In spite of the genomic rearrangements in the mtDNAs of plants, the number of genes and the coding sequences are conserved among different mtDNAs in angiosperms (i.e. ~60 genes encoding different tRNAs, rRNAs, ribosomal proteins and subunits of the respiratory system). Yet, in addition to the known genes, plant mtDNAs also harbor numerous ORFs, most of which are not conserved among species and are currently of unknown function. Remarkably, and relevant to our study, CMS in plants is primarily associated with the expression of novel chimericORFs, which likely derive from recombination events within the mtDNAs. Whereas the CMS loci are localized to the mtDNAs, the factors that restore fertility (Rfs) are identified as nuclear-encoded RNA-binding proteins. Interestingly, nearly all of the Rf’s are identified as pentatricopeptide repeat (PPR) proteins, a large family of modular RNA-binding proteins that mediate several aspects of gene expression primarily in plant organelles. In this project we proposed to develop a system to test the ability of mtORFs in plants, which are closely related to known CMS factors. We will induce male fertility in various species of Brassicaceae, and test whether a down-relation in the expression of the recombinantCMS-genes restores fertility, using synthetically designed PPR proteins.
8
Mawassi, Munir, e Valerian Dolja. Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A. United States Department of Agriculture, gennaio 2010. http://dx.doi.org/10.32747/2010.7592114.bard.
Abstract (sommario):
RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.
9
Mawassi, Munir, Adib Rowhani, Deborah A. Golino, Avichai Perl e Edna Tanne. Rugose Wood Disease of Grapevine, Etiology and Virus Resistance in Transgenic Vines. United States Department of Agriculture, novembre 2003. http://dx.doi.org/10.32747/2003.7586477.bard.
Abstract (sommario):
Rugose wood is a complex disease of grapevines, which occurs in all growing areas. The disease is spread in the field by vector transmission (mealybugs). At least five elongated-phloem- limited viruses are implicated in the various rugose wood disorders. The most fully characterized of these are Grapevine virus A (GV A) and GVB, members of a newly established genus, the vitivirus. GVC, a putative vitivirus, is much less well characterized than GV A or GVB. The information regarding the role of GVC in the etiology and epidemiology of rugose wood is fragmentary and no sequence data for GVC are available. The proposed research is aimed to study the etiology and epidemiology of rugose wood disease, and to construct genetically engineered virus-resistant grapevines. The objectives of our proposed research were to construct transgenic plants with coat protein gene sequences designed to induce post-transcriptional gene silencing (pTGS); to study the epidemiology and etiology of rugose wood disease by cloning and sequencing of GVC; and surveying of rugose wood- associated viruses in Californian and Israeli vineyards. In an attempt to experimentally define the role of the various genes of GV A, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. We explored the production of viral RNAs in a GV A-infected Nicotiana benthamiana herbaceous host, and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5 and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8 and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Several GV A constructs have been assembled into pCAMBIA 230 I, a binary vector which is used for Angrobacterium mediated transformation: GV A CP gene; two copies of the GV A CP gene arranged in the same antisense orientation; two copies of the GV A CP gene in which the downstream copy is in an antigens orientation; GV A replicase gene; GV A replicase gene plus the 3' UTR sequence; and the full genome of GV A. Experiments for transformation of N. benthamiana and grapevine cell suspension with these constructs have been initiated. Transgenic N. benthamiana plants that contained the CP gene, the replicase gene and the entire genome of GV A were obtained. For grapevine transformation, we have developed efficient protocols for transformation and successfully grapevine plantlets that contained the CP gene and the replicase genes of GV A were obtained. These plants are still under examination for expression of the trans genes. The construction of transgenic plants with GV A sequences will provide, in the long run, a means to control one of the most prevalent viruses associated with grapevines. Our many attempts to produce a cDNA library from the genome of GVC failed. For surveying of rugose wood associated viruses in California vineyards, samples were collected from different grape growing areas and tested by RT-PCR for GV A, GVB and GVD. The results indicated that some of the samples were infected with multiple viruses, but overall, we found higher incidence of GVB and GV A infection in California vineyards and new introduction varieties, respectively. In this research we also conducted studies to increase our understanding of virus - induced rootstock decline and its importance in vineyard productivity. Our results provided supporting evidence that the rootstock response to virus infection depends on the rootstock genotype and the virus type. In general, rootstocks are differ widely in virus susceptibility. Our data indicated that a virus type or its combination with other viruses was responsible in virus-induced rootstock decline. As the results showed, the growth of the rootstocks were severely affected when the combination of more than one virus was present.
