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Sample records for coronavirus rna synthesis

  1. Continuous and Discontinuous RNA Synthesis in Coronaviruses.

    PubMed

    Sola, Isabel; Almazán, Fernando; Zúñiga, Sonia; Enjuanes, Luis

    2015-11-01

    Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein-RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5' and 3' untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.

  2. The Nonstructural Proteins Directing Coronavirus RNA Synthesis and Processing.

    PubMed

    Snijder, E J; Decroly, E; Ziebuhr, J

    2016-01-01

    Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies. © 2016 Elsevier Inc. All rights reserved.

  3. Insights into RNA synthesis, capping, and proofreading mechanisms of SARS-coronavirus.

    PubMed

    Sevajol, Marion; Subissi, Lorenzo; Decroly, Etienne; Canard, Bruno; Imbert, Isabelle

    2014-12-19

    The successive emergence of highly pathogenic coronaviruses (CoVs) such as the Severe Acute Respiratory Syndrome (SARS-CoV) in 2003 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in 2012 has stimulated a number of studies on the molecular biology. This research has provided significant new insight into functions and activities of the replication/transcription multi-protein complex. The latter directs both continuous and discontinuous RNA synthesis to replicate and transcribe the large coronavirus genome made of a single-stranded, positive-sense RNA of ∼30 kb. In this review, we summarize our current understanding of SARS-CoV enzymes involved in RNA biochemistry, such as the in vitro characterization of a highly active and processive RNA polymerase complex which can associate with methyltransferase and 3'-5' exoribonuclease activities involved in RNA capping, and RNA proofreading, respectively. The recent discoveries reveal fascinating RNA-synthesizing machinery, highlighting the unique position of coronaviruses in the RNA virus world. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

    PubMed Central

    Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R.; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A.; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-01-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections. PMID:24874215

  5. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    PubMed

    Lundin, Anna; Dijkman, Ronald; Bergström, Tomas; Kann, Nina; Adamiak, Beata; Hannoun, Charles; Kindler, Eveline; Jónsdóttir, Hulda R; Muth, Doreen; Kint, Joeri; Forlenza, Maria; Müller, Marcel A; Drosten, Christian; Thiel, Volker; Trybala, Edward

    2014-05-01

    Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  6. Coronavirus cis-Acting RNA Elements.

    PubMed

    Madhugiri, R; Fricke, M; Marz, M; Ziebuhr, J

    2016-01-01

    Coronaviruses have exceptionally large RNA genomes of approximately 30 kilobases. Genome replication and transcription is mediated by a multisubunit protein complex comprised of more than a dozen virus-encoded proteins. The protein complex is thought to bind specific cis-acting RNA elements primarily located in the 5'- and 3'-terminal genome regions and upstream of the open reading frames located in the 3'-proximal one-third of the genome. Here, we review our current understanding of coronavirus cis-acting RNA elements, focusing on elements required for genome replication and packaging. Recent bioinformatic, biochemical, and genetic studies suggest a previously unknown level of conservation of cis-acting RNA structures among different coronavirus genera and, in some cases, even beyond genus boundaries. Also, there is increasing evidence to suggest that individual cis-acting elements may be part of higher-order RNA structures involving long-range and dynamic RNA-RNA interactions between RNA structural elements separated by thousands of nucleotides in the viral genome. We discuss the structural and functional features of these cis-acting RNA elements and their specific functions in coronavirus RNA synthesis. © 2016 Elsevier Inc. All rights reserved.

  7. [Coronaviruses].

    PubMed

    Taguchi, Fumihiro

    2011-12-01

    Coronaviruses contain positive-stranded RNA with ca. 30 kb as a genome, which is wrapped by the envelope, and constitute Nidovirales together with Arteriviridae. The feature of viruses in Nidovirales is the unique structure of the mRNA set, called 3' co-terminal nested set. Coronaviruses have several to more than 10 different species of subgenomic mRNA and generally only the OFR located in the 5' end of each mRNA is translated. The 5' 20 kb of the coronavirus genome or mRNA-1 consists of two ORFs, 1a and 1b, between that there is a unique RNA structure called pseudoknot. From mRNA-1, 1a as well as 1a+1b are translated; the latter 1a+1b results from the translation due to ribosomal frame-shifting facilitated by the pseudoknot structure. From those two proteins, totally 16 proteins are produced as a result of auto-cleavage by the proteases included in la protein. Those proteins exhibit different functions, such as RNA-dependent RNA polymerase, helicase, proteases and proteins that regulate cellular functions, mRNAs smaller than mRNA-2 translate in general the structural proteins, nucleocapsid (N) protein, spike (S) protein, integrated membrane (M) protein and envelope (E) proteins. Those proteins assemble to the vesicles located from ER to Golgi (ER Golgi intermediate compartment) and virions bud into the vesicles. Those virions are released from infected cells via exocytosis.

  8. Molecular mechanisms of coronavirus RNA capping and methylation.

    PubMed

    Chen, Yu; Guo, Deyin

    2016-02-01

    The 5'-cap structures of eukaryotic mRNAs are important for RNA stability, pre-mRNA splicing, mRNA export, and protein translation. Many viruses have evolved mechanisms for generating their own cap structures with methylation at the N7 position of the capped guanine and the ribose 2'-Oposition of the first nucleotide, which help viral RNAs escape recognition by the host innate immune system. The RNA genomes of coronavirus were identified to have 5'-caps in the early 1980s. However, for decades the RNA capping mechanisms of coronaviruses remained unknown. Since 2003, the outbreak of severe acute respiratory syndrome coronavirus has drawn increased attention and stimulated numerous studies on the molecular virology of coronaviruses. Here, we review the current understanding of the mechanisms adopted by coronaviruses to produce the 5'-cap structure and methylation modification of viral genomic RNAs.

  9. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells

    PubMed Central

    Nakagawa, K.; Lokugamage, K.G.; Makino, S.

    2017-01-01

    Coronaviruses have large positive-strand RNA genomes that are 5′ capped and 3′ polyadenylated. The 5′-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15–16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3′-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5′ leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells. PMID:27712623

  10. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells.

    PubMed

    Nakagawa, K; Lokugamage, K G; Makino, S

    2016-01-01

    Coronaviruses have large positive-strand RNA genomes that are 5' capped and 3' polyadenylated. The 5'-terminal two-thirds of the genome contain two open reading frames (ORFs), 1a and 1b, that together make up the viral replicase gene and encode two large polyproteins that are processed by viral proteases into 15-16 nonstructural proteins, most of them being involved in viral RNA synthesis. ORFs located in the 3'-terminal one-third of the genome encode structural and accessory proteins and are expressed from a set of 5' leader-containing subgenomic mRNAs that are synthesized by a process called discontinuous transcription. Coronavirus protein synthesis not only involves cap-dependent translation mechanisms but also employs regulatory mechanisms, such as ribosomal frameshifting. Coronavirus replication is known to affect cellular translation, involving activation of stress-induced signaling pathways, and employing viral proteins that affect cellular mRNA translation and RNA stability. This chapter describes our current understanding of the mechanisms involved in coronavirus mRNA translation and changes in host mRNA translation observed in coronavirus-infected cells.

  11. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

    PubMed

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H; Züst, Roland; Hwang, Mihyun; V'kovski, Philip; Stalder, Hanspeter; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; van Kuppeveld, Frank J M; Silverman, Robert H; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C; Ziebuhr, John; Weiss, Susan R; Kalinke, Ulrich; Thiel, Volker

    2017-02-01

    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

  12. RNA-RNA and RNA-protein interactions in coronavirus replication and transcription

    PubMed Central

    Sola, Isabel; Mateos-Gomez, Pedro A; Almazan, Fernando; Zuñiga, Sonia

    2011-01-01

    Coronavirus (CoV) RNA synthesis includes the replication of the viral genome, and the transcription of sgRNAs by a discontinuous mechanism. Both processes are regulated by RNA sequences such as the 5′ and 3′ untranslated regions (UTRs), and the transcription regulating sequences (TRSs) of the leader (TRS-L) and those preceding each gene (TRS-Bs). These distant RNA regulatory sequences interact with each other directly and probably through protein-RNA and protein-protein interactions involving viral and cellular proteins.1 By analogy to other plus-stranded RNA viruses, such as polioviruses, in which translation and replication switch involves a cellular factor (PCBP) and a viral protein (3CD),2 it is conceivable that in CoVs the switch between replication and transcription is also associated with the binding of proteins that are specifically recruited by the replication or transcription complexes. Complexes between RNA motifs such as TRS-L and the TRS-Bs located along the CoV genome are probably formed previously to the transcription start, and most likely promote template-switch of the nascent minus RNA to the TRS-L region.3 Many cellular proteins interacting with regulatory CoV RNA sequences4 are members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of RNA-binding proteins, involved in mRNA processing and transport, which shuttle between the nucleus and the cytoplasm. In the context of CoV RNA synthesis, these cellular ribonucleoproteins might also participate in RNA-protein complexes to bring into physical proximity TRS-L and distant TRS-B, as proposed for CoV discontinuous transcription.5–7 In this review, we summarize RNA-RNA and RNA-protein interactions that represent modest examples of complex quaternary RNA-protein structures required for the fine-tuning of virus replication. Design of chemically defined replication and transcription systems will help to clarify the nature and activity of these structures. PMID:21378501

  13. The RNA polymerase activity of SARS-coronavirus nsp12 is primer dependent

    PubMed Central

    te Velthuis, Aartjan J. W.; Arnold, Jamie J.; Cameron, Craig E.; van den Worm, Sjoerd H. E.; Snijder, Eric J.

    2010-01-01

    An RNA-dependent RNA polymerase (RdRp) is the central catalytic subunit of the RNA-synthesizing machinery of all positive-strand RNA viruses. Usually, RdRp domains are readily identifiable by comparative sequence analysis, but biochemical confirmation and characterization can be hampered by intrinsic protein properties and technical complications. It is presumed that replication and transcription of the ∼30-kb severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) RNA genome are catalyzed by an RdRp domain in the C-terminal part of nonstructural protein 12 (nsp12), one of 16 replicase subunits. However, thus far full-length nsp12 has proven refractory to expression in bacterial systems, which has hindered both the biochemical characterization of coronavirus RNA synthesis and RdRp-targeted antiviral drug design. Here, we describe a combined strategy involving bacterial expression of an nsp12 fusion protein and its in vivo cleavage to generate and purify stable SARS-CoV nsp12 (106 kDa) with a natural N-terminus and C-terminal hexahistidine tag. This recombinant protein possesses robust in vitro RdRp activity, as well as a significant DNA-dependent activity that may facilitate future inhibitor studies. The SARS-CoV nsp12 is primer dependent on both homo- and heteropolymeric templates, supporting the likeliness of a close enzymatic collaboration with the intriguing RNA primase activity that was recently proposed for coronavirus nsp8. PMID:19875418

  14. Manipulation of the coronavirus genome using targeted RNA recombination with interspecies chimeric coronaviruses.

    PubMed

    de Haan, Cornelis A M; Haijema, Bert Jan; Masters, Paul S; Rottier, Peter J M

    2008-01-01

    Targeted RNA recombination has proven to be a powerful tool for the genetic engineering of the coronavirus genome, particularly in its 3' part. Here we describe procedures for the generation of recombinant and mutant mouse hepatitis virus and feline infectious peritonitis virus. Key to the two-step method is the efficient selection of recombinant viruses based on host cell switching. The first step consists of the preparation---using this selection principle--of an interspecies chimeric coronavirus. In this virus the ectodomain of the spike glycoprotein is replaced by that of a coronavirus with a different species tropism. In the second step this chimeric virus is used as the recipient for recombination with synthetic donor RNA carrying the original spike gene. Recombinant viruses are then isolated on the basis of their regained natural (e.g., murine or feline) cell tropism. Additional mutations created in the donor RNA can be co-incorporated into the recombinant virus in order to generate mutant viruses.

  15. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities.

    PubMed

    Subissi, Lorenzo; Posthuma, Clara C; Collet, Axelle; Zevenhoven-Dobbe, Jessika C; Gorbalenya, Alexander E; Decroly, Etienne; Snijder, Eric J; Canard, Bruno; Imbert, Isabelle

    2014-09-16

    In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3'-5' exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5'-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

  16. One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

    PubMed Central

    Subissi, Lorenzo; Posthuma, Clara C.; Collet, Axelle; Zevenhoven-Dobbe, Jessika C.; Gorbalenya, Alexander E.; Decroly, Etienne; Snijder, Eric J.; Canard, Bruno; Imbert, Isabelle

    2014-01-01

    In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities. PMID:25197083

  17. Specific interaction between coronavirus leader RNA and nucleocapsid protein

    SciTech Connect

    Stohlman, S.A.; Baric, R.S.; Nelson, G.N.; Soe, L.H.; Welter, L.M.; Deans, R.J.

    1988-11-01

    Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. The authors accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article the authors report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. They have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.

  18. In Vitro Reconstitution of SARS-Coronavirus mRNA Cap Methylation

    PubMed Central

    Bouvet, Mickaël; Debarnot, Claire; Imbert, Isabelle; Selisko, Barbara; Snijder, Eric J.; Canard, Bruno; Decroly, Etienne

    2010-01-01

    SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5′ end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2′O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7MeGpppA-RNAs. The latter are then selectively 2′O-methylated by the 2′O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7MeGpppA2′OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design. PMID:20421945

  19. In vitro reconstitution of SARS-coronavirus mRNA cap methylation.

    PubMed

    Bouvet, Mickaël; Debarnot, Claire; Imbert, Isabelle; Selisko, Barbara; Snijder, Eric J; Canard, Bruno; Decroly, Etienne

    2010-04-22

    SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 5' end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-2'O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 (7Me)GpppA-RNAs. The latter are then selectively 2'O-methylated by the 2'O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 (7Me)GpppA(2'OMe)-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC(50) values in the micromolar range, providing a validated basis for anti-coronavirus drug design.

  20. RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

    PubMed

    Ishimaru, Daniella; Plant, Ewan P; Sims, Amy C; Yount, Boyd L; Roth, Braden M; Eldho, Nadukkudy V; Pérez-Alvarado, Gabriela C; Armbruster, David W; Baric, Ralph S; Dinman, Jonathan D; Taylor, Deborah R; Hennig, Mirko

    2013-02-01

    Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

  1. Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication

    PubMed Central

    Kindler, Eveline; Gil-Cruz, Cristina; Spanier, Julia; Li, Yize; Wilhelm, Jochen; Rabouw, Huib H.; Züst, Roland; Marti, Sabrina; Habjan, Matthias; Cervantes-Barragan, Luisa; Elliot, Ruth; Karl, Nadja; Gaughan, Christina; Silverman, Robert H.; Keller, Markus; Ludewig, Burkhard; Bergmann, Cornelia C.; Ziebuhr, John; Kalinke, Ulrich

    2017-01-01

    Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis–within the replicase complex—suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses. PMID:28158275

  2. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling.

    PubMed

    Irigoyen, Nerea; Firth, Andrew E; Jones, Joshua D; Chung, Betty Y-W; Siddell, Stuart G; Brierley, Ian

    2016-02-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global "snap-shot" of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  3. High-Resolution Analysis of Coronavirus Gene Expression by RNA Sequencing and Ribosome Profiling

    PubMed Central

    Jones, Joshua D.; Chung, Betty Y.-W.; Siddell, Stuart G.; Brierley, Ian

    2016-01-01

    Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal

  4. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

    PubMed

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

    2015-09-01

    RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression

  5. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells

    PubMed Central

    Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi

    2015-01-01

    ABSTRACT RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic

  6. Altering SARS coronavirus frameshift efficiency affects genomic and subgenomic RNA production.

    PubMed

    Plant, Ewan P; Sims, Amy C; Baric, Ralph S; Dinman, Jonathan D; Taylor, Deborah R

    2013-01-18

    In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. It was not clear if these differences were due to changes in genomic sequence, the protein sequence or the frequency of frameshifting. Here, viruses with synonymous codon changes are shown to produce different ratios of genomic and subgenomic RNA. These findings demonstrate that the protein sequence is not the primary cause of altered genomic and subgenomic RNA production. The synonymous codon changes affect both the structure of the frameshift signal and frameshifting efficiency. Small differences in frameshifting efficiency result in dramatic differences in genomic RNA production and TCID50 suggesting that the frameshifting frequency must stay above a certain threshold for optimal virus production. The data suggest that either the RNA sequence or the ratio of viral proteins resulting from different levels of frameshifting affects viral replication.

  7. Mouse Hepatitis Coronavirus RNA Replication Depends on GBF1-Mediated ARF1 Activation

    PubMed Central

    Verheije, Monique H.; Raaben, Matthijs; Mari, Muriel; te Lintelo, Eddie G.; Reggiori, Fulvio; van Kuppeveld, Frank J. M.; Rottier, Peter J. M.; de Haan, Cornelis A. M.

    2008-01-01

    Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected. PMID:18551169

  8. Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition

    SciTech Connect

    Versteeg, Gijs A.; Bredenbeek, Peter J.; Worm, Sjoerd H.E. van den; Spaan, Willy J.M. . E-mail: w.j.m.spaan@lumc.nl

    2007-04-25

    Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction.

  9. Coronavirus diversity, phylogeny and interspecies jumping.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Huang, Yi; Yuen, Kwok-Yung

    2009-10-01

    The SARS epidemic has boosted interest in research on coronavirus biodiversity and genomics. Before 2003, there were only 10 coronaviruses with complete genomes available. After the SARS epidemic, up to December 2008, there was an addition of 16 coronaviruses with complete genomes sequenced. These include two human coronaviruses (human coronavirus NL63 and human coronavirus HKU1), 10 other mammalian coronaviruses [bat SARS coronavirus, bat coronavirus (bat-CoV) HKU2, bat-CoV HKU4, bat-CoV HKU5, bat-CoV HKU8, bat-CoV HKU9, bat-CoV 512/2005, bat-CoV 1A, equine coronavirus, and beluga whale coronavirus] and four avian coronaviruses (turkey coronavirus, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13). Two novel subgroups in group 2 coronavirus (groups 2c and 2d) and two novel subgroups in group 3 coronavirus (groups 3b and 3c) have been proposed. The diversity of coronaviruses is a result of the infidelity of RNA-dependent RNA polymerase, high frequency of homologous RNA recombination, and the large genomes of coronaviruses. Among all hosts, the diversity of coronaviruses is most evidenced in bats and birds, which may be a result of their species diversity, ability to fly, environmental pressures, and habits of roosting and flocking. The present evidence supports that bat coronaviruses are the gene pools of group 1 and 2 coronaviruses, whereas bird coronaviruses are the gene pools of group 3 coronaviruses. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed, which were results of recent interspecies jumping and may be the cause of disastrous outbreaks of zoonotic diseases.

