Download or read book Functional Studies on the PB2 Subunit of the RNA Polymerase Complex of Influenza Viruses written by Simon M. Carr and published by . This book was released on 2005 with total page 278 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 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 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 Phosphorylation Controls Assembly and Activity of the Influenza Virus Replication Machinery written by Anthony Rohit Dawson and published by . This book was released on 2020 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Successful influenza virus replication requires that viral gene expression and genome replication are balanced during viral infection. Both of these tasks occur via the viral ribonucleoprotein (RNP) containing genomic RNA encapsidated by viral nucleoprotein (NP) and bound by the viral RNA-dependent RNA polymerase consisting of subunits PB1, PB2 and PA. RNP function is regulated by multiple host factors and post-translational modifications. In particular, studies presented here provide evidence that phosphorylation by host kinases controls the activities of all RNP proteins. The overall goal of this thesis is to define how host-mediated phosphorylation controls RNA synthesis of the influenza virus replication machinery. Initial studies sought to understand how RNP assembly is regulated. RNP assembly is required only for replication and assembly requires NP to oligomerizes along genomic RNA. We determined that phosphorylation of the oligomeric interface inhibits RNP assembly. These phosphorylation sites are conserved among influenza virus genera suggesting a common regulatory scheme. These studies also identified the host kinase PKC Îþ as a key modifier of NP and regulator of influenza virus RNA synthesis. Additional studies explored how phosphorylation controls the influenza virus polymerase. Each polymerase subunit possesses unique functions to enable transcription and genome replication. PB1 harbors the catalytic activity of the polymerase. We established that phosphorylation of PB1 controls both RNA binding and transcription by the viral polymerase. Moreover, prior studies determined that transcription by the polymerase occurs via a cap-snatching mechanism wherein the polymerase binds capped host mRNA, cleaves the 5' end to produce a short capped oligonucleotide, and uses the cleaved product to prime transcription of viral genes. The PB2 subunit harbors the required cap binding activity. We provide evidence that phosphorylation controls PB2 cap binding. The endonuclease activity required for cap snatching resides in PA. Experiments with PA phospho-mutants display severe defects in transcription, likely caused by impaired endonuclease activity. Some identified phosphorylated residues appear essential for total polymerase function, whereas the function of other phosphorylation events remains elusive. Thus, phosphorylation indirectly regulates genome replication through NP modifications and changes in RNP assembly, and directly regulates transcription and replication by modifying the polymerase. We also analyzed the phosphoproteome of synchronously influenza virus-infected cells. Viral phosphoproteins generally increase throughout infection commensurate with the abundance of these proteins, suggesting that viral protein phosphorylation does not change en masse to segregate functions of viral protein at discrete times during infection. In contrast, host phosphoprotein abundances show multiple patterns, potentially enabling key viral replication events. In sum, directed studies of specific phosphorylation events coupled with broad analysis of the host phosphoproteome during infection provide a larger framework to understand post translational control of the influenza virus replication cycle.

Download or read book Conserved Polymerase Structural Features and Ubiquitination Regulate the Influenza Virus RNA Replication Machinery written by and published by . This book was released on 2016 with total page 158 pages. Available in PDF, EPUB and Kindle. Book excerpt: The influenza A virus polymerase is essential for the virus life cycle. The polymerase is a trimeric complex composed of subunits PA, PB1, and PB2 and associates with viral RNAs and nucleoprotein (NP) to form higher order ribonucleoprotein (RNP) complexes. In the context of these RNPs, the polymerase expresses viral genes and replicates the viral genome. The polymerase is also a major determinant of influenza virus host tropism and pathogenicity. It is a target for species-specific restriction of influenza viruses in mammals. Polymerases encoded by avian influenza viruses do not function efficiently in mammals. This restriction has been mapped to position 627 in the PB2 subunit, which is normally a glutamic acid in avian isolates. Conversely, a lysine is present at position 627 in most mammalian viral isolates and creates a basic face on the domain surface that confers high activity in mammals. In addition to species-specific regulation, the polymerase is also regulated temporally over the course of infection to ensure coordinated expression of viral genes as well as replication of the viral genome. Various host factors and processes have been implicated in regulation of the IAV polymerase function, including post-translational modifications, however the mechanisms are not fully understood. We created a series of mutants in the 627 domain of the PB2 subunit to alter the conserved “P[F/P]AAAPP” sequence motif and a number of conserved basic residues that give the domain surface a basic face. Mutating the basic face or the P[F/P]AAAP motif impaired polymerase activity, assembly of replication complexes and viral replication. We found that the P[F/P]AAAP motif residues were important for polymerase function in both human and chicken cells, suggesting that they play a structural role and are essential for overall polymerase function. We also identified PB2 positions 586 and 589 on the basic surface of the 627 domain to be species-specific determinants of polymerase function that are preferentially required for function in human versus avian cells. Thus, we identified new residues in the 627 domain that regulate overall polymerase function and those that function in a species-specific fashion. This work highlights the importance of the surface charge and structure of the PB2 627 domain for virus replication and host adaptation. We assessed the ubiquitination status of the RNP complex and investigated the effect of ubiquitin expression on polymerase function. We show that all protein subunits in the RNP complex are ubiquitinated and that their levels are not significantly affected, despite the well known activity of ubiquitin-mediated protein degradation. Instead, we found that ubiquitination and an active proteasome enhance polymerase activity. Ubiquitin expression up-regulates polymerase function causing increased accumulation of vRNA, cRNA and mRNA and enhanced viral gene expression during infection. We show that ubiquitin expression enhances polymerase activity independent of NP or RNP assembly. Ubiquitination and the proteasome pathway play multiple roles in the influenza virus cycle, and we now demonstrate that ubiquitination also modulates polymerase activity independent of protein degradation. Overall, we describe here key features of the influenza virus polymerase that regulate its function in a species-specific fashion and a new way in which a host cell process can be co-opted by the virus to enhance its polymerase function.

