Download or read book The Multi functional Influenza A Virus RNA dependent RNA Polymerase written by Thomas M. Bechert and published by . This book was released on 2003 with total page 63 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Study of the Structure and Function of the Influenza Virus RNA dependent RNA Polymerase written by Itziar Serna Martin and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Influenza A Virus A Role for the RNA Polymerase in Viral Particle Assembly written by John F. Regan and published by . This book was released on 2005 with total page 660 pages. Available in PDF, EPUB and Kindle. Book excerpt: Influenza is an RNA virus whose segmented genome is encapsidated into viral ribonucleoprotein complexes (vRNPs). Upon infection, the vRNPs migrate into the nucleus where transcription and replication take place. The vRNPs contain a RNA-dependent RNA polymerase that is responsible for viral transcription and replication. The polymerase is composed of three subunits, PB1, PB2, and PA. PBI has polymerase activity and PB2 is involved in viral transcription. The function of PA is unclear. To help elucidate the role of PA in the viral life cycle, 16 conserved regions of PA were targeted for alanine substitution. A plasmid-based transfection system was used to generate recombinant influenza particles bearing each mutation, which were tested for viral viability and the ability of each mutant polymerase to transcribe and replicate a reporter. Mutations in the N-terminus were not well tolerated and resulted in either non-viable or attenuated viruses. One of the mutants, J10, was capable of RNA synthesis, yet did not create viral particles capable of plaque formation in MDCK cells. Specifically, when compared to wild-type, this mutant synthesized 50+/-7% vRNA, 86+/-12% mRNA, and 128+/-18% cRNA. These levels are compatible with viability, as mutants J8 (27%) and J12 (23%), produced significantly less vRNA than J10, yet were viable by plaque assay. The mRNAs generated from J10 polymerase were found to be translationally-active, and both the mutant protein and its RNA products were appropriately localized in the cytoplasm, where influenza assembly occurs. Nevertheless, J10 failed to generate infectious particles from cells in a plasmid-based influenza assembly assay, and hemagglutinating material from the supernatants of such cells contained little or no nuclease-resistant genomic RNA. These findings suggest that PA has a previously unrecognized role in assembly or release of influenza virions, perhaps influencing core structure or the packaging of vRNAs or other essential components into nascent influenza particles.

Download or read book Viral Polymerases written by Satya Prakash Gupta and published by Academic Press. This book was released on 2018-10-29 with total page 498 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viral Polymerases: Structures, Functions and Roles as Antiviral Drug Targets presents in-depth study information on the structure and functions of polymerases and their roles in the lifecycle of viruses, and as drug targets. Viral polymerases constitute a vital component in the lifecycle of many viruses, such as human immunodeficiency virus (HIV), hepatitis viruses, influenza virus, and several others. They are essentially required for the replication of viruses. Thus, the polymerases that can be found in viruses (called viral polymerases) represent favorable targets for the design and development of antiviral drugs. - Provides comprehensive, state-of-the-art coverage on virus infections, the virus lifecycle, and mechanisms of polymerase inhibition - Analyzes the structure-activity relationships of inhibitors of each viral polymerase - Presents a consistent and comprehensive coverage of all aspects of viral polymerases, including structure, function and their role as antiviral drug targets