  10. Detection of ascitic feline coronavirus RNA from cats with clinically suspected feline infectious peritonitis.

    PubMed

    Soma, Takehisa; Wada, Makoto; Taharaguchi, Satoshi; Tajima, Tomoko

    2013-10-01

    Ascitic feline coronavirus (FCoV) RNA was examined in 854 cats with suspected feline infectious peritonitis (FIP) by RT-PCR. The positivity was significantly higher in purebreds (62.2%) than in crossbreds (34.8%) (P<0.0001). Among purebreds, the positivities in the Norwegian forest cat (92.3%) and Scottish fold (77.6%) were significantly higher than the average of purebreds (P=0.0274 and 0.0251, respectively). The positivity was significantly higher in males (51.5%) than in females (35.7%) (P<0.0001), whereas no gender difference has generally been noted in FCoV antibody prevalence, indicating that FIP more frequently develops in males among FCoV-infected cats. Genotyping was performed for 377 gene-positive specimens. Type I (83.3%) was far more predominantly detected than type II (10.6%) (P<0.0001), similar to previous serological and genetic surveys.

  11. Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

    SciTech Connect

    Sardinia, L.M.

    1985-01-01

    The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with /sup 35/S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with /sup 32/P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships.

  12. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    PubMed

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

  13. Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

    PubMed Central

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  14. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences.

    PubMed

    Drexler, Jan Felix; Gloza-Rausch, Florian; Glende, Jörg; Corman, Victor Max; Muth, Doreen; Goettsche, Matthias; Seebens, Antje; Niedrig, Matthias; Pfefferle, Susanne; Yordanov, Stoian; Zhelyazkov, Lyubomir; Hermanns, Uwe; Vallo, Peter; Lukashev, Alexander; Müller, Marcel Alexander; Deng, Hongkui; Herrler, Georg; Drosten, Christian

    2010-11-01

    Bats may host emerging viruses, including coronaviruses (CoV). We conducted an evaluation of CoV in rhinolophid and vespertilionid bat species common in Europe. Rhinolophids carried severe acute respiratory syndrome (SARS)-related CoV at high frequencies and concentrations (26% of animals are positive; up to 2.4×10(8) copies per gram of feces), as well as two Alphacoronavirus clades, one novel and one related to the HKU2 clade. All three clades present in Miniopterus bats in China (HKU7, HKU8, and 1A related) were also present in European Miniopterus bats. An additional novel Alphacoronavirus clade (bat CoV [BtCoV]/BNM98-30) was detected in Nyctalus leisleri. A CoV grouping criterion was developed by comparing amino acid identities across an 816-bp fragment of the RNA-dependent RNA polymerases (RdRp) of all accepted mammalian CoV species (RdRp-based grouping units [RGU]). Criteria for defining separate RGU in mammalian CoV were a >4.8% amino acid distance for alphacoronaviruses and a >6.3% distance for betacoronaviruses. All the above-mentioned novel clades represented independent RGU. Strict associations between CoV RGU and host bat genera were confirmed for six independent RGU represented simultaneously in China and Europe. A SARS-related virus (BtCoV/BM48-31/Bulgaria/2008) from a Rhinolophus blasii (Rhi bla) bat was fully sequenced. It is predicted that proteins 3b and 6 were highly divergent from those proteins in all known SARS-related CoV. Open reading frame 8 (ORF8) was surprisingly absent. Surface expression of spike and staining with sera of SARS survivors suggested low antigenic overlap with SARS CoV. However, the receptor binding domain of SARS CoV showed higher similarity with that of BtCoV/BM48-31/Bulgaria/2008 than with that of any Chinese bat-borne CoV. Critical spike domains 472 and 487 were identical and similar, respectively. This study underlines the importance of assessments of the zoonotic potential of widely distributed bat-borne CoV.

  15. Detection and genotyping of canine coronavirus RNA in diarrheic dogs in Japan.

    PubMed

    Soma, Takehisa; Ohinata, Tsuyoshi; Ishii, Hiroshi; Takahashi, Toshikazu; Taharaguchi, Satoshi; Hara, Motonobu

    2011-04-01

    To clarify the prevalence of canine coronavirus (CCoV) infection in Japan, faecal samples from 109 dogs with diarrhoea were examined for CCoV RNA together with canine parvovirus type 2 (CPV-2) DNA. The detection rates of CCoV and CPV-2 for dogs aged less than 1 year were 66.3% and 43.8%, while those for dogs aged 1 year or older were 6.9% and 10.3%, respectively, which were significantly different (p<0.0001 and p=0.0003, respectively), indicating not CPV-2 but CCoV is an important diarrhoea-causing organism in juvenile dogs. Among the CCoV-positive dogs, 65.5% and 72.7% showed to be positive for CCoV types I and II, respectively, and simultaneous detection rate of both types was high at 40.0%. Furthermore, transmissible gastroenteritis virus (TGEV)-like CCoV RNA was detected from 8 dogs. These findings indicate that CCoV type I and TGEV-like CCoV are already circulating in Japan, though no reports have been presented to date. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Bovine coronavirus nonstructural protein 1 (p28) is an RNA binding protein that binds terminal genomic cis-replication elements.

    PubMed

    Gustin, Kortney M; Guan, Bo-Jhih; Dziduszko, Agnieszka; Brian, David A

    2009-06-01

    Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5'-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of approximately 60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5' untranslated region (UTR)- and one 3' UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5' UTR with approximately 2.5 muM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.

  17. Flavivirus RNA Synthesis in vitro

    PubMed Central

    Padmanabhan, Radhakrishnan; Takhampunya, Ratree; Teramoto, Tadahisa; Choi, Kyung H.

    2015-01-01

    Summary Establishment of in vitro systems to study mechanisms of RNA synthesis for positive strand RNA viruses have been very useful in the past and have shed light on the composition of protein and RNA components, optimum conditions, the nature of the products formed, cis-acting RNA elements and trans-acting protein factors required for efficient synthesis. In this review, we summarize our current understanding regarding the requirements for flavivirus RNA synthesis in vitro. We describe details of reaction conditions, the specificity of template used by either the multi-component membrane-bound viral replicase complex or by purified, recombinant RNA-dependent RNA polymerase. We also discuss future perspectives to extend the boundaries of our knowledge. PMID:26272247

  18. The 7a accessory protein of severe acute respiratory syndrome coronavirus acts as an RNA silencing suppressor.

    PubMed

    Karjee, Sumona; Minhas, Ankita; Sood, Vikas; Ponia, Sanket S; Banerjea, Akhil C; Chow, Vincent T K; Mukherjee, Sunil K; Lal, Sunil K

    2010-10-01

    RNA silencing suppressors (RSSs) are well studied for plant viruses but are not well defined to date for animal viruses. Here, we have identified an RSS from a medically important positive-sense mammalian virus, Severe acute respiratory syndrome coronavirus. The viral 7a accessory protein suppressed both transgene and virus-induced gene silencing by reducing the levels of small interfering RNA (siRNA). The suppression of silencing was analyzed by two independent assays, and the middle region (amino acids [aa] 32 to 89) of 7a was responsible for suppression. Finally, the RNA suppression property and the enhancement of heterologous replicon activity by the 7a protein were confirmed for animal cell lines.

  19. Catalysis and prebiotic RNA synthesis

    NASA Technical Reports Server (NTRS)

    Ferris, James P.

    1993-01-01

    The essential role of catalysis for the origins of life is discussed. The status of the prebiotic synthesis of 2',5'- and 3'5'-linked oligomers of RNA is reviewed. Examples of the role of metal ion and mineral catalysis in RNA oligomer formation are discussed.

  20. Coronavirus Infections

    MedlinePlus

    Coronaviruses are common viruses that most people get some time in their life. They are common throughout the world, and they can infect people and animals. Several different coronaviruses can infect people ...

  1. Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

    PubMed

    Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

    2010-05-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

  2. Achieving a Golden Mean: Mechanisms by Which Coronaviruses Ensure Synthesis of the Correct Stoichiometric Ratios of Viral Proteins▿

    PubMed Central

    Plant, Ewan P.; Rakauskaitė, Rasa; Taylor, Deborah R.; Dinman, Jonathan D.

    2010-01-01

    In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed −1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the −1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a “golden mean” model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins. PMID:20164235

  3. Human coronavirus 229E nonstructural protein 13: characterization of duplex-unwinding, nucleoside triphosphatase, and RNA 5'-triphosphatase activities.

    PubMed

    Ivanov, Konstantin A; Ziebuhr, John

    2004-07-01

    The human coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5'-triphosphatase activity, which may be involved in the capping of viral RNAs.

  4. Tagetitoxin inhibits chloroplast RNA synthesis

    SciTech Connect

    Mathews, D.E.; Durbin, R.D.

    1987-04-01

    Tagetitoxin is a non-host specific phytotoxin which inhibits chloroplast development. Chloroplast encoded gene products as well as their transcripts are conspicuously depleted in toxin-treated tissue. Intact chloroplasts from 8-9 day old peas were incubated for 60 min. in the presence of tagetitoxin. This treatment reduced RNA synthesis but did not affect protein synthesis as measured by the incorporation of radiolabeled uridine or methionine, respectively. Tagetitoxin also inhibited chloroplast RNA synthesis in vitro. Total UTP incorporation was reduced 50% by 0.5..mu..M tagetitoxin in transcriptionally active chloroplast extracts containing 5mg/ml protein. In vitro transcription with purified E. coli RNA polymerase was also inhibited by tagetitoxin, yet wheat germ RNA polymerase II and several bacteriophage RNA polymerase enzymes were unaffected. Recent evidence suggests that RNA polymerase from chloroplasts and prokaryotes may share extensive homology. In light of this evidence and the authors own data, they propose that tagetitoxin directly inhibits chloroplast RNA polymerase.

  5. Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

    PubMed

    Berto, A; Anh, P H; Carrique-Mas, J J; Simmonds, P; Van Cuong, N; Tue, N T; Van Dung, N; Woolhouse, M E; Smith, I; Marsh, G A; Bryant, J E; Thwaites, G E; Baker, S; Rabaa, M A

    2017-04-18

    Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

  6. RNase L-Independent Specific 28S rRNA Cleavage in Murine Coronavirus-Infected Cells

    PubMed Central

    Banerjee, Sangeeta; An, Sungwhan; Zhou, Aimin; Silverman, Robert H.; Makino, Shinji

    2000-01-01

    We characterized a novel 28S rRNA cleavage in cells infected with the murine coronavirus mouse hepatitis virus (MHV). The 28S rRNA cleavage occurred as early as 4 h postinfection (p.i.) in MHV-infected DBT cells, with the appearance of subsequent cleavage products and a decrease in the amount of intact 28S rRNA with increasing times of infection; almost all of the intact 28S rRNA disappeared by 24 h p.i. In contrast, no specific 18S rRNA cleavage was detected in infected cells. MHV-induced 28S rRNA cleavage was detected in all MHV-susceptible cell lines and all MHV strains tested. MHV replication was required for the 28S rRNA cleavage, and mature cytoplasmic 28S rRNA underwent cleavage. In certain combination of cells and viruses, pretreatment of virus-infected cells with interferon activates a cellular endoribonuclease, RNase L, that causes rRNA degradation. No interferon was detected in the inoculum used for MHV infection. Addition of anti-interferon antibody to MHV-infected cells did not inhibit 28S rRNA cleavage. Furthermore, 28S rRNA cleavage occurred in an MHV-infected mouse embryonic fibroblast cell line derived from RNase L knockout mice. Thus, MHV-induced 28S rRNA cleavage was independent of the activation of RNase L. MHV-induced 28S rRNA cleavage was also different from apoptosis-related rRNA degradation, which usually occurs concomitantly with DNA fragmentation. In MHV-infected 17Cl-1 cells, 28S rRNA cleavage preceded DNA fragmentation by at least 18 h. Blockage of apoptosis in MHV-infected 17Cl-1 cells by treatment with a caspase inhibitor did not block 28S rRNA cleavage. Furthermore, MHV-induced 28S rRNA cleavage occurred in MHV-infected DBT cells that do not show apoptotic signs, including activation of caspase-3 and DNA fragmentation. Thus, MHV-induced 28S rRNA cleavage appeared to differ from any rRNA degradation mechanism described previously. PMID:10982321

  7. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex

    PubMed Central

    Bouvet, Mickaël; Imbert, Isabelle; Subissi, Lorenzo; Gluais, Laure; Canard, Bruno; Decroly, Etienne

    2012-01-01

    The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1–16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3′ to 5′ direction as well as a single mismatched nucleotide at the 3′-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3′-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2′-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism. PMID:22635272

  8. RNA 3'-end mismatch excision by the severe acute respiratory syndrome coronavirus nonstructural protein nsp10/nsp14 exoribonuclease complex.

    PubMed

    Bouvet, Mickaël; Imbert, Isabelle; Subissi, Lorenzo; Gluais, Laure; Canard, Bruno; Decroly, Etienne

    2012-06-12

    The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1-16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3' to 5' direction as well as a single mismatched nucleotide at the 3'-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3'-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2'-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism.

  9. Genotyping bovine coronaviruses.

    USDA-ARS?s Scientific Manuscript database

    Bovine coronaviruses (BoCV) are enveloped, single-stranded, positive-sense RNA viruses of the Coronaviridae family. Infection is associated with enteritis and pneumonia in calves and Winter Dysentery in adult cattle. Strains, isolated more than 50 years ago, are used in vaccines and as laboratory ...

  10. Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro.

    PubMed

    McDonagh, Phillip; Sheehy, Paul A; Norris, Jacqueline M

    2015-03-23

    Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure.

    PubMed Central

    Rowe, C L; Fleming, J O; Nathan, M J; Sgro, J Y; Palmenberg, A C; Baker, S C

    1997-01-01

    Coronavirus RNA evolves in the central nervous systems (CNS) of mice during persistent infection. This evolution can be monitored by detection of a viral quasispecies of spike deletion variants (SDVs) (C. L. Rowe, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-2969, 1997). We and others have found that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike gene sequence, termed the "hypervariable" region. To address how SDVs might arise, we generated the predicted folding structures of the positive- and negative-strand senses of the entire 4,139-nt spike RNA sequence. We found that a prominent, isolated stem-loop structure is coincident with the hypervariable region in each structure. To determine if this predicted stem-loop is a "hot spot" for RNA recombination, we assessed whether this region of the spike is more frequently deleted than three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of the spike sequence were exclusively deleted. Furthermore, sequence analysis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-frequency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely generated by intramolecular recombination. Overall, our results are consistent with there being an important role for the spike RNA secondary structure as a contributing factor in the generation of SDVs during persistent infection. PMID:9223514

  12. Detection of group 1 coronaviruses in bats in North America

    USGS Publications Warehouse

    Dominguez, S.R.; O'Shea, T.J.; Oko, L.M.; Holmes, K.V.

    2007-01-01

    The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People's Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription-PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

  13. Homology-Based Identification of a Mutation in the Coronavirus RNA-Dependent RNA Polymerase That Confers Resistance to Multiple Mutagens

    PubMed Central

    Sexton, Nicole R.; Smith, Everett Clinton; Blanc, Hervé; Vignuzzi, Marco; Peersen, Olve B.

    2016-01-01

    ABSTRACT Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity

  14. About Coronavirus

    MedlinePlus

    ... to moderate upper-respiratory tract illnesses, like the common cold. Coronaviruses are named for the crown-like spikes ... to moderate upper-respiratory tract illnesses, like the common cold, that last for a short amount of time. ...

  15. Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014.

    PubMed

    Reusken, C B; Farag, E A; Jonges, M; Godeke, G J; El-Sayed, A M; Pas, S D; Raj, V S; Mohran, K A; Moussa, H A; Ghobashy, H; Alhajri, F; Ibrahim, A K; Bosch, B J; Pasha, S K; Al-Romaihi, H E; Al-Thani, M; Al-Marri, S A; AlHajri, M M; Haagmans, B L; Koopmans, M P

    2014-06-12

    Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral RNA was detected in milk of five of these seven camels. The presence of MERS-CoV RNA in milk of camels actively shedding the virus warrants measures to prevent putative food-borne transmission of MERS-CoV.

  16. Coronaviruses: an overview of their replication and pathogenesis.

    PubMed

    Fehr, Anthony R; Perlman, Stanley

    2015-01-01

    Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the recently identified Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV).

  17. Coronaviruses: An Overview of Their Replication and Pathogenesis

    PubMed Central

    Fehr, Anthony R.; Perlman, Stanley

    2015-01-01

    Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We will also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the recently identified Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV). PMID:25720466

  18. Design, synthesis and evaluation of a series of acyclic fleximer nucleoside analogues with anti-coronavirus activity.

    PubMed

    Peters, Hannah L; Jochmans, Dirk; de Wilde, Adriaan H; Posthuma, Clara C; Snijder, Eric J; Neyts, Johan; Seley-Radtke, Katherine L

    2015-08-01

    A series of doubly flexible nucleoside analogues were designed based on the acyclic sugar scaffold of acyclovir and the flex-base moiety found in the fleximers. The target compounds were evaluated for their antiviral potential and found to inhibit several coronaviruses. Significantly, compound 2 displayed selective antiviral activity (CC50 >3× EC50) towards human coronavirus (HCoV)-NL63 and Middle East respiratory syndrome-coronavirus, but not severe acute respiratory syndrome-coronavirus. In the case of HCoV-NL63 the activity was highly promising with an EC50 <10 μM and a CC50 >100 μM. As such, these doubly flexible nucleoside analogues are viewed as a novel new class of drug candidates with potential for potent inhibition of coronaviruses.

  19. Biochemical and structural insights into the mechanisms of SARS coronavirus RNA ribose 2'-O-methylation by nsp16/nsp10 protein complex.

    PubMed

    Chen, Yu; Su, Ceyang; Ke, Min; Jin, Xu; Xu, Lirong; Zhang, Zhou; Wu, Andong; Sun, Ying; Yang, Zhouning; Tien, Po; Ahola, Tero; Liang, Yi; Liu, Xinqi; Guo, Deyin

    2011-10-01

    The 5'-cap structure is a distinct feature of eukaryotic mRNAs, and eukaryotic viruses generally modify the 5'-end of viral RNAs to mimic cellular mRNA structure, which is important for RNA stability, protein translation and viral immune escape. SARS coronavirus (SARS-CoV) encodes two S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTase) which sequentially methylate the RNA cap at guanosine-N7 and ribose 2'-O positions, catalyzed by nsp14 N7-MTase and nsp16 2'-O-MTase, respectively. A unique feature for SARS-CoV is that nsp16 requires non-structural protein nsp10 as a stimulatory factor to execute its MTase activity. Here we report the biochemical characterization of SARS-CoV 2'-O-MTase and the crystal structure of nsp16/nsp10 complex bound with methyl donor SAM. We found that SARS-CoV nsp16 MTase methylated m7GpppA-RNA but not m7GpppG-RNA, which is in contrast with nsp14 MTase that functions in a sequence-independent manner. We demonstrated that nsp10 is required for nsp16 to bind both m7GpppA-RNA substrate and SAM cofactor. Structural analysis revealed that nsp16 possesses the canonical scaffold of MTase and associates with nsp10 at 1∶1 ratio. The structure of the nsp16/nsp10 interaction interface shows that nsp10 may stabilize the SAM-binding pocket and extend the substrate RNA-binding groove of nsp16, consistent with the findings in biochemical assays. These results suggest that nsp16/nsp10 interface may represent a better drug target than the viral MTase active site for developing highly specific anti-coronavirus drugs.

