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Sarah J Poynter, Stephanie J DeWitte-Orr
Long double-stranded (ds) RNA molecules are produced as a byproduct of viral replication. Studying virus-derived dsRNA is important for understanding virus replication, understanding host responses to virus infections, and as a diagnostic tool for virus presence and replication. Here, we describe four different techniques for visualizing dsRNA; two antibody-dependent methods (immunoblotting and immunocytochemistry), as well as two antibody-independent methods (differential digestion and acridine orange staining)...
2017: Methods in Molecular Biology
Jesse Donovan, Sneha Rath, David Kolet-Mandrikov, Alexei Korennykh
Mammalian cells respond to double-stranded RNA (dsRNA) by activating a translation-inhibiting endoribonuclease, RNase L. Consensus in the field indicates that RNase L arrests protein synthesis by degrading ribosomal RNAs (rRNAs) and messenger RNAs (mRNAs). However, here we provide evidence for a different and far more efficient mechanism. By sequencing abundant RNA fragments generated by RNase L in human cells, we identify site-specific cleavage of two groups of non-coding RNAs: Y-RNAs, whose function is poorly understood, and cytosolic tRNAs, which are essential for translation...
August 14, 2017: RNA
Mohammad Vatanparast, Yonggyun Kim
Double-stranded RNA (dsRNA) has been applied to control insect pests due to its induction of RNA interference (RNAi) of a specific target gene expression. However, developing dsRNA-based insecticidal agent has been a great challenge especially against lepidopteran insect pests due to variations in RNAi efficiency. The objective of this study was to screen genes of chymotrypsins (SeCHYs) essential for the survival of the beet armyworm, Spodoptera exigua, to construct insecticidal dsRNA. In addition, an optimal oral delivery method was developed using recombinant bacteria...
2017: PloS One
Xiaofeng Su, Latifur Rehman, Huiming Guo, Xiaokang Li, Hongmei Cheng
Verticillium dahliae is the most overwhelming plant pathogen, causing Verticillium wilt in a number of economic crops. The molecular mechanism is still unclear and identification of the genes involved in the pathogenicity or virulence of this fungus would benefit to uncover such mechanism. STT3 is a catalytic subunit of the multi-subunit oligosaccharyl transferase (OST) and plays an essential role in glycoprotein modification. Here, we characterized STT3 gene (VDAG_03232.1) of V. dahliae to explore its regulatory role in the development and virulence by deletion and complementation of this gene, as well as its silence in transgenic plants...
August 10, 2017: Current Genetics
Marianna Pauletto, Amélie Segarra, Caroline Montagnani, Virgile Quillien, Nicole Faury, Jacqueline Le Grand, Philippe Miner, Bruno Petton, Yannick Labreuche, Elodie Fleury, Caroline Fabioux, Luca Bargelloni, Tristan Renault, Arnaud Huvet
Double stranded RNA-mediated genetic interference (RNAi) is a widely used reverse genetic tool for determining the loss-of-function phenotype of a gene. Here, the possible induction of an immune response by long dsRNA was tested in a marine bivalve, i.e. Crassostrea gigas, as well as the specific role of the subunit 2 of the nuclear factor κB inhibitor (IκB2). This gene is a candidate of particular interest for functional investigations in the context of massive mortality oyster events as Cg-IκB2 mRNA levels exhibited significant variation depending on the amount of ostreid herpesvirus 1 (OsHV-1) DNA detected...
August 10, 2017: Journal of Experimental Biology
Jamie W L Ong, Hua Li, Krishnapillai Sivasithamparam, Kingsley W Dixon, Michael G K Jones, Stephen J Wylie
The bipartite alpha- and betapartitiviruses are recorded from a wide range of fungi and plants. Using a combination of dsRNA-enrichment, high-throughput shotgun sequencing and informatics, we report the occurrence of multiple new partitiviruses associated with mycorrhizal Ceratobasidium fungi, themselves symbiotically associated with a small wild population of Pterostylis sanguinea orchids in Australia, over two consecutive years. Twenty-one partial or near-complete sequences representing 16 definitive alpha- and betapartitivirus species, and further possible species, were detected from two fungal isolates...
August 7, 2017: Virology
Cheng Huang, Olga A Kolokoltsova, Elizabeth J Mateer, Takaaki Koma, Slobodan Paessler
The arenavirus family consists of several highly pathogenic viruses including the Old World (OW) arenavirus Lassa fever virus (LASV) and the New World (NW) Junín virus (JUNV) and Machupo virus (MACV). Host response to infection by these pathogenic arenaviruses is distinct in many aspects. NW JUNV and MACV infections readily induce an IFN response in human cells, while OW LASV infection usually triggers an undetectable or weak IFN response. JUNV induces IFN response through RIG-I, suggesting that the host non-self RNA sensor readily detects JUNV viral RNAs during infection and activates IFN response...