10
Bercovier, Herve, e Ronald P. Hedrick. Diagnostic, eco-epidemiology and control of KHV, a new viral pathogen of koi and common carp. United States Department of Agriculture, dicembre 2007. http://dx.doi.org/10.32747/2007.7695593.bard.
Abstract (sommario):
Original objectives and revisions-The proposed research included these original objectives: field validation of diagnostic tests (PCR), the development and evaluation of new sensitive tools (LC-PCR/TaqManPCR, antibody detection by ELISA) including their use to study the ecology and the epidemiology of KHV (virus distribution in the environment and native cyprinids) and the carrier status of fish exposed experimentally or naturally to KHV (sites of virus replication and potential persistence or latency). In the course of the study we completed the genome sequence of KHV and developed a DNA array to study the expression of KHV genes in different conditions. Background to the topics-Mass mortality of koi or common carp has been observed in Israel, USA, Europe and Asia. These outbreaks have reduced exports of koi from Israel and have created fear about production, import, and movements of koi and have raised concerns about potential impacts on native cyprinid populations in the U.S.A. Major conclusions-A suite of new diagnostic tools was developed that included 3 PCR assays for detection of KHV DNA in cell culture and fish tissues and an ELISA assay capable of detecting anti-KHV antibodies in the serum of koi and common carp. The TKPCR assay developed during the grant has become an internationally accepted gold standard for detection of viral DNA. Additionally, the ELISA developed for detecting serum anti-KHV antibodies is now in wide use as a major nonlethal screening tool for evaluating virus status of koi and common carp populations. Real time PCR assays have been able to detect viral DNA in the internal organs of survivors of natural and wild type vaccine exposures at 1 and 10³ genome equivalents at 7 months after exposure. In addition, vaccinated fish were able to transmit the virus to naive fish. Potential control utilizing hybrids of goldfish and common carp for production demonstrated they were considerably more resistant than pure common carp or koi to both KHV (CyHV-3). There was no evidence that goldfish or other tested endemic cyprinids species were susceptible to KHV. The complete genomic sequencing of 3 strains from Japan, the USA, and Israel revealed a 295 kbp genome containing a 22 kbp terminal direct repeat encoding clear gene homologs to other fish herpesviruses in the family Herpesviridae. The genome encodes156 unique protein-coding genes, eight of which are duplicated in the terminal repeat. Four to seven genes are fragmented and the loss of these genes may be associated with the high virulence of the virus. Viral gene expression was studies by a newly developed chip which has allowed verification of transcription of most all hypothetical genes (ORFs) as well as their kinetics. Implications, both scientific and agricultural- The results from this study have immediate application for the control and management of KHV. The proposal provides elements key to disease management with improved diagnostic tools. Studies on the ecology of the virus also provide insights into management of the virus at the farms that farmers will be able to apply immediately to reduce risks of infections. Lastly, critical issues that surround present procedures used to create “resistant fish” must be be resolved (e.g. carriers, risks, etc.). Currently stamping out may be effective in eradicating the disease. The emerging disease caused by KHV continues to spread. With the economic importance of koi and carp and the vast international movements of koi for the hobby, this disease has the potential for even further spread. The results from our studies form a critical component of a comprehensive program to curtail this emerging pathogen at the local, regional and international levels.