  20. The polypyrimidine tract-binding protein affects coronavirus RNA accumulation levels and relocalizes viral RNAs to novel cytoplasmic domains different from replication-transcription sites.

    PubMed

    Sola, Isabel; Galán, Carmen; Mateos-Gómez, Pedro A; Palacio, Lorena; Zúñiga, Sonia; Cruz, Jazmina L; Almazán, Fernando; Enjuanes, Luis

    2011-05-01

    The coronavirus (CoV) discontinuous transcription mechanism is driven by long-distance RNA-RNA interactions between transcription-regulating sequences (TRSs) located at the 5' terminal leader (TRS-L) and also preceding each mRNA-coding sequence (TRS-B). The contribution of host cell proteins to CoV transcription needs additional information. Polypyrimidine tract-binding protein (PTB) was reproducibly identified in association with positive-sense RNAs of transmissible gastroenteritis coronavirus (TGEV) TRS-L and TRS-B by affinity chromatography and mass spectrometry. A temporal regulation of PTB cytoplasmic levels was observed during infection, with a significant increase from 7 to 16 h postinfection being inversely associated with a decrease in viral replication and transcription. Silencing the expression of PTB with small interfering RNA in two cell lines (Huh7 and HEK 293T) led to a significant increase of up to 4-fold in mRNA levels and virus titer, indicating a negative effect of PTB on CoV RNA accumulation. During CoV infection, PTB relocalized from the nucleus to novel cytoplasmic structures different from replication-transcription sites in which stress granule markers T-cell intracellular antigen-1 (TIA-1) and TIA-1-related protein (TIAR) colocalized. PTB was detected in these modified stress granules in TGEV-infected swine testis cells but not in stress granules induced by oxidative stress. Furthermore, viral genomic and subgenomic RNAs were detected in association with PTB and TIAR. These cytoplasmic ribonucleoprotein complexes might be involved in posttranscriptional regulation of virus gene expression.

  1. Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

    PubMed Central

    Chen, I-Jung; Yuann, Jeu-Ming P.; Chang, Yu-Ming; Lin, Shing-Yen; Zhao, Jincun; Perlman, Stanley; Shen, Yo-Yu; Huang, Tai-Huang; Hou, Ming-Hon

    2013-01-01

    Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication. PMID:23501675

  2. Regulation of Flavivirus RNA Synthesis and Capping

    PubMed Central

    Saeedi, Bejan J.; Geiss, Brian J.

    2013-01-01

    RNA viruses, such as flaviviruses, are able to efficiently replicate and cap their RNA genomes in vertebrate and invertebrate cells. Flaviviruses use several specialized proteins to first make an uncapped negative strand copy of the viral genome that is used as a template for the synthesis of large numbers of capped genomic RNAs. Despite using relatively simple mechanisms to replicate their RNA genomes, there are significant gaps in our understanding of how flaviviruses switch between negative and positive strand RNA synthesis and how RNA capping is regulated. Recent work has begun to provide a conceptual framework for flavivirus RNA replication and capping and shown some surprising roles for genomic RNA during replication and pathogenesis. PMID:23929625

  3. Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.

    PubMed

    Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

    2010-10-22

    Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

  4. Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

    PubMed Central

    Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

    2010-01-01

    Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses. PMID:20699222

  5. Aminoacyl-tRNA synthesis in Archaea.

    PubMed

    Ibba, M; Celic, I; Curnow, A; Kim, H; Pelaschier, J; Tumbula, D; Vothknecht, U; Woese, C; Söll, D

    1997-01-01

    The mechanism of aminoacyl-tRNA synthesis differs substantially between Archaea, Bacteria and Eukarya. Sequencing of archaeal genomes has suggested that the asparaginyl-, cysteinyl-, glutaminyl- and lysyl-tRNA synthetases are absent from a number of organisms in this kingdom. The absence of the asparaginyl- and glutaminyl-tRNA synthetases is in agreement with the observation that Asn-tRNA and Gln-tRNA are synthesized by tRNA-dependent transamidation of Asp-tRNA and Glu-tRNA respectively in the archaeon Haloferax volcanii. Biochemical and genetic studies have now shown that while the cysteinyl- and lysyl-tRNA synthetases are present, the enzymes responsible for these activities are unique to Archaea.

  6. Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

    PubMed Central

    Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

    2013-01-01

    Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

  7. More and More Coronaviruses: Human Coronavirus HKU1.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Yip, Cyril C Y; Huang, Yi; Yuen, Kwok-Yung

    2009-06-01

    After human coronaviruses OC43, 229E and NL63, human coronavirus HKU1 (HCoV-HKU1) is the fourth human coronavirus discovered. HCoV-HKU1 is a group 2a coronavirus that is still not cultivable. The G + C contents of HCoV-HKU1 genomes are 32%, the lowest among all known coronaviruses with complete genome sequences available. Among all coronaviruses, HCoV-HKU1 shows the most extreme codon usage bias, attributed most importantly to severe cytosine deamination. All HCoV-HKU1 genomes contain unique tandem copies of a 30-base acidic tandem repeat of unknown function at the N-terminus of nsp3 inside the acidic domain upstream of papain-like protease 1. Three genotypes, A, B and C, of HCoV-HKU1 and homologous recombination among their genomes, are observed. The incidence of HCoV-HKU1 infections is the highest in winter. Similar to other human coronaviruses, HCoV-HKU1 infections have been reported globally, with a median (range) incidence of 0.9 (0 - 4.4) %. HCoV-HKU1 is associated with both upper and lower respiratory tract infections that are mostly self-limiting. The most common method for diagnosing HCoV-HKU1 infection is RT-PCR or real-time RT-PCR using RNA extracted from respiratory tract samples such as nasopharyngeal aspirates (NPA). Both the pol and nucleocapsid genes have been used as the targets for amplification. Monoclonal antibodies have been generated for direct antigen detection in NPA. For antibody detection, Escherichia coli BL21 and baculovirus-expressed recombinant nucleocapsid of HCoV-HKU1 have been used for IgG and IgM detection in sera of patients and normal individuals, using Western blot and enzyme-linked immunoassay.

  8. Prebiotic RNA Synthesis by Montmorillonite Catalysis

    NASA Astrophysics Data System (ADS)

    Jheeta, Sohan; Joshi, Prakash C.

    2014-08-01

    This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the "Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA)" conference at the Open University, Milton Keynes, UK, 5-6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1). Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7) produced only dimers from its monomers in water, addition of sodium chloride (1 M) enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl- > Br- > I-. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt.

  9. Prebiotic RNA Synthesis by Montmorillonite Catalysis

    PubMed Central

    Jheeta, Sohan; Joshi, Prakash C.

    2014-01-01

    This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the “Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA)” conference at the Open University, Milton Keynes, UK, 5–6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1). Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7) produced only dimers from its monomers in water, addition of sodium chloride (1 M) enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl− > Br− > I−. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt. PMID:25370375

  10. Prebiotic RNA synthesis by montmorillonite catalysis.

    PubMed

    Jheeta, Sohan; Joshi, Prakash C

    2014-08-05

    This review summarizes our recent findings on the role of mineral salts in prebiotic RNA synthesis, which is catalyzed by montmorillonite clay minerals. The clay minerals not only catalyze the synthesis of RNA but also facilitate homochiral selection. Preliminary data of these findings have been presented at the "Horizontal Gene Transfer and the Last Universal Common Ancestor (LUCA)" conference at the Open University, Milton Keynes, UK, 5-6 September 2013. The objective of this meeting was to recognize the significance of RNA in LUCA. We believe that the prebiotic RNA synthesis from its monomers must have been a simple process. As a first step, it may have required activation of the 5'-end of the mononucleotide with a leaving group, e.g., imidazole in our model reaction (Figure 1). Wide ranges of activating groups are produced from HCN under plausible prebiotic Earth conditions. The final step is clay mineral catalysis in the presence of mineral salts to facilitate selective production of functional RNA. Both the clay minerals and mineral salts would have been abundant on early Earth. We have demonstrated that while montmorillonite (pH 7) produced only dimers from its monomers in water, addition of sodium chloride (1 M) enhanced the chain length multifold, as detected by HPLC. The effect of monovalent cations on RNA synthesis was of the following order: Li+ > Na+ > K+. A similar effect was observed with the anions, enhancing catalysis in the following order: Cl- > Br- > I-. The montmorillonite-catalyzed RNA synthesis was not affected by hydrophobic or hydrophilic interactions. We thus show that prebiotic synthesis of RNA from its monomers was a simple process requiring only clay minerals and a small amount of salt.

  11. Comprehensive detection and identification of seven animal coronaviruses and human respiratory coronavirus 229E with a microarray hybridization assay.

    PubMed

    Chen, Qin; Li, Jian; Deng, Zhirui; Xiong, Wei; Wang, Quan; Hu, Yong-Qiang

    2010-01-01

    Based on microarray hybridization, a diagnostic test for coronavirus infection was developed using eight coronavirus strains: canine coronavirus (CCoV), feline infectious peritonitis virus (FIPV), feline coronavirus (FCoV), bovine coronavirus (BCoV), porcine respiratory coronavirus (PRCoV), turkey enteritis coronavirus (TCoV), transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HRCoV). Up to 104 cDNA clones of eight viruses were obtained by reverse transcription PCR with different pairs of primers designed for each virus and a pair of universal primers designed for the RNA polymerase gene of coronavirus. Total RNAs extracted from virus were reverse transcribed, followed by multi-PCR amplification and labeled with Cy3-dCTP. All labeled cDNAs and prepared gene chips were subjected to specific hybridization. The results showed that extensive cross-reaction existed between CCoV, FCoV, FIPV, TGEV and PRCoV, while there was no cross-reaction between BCoV, TCoV and HRCoV. The ultimate specific gene chip was developed with DNA fragments reamplified from the chosen recombinant plasmids without cross-reaction between different coronaviruses. The hybridization results showed that this gene chip could specifically identify and distinguish the eight coronaviruses and the sensitivity of the chip may be 1,000x more sensitive than PCR, indicating that it can be used for the diagnosis of eight coronavirus infections at the same time.

  12. The RNA synthesis machinery of negative-stranded RNA viruses

    SciTech Connect

    Ortín, Juan; Martín-Benito, Jaime

    2015-05-15

    The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure of their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.

  13. Detection and Phylogenetic Analysis of Group 1 Coronaviruses in South American Bats

    PubMed Central

    Foster, Jerome E.; Zhu, Hua Chen; Zhang, Jin Xia; Smith, Gavin J.D.; Thompson, Nadin; Auguste, Albert J.; Ramkissoon, Vernie; Adesiyun, Abiodun A.; Guan, Yi

    2008-01-01

    Bat coronaviruses (Bt-CoVs) are thought to be the precursors of severe acute respiratory syndrome coronavirus. We detected Bt-CoVs in 2 bat species from Trinidad. Phylogenetic analysis of the RNA-dependent RNA polymerase gene and helicase confirmed them as group 1 coronaviruses. PMID:19046513

  14. Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs.

    PubMed

    Decaro, Nicola; Martella, Vito; Ricci, Dominga; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Cavaliere, Nicola; Di Trani, Livia; Tempesta, Maria; Buonavoglia, Canio

    2005-12-01

    Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

  15. Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT-PCR amplification coupled with oligonucleotide microarray hybridization.

    PubMed

    Zhang, Zhi-wei; Zhou, Yi-ming; Zhang, Yan; Guo, Yong; Tao, Sheng-ce; Li, Ze; Zhang, Qiong; Cheng, Jing

    2005-01-01

    We have developed a sensitive method for the detection of specific genes simultaneously. First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 10(0) copies/microL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.

  16. Dissection of Amino-Terminal Functional Domains of Murine Coronavirus Nonstructural Protein 3

    PubMed Central

    Hurst-Hess, Kelley R.; Kuo, Lili

    2015-01-01

    ABSTRACT Coronaviruses, the largest RNA viruses, have a complex program of RNA synthesis that entails genome replication and transcription of subgenomic mRNAs. RNA synthesis by the prototype coronavirus mouse hepatitis virus (MHV) is carried out by a replicase-transcriptase composed of 16 nonstructural protein (nsp) subunits. Among these, nsp3 is the largest and the first to be inserted into the endoplasmic reticulum. nsp3 comprises multiple structural domains, including two papain-like proteases (PLPs) and a highly conserved ADP-ribose-1″-phosphatase (ADRP) macrodomain. We have previously shown that the ubiquitin-like domain at the amino terminus of nsp3 is essential and participates in a critical interaction with the viral nucleocapsid protein early in infection. In the current study, we exploited atypical expression schemes to uncouple PLP1 from the processing of nsp1 and nsp2 in order to investigate the requirements of nsp3 domains for viral RNA synthesis. In the first strategy, a mutant was created in which replicase polyprotein translation initiated with nsp3, thereby establishing that complete elimination of nsp1 and nsp2 does not abolish MHV viability. In the second strategy, a picornavirus autoprocessing element was used to separate a truncated nsp1 from nsp3. This provided a platform for further dissection of amino-terminal domains of nsp3. From this, we found that catalytic mutation of PLP1 or complete deletion of PLP1 and the adjacent ADRP domain was tolerated by the virus. These results showed that neither the PLP1 domain nor the ADRP domain of nsp3 provides integral activities essential for coronavirus genomic or subgenomic RNA synthesis. IMPORTANCE The largest component of the coronavirus replicase-transcriptase complex, nsp3, contains multiple modules, many of which do not have clearly defined functions in genome replication or transcription. These domains may play direct roles in RNA synthesis, or they may have evolved for other purposes, such as

  17. Epigenetic Landscape during Coronavirus Infection

    PubMed Central

    Schäfer, Alexandra; Baric, Ralph S.

    2017-01-01

    Coronaviruses (CoV) comprise a large group of emerging human and animal pathogens, including the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) strains. The molecular mechanisms regulating emerging coronavirus pathogenesis are complex and include virus–host interactions associated with entry, replication, egress and innate immune control. Epigenetics research investigates the genetic and non-genetic factors that regulate phenotypic variation, usually caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. Epigenetic modifications, such as histone modifications, DNA methylation, chromatin remodeling, and non-coding RNAs, function as important regulators that remodel host chromatin, altering host expression patterns and networks in a highly flexible manner. For most of the past two and a half decades, research has focused on the molecular mechanisms by which RNA viruses antagonize the signaling and sensing components that regulate induction of the host innate immune and antiviral defense programs upon infection. More recently, a growing body of evidence supports the hypothesis that viruses, even lytic RNA viruses that replicate in the cytoplasm, have developed intricate, highly evolved, and well-coordinated processes that are designed to regulate the host epigenome, and control host innate immune antiviral defense processes, thereby promoting robust virus replication and pathogenesis. In this article, we discuss the strategies that are used to evaluate the mechanisms by which viruses regulate the host epigenome, especially focusing on highly pathogenic respiratory RNA virus infections as a model. By combining measures of epigenome reorganization with RNA and proteomic datasets, we articulate a spatial-temporal data integration approach to identify regulatory genomic clusters and regions that

  18. Epigenetic Landscape during Coronavirus Infection.

    PubMed

    Schäfer, Alexandra; Baric, Ralph S

    2017-02-15

    Coronaviruses (CoV) comprise a large group of emerging human and animal pathogens, including the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) strains. The molecular mechanisms regulating emerging coronavirus pathogenesis are complex and include virus-host interactions associated with entry, replication, egress and innate immune control. Epigenetics research investigates the genetic and non-genetic factors that regulate phenotypic variation, usually caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. Epigenetic modifications, such as histone modifications, DNA methylation, chromatin remodeling, and non-coding RNAs, function as important regulators that remodel host chromatin, altering host expression patterns and networks in a highly flexible manner. For most of the past two and a half decades, research has focused on the molecular mechanisms by which RNA viruses antagonize the signaling and sensing components that regulate induction of the host innate immune and antiviral defense programs upon infection. More recently, a growing body of evidence supports the hypothesis that viruses, even lytic RNA viruses that replicate in the cytoplasm, have developed intricate, highly evolved, and well-coordinated processes that are designed to regulate the host epigenome, and control host innate immune antiviral defense processes, thereby promoting robust virus replication and pathogenesis. In this article, we discuss the strategies that are used to evaluate the mechanisms by which viruses regulate the host epigenome, especially focusing on highly pathogenic respiratory RNA virus infections as a model. By combining measures of epigenome reorganization with RNA and proteomic datasets, we articulate a spatial-temporal data integration approach to identify regulatory genomic clusters and regions that

  19. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

    PubMed

    Decroly, Etienne; Debarnot, Claire; Ferron, François; Bouvet, Mickael; Coutard, Bruno; Imbert, Isabelle; Gluais, Laure; Papageorgiou, Nicolas; Sharff, Andrew; Bricogne, Gérard; Ortiz-Lombardia, Miguel; Lescar, Julien; Canard, Bruno

    2011-05-01

    Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2'-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in +RNA viruses.

  20. Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex

    PubMed Central

    Decroly, Etienne; Debarnot, Claire; Ferron, François; Bouvet, Mickael; Coutard, Bruno; Imbert, Isabelle; Gluais, Laure; Papageorgiou, Nicolas; Sharff, Andrew; Bricogne, Gérard; Ortiz-Lombardia, Miguel; Lescar, Julien; Canard, Bruno

    2011-01-01

    Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Å2 surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses. PMID:21637813

  1. Bovine coronavirus associated syndromes.

    PubMed

    Boileau, Mélanie J; Kapil, Sanjay

    2010-03-01

    Bovine coronaviruses, like other animal coronaviruses, have a predilection for intestinal and respiratory tracts. The viruses responsible for enteric and respiratory symptoms are closely related antigenically and genetically. Only 4 bovine coronavirus isolates have been completely sequenced and thus, the information about the genetics of the virus is still limited. This article reviews the clinical syndromes associated with bovine coronavirus, including pneumonia in calves and adult cattle, calf diarrhea, and winter dysentery; diagnostic methods; prevention using vaccination; and treatment, with adjunctive immunotherapy.