Download or read book DEVELOPMENT OF ANTIVIRAL AGENT written by Shuofeng Yuan and published by Open Dissertation Press. This book was released on 2017-01-26 with total page 220 pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Development of Antiviral Agents Targeting the RNA Polymerase of Influenza Virus" by Shuofeng, Yuan, 袁碩峰, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: The rapid mutability of influenza virus in conjunction with genomic reassortment between viral strains promotes the virus' ability to evade vaccines and to become resistant to antiviral drugs. Therefore, novel anti-influenza therapeutics utilizing new targets and creative strategies are essential. The RNA-dependent RNA polymerase of the virus consists of PA, PB1, and PB2 subunits. Biological and structural investigations of the functional domains of these subunits have broadened the target reservoir for drug screening. With the wealth of knowledge from these studies, identification of small-molecule inhibitors that specifically disrupt the polymerase assembly or abrogate polymerase activities has emerged as an innovative and promising approach. In an attempt to facilitate the discovery of antiviral agents that target viral polymerase, isolated functional domains such as the PA endonuclease domain, the PB2 cap-binding domain, and the PA-PB1 interaction domains were expressed as screening targets. Based on the biochemical and structural properties of individual targets, a variety of platforms were established for the effective screening of inhibitors, including systematic evolution of ligands by exponential enrichment (SELEX), fluorescence resonance energy transfer (FRET) assay, fluorescence polarization (FP) assay, and enzyme-linked immunosorbent assay (ELISA). The antiviral efficacies of selected inhibitors were examined in vitro and in vivo, followed by verification of their antiviral mechanisms. Clinical merits of selected inhibitors were further evaluated, focusing mainly on their cross-protection abilities among influenza virus subtypes and their potential synergetic antiviral effects when used in combination with other drugs. A number of small-molecule compounds, i.e. PA-30, ANA-0, PB2-19, PAC-3 and ANA-1, together with the aptamer PAN-2, were identified as potent inhibitors against the replication of multiple subtypes of influenza A virus, including H1N1, H3N2, H5N1, H7N7, H7N9, and H9N2, in Madin-Darby canine kidney (MDCK) cell cultures. The intranasal administration of the identified compounds enhanced survival rates and reduced lung viral loads in BALB/c mice infected with H1N1 virus. The docking analyses predicted the compounds targeting PA or PB2 interacted with enzyme active sites to abolish endonuclease or cap-binding activity of the polymerase, whereas the compound targeting the PA-PB1 interaction likely induced configurational changes that impeded polymerase assembly. In addition, the combined treatment of zanamivir with the PA- or PB2-targeted compounds exerted synergistic antiviral effects in vitro. This study underscores the medical importance of polymerase functional domains as druggable targets, which may be due to the fact that these targeted areas are not only highly conserved among virus subtypes but also key to viral fitness. The identified antivirals exhibit substantial promise for clinical applications and provide new additions to the arsenal of drugs that are already used for chemoprophylaxis and treatment of influenza. Importantly, the established screening platforms for PA endonuclease inhibitors, PB2 cap-binding inhibitors, and PA-PB1 interaction disrupters should advance the development of a category of anti-influenza drugs that target viral polymerase. DOI: 10.5353/th_b5699888 Subjects: Antiviral agents RNA po

Download or read book Functional Mapping of the C terminus of Influenza Virus RNA Polymerase Subunit PBl written by Philip S. Kerry and published by . This book was released on 2008 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book RNA binding Proteins Regulate Influenza Virus Infection written by Mitchell Ledwith and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viruses are obligate intracellular parasites that depend on host resources andmacromolecular machinery to selfishly replicate and spread genetic material. This genetic material can exist in multiple forms, with viruses utilizing RNA, DNA, and diverse combinations thereof to stably transmit between hosts. These diverse strategies require diverse replication schemes, e.g., DNA viruses must either encode or co-opt host DNA-dependent DNA polymerases to replicate a genome, and negative sense RNA viruses must encode and package RNA-dependent RNA polymerases in virons in order to replicate an RNA genome in the next host. Regardless of scheme, all viruses converge and are absolutely reliant on RNA, and more specifically the host ability to translate RNA molecules into proteins. Thus, both viruses and hosts have devoted significant coding capital in regulating RNA, with emphasis on the evolutionary struggle that exists at this nexus. The over-arching theme of this thesis is to illuminate how and why influenza viruses co- opt, manipulate, and embrace RNA regulation strategies of human hosts. In the first chapter of this thesis, we profiled the RNA-binding characteristics of IFIT2, a canonically anti-viral and interferon-induced host protein, which influenza virus utilizes in a pro- viral manner. Through genome-wide cross-linking immunopreciptation sequencing (CLIP-seq) experiments, we found that IFIT2 binds mRNAs including those of influenza virus. We validated IFIT2 as an mRNA-binding protein, and found that IFIT2 specifically binds AU-rich regions in human RNAs with a preference for binding the mRNAs of other interferon-induced proteins. To accomplish a broadly anti-viral and selectively pro-viral function, we found that IFIT2 binds mRNAs to enhance their translation. Polysome and ribosome-profiling revealed that IFIT2-bound RNAs are poorly translated in the absence of IFIT2, including influenza virus mRNAs. This links the RNA-binding ability of IFIT2 to a functional role in translation, potentially explaining thexi contradiction between the previously observed anti-viral phenotypic role for IFIT2 and the mechanistically observed pro-viral role for IFIT2 in the context of influenza virus. Altogether, these data describe a new node for the regulation of translation during interferon responses and highlight the regulatory volatility that exists at the mRNA interface during infection. In the second chapter of this thesis, we sought to understand the non-specific RNA-binding capability of the influenza virus nucleoprotein, which binds and protects the negative-sense RNA genome to facilitate gene expression and genome replication. Despite this well-established role, the incongruity of the specificity of NP for the viral genome and known biochemical promiscuity of NP is not reconciled. To address this, we perfomed cross-linking immunoprecipitation sequencing to unbiasedly determine the identity of NP-bound RNAs during infection. NP binds the viral genome and anti-genome, but also engages with a number of discrete classes of host small non-coding RNAs. Many of these RNAs have been shown to be host-derived RIG-I agonists during DNA virus infections, or structurally resemble validated RIG-I agonists. We profiled the RNAs bound by RIG-I during infection with quantitative CLIP-seq and found that host RNAs are bound and sampled by RIG-I, and NP likely interferes with the ability of RIG-I to sample endogenous and exogenous agonists. These data show that NP not only acts as a structural component of the influenza gene expression and replication machinery, but also acts as a viral countermeasure for innate sensing of immunogenic RNAs. As a whole, this thesis describes two dissimilar instances by which influenza manipulates host RNA regulation. While the context of these host protein:viral mRNA and viral protein:host non-coding RNA interactions are different, they collectively suggest that RNA regulation during the host innate response to viral infection is an essential and commonplace strategy, but often leaves the host exposed to quickly adapting viral pathogens.