Download or read book Influenza written by Gabriele Neumann (Professor at the Influenza Research Institute) and published by . This book was released on 2020 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: "Influenza is a serious disease that affects millions worldwide every year. This book discusses cutting edge research on the viruses that cause the disease, its effects on the host, and current vaccine design strategies"--

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 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 Viral Molecular Machines written by Michael G. Rossmann and published by Springer Science & Business Media. This book was released on 2012-02-02 with total page 685 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book will contain a series of solicited chapters that concern with the molecular machines required by viruses to perform various essential functions of virus life cycle. The first three chapters (Introduction, Molecular Machines and Virus Architecture) introduce the reader to the best known molecular machines and to the structure of viruses. The remainder of the book will examine in detail various stages of the viral life cycle. Beginning with the viral entry into a host cell, the book takes the reader through replication of the genome, synthesis and assembly of viral structural components, genome packaging and maturation into an infectious virion. Each chapter will describe the components of the respective machine in molecular or atomic detail, genetic and biochemical analyses, and mechanism. Topics are carefully selected so that the reader is exposed to systems where there is a substantial infusion of new knowledge in recent years, which greatly elevated the fundamental mechanistic understanding of the respective molecular machine. The authors will be encouraged to simplify the detailed knowledge to basic concepts, include provocative new ideas, as well as design colorful graphics, thus making the cutting-edge information accessible to broad audience.

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 Studies on the Structure and Function of the Influenza Virus Ribonucleoprotein and Polymerase Complex written by Klaus Klumpp and published by . This book was released on 1997 with total page 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 Virus written by Yoshihiro Kawaoka and published by Humana Press. This book was released on 2016-08-23 with total page 234 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reports of influenza-like illnesses date back to the Middle Ages, and outbreaks of influenza likely afflicted humans long before that. Over the last half century, influenza virus research has led to the development of two classes of antivirals – ion channel and neuraminidase inhibitors. Recently, a method of the artificial generation of an influenza virus was established. This system has been instrumental in the development of novel influenza vaccines and in the understanding of viral pathogenicity and the functions of viral proteins. Influenza Virus: Methods and Protocols summarizes the current techniques that have made this progress possible, ranging from protocols for virus isolation, growth, and subtyping to procedures for the efficient generation of any influenza virus. Written in the successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Influenza Virus: Methods and Protocols seeks to serve both professionals and novices with the techniques used in numerous laboratories around the world that are, thus, the building blocks that underpin almost all influenza virus research.

Download or read book Polymerase Activity of Chimeric Polymerase written by Wing-Hong Chin and published by Open Dissertation Press. This book was released on 2017-01-26 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation, "Polymerase Activity of Chimeric Polymerase: a Determining Factor for an Influenza Virus to Be a Pandemic Strain" by Wing-hong, Chin, 錢永康, 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 influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza. DOI: 10.5353/th_b4979918 Subjects: RNA polymerases Influenza viruses Nucleoproteins

Download or read book Textbook of Influenza written by Robert G. Webster and published by John Wiley & Sons. This book was released on 2014-01-06 with total page 684 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Textbook of Influenza is a comprehensive resource covering all aspects of influenza, from the genetic and molecular biology of the virus through to clinical aspects of the disease and the latest drug developments and treatments. This new edition has been completely revised and reflects the integration of disciplines concerning the emergence, evolution, pathogenesis and control of influenza viruses in the field of human and veterinary public health. Textbook of Influenza examines the lessons learnt from the latest pandemic and provides the current state of knowledge for many yet unresolved issues related to virus origin, spread, pathogenesis and disease severity to better prepare for future pandemics. It covers the background to recent advances in influenza genomics and reverse genetics which have allowed the identification of virus virulence factors and the analysis and reconstruction of influenza viruses such as the 1918 Spanish flu strain. This new edition is divided into eight key sections, containing chapters co-written by international experts from both the clinical and scientific communities, covering: • Influenza Perspectives • Structure and Replication • Evolution and Ecology • Epidemiology and Surveillance • Immunology • Vaccines and Vaccine Development • Clinical Aspects and Antivirals • Public Health Textbook of Influenza is for all those working in the area of influenza including clinical and basic scientists, immunologists, molecular and structural virologists, public health officials and global pandemic control planners.

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