  2. A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

    PubMed Central

    de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

    2015-01-01

    ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although

  3. Synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent SARS coronavirus 3CL protease inhibitor.

    PubMed

    Yang, Syaulan; Chen, Shu-Jen; Hsu, Min-Feng; Wu, Jen-Dar; Tseng, Chien-Te K; Liu, Yu-Fan; Chen, Hua-Chien; Kuo, Chun-Wei; Wu, Chi-Shen; Chang, Li-Wen; Chen, Wen-Chang; Liao, Shao-Ying; Chang, Teng-Yuan; Hung, Hsin-Hui; Shr, Hui-Lin; Liu, Cheng-Yuan; Huang, Yu-An; Chang, Ling-Yin; Hsu, Jen-Chi; Peters, Clarence J; Wang, Andrew H-J; Hsu, Ming-Chu

    2006-08-10

    A potent SARS coronavirus (CoV) 3CL protease inhibitor (TG-0205221, Ki = 53 nM) has been developed. TG-0205221 showed remarkable activity against SARS CoV and human coronavirus (HCoV) 229E replications by reducing the viral titer by 4.7 log (at 5 microM) for SARS CoV and 5.2 log (at 1.25 microM) for HCoV 229E. The crystal structure of TG-0205221 (resolution = 1.93 A) has revealed a unique binding mode comprising a covalent bond, hydrogen bonds, and numerous hydrophobic interactions. Structural comparisons between TG-0205221 and a natural peptide substrate were also discussed. This information may be applied toward the design of other 3CL protease inhibitors.

  4. Synthesis of RNA oligomers on heterogeneous templates

    NASA Technical Reports Server (NTRS)

    Ertem, G.; Ferris, J. P.

    1996-01-01

    The concept of an RNA world in the chemical origin of life is appealing, as nucleic acids are capable of both information storage and acting as templates that catalyse the synthesis of complementary molecules. Template-directed synthesis has been demonstrated for homogeneous oligonucleotides that, like natural nucleic acids, have 3',5' linkages between the nucleotide monomers. But it seems likely that prebiotic routes to RNA-like molecules would have produced heterogeneous molecules with various kinds of phosphodiester linkages and both linear and cyclic nucleotide chains. Here we show that such heterogeneity need be no obstacle to the templating of complementary molecules. Specifically, we show that heterogeneous oligocytidylates, formed by the montmorillonite clay-catalysed condensation of actuated monomers, can serve as templates for the synthesis of oligoguanylates. Furthermore, we show that oligocytidylates that are exclusively 2',5'-linked can also direct synthesis of oligoguanylates. Such heterogeneous templating reactions could have increased the diversity of the pool of protonucleic acids from which life ultimately emerged.

  5. Determination of Ferret Enteric Coronavirus Genome in Laboratory Ferrets.

    PubMed

    Li, Tian-Cheng; Yoshizaki, Sayaka; Kataoka, Michiyo; Doan, Yen Hai; Ami, Yasushi; Suzaki, Yuriko; Nakamura, Tomofumi; Takeda, Naokazu; Wakita, Takaji

    2017-09-01

    Ferret enteric coronavirus (FRECV) RNA was detected in laboratory ferrets. Analysis of the complete genome sequence of 2 strains, FRCoV4370 and FRCoV063, revealed that FRECV shared 49.9%-68.9% nucleotide sequence identity with known coronaviruses. These results suggest that FRECV might be classified as a new species in the genus Alphacoronavirus.

  6. Coronavirus genotype diversity and prevalence of infection in wild carnivores in the Serengeti National Park, Tanzania.

    PubMed

    Goller, Katja V; Fickel, Jörns; Hofer, Heribert; Beier, Sandra; East, Marion L

    2013-04-01

    Knowledge of coronaviruses in wild carnivores is limited. This report describes coronavirus genetic diversity, species specificity and infection prevalence in three wild African carnivores. Coronavirus RNA was recovered from fresh feces from spotted hyena and silver-backed jackal, but not bat-eared fox. Analysis of sequences of membrane (M) and spike (S) gene fragments revealed strains in the genus Alphacoronavirus, including three distinct strains in hyenas and one distinct strain in a jackal. Coronavirus RNA prevalence was higher in feces from younger (17 %) than older (3 %) hyenas, highlighting the importance of young animals for coronavirus transmission in wild carnivores.

  7. The structure and functions of coronavirus genomic 3' and 5' ends.

    PubMed

    Yang, Dong; Leibowitz, Julian L

    2015-08-03

    Coronaviruses (CoVs) are an important cause of illness in humans and animals. Most human coronaviruses commonly cause relatively mild respiratory illnesses; however two zoonotic coronaviruses, SARS-CoV and MERS-CoV, can cause severe illness and death. Investigations over the past 35 years have illuminated many aspects of coronavirus replication. The focus of this review is the functional analysis of conserved RNA secondary structures in the 5' and 3' of the betacoronavirus genomes. The 5' 350 nucleotides folds into a set of RNA secondary structures which are well conserved, and reverse genetic studies indicate that these structures play an important role in the discontinuous synthesis of subgenomic RNAs in the betacoronaviruses. These cis-acting elements extend 3' of the 5'UTR into ORF1a. The 3'UTR is similarly conserved and contains all of the cis-acting sequences necessary for viral replication. Two competing conformations near the 5' end of the 3'UTR have been shown to make up a potential molecular switch. There is some evidence that an association between the 3' and 5'UTRs is necessary for subgenomic RNA synthesis, but the basis for this association is not yet clear. A number of host RNA proteins have been shown to bind to the 5' and 3' cis-acting regions, but the significance of these in viral replication is not clear. Two viral proteins have been identified as binding to the 5' cis-acting region, nsp1 and N protein. A genetic interaction between nsp8 and nsp9 and the region of the 3'UTR that contains the putative molecular switch suggests that these two proteins bind to this region.

  8. Analytical and Clinical Validation of Six Commercial Middle East Respiratory Syndrome Coronavirus RNA Detection Kits Based on Real-Time Reverse-Transcription PCR

    PubMed Central

    Ko, Young Jin; Seong, Moon-Woo; Kim, Jae-Seok; Shin, Bo-Moon; Sung, Heungsup

    2016-01-01

    Background During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. Methods PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. Results The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. Conclusions The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls. PMID:27374710

  9. Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR).

    PubMed

    Hornyák, Akos; Bálint, Adám; Farsang, Attila; Balka, Gyula; Hakhverdyan, Mikhayil; Rasmussen, Thomas Bruun; Blomberg, Jonas; Belák, Sándor

    2012-05-01

    Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCoV) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection and the quantitation of the genome copies of FCoV. In order to detect the broadest spectrum of potential FCoV variants and to achieve the most accurate results in the detection ability the new assay is applying the primer-probe energy transfer (PriProET) principle. This technology was chosen since PriProET is very robust to tolerate the nucleotide substitutions in the target area. Therefore, this technology provides a very broad-range system, which is able to detect simultaneously many variants of the virus(es) even if the target genomic regions show large scale of variations. The detection specificity of the new assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from uncertain clinical cases were also subjected to the assay. The sensitivity of the P-sg-QPCR method was high, since as few as 10 genome copies of FCoV were detected. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of

  10. Eucaryotic RNA polymerase conditional mutant that rapidly ceases mRNA synthesis.

    PubMed Central

    Nonet, M; Scafe, C; Sexton, J; Young, R

    1987-01-01

    We have isolated a yeast conditional mutant which rapidly ceases synthesis of mRNA when subjected to the nonpermissive temperature. This mutant (rpb1-1) was constructed by replacing the wild-type chromosomal copy of the gene encoding the largest subunit of RNA polymerase II with one mutagenized in vitro. The rapid cessation of mRNA synthesis in vivo and the lack of RNA polymerase II activity in crude extracts indicate that the mutant possesses a functionally defective, rather than an assembly-defective, RNA polymerase II. The shutdown in mRNA synthesis in the rpb1-1 mutant has pleiotropic effects on the synthesis of other RNAs and on the heat shock response. This mutant provides direct evidence that the RPB1 protein has a functional role in mRNA synthesis. Images PMID:3299050

  11. Coronavirus infection of spotted hyenas in the Serengeti ecosystem.

    PubMed

    East, Marion L; Moestl, Karin; Benetka, Viviane; Pitra, Christian; Höner, Oliver P; Wachter, Bettina; Hofer, Heribert

    2004-08-19

    Sera from 38 free-ranging spotted hyenas (Crocuta crocuta) in the Serengeti ecosystem, Tanzania, were screened for exposure to coronavirus of antigenic group 1. An immunofluorescence assay indicated high levels of exposure to coronavirus among Serengeti hyenas: 95% when considering sera with titer levels of > or = 1:10 and 74% when considering sera with titer levels of > or = 1:40. Cubs had generally lower mean titer levels than adults. Exposure among Serengeti hyenas to coronavirus was also confirmed by a serum neutralisation assay and an ELISA. Application of RT-PCR to 27 fecal samples revealed viral RNA in three samples (11%). All three positive fecal samples were from the 15 juvenile animals (<24 months of age) sampled, and none from the 12 adults sampled. No viral RNA was detected in tissue samples (lymph node, intestine, lung) from 11 individuals. Sequencing of two amplified products from the S protein gene of a positive sample revealed the presence of coronavirus specific RNA with a sequence homology to canine coronavirus of 76 and 78% and to feline coronavirus type II of 80 and 84%, respectively. Estimation of the phylogenetic relationship among coronavirus isolates indicated considerable divergence of the hyena variant from those in European, American and Japanese domestic cats and dogs. From long-term observations of several hundred known individuals, the only clinical sign in hyenas consistent with those described for coronavirus infections in dogs and cats was diarrhea. There was no evidence that coronavirus infection in hyenas caused clinical signs similar to feline infectious peritonitis in domestic cats or was a direct cause of mortality in hyenas. To our knowledge, this is the first report of coronavirus infection in Hyaenidae.

  12. Translating Ribosomes Inhibit Poliovirus Negative-Strand RNA Synthesis

    PubMed Central

    Barton, David J.; Morasco, B. Joan; Flanegan, James B.

    1999-01-01

    Poliovirus has a single-stranded RNA genome of positive polarity that serves two essential functions at the start of the viral replication cycle in infected cells. First, it is translated to synthesize viral proteins and, second, it is copied by the viral polymerase to synthesize negative-strand RNA. We investigated these two reactions by using HeLa S10 in vitro translation-RNA replication reactions. Preinitiation RNA replication complexes were isolated from these reactions and then used to measure the sequential synthesis of negative- and positive-strand RNAs in the presence of different protein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cycloheximide, anisomycin, and ricin A chain. Dose-response experiments showed that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replication. This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ribosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive-strand synthesis in normal RNA replication complexes. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA. Cycloheximide and other inhibitors of polypeptide chain elongation “freeze” ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase was not able to dislodge translating ribosomes from viral RNA templates and mediate the switch from translation to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated

  13. Role of RNA and Protein Synthesis in Abscission

    PubMed Central

    Abeles, F. B.

    1968-01-01

    The cell separation aspect of abscission is thought to involve the action of specific cell wall degrading enzymes. Enzymes represent synthesis which in turn is preceded by the synthesis of specific RNA molecules, and it follows that inhibition of either of these processes would also block abscission. Since abscission is a localized phenomenon usually involving 2 or 3 cell layers, RNA and protein synthesis should also be localized. Manipulations of plant material which either accelerate or retard abscission may be due to the regulation of RNA and protein synthesis. This paper is a review of literature concerned with these and related questions. Images PMID:16657020

  14. Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes.

    PubMed

    Posthuma, Clara C; Te Velthuis, Aartjan J W; Snijder, Eric J

    2017-02-06

    Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26-34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3'-to-5' exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.

  15. Drug targets for rational design against emerging coronaviruses.

    PubMed

    Zhao, Qi; Weber, Erin; Yang, Haitao

    2013-04-01

    The recent, fatal outbreak of the novel coronavirus strain in the Middle East highlights the real threat posed by this unique virus family. Neither pharmaceutical cures nor preventive vaccines are clinically available to fight against coronavirus associated syndromes, not to mention a lack of symptom soothing drugs. Development of treatment options is complicated by the unpredictable, recurring instances of cross-species viral transmission. The vastly distributing virus reservoir and the rapid rate of host-species exchange of coronavirus demands wide spectrum potency in an ideal therapeutic. Through summarizing the available information and progress in coronavirus research, this review provides a systematic assessment of the potential wide-spectrum features on the most popular drug targets including viral proteases, spike protein, RNA polymerases and editing enzymes as well as host-virus interaction pathways associated with coronaviruses.

  16. Synthesis and Characterization of a Native, Oligomeric Form of Recombinant Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein

    PubMed Central

    Song, Hyun Chul; Seo, Mi-Young; Stadler, Konrad; Yoo, Byoung J.; Choo, Qui-Lim; Coates, Stephen R.; Uematsu, Yasushi; Harada, Takashi; Greer, Catherine E.; Polo, John M.; Pileri, Piero; Eickmann, Markus; Rappuoli, Rino; Abrignani, Sergio; Houghton, Michael; Han, Jang H.

    2004-01-01

    We have expressed and characterized the severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein in cDNA-transfected mammalian cells. The full-length spike protein (S) was newly synthesized as an endoglycosidase H (endo H)-sensitive glycoprotein (gp170) that is further modified into an endo H-resistant glycoprotein (gp180) in the Golgi apparatus. No substantial proteolytic cleavage of S was observed, suggesting that S is not processed into head (S1) and stalk (S2) domains as observed for certain other coronaviruses. While the expressed full-length S glycoprotein was exclusively cell associated, a truncation of S by excluding the C-terminal transmembrane and cytoplasmic tail domains resulted in the expression of an endoplasmic reticulum-localized glycoprotein (gp160) as well as a Golgi-specific form (gp170) which was ultimately secreted into the cell culture medium. Chemical cross-linking, thermal denaturation, and size fractionation analyses suggested that the full-length S glycoprotein of SARS-CoV forms a higher order structure of ∼500 kDa, which is consistent with it being an S homotrimer. The latter was also observed in purified virions. The intracellular form of the C-terminally truncated S protein (but not the secreted form) also forms trimers, but with much less efficiency than full-length S. Deglycosylation of the full-length homotrimer with peptide N-glycosidase-F under native conditions abolished recognition of the protein by virus-neutralizing antisera raised against purified virions, suggesting the importance of the carbohydrate in the correct folding of the S protein. These data should aid in the design of recombinant vaccine antigens to prevent the spread of this emerging pathogen. PMID:15367599

  17. Extraordinary GU-rich single-strand RNA identified from SARS coronavirus contributes an excessive innate immune response.

    PubMed

    Li, Yan; Chen, Ming; Cao, Hongwei; Zhu, Yuanfeng; Zheng, Jiang; Zhou, Hong

    2013-02-01

    A dangerous cytokine storm occurs in the SARS involving in immune disorder, but many aspects of the pathogenetic mechanism remain obscure since its outbreak. To deeply reveal the interaction of host and SARS-CoV, based on the basic structural feature of pathogen-associated molecular pattern, we created a new bioinformatics method for searching potential pathogenic molecules and identified a set of SARS-CoV specific GU-rich ssRNA fragments with a high-density distribution in the genome. In vitro experiments, the result showed the representative SARS-CoV ssRNAs had powerful immunostimulatory activities to induce considerable level of pro-inflammatory cytokine TNF-a, IL-6 and IL-12 release via the TLR7 and TLR8, almost 2-fold higher than the strong stimulatory ssRNA40 that was found previously from other virus. Moreover, SARS-CoV ssRNA was able to cause acute lung injury in mice with a high mortality rate in vivo experiment. It suggests that SARS-CoV specific GU-rich ssRNA plays a very important role in the cytokine storm associated with a dysregulation of the innate immunity. This study not only presents new evidence about the immunopathologic damage caused by overactive inflammation during the SARS-CoV infection, but also provides a useful clue for a new therapeutic strategy. Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  18. 2-Selenouridine triphosphate synthesis and Se-RNA transcription.

    PubMed

    Sun, Huiyan; Jiang, Sibo; Caton-Williams, Julianne; Liu, Hehua; Huang, Zhen

    2013-09-01

    2-Selenouridine ((Se)U) is one of the naturally occurring modifications of Se-tRNAs ((Se)U-RNA) at the wobble position of the anticodon loop. Its role in the RNA-RNA interaction, especially during the mRNA decoding, is elusive. To assist the research exploration, herein we report the enzymatic synthesis of the (Se)U-RNA via 2-selenouridine triphosphate ((Se)UTP) synthesis and RNA transcription. Moreover, we have demonstrated that the synthesized (Se)UTP is stable and recognizable by T7 RNA polymerase. Under the optimized conditions, the transcription yield of (Se)U-RNA can reach up to 85% of the corresponding native RNA. Furthermore, the transcribed (Se)U-hammerhead ribozyme has the similar activity as the corresponding native, which suggests usefulness of (Se)U-RNAs in function and structure studies of noncoding RNAs, including the Se-tRNAs.

  19. Specific mRNA destabilization in Dictyostelium discoideum requires RNA synthesis.

    PubMed Central

    Amara, J F; Lodish, H F

    1987-01-01

    We tested the effects of inhibitors of protein and RNA synthesis on the disaggregation-mediated destabilization of prespore mRNAs in Dictyostelium discoideum. Incubating disaggregated cells with daunomycin to inhibit RNA synthesis prevented the loss of prespore mRNAs, whereas the inhibitor decreased or did not affect levels of the common mRNAs CZ22 and actin. Protein synthesis inhibitors varied in their effects. Cycloheximide, which inhibited protein synthesis almost completely, prevented the loss of the prespore mRNAs, but puromycin, which inhibited protein synthesis less well, did not. These results indicate that the process of specific mRNA destabilization requires the synthesis of RNA and possibly of protein. Images PMID:3437899

  20. Aminoacyl-RNA synthesis catalyzed by an RNA.

    PubMed

    Illangasekare, M; Sanchez, G; Nickles, T; Yarus, M

    1995-02-03

    An RNA has been selected that rapidly aminoacylates its 2'(3') terminus when provided with phenylalanyl-adenosine monophosphate. That is, the RNA accelerates the same aminoacyl group transfer catalyzed by protein aminoacyl-transfer RNA synthetases. The best characterized RNA reaction requires both Mg2+ and Ca2+. These results confirm a necessary prediction of the RNA world hypothesis and represent efficient RNA reaction (> or = 10(5) times accelerated) at a carbonyl carbon, exemplifying a little explored type of RNA catalysis.