Download or read book RNA binding Proteins Regulate Influenza Virus Infection written by Mitchell Ledwith and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viruses are obligate intracellular parasites that depend on host resources andmacromolecular machinery to selfishly replicate and spread genetic material. This genetic material can exist in multiple forms, with viruses utilizing RNA, DNA, and diverse combinations thereof to stably transmit between hosts. These diverse strategies require diverse replication schemes, e.g., DNA viruses must either encode or co-opt host DNA-dependent DNA polymerases to replicate a genome, and negative sense RNA viruses must encode and package RNA-dependent RNA polymerases in virons in order to replicate an RNA genome in the next host. Regardless of scheme, all viruses converge and are absolutely reliant on RNA, and more specifically the host ability to translate RNA molecules into proteins. Thus, both viruses and hosts have devoted significant coding capital in regulating RNA, with emphasis on the evolutionary struggle that exists at this nexus. The over-arching theme of this thesis is to illuminate how and why influenza viruses co- opt, manipulate, and embrace RNA regulation strategies of human hosts. In the first chapter of this thesis, we profiled the RNA-binding characteristics of IFIT2, a canonically anti-viral and interferon-induced host protein, which influenza virus utilizes in a pro- viral manner. Through genome-wide cross-linking immunopreciptation sequencing (CLIP-seq) experiments, we found that IFIT2 binds mRNAs including those of influenza virus. We validated IFIT2 as an mRNA-binding protein, and found that IFIT2 specifically binds AU-rich regions in human RNAs with a preference for binding the mRNAs of other interferon-induced proteins. To accomplish a broadly anti-viral and selectively pro-viral function, we found that IFIT2 binds mRNAs to enhance their translation. Polysome and ribosome-profiling revealed that IFIT2-bound RNAs are poorly translated in the absence of IFIT2, including influenza virus mRNAs. This links the RNA-binding ability of IFIT2 to a functional role in translation, potentially explaining thexi contradiction between the previously observed anti-viral phenotypic role for IFIT2 and the mechanistically observed pro-viral role for IFIT2 in the context of influenza virus. Altogether, these data describe a new node for the regulation of translation during interferon responses and highlight the regulatory volatility that exists at the mRNA interface during infection. In the second chapter of this thesis, we sought to understand the non-specific RNA-binding capability of the influenza virus nucleoprotein, which binds and protects the negative-sense RNA genome to facilitate gene expression and genome replication. Despite this well-established role, the incongruity of the specificity of NP for the viral genome and known biochemical promiscuity of NP is not reconciled. To address this, we perfomed cross-linking immunoprecipitation sequencing to unbiasedly determine the identity of NP-bound RNAs during infection. NP binds the viral genome and anti-genome, but also engages with a number of discrete classes of host small non-coding RNAs. Many of these RNAs have been shown to be host-derived RIG-I agonists during DNA virus infections, or structurally resemble validated RIG-I agonists. We profiled the RNAs bound by RIG-I during infection with quantitative CLIP-seq and found that host RNAs are bound and sampled by RIG-I, and NP likely interferes with the ability of RIG-I to sample endogenous and exogenous agonists. These data show that NP not only acts as a structural component of the influenza gene expression and replication machinery, but also acts as a viral countermeasure for innate sensing of immunogenic RNAs. As a whole, this thesis describes two dissimilar instances by which influenza manipulates host RNA regulation. While the context of these host protein:viral mRNA and viral protein:host non-coding RNA interactions are different, they collectively suggest that RNA regulation during the host innate response to viral infection is an essential and commonplace strategy, but often leaves the host exposed to quickly adapting viral pathogens.

