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Author: Jing Zhong
Publisher:
ISBN: 9781321321029
Category : Caenorhabditis elegans
Languages : en
Pages : 129
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Book Description
RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode Caenorhabditis elegans mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that C. elegans mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed.
Author: Jing Zhong
Publisher:
ISBN: 9781321321029
Category : Caenorhabditis elegans
Languages : en
Pages : 129
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Book Description
RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode Caenorhabditis elegans mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that C. elegans mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed.
Author: Stephanie Renee Coffman
Publisher:
ISBN: 9781339467023
Category : Antiviral agents
Languages : en
Pages : 161
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Book Description
Both RIG-I and DRH-1 function in antiviral immunity through the activation of type-I interferon signaling in mammals and antiviral RNAi in C. elegans, respectively. This work investigates the function of DRH-1 in the production of virus-derived siRNAs (vsiRNAs). Deep sequencing of wild-type and drh-1 mutants challenged with Orsay Virus demonstrated that drh-1 mutants produced primary vsiRNAs that are dependent on DCR-1 and the dsRNA-binding protein RDE-4. However, these DRH-1 independent vsiRNAs are predominantly derived from the 5' terminal regions of both OrV RNA1 and RNA2 and were depleted from the internal regions of viral RNA. In contrast, wild-type and other mutant animals did not exhibit a bias for primary vsiRNA biogenesis from the 5' terminal regions. Mammalian RIG-I acts as an ATP-powered translocase, though the biological function of this activity is unknown. Therefore, DRH-1 may function to promote the production of primary vsiRNAs spreading from the 5' terminal regions to the internal and 3' regions of the viral RNA using translocase activity.
Author: Jinfeng Lu
Publisher:
ISBN: 9781339467412
Category : Antiviral agents
Languages : en
Pages : 133
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Book Description
Diverse eukaryotic hosts produce virus-derived small interfering RNAs (siRNAs) to direct antiviral immunity by RNA interference (RNAi). However, it has been controversial in the past decade whether the mammalian RNAi pathway has a natural antiviral function. In this dissertation, I demonstrate the production of canonical virus-derived siRNAs processed by endoribonuclease Dicer from viral double-stranded RNA (dsRNA) precursors in cultured hamster and human somatic cells and in mice infected by two distinct RNA viruses after their cognate viral suppressor of RNA silencing (VSR) is rendered inactive. These mammalian viral siRNAs are predominantly 22 nucleotides long and contain mostly uridine as the 5'-terminal nucleotide, and are therefore similar to mammalian microRNAs. I show that canonical viral siRNAs are loaded into mouse and human Argonaute proteins at high levels in the infected cells. Further analysis reveals that the VSR protein NS1 of Influenza A virus inhibits the biogenesis of viral siRNAs whereas the VSR protein B2 of Nodamura virus suppresses both the biogenesis and the Argonaute loading of viral siRNAs during infection. The results together demonstrate that the antiviral RNAi response is conserved in mammals as found in fungi, plants, insects, and nematodes.
Author: Guoping (Sam) Gu
Publisher:
ISBN:
Category :
Languages : en
Pages : 232
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Author: Michael Philip Dybbs
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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Author: Ronald P. van Rij
Publisher: Humana Press
ISBN: 9781617790362
Category : Medical
Languages : en
Pages : 0
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Book Description
Viruses and RNAi share an intricate relationship at many levels. RNAi is an important antiviral defense mechanism in plants and invertebrates, microRNAs – of viral or cellular origin – affect many aspects of virus biology, and replication of many, if not all, mammalian viruses can be suppressed by RNAi. Antiviral RNAi: Concepts, Methods, and Applications provides a collection of protocols for the analysis of viral small RNAs and natural antiviral RNAi responses as well as for the development and optimization of RNAi-based antiviral drugs. As RNAi is a central regulatory mechanism in the cell, the methods in this volume can also be applied out of the context of a virus infection. Divided into five convenient parts, this detailed volume reviews important basic concepts in the field of antiviral RNAi, provides experimental and bio-informatic tools for the analysis of small silencing RNAs, covers methods to biochemically dissect RNAi-based antiviral defense and viral counter-defense mechanisms, describes methods for the design, expression, and delivery of therapeutic antiviral siRNAs, and finally presents genome-wide RNAi approaches for the identification of factors involved in virus replication. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Antiviral RNAi: Concepts, Methods, and Applications serves as an ideal guide for both novice and experienced researchers alike striving to dissect the role of RNAi in the viral life cycle or to further boost the development of novel therapeutics and experimental tools based on RNAi technology.