  1. Archaeal aminoacyl-tRNA synthesis: diversity replaces dogma.

    PubMed Central

    Tumbula, D; Vothknecht, U C; Kim, H S; Ibba, M; Min, B; Li, T; Pelaschier, J; Stathopoulos, C; Becker, H; Söll, D

    1999-01-01

    Accurate aminoacyl-tRNA synthesis is essential for faithful translation of the genetic code and consequently has been intensively studied for over three decades. Until recently, the study of aminoacyl-tRNA synthesis in archaea had received little attention. However, as in so many areas of molecular biology, the advent of archaeal genome sequencing has now drawn researchers to this field. Investigations with archaea have already led to the discovery of novel pathways and enzymes for the synthesis of numerous aminoacyl-tRNAs. The most surprising of these findings has been a transamidation pathway for the synthesis of asparaginyl-tRNA and a novel lysyl-tRNA synthetase. In addition, seryl- and phenylalanyl-tRNA synthetases that are only marginally related to known examples outside the archaea have been characterized, and the mechanism of cysteinyl-tRNA formation in Methanococcus jannaschii and Methanobacterium thermoautotrophicum is still unknown. These results have revealed completely unexpected levels of complexity and diversity, questioning the notion that aminoacyl-tRNA synthesis is one of the most conserved functions in gene expression. It has now become clear that the distribution of the various mechanisms of aminoacyl-tRNA synthesis in extant organisms has been determined by numerous gene transfer events, indicating that, while the process of protein biosynthesis is orthologous, its constituents are not. PMID:10430557

  2. Initiation of protein-primed picornavirus RNA synthesis

    PubMed Central

    Paul, Aniko V.; Wimmer, Eckard

    2015-01-01

    Plus strand RNA viruses use different mechanisms to initiate the synthesis of their RNA chains. The Picornaviridae family constitutes a large group of plus strand RNA viruses that possess a small terminal protein (VPg) covalently linked to the 5’-end of their genomes. The RNA polymerases of these viruses use VPg as primer for both minus and plus strand RNA synthesis. In the first step of the initiation reaction the RNA polymerase links a UMP to the hydroxyl group of a tyrosine in VPg using as template a cis-replicating element (cre) positioned in different regions of the viral genome. In this review we will summarize what is known about the intiation reaction of protein-primed RNA synthesis by the RNA polymerases of the Picornaviridae. As an example we will use the RNA polymerase of poliovirus, the prototype of Picornaviridae. We will also discuss models of how these nucleotidylylated protein primers might be used, together with viral and cellular replication proteins and other cis-replicating RNA elements, during minus and plus strand RNA synthesis. PMID:25592245

  3. RNA synthesis in the thymus of the immunologically mature mouse

    PubMed Central

    Patt, D. J.; Cohen, E. P.

    1974-01-01

    RNA synthesis was investigated in the thymus glands of adult immunized mice. After the intraperitoneal injection of mice with sheep red blood cells (SRBC), the net synthesis of RNA in the gland increased. A small but consistent amount of the RNA synthesized was distinguished by RNA:DNA hybridizations from that found in the glands of mice not injected with antigen. The RNA formed after immunization did not appear by hybridization to be specific for different antigens since the species of RNA formed in the glands of mice injected with SRBC was indistinguishable from RNA formed in the thymuses of mice injected with chicken red blood cells. RNA synthesized in the thymus glands of mice pharmacologically `stressed' by the injections of hydrocortisone, however, was distinguishable from that formed in the glands of mice injected with antigen. PMID:4854183

  4. Synthesis of RNA using 2'-O-DTM protection.

    PubMed

    Semenyuk, Andrey; Földesi, Andras; Johansson, Tommy; Estmer-Nilsson, Camilla; Blomgren, Peter; Brännvall, Mathias; Kirsebom, Leif A; Kwiatkowski, Marek

    2006-09-27

    tert-Butyldithiomethyl (DTM), a novel hydroxyl protecting group, cleavable under reductive conditions, was developed and applied for the protection of 2'-OH during solid-phase RNA synthesis. This function is compatible with all standard protecting groups used in oligonucleotide synthesis, and allows for fast and high-yield synthesis of RNA. Oligonucleotides containing the 2'-O-DTM groups can be easily deprotected under the mildest possible aqueous and homogeneous conditions. The preserved 5'-O-DMTr function can be used for high-throughput cartridge RNA purification.

  5. RNA-catalysed synthesis of complementary-strand RNA

    NASA Astrophysics Data System (ADS)

    Doudna, Jennifer A.; Szostak, Jack W.

    1989-06-01

    The Tetrahymena ribozyme can splice together multiple oligonucleotides aligned on a template strand to yield a fully complementary product strand. This reaction demonstrates the feasibility of RNA-catalysed RNA replications.

  6. De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

    PubMed Central

    Luo, Guangxiang; Hamatake, Robert K.; Mathis, Danielle M.; Racela, Jason; Rigat, Karen L.; Lemm, Julie; Colonno, Richard J.

    2000-01-01

    Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo. PMID:10623748

  7. Cisplatin inhibits synthesis of ribosomal RNA in vivo.

    PubMed Central

    Jordan, P; Carmo-Fonseca, M

    1998-01-01

    Cis -diammininedichloroplatinum(II) (cisplatin or cis -DDP) is a DNA-damaging agent that is widely used in cancer chemotherapy. Cisplatin crosslinks DNA and the resulting adducts interact with proteins that contain high-mobility-group (HMG) domains, such as UBF(upstream binding factor). UBF is a transcription factor that binds to the promoter of ribosomal RNA (rRNA) genes thereby supporting initiation of transcription by RNA polymerase I. Here we report that cisplatin causes a redistribution of UBF in the nucleolus of human cells, similar to that observed after inhibition of rRNA synthesis. A similar redistribution was observed for the major components of the rRNA transcription machinery, namely TBP, TAFIs and RNA polymerase I. Furthermore, we provide for the first time direct in vivo evidence that cisplatin blocks synthesis of rRNA, while activity of RNA polymerase II continues to be detected throughout the nucleus. The clinically ineffective trans isomer (trans -DDP) does not alter the localization of either UBF or other components of the RNA polymerase I transcription machinery. These results suggest that disruption of rRNA synthesis, which is stimulated in proliferating cells, plays an important role in the clinical success of cisplatin. PMID:9611224

  8. Synthesis of 2′-Fluoro RNA by Syn5 RNA polymerase

    PubMed Central

    Zhu, Bin; Hernandez, Alfredo; Tan, Min; Wollenhaupt, Jan; Tabor, Stanley; Richardson, Charles C.

    2015-01-01

    The substitution of 2′-fluoro for 2′-hydroxyl moieties in RNA substantially improves the stability of RNA. RNA stability is a major issue in RNA research and applications involving RNA. We report that the RNA polymerase from the marine cyanophage Syn5 has an intrinsic low discrimination against the incorporation of 2′-fluoro dNMPs during transcription elongation. The presence of both magnesium and manganese ions at high concentrations further reduce this discrimination without decreasing the efficiency of incorporation. We have constructed a Syn5 RNA polymerase in which tyrosine 564 is replaced with phenylalanine (Y564F) that further decreases the discrimination against 2′-fluoro-dNTPs during RNA synthesis. Sequence elements in DNA templates that affect the yield of RNA and incorporation of 2′-fluoro-dNMPs by Syn5 RNA polymerase have been identified. PMID:25897116

  9. Characterization of the Role of Hexamer AGUAAA and Poly(A) Tail in Coronavirus Polyadenylation

    PubMed Central

    Peng, Yu-Hui; Lin, Ching-Houng; Lin, Chao-Nan; Lo, Chen-Yu; Tsai, Tsung-Lin; Wu, Hung-Yi

    2016-01-01

    Similar to eukaryotic mRNA, the positive-strand coronavirus genome of ~30 kilobases is 5’-capped and 3’-polyadenylated. It has been demonstrated that the length of the coronaviral poly(A) tail is not static but regulated during infection; however, little is known regarding the factors involved in coronaviral polyadenylation and its regulation. Here, we show that during infection, the level of coronavirus poly(A) tail lengthening depends on the initial length upon infection and that the minimum length to initiate lengthening may lie between 5 and 9 nucleotides. By mutagenesis analysis, it was found that (i) the hexamer AGUAAA and poly(A) tail are two important elements responsible for synthesis of the coronavirus poly(A) tail and may function in concert to accomplish polyadenylation and (ii) the function of the hexamer AGUAAA in coronaviral polyadenylation is position dependent. Based on these findings, we propose a process for how the coronaviral poly(A) tail is synthesized and undergoes variation. Our results provide the first genetic evidence to gain insight into coronaviral polyadenylation. PMID:27760233

  10. RNA Synthesis by in Vitro Selected Ribozymes for Recreating an RNA World

    PubMed Central

    Martin, Lyssa L.; Unrau, Peter J.; Müller, Ulrich F.

    2015-01-01

    The RNA world hypothesis states that during an early stage of life, RNA molecules functioned as genome and as the only genome-encoded catalyst. This hypothesis is supported by several lines of evidence, one of which is the in vitro selection of catalytic RNAs (ribozymes) in the laboratory for a wide range of reactions that might have been used by RNA world organisms. This review focuses on three types of ribozymes that could have been involved in the synthesis of RNA, the core activity in the self-replication of RNA world organisms. These ribozyme classes catalyze nucleoside synthesis, triphosphorylation, and the polymerization of nucleoside triphosphates. The strengths and weaknesses regarding each ribozyme’s possible function in a self-replicating RNA network are described, together with the obstacles that need to be overcome before an RNA world organism can be generated in the laboratory. PMID:25610978

  11. Regulation of Stress Responses and Translational Control by Coronavirus

    PubMed Central

    Fung, To Sing; Liao, Ying; Liu, Ding Xiang

    2016-01-01

    Similar to other viruses, coronavirus infection triggers cellular stress responses in infected host cells. The close association of coronavirus replication with the endoplasmic reticulum (ER) results in the ER stress responses, which impose a challenge to the viruses. Viruses, in turn, have come up with various mechanisms to block or subvert these responses. One of the ER stress responses is inhibition of the global protein synthesis to reduce the amount of unfolded proteins inside the ER lumen. Viruses have evolved the capacity to overcome the protein translation shutoff to ensure viral protein production. Here, we review the strategies exploited by coronavirus to modulate cellular stress response pathways. The involvement of coronavirus-induced stress responses and translational control in viral pathogenesis will also be briefly discussed. PMID:27384577

  12. The nucleocapsid protein gene of bovine coronavirus is bicistronic.

    PubMed Central

    Senanayake, S D; Hofmann, M A; Maki, J L; Brian, D A

    1992-01-01

    For animal RNA viruses that replicate through an RNA intermediate, reported examples of bicistronic mRNAs with overlapping open reading frames in which one cistron is contained entirely within another have been made only for those with negative-strand or double-stranded genomes. In this report, we demonstrate for the positive-strand bovine coronavirus that an overlapping open reading frame potentially encoding a 23-kDa protein (names the I [for internal open reading frame] protein) and lying entirely within the gene for the 49-kDa nucleocapsid phosphoprotein is expressed during virus replication from a single species of unedited mRNA. The I protein was specifically immunoprecipitated from virus-infected cells with an I-specific antipeptide serum and was shown to be membrane associated. Many features of I protein synthesis conform to the leaky ribosomal scanning model for regulation of translation. This, to our knowledge, is the first example of a bicistronic mRNA for a cytoplasmically replicating, positive-strand animal RNA virus in which one cistron entirely overlaps another. Images PMID:1501275

  13. Coronavirus Attachment and Replication

    DTIC Science & Technology

    1988-03-28

    synthesis during RNA replication of vesicular stomatitis virus. J. Virol. 49:303-309. Pedersen, N.C. 1976a. Feline infectious peritonitis: Something old...receptors on intestinal brush border membranes from normal host species were developed for canine (CCV), feline (FIPV), porcine (TGEV), human (HCV...gastroenteritis receptor on pig BBMs ...... ................. ... 114 Feline infectious peritonitis virus receptor on cat BBMs ... .............. 117 Human

  14. The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world

    PubMed Central

    Egloff, Marie-Pierre; Ferron, François; Campanacci, Valérie; Longhi, Sonia; Rancurel, Corinne; Dutartre, Hélène; Snijder, Eric J.; Gorbalenya, Alexander E.; Cambillau, Christian; Canard, Bruno

    2004-01-01

    The recently identified etiological agent of the severe acute respiratory syndrome (SARS) belongs to Coronaviridae (CoV), a family of viruses replicating by a poorly understood mechanism. Here, we report the crystal structure at 2.7-Å resolution of nsp9, a hitherto uncharacterized subunit of the SARS-CoV replicative polyproteins. We show that SARS-CoV nsp9 is a single-stranded RNA-binding protein displaying a previously unreported, oligosaccharide/oligonucleotide fold-like fold. The presence of this type of protein has not been detected in the replicative complexes of RNA viruses, and its presence may reflect the unique and complex CoV viral replication/transcription machinery. PMID:15007178

  15. Antiviral Drugs Specific for Coronaviruses in Preclinical Development

    PubMed Central

    Adedeji, Adeyemi O.; Sarafianos, Stefan G.

    2014-01-01

    Coronaviruses are positive stranded RNA viruses that cause respiratory, enteric and central nervous system diseases in many species, including humans. Until recently, the relatively low burden of disease in humans caused by few of these viruses impeded the development of coronavirus specific therapeutics. However, the emergence of severe acute respiratory syndrome coronavirus (SARS-CoV), and more recently, Middle East respiratory syndrome coronavirus (MERS-CoV), has impelled the development of such drugs. This review focuses on some newly identified SARS-CoV inhibitors, with known mechanisms of action and their potential to inhibit the novel MERS-CoV. The clinical development of optimized versions of such compounds could be beneficial for the treatment and control of SARS-CoV, the current MERS-CoV and other future SARS-like epidemics. PMID:24997250

  16. Extensive coronavirus-induced membrane rearrangements are not a determinant of pathogenicity

    PubMed Central

    Maier, Helena J.; Neuman, Benjamin W.; Bickerton, Erica; Keep, Sarah M.; Alrashedi, Hasan; Hall, Ross; Britton, Paul

    2016-01-01

    Positive-strand RNA (+RNA) viruses rearrange cellular membranes during replication, possibly in order to concentrate and arrange viral replication machinery for efficient viral RNA synthesis. Our previous work showed that in addition to the conserved coronavirus double membrane vesicles (DMVs), Beau-R, an apathogenic strain of avian Gammacoronavirus infectious bronchitis virus (IBV), induces regions of ER that are zippered together and tethered open-necked double membrane spherules that resemble replication organelles induced by other +RNA viruses. Here we compared structures induced by Beau-R with the pathogenic lab strain M41 to determine whether membrane rearrangements are strain dependent. Interestingly, M41 was found to have a low spherule phenotype. We then compared a panel of pathogenic, mild and attenuated IBV strains in ex vivo tracheal organ culture (TOC). Although the low spherule phenotype of M41 was conserved in TOCs, each of the other tested IBV strains produced DMVs, zippered ER and spherules. Furthermore, there was a significant correlation for the presence of DMVs with spherules, suggesting that these structures are spatially and temporally linked. Our data indicate that virus induced membrane rearrangements are fundamentally linked to the viral replicative machinery. However, coronavirus replicative apparatus clearly has the plasticity to function in different structural contexts. PMID:27255716

  17. [Advances in recently identified coronaviruses].

    PubMed

    Geng, He-Yuan; Tan, Wen-Jie

    2013-01-01

    Coronaviruses are a large family of viruses which include viruses that cause the common cold and severe acute respiratory syndrome (SARS) in humans and other diseases in animals. There are considerable genetic diversities within coronaviruses due to their wide rang hosts and their special gene replication and transcription mechanisms. During this process, gene recombinations often occur, resulting in novel subtype or coronavirus emerge constantly. Of note are SARS-like-CoVs and novel HCoV-EMC identified in 2012. This minireview summarized major advances of recently identified coronaviruses, focusing on the genome structures and interspecies jumping mechanism of coronavirus.

  18. [The first steps of chlorophyll synthesis: RNA involvement and regulation

    SciTech Connect

    Soell, D.

    1992-01-01

    Glu-tRNA[sup Glu] is synthesized from glutamate and tRNA[sup Glu] by glutamyl-tRNA synthetase (GluRS). Recent work has demonstrated that Glu-tRNA[sup Glu] has dual functions and is a precursor for protein and 5-aminolevulinate (ALA) synthesis. Current data does not provide compelling evidence for the notion that GluRS is regulated by chlorophyll precursors or in concert with the other enzymes of ALA synthesis. We have redefined the C5-pathway as a two-step route to ALA starting with Glu-tRNA[sup Glu]. Only two enzymes, Glu-tRNA reductase (GluTR) and GSA-2,1-amino-mutase (GSA-AM), are specifically involved in ALA synthesis. We have purified these enzymatic activities from Chlamydomonas and demonstrated that the two purified proteins in the presence of their cofactors NADPH and pyridoxal phosphate are sufficient for the in vitro Glu-tRNA [yields] ALA conversion. We have cloned the genes encoding GluTR. The sequences of the GluTR proteins deduced from these genes share highly conserved regions with those of bacterial origin. We havealso cloned and analyzed the gene encoding GSA-AM from Arabidopsis. As in Salmonella typhimurium, there are indications of the existence of an additional pathway for ALA formation in E. coli. To shed light on the recognition of the single tRNA[sup Glu] by the chloroplast enzymes GluTR, GluRS we characterized a chlorophyll-deficient mutant of Euglena having tRNA[sup Glu] with a point mutation in the T[Psi]C-loop. The altered tRNA supports protein but not ALA synthesis.

  19. The Coronavirus Nucleocapsid Is a Multifunctional Protein

    PubMed Central

    McBride, Ruth; van Zyl, Marjorie; Fielding, Burtram C.

    2014-01-01

    The coronavirus nucleocapsid (N) is a structural protein that forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. Recent studies have confirmed that N is a multifunctional protein. The aim of this review is to highlight the properties and functions of the N protein, with specific reference to (i) the topology; (ii) the intracellular localization and (iii) the functions of the protein. PMID:25105276

  20. Total RNA-seq to identify pharmacological effects on specific stages of mRNA synthesis.

    PubMed

    Boswell, Sarah A; Snavely, Andrew; Landry, Heather M; Churchman, L Stirling; Gray, Jesse M; Springer, Michael

    2017-03-06

    Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

  1. The 5' nontranslated region of potato virus X RNA affects both genomic and subgenomic RNA synthesis.

    PubMed Central

    Kim, K H; Hemenway, C

    1996-01-01

    A tobacco protoplast system was developed to analyze cis-acting sequences required for potato virus X (PVX) replication. Protoplasts inoculated with transcripts derived from a PVX cDNA clone or from clones containing mutations in their 5' nontranslated regions (NTRs) were assayed for RNA production by S1 nuclease protection assays. A time course of plus- and minus-strand-RNA accumulation indicated that both minus- and plus-strand PVX RNAs were detectable at 0.5 h postinoculation. Although minus-strand RNAs accumulated more rapidly than plus-strand RNAs, maximum levels of plus-strand RNAs were 40- to 80-fold higher. On the basis of these data, time points were chosen for determination of RNA levels in protoplasts inoculated with PVX clones containing deletions or an insertion in their 5' NTRs. Deletions of more than 12 nucleotides from the 5' end, internal deletions, and one insertion in the 5' NTR resulted in substantially decreased levels of plus-strand-RNA production. In contrast, all modified transcripts were functional for minus-strand-RNA synthesis, suggesting that elements in the 5' NTR were not essential for minus-strand-RNA synthesis. Further analysis of the 5' NTR deletion mutants indicated that all mutations that decreased genomic plus-strand-RNA synthesis also decreased synthesis of the two major subgenomic RNAs. These data indicate that cis-acting elements from different regions of the 5' NTR are required for plus-strand-RNA synthesis and that this process may be linked to synthesis of subgenomic RNAs. PMID:8764066

  2. Alphavirus RNA synthesis and non-structural protein functions.

    PubMed

    Rupp, Jonathan C; Sokoloski, Kevin J; Gebhart, Natasha N; Hardy, Richard W

    2015-09-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field.