August 9, 2017: Journal of Virology
Ana María Fernández-Presas, Luis Padilla-Noriega, Ingeborg Becker, Lilia Robert, José Agustín Jiménez, Sandra Solano, Jose Delgado, Patricia Tato, José Luis Molinari
Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed...
August 7, 2017: Revista do Instituto de Medicina Tropical de São Paulo
Neena Mitter, Elizabeth A Worrall, Karl E Robinson, Zhi Ping Xu, Bernard J Carroll
Exogenous application of double-stranded RNA (dsRNA) for virus resistance in plants represents a very attractive alternative to virus resistant transgenic crops or pesticides targeting virus vectors. However, the instability of dsRNA sprayed onto plants is a major challenge as spraying naked dsRNA onto plants provides protection against homologous viruses for only 5 days. Innovative approaches, such as the use of nanoparticles as carriers of dsRNA for improved stability and sustained release, are emerging as key disruptive technologies...
August 1, 2017: Current Opinion in Virology
Elsie Yekwa, Joelle Khourieh, Bruno Canard, Nicolas Papageorgiou, François Ferron
The Arenaviridae family is one of the two RNA viral families that encode a 3'-5' exonuclease in their genome. An exonuclease domain is found in the Arenaviridae nucleoprotein and targets dsRNA specifically. This domain is directly involved in suppression of innate immunity in the host cell. Like most phosphate-processing enzymes, it requires a divalent metal ion such as Mg(2+) (or Mn(2+)) as a cofactor to catalyse nucleotide-cleavage and nucleotide-transfer reactions. On the other hand, calcium (Ca(2+)) inhibits this enzymatic activity, in spite of the fact that Mg(2+) and Ca(2+) present comparable binding affinities and biological availabilities...
August 1, 2017: Acta Crystallographica. Section D, Structural Biology
Surender Kumar, Burra L Subbarao, Reenu Kumari, Vipin Hallan
A new member of the genus Deltapartitivirus was identified containing three dsRNAs with an estimated size of 1.71, 1.49 and 1.43 kb. The dsRNAs were extracted from symptomless pigeonpea [Cajanus cajan (L.) Millspaugh] plants cv. Erra Kandulu. This new virus with 4.64 kb genome was tentatively named Arhar cryptic virus-1 (ArCV-1). The genomic RNAs were amplified and characterized by sequence independent single primer amplification. The dsRNAs shared a highly conserved 16 nt 5' non-coding region (5'-GATAATGATCCAAGGA-3')...
2017: PloS One
Christine Pourcel, Cédric Midoux, Gilles Vergnaud, Libera Latino
ssRNA bacteriophages are very abundant but poorly studied, particularly in relation to their effect on bacterial evolution. We isolated a new Pseudomonas aeruginosa levivirus, vB_PaeL_PcyII-10_LeviOr01, from hospital waste water. Its genome comprises 3669 nucleotides and encodes four putative proteins. Following bacterial infection, a carrier state is established in a fraction of the cells, conferring superinfection immunity. Such cells also resist other phages that use type IV pili as a receptor. The carrier population is composed of a mixture of cells producing phage, and susceptible cells that are non-carriers...
August 4, 2017: Journal of General Virology
Nina Hahn, Debbra Y Knorr, Johannes Liebig, Liane Wüstefeld, Karsten Peters, Marita Büscher, Gregor Bucher, Hannelore Ehrenreich, Ralf Heinrich
The cytokine erythropoietin (Epo) mediates various cell homeostatic responses to environmental challenges and pathological insults. While stimulation of vertebrate erythrocyte production is mediated by homodimeric "classical" Epo receptors, alternative receptors are involved in neuroprotection. However, their identity remains enigmatic due to complex cytokine ligand and receptor interactions and conflicting experimental results. Besides the classical Epo receptor, the family of type I cytokine receptors also includes the poorly characterized orphan cytokine receptor-like factor 3 (CRLF3) present in vertebrates including human and various insect species...
2017: Frontiers in Molecular Neuroscience
Guangmei Zhang, Sultan Asad, Alexander A Khromykh, Sassan Asgari
The genus Flavivirus contains more than 70 single-stranded, positive-sense arthropod-borne RNA viruses. Some flaviviruses are particularly medically important to humans and other vertebrates including dengue virus (DENV), West Nile virus, and yellow fever virus. These viruses are transmitted to vertebrates by mosquitoes and other arthropod species. Mosquitoes are also infected by insect-specific flaviviruses (ISFs) that do not appear to be infective to vertebrates. Cell fusing agent virus (CFAV) was the first described ISF, which was discovered in an Aedes aegypti cell culture...