Download or read book Structure of the RNA dependent RNA Polymerase from Influenza C Virus written by Narin Hengrung and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Nucleic Acid Polymerases written by Katsuhiko S. Murakami and published by Springer Science & Business Media. This book was released on 2013-10-22 with total page 342 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides a review of the multitude of nucleic acid polymerases, including DNA and RNA polymerases from Archea, Bacteria and Eukaryota, mitochondrial and viral polymerases, and other specialized polymerases such as telomerase, template-independent terminal nucleotidyl transferase and RNA self-replication ribozyme. Although many books cover several different types of polymerases, no book so far has attempted to catalog all nucleic acid polymerases. The goal of this book is to be the top reference work for postgraduate students, postdocs, and principle investigators who study polymerases of all varieties. In other words, this book is for polymerase fans by polymerase fans. Nucleic acid polymerases play a fundamental role in genome replication, maintenance, gene expression and regulation. Throughout evolution these enzymes have been pivotal in transforming life towards RNA self-replicating systems as well as into more stable DNA genomes. These enzymes are generally extremely efficient and accurate in RNA transcription and DNA replication and share common kinetic and structural features. How catalysis can be so amazingly fast without loss of specificity is a question that has intrigued researchers for over 60 years. Certain specialized polymerases that play a critical role in cellular metabolism are used for diverse biotechnological applications and are therefore an essential tool for research.

Download or read book Proteomic and Functional Studies of the Influenza A Virus PA X Protein written by Brittany Porter and published by . This book was released on 2019 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Viral RNA endonuclease activity is required for influenza A virus (IAV) replication. This activity resides in the polymerase acidic (PA) protein, which assembles into viral RNA-dependent RNA polymerase (RdRp) complexes and cleaves nascent host pre-mRNAs proximal to 5'-m7G caps, creating primers for viral mRNA synthesis. A rare (+1) ribosomal frameshifting event during translation of the PA open reading frame (ORF) creates the polymerase acidic-X (PA-X) protein. PA-X retains the amino-terminal PA RNA endonuclease domain, but contains a novel short carboxy-terminus, dubbed the X-ORF. Accumulating evidence indicates PA-X is a host shutoff protein functioning in the nucleus, selectively cleaving RNAs transcribed by host RNA polymerase II (pol II) while sparing RNA pol I, III, and viral transcripts. The molecular mechanism for this specificity remains to be elucidated. I hypothesize that PA-X gains access to target RNAs by X-ORF-mediated interaction with host proteins. In this study, I used a proximity labeling proteomic method known as BioID to identify host proteins that interact with the X-ORF. In BioID, fusion of the bait protein to a promiscuous biotin ligase allows efficient biotinylation of lysine residues on nearby proteins. X-ORF baits subjected to BioID included a 61-amino acid variant from A/Puerto Rico/8/1934 (H1N1) and a truncated 41-amino acid variant from A/California/7/2009 (H1N1), as well as a mutant X-ORF lacking basic residues required for nuclear localization. Affinity-purified proteins were trypsinized, subjected to reductive dimethylation with stable isotope tags, and identified by mass spectrometry. Using quantitative analysis, 29 high-confidence candidate X-ORF-interacting proteins were identified. X-ORF interacting proteins were validated using a luciferase-based functional assay in cells where each candidate host gene was silenced by short-hairpin RNAs. Through this study, the cleavage factor I (CFIm) complex proteins, cleavage and polyadenylation specificity factor subunit 5 (CPSF5) and CPSF6, were identified as required for PA-X function. The CFIm complex is poorly characterized but is known to influence site selection for mRNA 3'-end cleavage and polyadenylation. My observations are concordant with the emerging model for PA-X host shutoff activity, which has been shown to require canonical mRNA 3'-end processing mechanisms.