  3. Design and synthesis of a series of serine derivatives as small molecule inhibitors of the SARS coronavirus 3CL protease.

    PubMed

    Konno, Hiroyuki; Wakabayashi, Masaki; Takanuma, Daiki; Saito, Yota; Akaji, Kenichi

    2016-03-15

    Synthesis of serine derivatives having the essential functional groups for the inhibitor of SARS 3CL protease and evaluation of their inhibitory activities using SARS 3CL R188I mutant protease are described. The lead compounds, functionalized serine derivatives, were designed based on the tetrapeptide aldehyde and Bai's cinnamoly inhibitor, and additionally performed with simulation on GOLD softwear. Structure activity relationship studies of the candidate compounds were given reasonable inhibitors ent-3 and ent-7k against SARS 3CL R188I mutant protease. These inhibitors showed protease selectivity and no cytotoxicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    SciTech Connect

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.

    1983-01-01

    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.

  5. Prevalence of canine coronavirus (CCoV) in dog in Japan: detection of CCoV RNA and retrospective serological analysis

    PubMed Central

    TAKANO, Tomomi; YAMASHITA, Saya; MURATA-OHKUBO, Michiko; SATOH, Kumi; DOKI, Tomoyoshi; HOHDATSU, Tsutomu

    2015-01-01

    We collected rectal swabs from dogs in Japan during 2011 to 2014, and canine coronavirus (CCoV) nucleocapsid gene was detected by RT-PCR. The relationship between CCoV infection and the manifestation of diarrhea symptoms was investigated, and a correlation was noted (df=1, χ2=8.90, P<0.005). The types of CCoV detected in samples from CCoV-infected dogs were CCoV-I in 88.9% and CCoV-II in 7.4%, respectively. We retrospectively investigated the seroprevalence of CCoV-I in dogs in Japan during 1998 to 2006. The sera were tested with a neutralizing antibody test. In the absence of CCoV-I laboratory strain, we used feline coronavirus (FCoV)-I that shares high sequence homology in the S protein with CCoV-I. 77.7% of the sera were positive for neutralizing anti-FCoV-I antibodies. PMID:26460314

  6. RNA synthesis and purification for structural studies.

    PubMed

    Ahmed, Yasar Luqman; Ficner, Ralf

    2014-01-01

    RNAs play pivotal roles in the cell, ranging from catalysis (e.g., RNase P), acting as adaptor molecule (tRNA) to regulation (e.g., riboswitches). Precise understanding of its three-dimensional structures has given unprecedented insight into the molecular basis for all of these processes. Nevertheless, structural studies on RNA are still limited by the very special nature of this polymer. The most common methods for the determination of 3D RNA structures are NMR and X-ray crystallography. Both methods have their own set of requirements and give different amounts of information about the target RNA. For structural studies, the major bottleneck is usually obtaining large amounts of highly pure and homogeneously folded RNA. Especially for X-ray crystallography it can be necessary to screen a large number of variants to obtain well-ordered single crystals. In this mini-review we give an overview about strategies for the design, in vitro production, and purification of RNA for structural studies.

  7. Guanosine tetraphosphate as a global regulator of bacterial RNA synthesis: a model involving RNA polymerase pausing and queuing.

    PubMed

    Bremer, H; Ehrenberg, M

    1995-05-17

    A recently reported comparison of stable RNA (rRNA, tRNA) and mRNA synthesis rates in ppGpp-synthesizing and ppGpp-deficient (delta relA delta spoT) bacteria has suggested that ppGpp inhibits transcription initiation from stable RNA promoters, as well as synthesis of (bulk) mRNA. Inhibition of stable RNA synthesis occurs mainly during slow growth of bacteria when cytoplasmic levels of ppGpp are high. In contrast, inhibition of mRNA occurs mainly during fast growth when ppGpp levels are low, and it is associated with a partial inactivation of RNA polymerase. To explain these observations it has been proposed that ppGpp causes transcriptional pausing and queuing during the synthesis of mRNA. Polymerase queuing requires high rates of transcription initiation in addition to polymerase pausing, and therefore high concentrations of free RNA polymerase. These conditions are found in fast growing bacteria. Furthermore, the RNA polymerase queues lead to a promoter blocking when RNA polymerase molecules stack up from the pause site back to the (mRNA) promoter. This occurs most frequently at pause sites close to the promoter. Blocking of mRNA promoters diverts RNA polymerase to stable RNA promoters. In this manner ppGpp could indirectly stimulate synthesis of stable RNA at high growth rates. In the present work a mathematical analysis, based on the theory of queuing, is presented and applied to the global control of transcription in bacteria. This model predicts the in vivo distribution of RNA polymerase over stable RNA and mRNA genes for both ppGpp-synthesizing and ppGpp-deficient bacteria in response to different environmental conditions. It also shows how small changes in basal ppGpp concentrations can produce large changes in the rate of stable RNA synthesis.

  8. Mutation in human selenocysteine transfer RNA selectively disrupts selenoprotein synthesis

    PubMed Central

    Schoenmakers, Erik; Carlson, Bradley; Agostini, Maura; Moran, Carla; Rajanayagam, Odelia; Bochukova, Elena; Tobe, Ryuta; Peat, Rachel; Gevers, Evelien; Muntoni, Francesco; Guicheney, Pascale; Schoenmakers, Nadia; Farooqi, Sadaf; Lyons, Greta; Hatfield, Dolph; Chatterjee, Krishna

    2016-01-01

    Selenium is a trace element that is essential for human health and is incorporated into more than 25 human selenocysteine-containing (Sec-containing) proteins via unique Sec-insertion machinery that includes a specific, nuclear genome–encoded, transfer RNA (tRNA[Ser]Sec). Here, we have identified a human tRNA[Ser]Sec mutation in a proband who presented with a variety of symptoms, including abdominal pain, fatigue, muscle weakness, and low plasma levels of selenium. This mutation resulted in a marked reduction in expression of stress-related, but not housekeeping, selenoproteins. Evaluation of primary cells from the homozygous proband and a heterozygous parent indicated that the observed deficit in stress-related selenoprotein production is likely mediated by reduced expression and diminished 2′-O-methylribosylation at uridine 34 in mutant tRNA[Ser]Sec. Moreover, this methylribosylation defect was restored by cellular complementation with normal tRNA[Ser]Sec. This study identifies a tRNA mutation that selectively impairs synthesis of stress-related selenoproteins and demonstrates the importance of tRNA modification for normal selenoprotein synthesis. PMID:26854926

  9. Synthesis of Geranyl-2-Thiouridine-Modified RNA.

    PubMed

    Wang, Rui; Haruehanroengra, Phensinee; Sheng, Jia

    2017-03-02

    This unit describes the chemical synthesis of the S-geranyl-2-thiouridine (ges(2) U) phosphoramidite and its incorporation into RNA oligonucleotides through solid-phase synthesis. Starting from the 2-thiouracil nucleobase and the protected ribose, the 2-thiouridine is synthesized and the geranyl functionality is introduced into the 2-thio position by using geranyl bromide as the geranylating reagent before the conversion of this modified nucleoside into a phosphoramidite building block. The modified phosphoramidite is used to make the geranyl-RNA oligonucleotides with a solid-phase DNA synthesizer. These RNA strands are then purified by ion-exchange HPLC before further structural and functional studies, such as base pairing and enzyme recognition, can be done. © 2017 by John Wiley & Sons, Inc.

  10. Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases.

    PubMed

    Chuck, Chi-Pang; Chen, Chao; Ke, Zhihai; Wan, David Chi-Cheong; Chow, Hak-Fun; Wong, Kam-Bo

    2013-01-01

    Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CL(pro)) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CL(pro) of severe-acute-respiratory-syndrome-coronavirus. The IC(50) values of the inhibitors were in the range of 4.6-49 μM, demonstrating that the nitrile warhead can effectively inactivate the 3CL(pro) autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was ~10x more potent than the other inhibitors tested. Crystal structures of the enzyme-inhibitor complexes showed that the nitrile warhead inhibits 3CL(pro) by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1-S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CL(pro) from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC(50) values ranging from 1.3 to 3.7 μM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CL(pro), and they inhibit 3CL(pro) from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection.

  11. Initiating nucleotide identity determines efficiency of RNA synthesis from 6S RNA templates in Bacillus subtilis but not Escherichia coli

    PubMed Central

    Cabrera-Ostertag, Ignacio J.; Cavanagh, Amy T.; Wassarman, Karen M.

    2013-01-01

    The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ70 in Escherichia coli, EσA in Bacillus subtilis) and regulates transcription. Efficient release of RNA polymerase from 6S RNA regulation during outgrowth from stationary phase is dependent on use of 6S RNA as a template to generate a product RNA (pRNA). Interestingly, B. subtilis has two 6S RNAs, 6S-1 and 6S-2, but only 6S-1 RNA appears to be used efficiently as a template for pRNA synthesis during outgrowth. Here, we demonstrate that the identity of the initiating nucleotide is particularly important for the B. subtilis RNA polymerase to use RNA templates. Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP). Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity. These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms. PMID:23761441

  12. Multifunctional Superparamagnetic Nanoparticles: From Synthesis to siRNA Delivery.

    PubMed

    Arami, Sanam; Mahdavi, Majid; Rashidi, Mohammad Reza; Fathi, Marziyeh; Hejazi, Mohammad Saeid; Samadi, Nasser

    2017-01-01

    Targeted delivery of small interfering RNA (siRNA) to the specific tumor tissues and cells is the key challenge in the development of RNA interference as a therapeutic application. To target breast cancer, we developed a cationic nanoparticle as a therapeutic delivery system. The successful synthesis of the magnetic nanoparticles modified by polyaspartate (PAA) and polyethyleneimine (PEI) was confirmed using fourier transform infrared (FT-IR) measurements. The designed nanoparticle has been characterized evaluating its size and charge before and after nanoplex formation with siRNA. The designed nanoparticle could effectively form nanoplex with siRNA in 2:1 w/w ratio. Survivin siRNA was used to suppress the antiapoptotic gene, survivin, in MCF-7 cells. According to the importance of combinational therapy, Mitoxantrone (MTX) was used as a chemotherapeutic agent as well. The multifunctional nanoparticles have been successfully entered into about 63% of the MCF-7 cells shown via microscopic and flowcytometric methods. This effective cellular uptake led to the cell apoptosis. Down regulation of survivin was determined in mRNA and protein levels using Real Time PCR and western blotting, respectively. Gathering all obtained data, it was concluded that Fe3O4-PAA-PEI nanoparticles can deliver siRNA effectively into the cytoplasm of the MCF-7 breast cancer cells and induce apoptosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Genetic modulation of RNA metabolism in Drosophilia. I. Increased rate of ribosomal RNA synthesis.

    PubMed

    Clark, S H; Strausbaugh, L D; Kiefer, B I

    1977-08-01

    It has been suggested that a particular Y chromosome which is rDNA-deficient (YbbSuVar-5) may be associated with an increased utilization of rDNA template in adult testes (Shermoen and Kiefer 1975). To extend the observations on this chromosome, experiments were designed to determine if the chromosome has an effect on rRNA synthesis in bobbed adults and on classic bobbed phenotypes (shortened and thinner scutellar bristles and delayed development). Specific activity measurements were made on rRNA extracted from adult males of the genotypes car bb/YbbSuVar-5, which are rDNA-deficient to the same extent, and from Samarkand+ isogenic (Sam+ iso), which is a wild-type stock. The resulting data demonstrated that the presence of the YbbSuVar-5 chromosome increases the rate of ribosomal RNA synthesis in adult flies. In addition, it was found that the presence of this particular Y chromosome restores wild-type bristle phenotype and development time. Appropriate genetic crosses indicate that the observed effects (increased rRNA synthesis, restoration of wild-type phenotype) are a function of this particular Y chromosome, and are not due to autosomal factors. The results of these experiments suggest that the rate of rRNA accumulation is under genetic control.

  14. Evolution of Protein Synthesis from an RNA World

    PubMed Central

    Noller, Harry F.

    2012-01-01

    SUMMARY Because of the molecular complexity of the ribosome and protein synthesis, it is a challenge to imagine how translation could have evolved from a primitive RNA World. Two specific suggestions are made here to help to address this, involving separate evolution of the peptidyl transferase and decoding functions. First, it is proposed that translation originally arose not to synthesize functional proteins, but to provide simple (perhaps random) peptides that bound to RNA, increasing its available structure space, and therefore its functional capabilities. Second, it is proposed that the decoding site of the ribosome evolved from a mechanism for duplication of RNA. This process involved homodimeric “duplicator RNAs,” resembling the anticodon arms of tRNAs, which directed ligation of trinucleotides in response to an RNA template. PMID:20610545

  15. Solid-Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages.

    PubMed

    Yang, Xianbin

    2017-09-18

    The oligoribonucleotide phosphorodithioate (PS2-RNA) modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2-RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2-aptamers (thioaptamers). Thus, the interest in and promise of PS2-RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA-thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid-phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  16. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  17. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  18. Mechanisms of coronavirus cell entry mediated by the viral spike protein.

    PubMed

    Belouzard, Sandrine; Millet, Jean K; Licitra, Beth N; Whittaker, Gary R

    2012-06-01

    Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes-A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

  19. Isolation of a coronavirus from a green-cheeked Amazon parrot (Amazon viridigenalis Cassin).

    PubMed

    Gough, Richard E; Drury, Sally E; Culver, Francesca; Britton, Paul; Cavanagh, Dave

    2006-04-01

    A virus (AV71/99) was isolated from a green-cheeked Amazon parrot by propagation and passage in both primary embryo liver cells derived from blue and yellow macaw (Ara ararauna) embryos and chicken embryo liver cells. Electron microscopic examination of cytopathic agents derived from both types of cell cultures suggested that it was a coronavirus. This was confirmed using a pan-coronavirus reverse transcriptase polymerase chain reaction that amplified part of gene 1 that encodes the RNA-dependent RNA polymerase. The deduced sequence of 66 amino acids had 66 to 74% amino acid identity with the corresponding sequence of coronaviruses in groups 1, 2 and 3. Several other oligonucleotide primer pairs that give PCR products corresponding to genes 3, 5, N and the 3'-untranslated region of infectious bronchitis virus, turkey coronavirus and pheasant coronavirus (all in group 3) failed to do so with RNA from the parrot coronavirus. This is the first demonstration of a coronavirus in a psittacine species.

  20. RNA Synthesis in Whole Cells and Protoplasts of Centaurea

    PubMed Central

    Kulikowski, Robert R.; Mascarenhas, Joseph P.

    1978-01-01

    Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [3H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells. PMID:16660339

  1. The Roles of RNA in the Synthesis of Protein

    PubMed Central

    Moore, Peter B.; Steitz, Thomas A.

    2011-01-01

    The crystal structures of ribosomes that have been obtained since 2000 have transformed our understanding of protein synthesis. In addition to proving that RNA is responsible for catalyzing peptide bond formation, these structures have provided important insights into the mechanistic details of how the ribosome functions. This review emphasizes what has been learned about the mechanism of peptide bond formation, the antibiotics that inhibit ribosome function, and the fidelity of decoding. PMID:21068149

  2. Semisynthetic tRNA complement mediates in vitro protein synthesis.

    PubMed

    Cui, Zhenling; Stein, Viktor; Tnimov, Zakir; Mureev, Sergey; Alexandrov, Kirill

    2015-04-08

    Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

  3. Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye.

    PubMed

    Cardoso, Tereza C; Ferrari, Heitor F; Bregano, Lívia C; Silva-Frade, Camila; Rosa, Ana Carolina G; Andrade, Alexandre L

    2010-12-01

    A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10(2) EID(50/50 μl) TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum-caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

  4. Improved Cell-Free RNA and Protein Synthesis System

    PubMed Central

    Li, Jun; Gu, Liangcai; Aach, John; Church, George M.

    2014-01-01

    Cell-free RNA and protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. Advances in reconstituted CFPS systems such as the Protein synthesis Using Recombinant Elements (PURE) system offer new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, protein microarrays, isotopic labeling, and incorporating unnatural amino acids. In this study, using firefly luciferase synthesis as a reporter system, we improved PURE system productivity up to 5 fold by adding or adjusting a variety of factors that affect transcription and translation, including Elongation factors (EF-Ts, EF-Tu, EF-G, and EF4), ribosome recycling factor (RRF), release factors (RF1, RF2, RF3), chaperones (GroEL/ES), BSA and tRNAs. The work provides a more efficient defined in vitro transcription and translation system and a deeper understanding of the factors that limit the whole system efficiency. PMID:25180701

  5. Facile synthesis of a 3-deazaadenosine phosphoramidite for RNA solid-phase synthesis

    PubMed Central

    2016-01-01

    Access to 3-deazaadenosine (c3A) building blocks for RNA solid-phase synthesis represents a severe bottleneck in modern RNA research, in particular for atomic mutagenesis experiments to explore mechanistic aspects of ribozyme catalysis. Here, we report the 5-step synthesis of a c3A phosphoramidite from cost-affordable starting materials. The key reaction is a silyl-Hilbert–Johnson nucleosidation using unprotected 6-amino-3-deazapurine and benzoyl-protected 1-O-acetylribose. The novel path is superior to previously described syntheses in terms of efficacy and ease of laboratory handling. PMID:28144324

  6. A Case for the Ancient Origin of Coronaviruses

    PubMed Central

    Chu, Daniel K. W.; Peiris, Joseph S. M.; Kosakovsky Pond, Sergei L.

    2013-01-01

    Coronaviruses are found in a diverse array of bat and bird species, which are believed to act as natural hosts. Molecular clock dating analyses of coronaviruses suggest that the most recent common ancestor of these viruses existed around 10,000 years ago. This relatively young age is in sharp contrast to the ancient evolutionary history of their putative natural hosts, which began diversifying tens of millions of years ago. Here, we attempted to resolve this discrepancy by applying more realistic evolutionary models that have previously revealed the ancient evolutionary history of other RNA viruses. By explicitly modeling variation in the strength of natural selection over time and thereby improving the modeling of substitution saturation, we found that the time to the most recent ancestor common for all coronaviruses is likely far greater (millions of years) than the previously inferred range. PMID:23596293

  7. Alphavirus RNA synthesis and non-structural protein functions

    PubMed Central

    Rupp, Jonathan C.; Sokoloski, Kevin J.; Gebhart, Natasha N.