July 31, 2017: Scientific Reports
Hengxia Jia, Kaili Dong, Lingling Zhou, Guoping Wang, Ni Hong, Daohong Jiang, Wenxing Xu
Viruses with double-stranded RNA genomes form isometric particles or are capsidless. Here we report a double-stranded RNA virus, Colletotrichum camelliae filamentous virus 1 (CcFV-1) isolated from a fungal pathogen, that forms filamentous particles. CcFV-1 has eight genomic double-stranded RNAs, ranging from 990 to 2444 bp, encoding 10 putative open reading frames, of which open reading frame 1 encodes an RNA-dependent RNA polymerase and open reading frame 4 a capsid protein. When inoculated, the naked CcFV-1 double-stranded RNAs are infectious and induce the accumulation of the filamentous particles in vivo...
August 1, 2017: Nature Communications
Pak-Yin Lui, Lok-Yin Roy Wong, Ting-Hin Ho, Shannon Wing Ngor Au, Chi-Ping Chan, Kin-Hang Kok, Dong-Yan Jin
MDA5 is a RIG-I-like cytoplasmic sensor of dsRNA and certain RNA viruses, such as encephalomyocarditis virus, for the initiation of the IFN signaling cascade in the innate antiviral response. The affinity of MDA5 toward dsRNA is low, and its activity becomes optimal in the presence of unknown cellular coactivators. In this article, we report an essential coactivator function of dsRNA-binding protein PACT in mediating the MDA5-dependent type I IFN response. Virus-induced and polyinosinic-polycytidylic acid-induced activation of MDA5 were severely impaired in PACT-knockout cells and attenuated in PACT-knockdown cells, but they were potentiated when PACT was overexpressed...
July 31, 2017: Journal of Immunology: Official Journal of the American Association of Immunologists
Kazuki Kubota, Yasushi Tsuchihashi, Toshihiro Kogure, Kaoru Maeyama, Fumihiro Hattori, Shigeharu Kinoshita, Shohei Sakuda, Hiromichi Nagasawa, Etsuro Yoshimura, Michio Suzuki
The bivalve hinge ligament is the hard tissue that functions to open and close shells. The ligament contains fibrous structures consisting of aragonite crystals surrounded by a dense organic matrix. This organic matrix may contribute to the formation of fibrous aragonite crystals, but the mechanism underlying this formation remains unclear. In this study, we identified a novel ligament-specific protein, Pinctada fucata tissue inhibitor of metalloproteinase (PfTIMP), from the fibrous organic matrix between aragonite crystals in the ligament using the amino acid sequence and cDNA cloning methods...
July 29, 2017: Journal of Structural Biology
Hideki Osaki, Atsuko Sasaki, Eiko Nakazono-Nagaoka, Nobuyoshi Ota, Ryoji Nakaune
According to previous studies, three double-stranded (ds) RNA molecules (dsRNA1, 2, and 3) detected in Japanese pear are transmitted to the next generation with high frequency through both ovules and pollen. Nucleotide sequence analysis of dsRNA1-encoding RNA-dependent RNA polymerase (RdRp) has suggested that these dsRNAs are related to a cryptovirus named Pyrus pyrifolia cryptic virus (PpCV). In this study, purified dsRNA prepared from a PpCV-infected Japanese pear cultivar was subjected to next-generation deep sequencing...
July 29, 2017: Virus Research
Nikolay Kovalev, Jun-Ichi Inaba, Zhenghe Li, Peter D Nagy
Reconstituted antiviral defense pathway in surrogate host yeast is used as an intracellular probe to further our understanding of virus-host interactions and the role of co-opted host factors in formation of membrane-bound viral replicase complexes in protection of the viral RNA against ribonucleases. The inhibitory effect of the RNA interference (RNAi) machinery of S. castellii, which only consists of the two-component DCR1 and AGO1 genes, was measured against tomato bushy stunt virus (TBSV) in wild type and mutant yeasts...
July 2017: PLoS Pathogens
Juliana Lukša, Bazilė Ravoitytė, Aleksandras Konovalovas, Lina Aitmanaitė, Anzhelika Butenko, Vyacheslav Yurchenko, Saulius Serva, Elena Servienė
Competitive and naturally occurring yeast killer phenotype is governed by coinfection with dsRNA viruses. Long-term relationship between the host cell and viruses appear to be beneficial and co-adaptive; however, the impact of viral dsRNA on the host gene expression has barely been investigated. Here, we determined the transcriptomic profiles of the host Saccharomyces cerevisiae upon the loss of the M-2 dsRNA alone and the M-2 along with the L-A-lus dsRNAs. We provide a comprehensive study based on the high-throughput RNA-Seq data, Gene Ontology and the analysis of the interaction networks...
July 25, 2017: Toxins
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