Author: Jimmy Jiajia Zhuang
Publisher:
ISBN:
Category :
Languages : en
Pages :
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RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.
Author: Carl Joseph Franz
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 165
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Book Description
The model organism, Caenorhabditis elegans, has been used in basic research for the last 50 years, providing a wealth of knowledge in a range of fields spanning genetics, development, neurobiology, and computational biology just naming a few. However, only in the last ~15 years has this model organism been broadly considered for investigating host-pathogen interactions. During this time a number of bacterial, fungal, and microsporidial pathogens were shown to infect C. elegans; however, until recently, no natural viruses were known, resulting in a limited understanding of virus-host interactions in C. elegans. In lieu of a known C. elegans virus, several studies were conducted by establishing an unnatural virus infection or creating transgenic viral replicon systems. In 2011, we along with our collaborators reported the discovery of the first known viruses that naturally infect Caenorhabditis nematodes. This dissertation encompasses the identification and characterization of the first viruses known to naturally infect C. elegans and its closest known relative, C. briggsae. Using Next generation 454 pyrosequencing, a novel virus (Orsay virus) was found in a wild isolate of C. elegans (JU1580) and 2 novel viruses (Santeuil and Le Blanc viruses) were found in wild isolates of C. briggsae (JU1264 and JU1498). Phylogenetic analysis revealed the closest known relatives of Orsay, Santeuil, and Le Blanc were viruses in the family Nodaviridae: viruses that naturally infect insects (alphanodaviruses) and fishes (betanodaviruses). Comparative genome analysis revealed substantial differences in genome size and genome organization between the Caenorhabditis viruses and nodaviruses. For instance, the Caenorhabditis viruses have bigger genomes partly due to the presence of a larger 5' untranslated region (UTR) in the RNA1 segment and an additional open reading frame (ORF delta) in the RNA2 segment. While ORF delta has no known function, evidence demonstrates it is translated fused to the capsid protein by a slippery sequence-mediated frameshift. An immunofluorescence assay (IFA) was established targeting viral proteins in infected Caenorhabditis nematodes. Viral protein could be detected as early as 6 hours post-infection (hpi) and the number of infected animals steadily rose between 6 to 12 hpi. In both Orsay virus-infected C. elegans and Santeuil and Le Blanc virus-infected C. briggsae, the site of infection localized primarily to the intestinal tissue. Strikingly, only 1 to 6 cells of the total 20 nonregenerating intestinal cells were positive by IFA for viral protein, suggesting that infection is confined to a subset of the intestine. A transcriptional profile analysis was carried out to investigate which host genes in Caenorhabditis nematodes were responding to viral infection, yielding ~200 genes that were specifically induced by virus in C. elegans. These candidate genes were then individually subjected to RNA interference revealing several characterized and uncharacterized genes that conferred an increased infection phenotype upon knockdown and may play a previously undescribed role in antiviral defense. The discovery and characterization of these novel Caenorhabditis nematode viruses lay the groundwork for the introduction of C. elegans as a powerful model organism to study virus-host interactions.
Author: Joseph Wawrzyniak
Publisher:
ISBN:
Category : Caenorhabditis elegans
Languages : en
Pages : 51
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Book Description
Author: Ravi Shanker Kamath
Publisher:
ISBN:
Category :
Languages : en
Pages :
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