    2015-01-01

    The members of the genus Alphavirus are positive-sense RNA viruses, which are predominantly transmitted to vertebrates by a mosquito vector. Alphavirus disease in humans can be severely debilitating, and depending on the particular viral species, infection may result in encephalitis and possibly death. In recent years, alphaviruses have received significant attention from public health authorities as a consequence of the dramatic emergence of chikungunya virus in the Indian Ocean islands and the Caribbean. Currently, no safe, approved or effective vaccine or antiviral intervention exists for human alphavirus infection. The molecular biology of alphavirus RNA synthesis has been well studied in a few species of the genus and represents a general target for antiviral drug development. This review describes what is currently understood about the regulation of alphavirus RNA synthesis, the roles of the viral non-structural proteins in this process and the functions of cis-acting RNA elements in replication, and points to open questions within the field. PMID:26219641

  8. Mutation of Host Δ9 Fatty Acid Desaturase Inhibits Brome Mosaic Virus RNA Replication between Template Recognition and RNA Synthesis

    PubMed Central

    Lee, Wai-Ming; Ishikawa, Masayuki; Ahlquist, Paul

    2001-01-01

    All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding Δ9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy. PMID:11160714

  9. In Vitro Synthesis of Rous Sarcoma Virus-Specific RNA is Catalyzed by a DNA-Dependent RNA Polymerase

    PubMed Central

    Rymo, L.; Parsons, J. T.; Coffin, J. M.; Weissmann, C.

    1974-01-01

    Synthesis of Rous sarcoma virus RNA was examined in vitro with a new assay for radioactive virus-specific RNA. Nuclei from infected and uninfected cells were incubated with ribonucleoside [α-32P]triphosphates, Mn++, Mg++ and (NH4)2SO4. Incorporation into total and viral RNA proceeded with similar kinetics for up to 25 min at 37°. About 0.5% of the RNA synthesized by the infected system was scored as virus-specific, compared to 0.03% of the RNA from the uninfected system and 0.005% of the RNA synthesized by monkey kidney cell nuclei. Preincubation with DNase or actinomycin D completely suppressed total and virus-specific RNA synthesis. α-Amanitin, a specific inhibitor of eukaryotic RNA polymerase II, completely inhibited virus-specific RNA synthesis, while reducing total RNA synthesis by only 50%. We conclude that tumor virus-specific RNA is synthesized on a DNA template, most probably by the host's RNA polymerase II. PMID:4368801

  10. Role of the lipid rafts in the life cycle of canine coronavirus.

    PubMed

    Pratelli, Annamaria; Colao, Valeriana

    2015-02-01

    Coronaviruses are enveloped RNA viruses that have evolved complex relationships with their host cells, and modulate their lipid composition, lipid synthesis and signalling. Lipid rafts, enriched in sphingolipids, cholesterol and associated proteins, are special plasma membrane microdomains involved in several processes in viral infections. The extraction of cholesterol leads to disorganization of lipid microdomains and to dissociation of proteins bound to lipid rafts. Because cholesterol-rich microdomains appear to be a general feature of the entry mechanism of non-eneveloped viruses and of several coronaviruses, the purpose of this study was to analyse the contribution of lipids to the infectivity of canine coronavirus (CCoV). The CCoV life cycle is closely connected to plasma membrane cholesterol, from cell entry to viral particle production. The methyl-β-cyclodextrin (MβCD) was employed to remove cholesterol and to disrupt the lipid rafts. Cholesterol depletion from the cell membrane resulted in a dose-dependent reduction, but not abolishment, of virus infectivity, and at a concentration of 15 mM, the reduction in the infection rate was about 68 %. MβCD treatment was used to verify if cholesterol in the envelope was required for CCoV infection. This resulted in a dose-dependent inhibitory effect, and at a concentration of 9 mM MβCD, infectivity was reduced by about 73 %. Since viral entry would constitute a target for antiviral strategies, inhibitory molecules interacting with viral and/or cell membranes, or interfering with lipid metabolism, may have strong antiviral potential. It will be interesting in the future to analyse the membrane microdomains in the CCoV envelope.

  11. pp32 and APRIL are host cell-derived regulators of influenza virus RNA synthesis from cRNA

    PubMed Central

    Sugiyama, Kenji; Kawaguchi, Atsushi; Okuwaki, Mitsuru; Nagata, Kyosuke

    2015-01-01

    Replication of influenza viral genomic RNA (vRNA) is catalyzed by viral RNA-dependent RNA polymerase (vRdRP). Complementary RNA (cRNA) is first copied from vRNA, and progeny vRNAs are then amplified from the cRNA. Although vRdRP and viral RNA are minimal requirements, efficient cell-free replication could not be reproduced using only these viral factors. Using a biochemical complementation assay system, we found a novel activity in the nuclear extracts of uninfected cells, designated IREF-2, that allows robust unprimed vRNA synthesis from a cRNA template. IREF-2 was shown to consist of host-derived proteins, pp32 and APRIL. IREF-2 interacts with a free form of vRdRP and preferentially upregulates vRNA synthesis rather than cRNA synthesis. Knockdown experiments indicated that IREF-2 is involved in in vivo viral replication. On the basis of these results and those of previous studies, a plausible role(s) for IREF-2 during the initiation processes of vRNA replication is discussed. DOI: http://dx.doi.org/10.7554/eLife.08939.001 PMID:26512887

  12. 6S RNA-dependent inhibition of RNA polymerase is released by RNA-dependent synthesis of small de novo products.

    PubMed

    Wurm, Reinhild; Neusser, Thomas; Wagner, Rolf

    2010-01-01

    6S RNA from Escherichia coli is known to bind to RNA polymerase, preventing interaction with many promoters during stationary growth. The resulting repression is released under conditions of nutritional upshift, when the growth situation improves. 6S RNA, which binds to the active site of RNA polymerase, has the particularly interesting feature to act as a template, causing the transcription of defined de novo RNAs (dnRNA) that are complementary to a specific sequence region of the 6S RNA. We analyzed the conditions of dnRNA synthesis and determined their effect on the 6S RNA-mediated inhibition of RNA polymerase in vitro and in vivo. Upon nutritional upshift the RNA polymerase/6S RNA complex induces the rapid synthesis of dnRNAs, which form stable hybrids with the 6S RNA template. The resulting structural change destabilizes the inactivated RNA polymerase complex, causing sigma subunit release. Both dnRNA and 6S RNA are rapidly degraded after complex disintegration. Experiments using the transcriptional inhibitor rifampicin demonstrate that active transcription is required for the disintegration of the RNA polymerase/6S RNA complex. Our results support the conclusion that 6S RNA not only inhibits transcription during stationary growth but also enables cells to resume rapid growth after starvation and help to escape from stationary phase.

  13. From SARS coronavirus to novel animal and human coronaviruses.

    PubMed

    To, Kelvin K W; Hung, Ivan F N; Chan, Jasper F W; Yuen, Kwok-Yung

    2013-08-01

    In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) caused one of the most devastating epidemics known to the developed world. There were two important lessons from this epidemic. Firstly, coronaviruses, in addition to influenza viruses, can cause severe and rapidly spreading human infections. Secondly, bats can serve as the origin and natural animal reservoir of deadly human viruses. Since then, researchers around the world, especially those in Asia where SARS-CoV was first identified, have turned their focus to find novel coronaviruses infecting humans, bats, and other animals. Two human coronaviruses, HCoV-HKU1 and HCoV-NL63, were identified shortly after the SARS-CoV epidemic as common causes of human respiratory tract infections. In 2012, a novel human coronavirus, now called Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in the Middle East to cause fatal human infections in three continents. MERS-CoV human infection is similar to SARS-CoV in having a high fatality rate and the ability to spread from person to person which resulted in secondary cases among close contacts including healthcare workers without travel history to the Middle East. Both viruses also have close relationships with bat coronaviruses. New cases of MERS-CoV infection in humans continue to occur with the origins of the virus still unknown in many cases. A multifaceted approach is necessary to control this evolving MERS-CoV outbreak. Source identification requires detailed epidemiological studies of the infected patients and enhanced surveillance of MERS-CoV or similar coronaviruses in humans and animals. Early diagnosis of infected patients and appropriate infection control measures will limit the spread in hospitals, while social distancing strategies may be necessary to control the outbreak in communities if it remained uncontrolled as in the SARS epidemic.

  14. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

  15. In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

    PubMed Central

    Reff, M E; Davidson, R L

    1979-01-01

    Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase. PMID:284320

  16. Coronaviruses lacking exoribonuclease activity are susceptible to lethal mutagenesis: evidence for proofreading and potential therapeutics.

    PubMed

    Smith, Everett Clinton; Blanc, Hervé; Surdel, Matthew C; Vignuzzi, Marco; Denison, Mark R

    2013-08-01

    No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002-2003, and the recent emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN-) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN- CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN- population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition

  17. The role of fluoride in montmorillonite-catalyzed RNA synthesis.

    PubMed

    Aldersley, Michael F; Joshi, Prakash C

    2014-05-01

    The montmorillonite-catalyzed reactions of the 5'-phosphorimidazolide of adenosine in the presence of fluoride were investigated to complete our study on the effect of salts on this type of reaction. Both anions and cations have been found to influence the oligomerization reactions of the activated nucleotides, being used here as a model system for pre-biotic RNA synthesis. However, in total contrast to the behavior of the activated nucleotides in the presence of montmorillonite and other salts, alkali metal fluorides did not yield any detectable oligomerization products except in very dilute (<0.005 M) solutions of fluoride. Instead, 5'-phosphorofluoridates were formed. Their identity was confirmed by a combination of HPLC, mass spectrometry, synthesis, and NMR.

  18. Infectious Bronchitis Coronavirus Limits Interferon Production by Inducing a Host Shutoff That Requires Accessory Protein 5b

    PubMed Central

    Kint, Joeri; Langereis, Martijn A.; Maier, Helena J.; Britton, Paul; van Kuppeveld, Frank J.; Koumans, Joseph; Wiegertjes, Geert F.

    2016-01-01

    ABSTRACT During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacoronavirus and Betacoronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), establish host shutoff via their nonstructural protein 1 (nsp1). The Gammacoronavirus and Deltacoronavirus genomes, however, do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here, we show that the Gammacoronavirus infectious bronchitis virus (IBV) does induce host shutoff, and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild-type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether, we demonstrate that 5b is a functional equivalent of nsp1, thereby answering the longstanding question of whether lack of nsp1 in gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies. IMPORTANCE Many viruses inhibit protein synthesis by their host cell to enhance virus replication and to antagonize antiviral defense mechanisms. This process is referred to as host shutoff. We studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff, however, does not require degradation of host RNA. Furthermore, we

  19. Neotropical Bats from Costa Rica harbour Diverse Coronaviruses.

    PubMed

    Moreira-Soto, A; Taylor-Castillo, L; Vargas-Vargas, N; Rodríguez-Herrera, B; Jiménez, C; Corrales-Aguilar, E

    2015-11-01

    Bats are hosts of diverse coronaviruses (CoVs) known to potentially cross the host-species barrier. For analysing coronavirus diversity in a bat species-rich country, a total of 421 anal swabs/faecal samples from Costa Rican bats were screened for CoV RNA-dependent RNA polymerase (RdRp) gene sequences by a pancoronavirus PCR. Six families, 24 genera and 41 species of bats were analysed. The detection rate for CoV was 1%. Individuals (n = 4) from four different species of frugivorous (Artibeus jamaicensis, Carollia perspicillata and Carollia castanea) and nectivorous (Glossophaga soricina) bats were positive for coronavirus-derived nucleic acids. Analysis of 440 nt. RdRp sequences allocated all Costa Rican bat CoVs to the α-CoV group. Several CoVs sequences clustered near previously described CoVs from the same species of bat, but were phylogenetically distant from the human CoV sequences identified to date, suggesting no recent spillover events. The Glossophaga soricina CoV sequence is sufficiently dissimilar (26% homology to the closest known bat CoVs) to represent a unique coronavirus not clustering near other CoVs found in the same bat species so far, implying an even higher CoV diversity than previously suspected.

  20. Synthesis, base pairing and structure studies of geranylated RNA

    PubMed Central

    Wang, Rui; Vangaveti, Sweta; Ranganathan, Srivathsan V.; Basanta-Sanchez, Maria; Haruehanroengra, Phensinee; Chen, Alan; Sheng, Jia

    2016-01-01

    Natural RNAs utilize extensive chemical modifications to diversify their structures and functions. 2-Thiouridine geranylation is a special hydrophobic tRNA modification that has been discovered very recently in several bacteria, such as Escherichia coli, Enterobacter aerogenes, Pseudomonas aeruginosa and Salmonella Typhimurium. The geranylated residues are located in the first anticodon position of tRNAs specific for lysine, glutamine and glutamic acid. This big hydrophobic terpene functional group affects the codon recognition patterns and reduces frameshifting errors during translation. We aimed to systematically study the structure, function and biosynthesis mechanism of this geranylation pathway, as well as answer the question of why nature uses such a hydrophobic modification in hydrophilic RNA systems. Recently, we have synthesized the deoxy-analog of S-geranyluridine and showed the geranylated T-G pair is much stronger than the geranylated T-A pair and other mismatched pairs in the B-form DNA duplex context, which is consistent with the observation that the geranylated tRNAGluUUC recognizes GAG more efficiently than GAA. In this manuscript we report the synthesis and base pairing specificity studies of geranylated RNA oligos. We also report extensive molecular simulation studies to explore the structural features of the geranyl group in the context of A-form RNA and its effect on codon–anticodon interaction during ribosome binding. PMID:27307604

  1. Synthesis and antiviral evaluation of 7-O-arylmethylquercetin derivatives against SARS-associated coronavirus (SCV) and hepatitis C virus (HCV).

    PubMed

    Park, Hye Ri; Yoon, Hyunjun; Kim, Mi Kyoung; Lee, Sung Dae; Chong, Youhoon

    2012-01-01

    Aryl diketoacid (ADK) is well known for antiviral activity which can be enhanced by introduction of an aromatic arylmethyl substituent. A natural flavonoid quercetin has a 3,5-dihydroxychromone pharmacophore which is in bioisosteric relationship with the 1,3-diketoacid moiety of the ADK. Thus, it was of our interest to test the antiviral activity of the quercetin derivatives with an arylmethyl group attached. In this study, we prepared a series of the 7-O-arylmethylquercetin derivatives with various aromatic substituents and evaluated their antiviral activity against the SARS-associated coronavirus (SARS-CoV, SCV) as well as hepatitis C virus (HCV). Single difference in the aromatic substituent fine-tuned the biological activity of the 7-O-arylmethylquercetin derivatives to result in two different classes of derivatives selectively active against SCV and HCV.

  2. Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses.

    PubMed

    Chan, Jasper Fuk-Woo; Choi, Garnet Kwan-Yue; Tsang, Alan Ka-Lun; Tee, Kah-Meng; Lam, Ho-Yin; Yip, Cyril Chik-Yan; To, Kelvin Kai-Wang; Cheng, Vincent Chi-Chung; Yeung, Man-Lung; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Chan, Kwok-Hung; Tang, Bone Siu-Fai; Yuen, Kwok-Yung

    2015-08-01

    Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

  3. Detection of group 1 coronaviruses in bats using universal coronavirus reverse transcription polymerase chain reactions.

    PubMed

    Poon, Leo L M; Peiris, J S Malik

    2008-01-01

    The zoonotic transmission of SARS coronavirus from animals to humans revealed the potential impact of coronaviruses on mankind. This incident also triggered several surveillance programs to hunt for novel coronaviruses in human and wildlife populations. Using classical RT-PCR assays that target a highly conserved sequence among coronaviruses, we identified the first coronaviruses in bats. These assays and the cloning and sequencing of the PCR products are described in this chapter. Using the same approach in our subsequent studies, we further detected several novel coronaviruses in bats. These findings highlighted the fact that bats are important reservoirs for coronaviruses.

  4. An intrinsically disordered peptide from Ebola virus VP35 controls viral RNA synthesis by modulating nucleoprotein-RNA interactions

    SciTech Connect

    Leung, Daisy  W.; Borek, Dominika; Luthra, Priya; Binning, Jennifer  M.; Anantpadma, Manu; Liu, Gai; Harvey, Ian B.; Su, Zhaoming; Endlich-Frazier, Ariel; Pan, Juanli; Shabman, Reed  S.; Chiu, Wah; Davey, Robert  A.; Otwinowski, Zbyszek; Basler, Christopher  F.; Amarasinghe, Gaya  K.

    2015-04-01

    During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20–48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludes a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.

  5. Protein expression redirects vesicular stomatitis virus RNA synthesis to cytoplasmic inclusions.

    PubMed

    Heinrich, Bianca S; Cureton, David K; Rahmeh, Amal A; Whelan, Sean P J

    2010-06-24

    Positive-strand and double-strand RNA viruses typically compartmentalize their replication machinery in infected cells. This is thought to shield viral RNA from detection by innate immune sensors and favor RNA synthesis. The picture for the non-segmented negative-strand (NNS) RNA viruses, however, is less clear. Working with vesicular stomatitis virus (VSV), a prototype of the NNS RNA viruses, we examined the location of the viral replication machinery and RNA synthesis in cells. By short-term labeling of viral RNA with 5'-bromouridine 5'-triphosphate (BrUTP), we demonstrate that primary mRNA synthesis occurs throughout the host cell cytoplasm. Protein synthesis results in the formation of inclusions that contain the viral RNA synthesis machinery and become the predominant sites of mRNA synthesis in the cell. Disruption of the microtubule network by treatment of cells with nocodazole leads to the accumulation of viral mRNA in discrete structures that decorate the surface of the inclusions. By pulse-chase analysis of the mRNA, we find that viral transcripts synthesized at the inclusions are transported away from the inclusions in a microtubule-dependent manner. Metabolic labeling of viral proteins revealed that inhibiting this transport step diminished the rate of translation. Collectively those data suggest that microtubule-dependent transport of viral mRNAs from inclusions facilitates their translation. Our experiments also show that during a VSV infection, protein synthesis is required to redirect viral RNA synthesis to intracytoplasmic inclusions. As viral RNA synthesis is initially unrestricted, we speculate that its subsequent confinement to inclusions might reflect a cellular response to infection.

  6. [Protein and RNA synthesis during the transition of potato tuber meristematic tissue from rest to growth].

    PubMed

    Korableva, N P; Ladyzhenskaia, E P; Metlitskiĭ, L V

    1976-01-01

    The rate of RNA and protein synthesis in resting and growing meristem tissues from isolated tuber growth points under their incubation with labelled precursors is studied. It is found that the label incorporation into RNA is low during deep resting, protein synthesis being rather intensive. Both RNA and protein synthesis is intensified with the beginning of growth period. More homogenous in their metabolic rate set of RNAs is found in resting tissues in contrast with growing ones. The data on fractionation of total RNA preparation on MAK column and in sucrose density gradient showed that synthesis of high molecular weight RNA with metabolic period of 4 hours is stimulated in growing meristems. It is possible that synthesis of certain fractions of RNA and protein is one of regulation mechanisms for resting in meristem tissues.

  7. A Bat-Derived Putative Cross-Family Recombinant Coronavirus with a Reovirus Gene

    PubMed Central

    Shi, Yi; Ji, Wei; Jia, Hao; Zhou, Yongming; Wen, Honghua; Zhao, Honglan; Liu, Huaxing; Li, Hong; Wang, Qihui; Wu, Ying; Wang, Liang; Liu, Di; Liu, Guang; Yu, Hongjie; Holmes, Edward C.; Lu, Lin; Gao, George F.