Download or read book RNAi written by Gregory J. Hannon and published by CSHL Press. This book was released on 2003 with total page 568 pages. Available in PDF, EPUB and Kindle. Book excerpt: In the past three years, the use of double-stranded RNA to silence gene activity has become widely and rapidly adopted. RNA interference is highly specific and remarkably potent, and it acts on cells and tissues far removed from the site of introduction. The principles behind RNAi are just being uncovered, but this laboratory technique has been applied effectively in a wide variety of animal and plant species. Variations on RNAi are revolutionizing many approaches to experimental biology, complementing traditional genetic technologies with a quicker and less expensive way of mimicking the effects of mutations both in cell cultures and in living animals. Recent advances in the use of RNAi to engineer heritable silencing in mammals, to alter stem cells for organ reconstitution, and to alter the course of disease in model systems indicate that RNAi may have a future in disease therapy. Written by pioneers in this new field and edited by Gregory Hannon, one of its leading figures, RNAi: A Guide to Gene Silencing presents the principles of RNAi and reliable protocols for its laboratory use in Caenorhabditis elegans, Drosophila, plants, avian embryos, mammalian cells, mouse oocytes, and more. This important and unique book is an essential laboratory resource for scientists studying gene regulation and for all experimental biologists interested in the emerging practical applications of RNAi.

Download or read book Virus Protein and Nucleoprotein Complexes written by J. Robin Harris and published by Springer. This book was released on 2018-06-13 with total page 443 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Subcellular Biochemistry series has recently embarked upon an almost encyclopaedic coverage of topics relating to the structure and function of macromolecular complexes (Volumes 82, 83 and 87). The present multi-author text covers numerous aspects of current research into molecular virology, with emphasis upon viral protein and nucleoprotein structure and function. Structural data from cryo-electron microscopy and X-ray crystallography is displayed throughout the book. The 17 chapters in the book cover diverse interesting topics, all currently under investigation, contributed by authors who are active actively involved in present-day research. Whilst structural aspects predominate, there is much consideration of the structure-function relationship. In addition, the book correlates with and extends from Volume 68 of the series “Structure and Physics of Viruses: An Integrated Textbook”. This book is directed primarily at professionals that work in the broad field of Structural Biology and will be of particular interest to Structural Virologists. The editors, David Bhella and Robin Harris, have much experience in virology and protein structure, respectively. Dr Bhella is Director of the Scottish Macromolecular Imaging Centre. Professor Robin Harris is the long-standing Series Editor of the Subcellular Biochemistry series. He has edited and contributed to several books in the series.

Download or read book Functional Interactions of Coxsackievirus B3 RNA dependent RNA Polymerase in Viral Replication written by Yolanda Corona Bell and published by . This book was released on 1999 with total page 406 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book A Study of Viral and Cellular Factors in the Regulation of the Influenza Virus RNA dependent RNA Polymerase written by Ashley D. York and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Structural and Functional Characterization of the Phosphoprotein Subunit of the Sendai Virus RNA dependent RNA Polymerase written by Mary Catherine Bowman and published by . This book was released on 1997 with total page 340 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Molecular Virology written by Susanne Modrow and published by Springer. This book was released on 2013-09-18 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The book gives a comprehensive overview on the knowledge of virus infection relevant for humans and animals. For each virus family the molecular details of the virus particle and the viral replication cycle are described. In the case of virus types with relevance for human and/or animal health the data on molecular biology, genetics and virus-cell interaction are combined with those concerning, pathogenesis, epidemiology, clinics, prevention and therapy.