    2016-01-01

    The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 has generated enormous interest in the biodiversity, genomics and cross-species transmission potential of coronaviruses, especially those from bats, the second most speciose order of mammals. Herein, we identified a novel coronavirus, provisionally designated Rousettus bat coronavirus GCCDC1 (Ro-BatCoV GCCDC1), in the rectal swab samples of Rousettus leschenaulti bats by using pan-coronavirus RT-PCR and next-generation sequencing. Although the virus is similar to Rousettus bat coronavirus HKU9 (Ro-BatCoV HKU9) in genome characteristics, it is sufficiently distinct to be classified as a new species according to the criteria defined by the International Committee of Taxonomy of Viruses (ICTV). More striking was that Ro-BatCoV GCCDC1 contained a unique gene integrated into the 3’-end of the genome that has no homologs in any known coronavirus, but which sequence and phylogeny analyses indicated most likely originated from the p10 gene of a bat orthoreovirus. Subgenomic mRNA and cellular-level observations demonstrated that the p10 gene is functional and induces the formation of cell syncytia. Therefore, here we report a putative heterologous inter-family recombination event between a single-stranded, positive-sense RNA virus and a double-stranded segmented RNA virus, providing insights into the fundamental mechanisms of viral evolution. PMID:27676249

  8. The RNA accordion model for template positioning by telomerase RNA during telomeric DNA synthesis

    PubMed Central

    Berman, Andrea J.; Akiyama, Benjamin M.; Stone, Michael D.; Cech, Thomas R.

    2011-01-01

    Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3′ end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation. PMID:22101935

  9. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter.

    PubMed

    Melton, D A; Krieg, P A; Rebagliati, M R; Maniatis, T; Zinn, K; Green, M R

    1984-09-25

    A simple and efficient method for synthesizing pure single stranded RNAs of virtually any structure is described. This in vitro transcription system is based on the unusually specific RNA synthesis by bacteriophage SP6 RNA polymerase which initiates transcription exclusively at an SP6 promoter. We have constructed convenient cloning vectors that contain an SP6 promoter immediately upstream from a polylinker sequence. Using these SP6 vectors, optimal conditions have been established for in vitro RNA synthesis. The advantages and uses of SP6 derived RNAs as probes for nucleic acid blot and solution hybridizations are demonstrated. We show that single stranded RNA probes of a high specific activity are easy to prepare and can significantly increase the sensitivity of nucleic acid hybridization methods. Furthermore, the SP6 transcription system can be used to prepare RNA substrates for studies on RNA processing (1,5,9) and translation (see accompanying paper).

  10. Origins of tmRNA: the missing link in the birth of protein synthesis?

    PubMed

    Macé, Kevin; Gillet, Reynald

    2016-09-30

    The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis.

  11. Origins of tmRNA: the missing link in the birth of protein synthesis?

    PubMed Central

    Macé, Kevin; Gillet, Reynald

    2016-01-01

    The RNA world hypothesis refers to the early period on earth in which RNA was central in assuring both genetic continuity and catalysis. The end of this era coincided with the development of the genetic code and protein synthesis, symbolized by the apparition of the first non-random messenger RNA (mRNA). Modern transfer-messenger RNA (tmRNA) is a unique hybrid molecule which has the properties of both mRNA and transfer RNA (tRNA). It acts as a key molecule during trans-translation, a major quality control pathway of modern bacterial protein synthesis. tmRNA shares many common characteristics with ancestral RNA. Here, we present a model in which proto-tmRNAs were the first molecules on earth to support non-random protein synthesis, explaining the emergence of early genetic code. In this way, proto-tmRNA could be the missing link between the first mRNA and tRNA molecules and modern ribosome-mediated protein synthesis. PMID:27484476

  12. Progress in demonstrating homochiral selection in prebiotic RNA synthesis

    NASA Astrophysics Data System (ADS)

    Joshi, Prakash C.; Aldersley, Michael F.; Ferris, James P.

    2013-03-01

    The dual properties of RNA as an enzyme catalyst (ribozyme) and its ability to store genetic information suggest that early life could have been based on RNA. We have synthesized RNA oligomers up to 50-mer chain lengths by Na+-montmorillonite catalyzed reactions of 5‧-activated mononucleotides. For studying chiral selectivity, the reactions of racemic mixtures of D, L-ImpA and D, L-ImpU were carried out on Na+-montmorillonite. The dimer, trimer, tetramer and pentamer fractions (yields 43.3%, 14.5%, 5.8% and 3.0%, respectively) were investigated for homochiral selection. These products were collected via ion exchange HPLC, their terminal 5‧-phosphate was cleaved by alkaline phosphatase and further analyzed by reverse phase HPLC. Twelve linear and three cyclic dimers were isolated and characterized. The homochirality of dimers was 63.5 ± 0.8%. Out of the sixteen trimers isolated, ten were homochiral with an overall homochirality of 74.2 ± 1.6%. The tetramers and pentamers were separated into 24 and 20 isomers, respectively. Their co-elution with those formed in the binary reactions of D-ImpA with D-ImpU on Na+-montmorillonite revealed 92.7 ± 2.0% and 97.2 ± 0.5% homochirality, respectively. These results suggest that Na+-montmorillonite not only catalyzes the prebiotic synthesis of RNA but it also facilitates homochiral selection. Work is in progress to determine chiral selectivity in the reaction mixtures of activated nucleotides of racemic A, U, G and C on Na+-montmorillonite.

  13. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    PubMed

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  14. Coexistence of multiple coronaviruses in several bat colonies in an abandoned mineshaft.

    PubMed

    Ge, Xing-Yi; Wang, Ning; Zhang, Wei; Hu, Ben; Li, Bei; Zhang, Yun-Zhi; Zhou, Ji-Hua; Luo, Chu-Ming; Yang, Xing-Lou; Wu, Li-Jun; Wang, Bo; Zhang, Yun; Li, Zong-Xiao; Shi, Zheng-Li

    2016-02-01

    Since the 2002-2003 severe acute respiratory syndrome (SARS) outbreak prompted a search for the natural reservoir of the SARS coronavirus, numerous alpha- and betacoronaviruses have been discovered in bats around the world. Bats are likely the natural reservoir of alpha- and betacoronaviruses, and due to the rich diversity and global distribution of bats, the number of bat coronaviruses will likely increase. We conducted a surveillance of coronaviruses in bats in an abandoned mineshaft in Mojiang County, Yunnan Province, China, from 2012-2013. Six bat species were frequently detected in the cave: Rhinolophus sinicus, Rhinolophus affinis, Hipposideros pomona, Miniopterus schreibersii, Miniopterus fuliginosus, and Miniopterus fuscus. By sequencing PCR products of the coronavirus RNA-dependent RNA polymerase gene (RdRp), we found a high frequency of infection by a diverse group of coronaviruses in different bat species in the mineshaft. Sequenced partial RdRp fragments had 80%-99% nucleic acid sequence identity with well-characterized Alphacoronavirus species, including BtCoV HKU2, BtCoV HKU8, and BtCoV1, and unassigned species BtCoV HKU7 and BtCoV HKU10. Additionally, the surveillance identified two unclassified betacoronaviruses, one new strain of SARS-like coronavirus, and one potentially new betacoronavirus species. Furthermore, coronavirus co-infection was detected in all six bat species, a phenomenon that fosters recombination and promotes the emergence of novel virus strains. Our findings highlight the importance of bats as natural reservoirs of coronaviruses and the potentially zoonotic source of viral pathogens.

  15. Biochemical and Cytological Analyses of RNA Synthesis in Kinetin-treated Pea Root Parenchyma 1

    PubMed Central

    Shininger, Terry L.

    1980-01-01

    Excised cortical parenchyma from the pea root (cv. Little Marvel) responds to kinetin/auxin treatment with an increased rate of RNA synthesis well before reinitiating DNA synthesis. Few cells synthesize RNA in the 1st hour of culture. In the presence of kinetin/auxin, the nuclear labeling index increases 2.5-fold as compared to control cultures. The RNA synthesis response has an apparent lag period of 2-4 hours as shown by double label ([3H]adenosine/[14C]adenosine) experiments. Qualitatively, the RNA synthesized at 4-6 hours sediments between 18S and 5S. The RNA synthesized at 14-16 hours and 24-26 hours is primarily ribosomal RNA when kinetin is present. In the absence of kinetin, no clear pattern of RNA synthesis emerges. The data are interpreted to mean that kinetin treatment elicits RNA synthesis in a small proportion of the population initially and this may involve messenger-like RNA. Later, more cells synthesize RNA and this is primarily rRNA. Images PMID:16661292

  16. The mechanism of montmorillonite catalysis in RNA synthesis

    NASA Astrophysics Data System (ADS)

    Joshi, Prakash

    The formation of complex prebiotic molecules on the early Earth is likely to have involved a component of mineral catalysis. Amongst the variety of clay minerals that have been investigated by us for their ability to catalyze the formation of RNA oligomers is montmorillonite. These are 2:1 layer silicates that have a wide range of chemical compositions [(Na,Ca)0.33(Al,Fe,Mg)2(Si,Al)4O10(OH)2.nH2O]. They are commonly produced by the weathering of silicic volcanic ashes to form Bentonite. Once formed, montmorillonites gradually transform to Illites at a modest pressure and temperature. Of the many samples of montmorillonite that we have experimentally examined, a selected subset has been observed to be catalytic for RNA synthesis (Joshi et. al., 2009; Aldersley et al., 2011). Those that have been observed to be excellent catalysts come from a restricted range of elemental compositions. The recent identification of phyllosilicates including montmorillonite on Mars (Bishop et al., 2008) raises the possibility that such processes may have taken place there too. The extent of catalysis depended not only upon the magnitude of the negative charge on the montmorillonite lattice and the number of cations associated with it, but also on the pH at which the reaction is promoted. The isotherm and catalysis studies were extended to provide binding information and catalytic outcomes over a wide pH range. When cations in raw montmorillonite are completely replaced by sodium ions, the resulting Na+-montmorillonite does not catalyze oligomer formation because the ions saturate the interlayer between the platelets of montmorillonite, which blocks the binding of the activated monomers. Acid washed montmorillonite titrated to pH 6-8 with alkali metal ions, serves as the model catalyst for this RNA synthesis (Aldersley et. al., 2011). The optimal binding occurred in the region of maximal oligomer formation. X-ray diffraction studies revealed changes in layer separations of

  17. Evolutionary insights into the ecology of coronaviruses.

    PubMed

    Vijaykrishna, D; Smith, G J D; Zhang, J X; Peiris, J S M; Chen, H; Guan, Y

    2007-04-01

    Although many novel members of the Coronaviridae have recently been recognized in different species, the ecology of coronaviruses has not been established. Our study indicates that bats harbor a much wider diversity of coronaviruses than any other animal species. Dating of different coronavirus lineages suggests that bat coronaviruses are older than those recognized in other animals and that the human severe acute respiratory syndrome (SARS) coronavirus was directly derived from viruses from wild animals in wet markets of southern China. Furthermore, the most closely related bat and SARS coronaviruses diverged in 1986, an estimated divergence time of 17 years prior to the outbreak, suggesting that there may have been transmission via an unknown intermediate host. Analysis of lineage-specific selection pressure also indicated that only SARS coronaviruses in civets and humans were under significant positive selection, also demonstrating a recent interspecies transmission. Analysis of population dynamics revealed that coronavirus populations in bats have constant population growth, while viruses from all other hosts show epidemic-like increases in population. These results indicate that diverse coronaviruses are endemic in different bat species, with repeated introductions to other animals and occasional establishment in other species. Our findings suggest that bats are likely the natural hosts for all presently known coronavirus lineages and that all coronaviruses recognized in other species were derived from viruses residing in bats. Further surveillance of bat and other animal populations is needed to fully describe the ecology and evolution of this virus family.

  18. A MicroRNA Precursor Surveillance System in Quality Control of MicroRNA Synthesis

    PubMed Central

    Liu, Xuhang; Zheng, Qi; Vrettos, Nicholas; Maragkakis, Manolis; Alexiou, Panagiotis; Gregory, Brian D.; Mourelatos, Zissimos

    2014-01-01

    Summary MicroRNAs (miRNAs) are essential for regulation of gene expression. Though numerous miRNAs have been identified by high throughput sequencing, few precursor miRNAs (pre-miRNAs) are experimentally validated. Here we report a strategy for constructing high-throughput sequencing libraries enriched for full-length pre-miRNAs. We find widespread and extensive uridylation of Argonaute bound pre-miRNAs, which is primarily catalyzed by two terminal uridylyltransferases: TUT7 and TUT4. Uridylation by TUT7/4 not only polishes pre-miRNA 3′ ends, but also facilitates their degradation by the exosome, preventing clogging of Ago with defective species. We show that the exosome exploits distinct substrate preferences of DIS3 and RRP6, its two catalytic subunits, to distinguish productive from defective pre-miRNAs. Furthermore, we identify a positive feedback loop formed by the exosome and TUT7/4 in triggering uridylation and degradation of Ago-bound pre-miRNAs. Our study reveals a pre-miRNA surveillance system that comprises TUT7, TUT4 and the exosome in quality control of miRNA synthesis. PMID:25175028

  19. Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS.

    PubMed

    Callendret, Benoît; Lorin, Valérie; Charneau, Pierre; Marianneau, Philippe; Contamin, Hugues; Betton, Jean-Michel; van der Werf, Sylvie; Escriou, Nicolas

    2007-07-05

    The SARS-CoV spike glycoprotein (S) is the main target of the protective immune response in humans and animal models of SARS. Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). The presence of both splice sites and WPRE markedly improved the immunogenicity of S-based DNA vaccines against SARS. Upon immunization of mice with low doses (2 microg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. Our observations are likely to be useful for the construction of plasmid and viral vectors designed for optimal expression of intronless genes derived from cytoplasmic RNA viruses.

  20. The Yin and Yang of Hepatitis C: Synthesis and Decay of HCV RNA

    PubMed Central

    Li, You; Yamane, Daisuke; Masaki, Takahiro; Lemon, Stanley M.

    2016-01-01

    PREFACE Hepatitis C virus (HCV) is an unusual RNA virus that possesses a striking capacity to persist for life in the majority of infected individuals. In order to persist, HCV must balance viral RNA synthesis and decay in infected cells. In this Review, we focus on interactions between the positive-sense RNA genome of HCV and host RNA-binding proteins and microRNAs (miRNAs) that influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. We discuss how these processes impact HCV pathogenesis and therapeutic strategies against the virus. PMID:26256788

  1. SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA.

    PubMed

    Liu, Yu; Wu, Na; Dong, Jie; Gao, Yaping; Zhang, Xin; Shao, Ningsheng; Yang, Guang

    2010-10-22

    SsrA RNA (small stable RNA A), also known as tmRNA and 10Sa RNA, functions both as tRNA and mRNA through its unique structure. The carotenoid pigment is the eponymous feature of human pathogen Staphylococcus aureus. Here, we found that the pigment of the mutant strain with ssrA deletion was increased. Furthermore, it was demonstrated that ssrA could act as an antisense RNA aside from its well-known biological function, and crtMN, encoding two essential enzymes for the pigment synthesis, was identified as target of ssrA. Further investigation showed ssrA could specifically base pair with the RBS (ribosomal binding site) region of the crtMN mRNA. Our results revealed a new mechanism by which ssrA regulated the biosynthesis of pigment in S. aureus.

  2. Detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses.

    PubMed

    Minami, Shohei; Kuroda, Yudai; Terada, Yutaka; Yonemitsu, Kenzo; Van Nguyen, Dung; Kuwata, Ryusei; Shimoda, Hiroshi; Takano, Ai; Maeda, Ken

    2016-12-01

    In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3'-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets.

  3. Pre-mRNA Processing Factor Prp18 Is a Stimulatory Factor of Influenza Virus RNA Synthesis and Possesses Nucleoprotein Chaperone Activity.

    PubMed

    Minakuchi, M; Sugiyama, K; Kato, Y; Naito, T; Okuwaki, M; Kawaguchi, A; Nagata, K

    2017-02-01

    The genome of influenza virus (viral RNA [vRNA]) is associated with the nucleoprotein (NP) and viral RNA-dependent RNA polymerases and forms helical viral ribonucleoprotein (vRNP) complexes. The NP-vRNA complex is the biologically active template for RNA synthesis by the viral polymerase. Previously, we identified human pre-mRNA processing factor 18 (Prp18) as a stimulatory factor for viral RNA synthesis using a Saccharomyces cerevisiae replicon system and a single-gene deletion library of Saccharomyces cerevisiae (T. Naito, Y. Kiyasu, K. Sugiyama, A. Kimura, R. Nakano, A. Matsukage, and K. Nagata, Proc Natl Acad Sci USA, 104:18235-18240, 2007, https://doi.org/10.1073/pnas.0705856104). In infected Prp18 knockdown (KD) cells, the synthesis of vRNA, cRNA, and viral mRNAs was reduced. Prp18 was found to stimulate in vitro viral RNA synthesis through its interaction with NP. Analyses using in vitro RNA synthesis reactions revealed that Prp18 dissociates newly synthesized RNA from the template after the early elongation step to stimulate the elongation reaction. We found that Prp18 functions as a chaperone for NP to facilitate the formation of NP-RNA complexes. Based on these results, it is suggested that Prp18 accelerates influenza virus RNA synthesis as an NP chaperone for the processive elongation reaction.

  4. Surveillance of Bat Coronaviruses in Kenya Identifies Relatives of Human Coronaviruses NL63 and 229E and Their Recombination History.

    PubMed

    Tao, Ying; Shi, Mang; Chommanard, Christina; Queen, Krista; Zhang, Jing; Markotter, Wanda; Kuzmin, Ivan V; Holmes, Edward C; Tong, Suxiang

    2017-03-01

    Bats harbor a large diversity of coronaviruses (CoVs), several of which are related to zoonotic pathogens that cause severe disease in humans. Our screening of bat samples collected in Kenya from 2007 to 2010 not only detected RNA from several novel CoVs but, more significantly, identified sequences that were closely related to human CoVs NL63 and 229E, suggesting that these two human viruses originate from bats. We also demonstrated that human CoV NL63 is a recombinant between NL63-like viruses circulating in Triaenops bats and 229E-like viruses circulating in Hipposideros bats, with the breakpoint located near 5' and 3' ends of the spike (S) protein gene. In addition, two further interspecies recombination events involving the S gene were identified, suggesting that this region may represent a recombination "hot spot" in CoV genomes. Finally, using a combination of phylogenetic and distance-based approaches, we showed that the genetic diversity of bat CoVs is primarily structured by host species and subsequently by geographic distances.IMPORTANCE Understanding the driving forces of cross-species virus transmission is central to understanding the nature of disease emergence. Previous studies have demonstrated that bats are the ultimate reservoir hosts for a number of coronaviruses (CoVs), including ancestors of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and human CoV 229E (HCoV-229E). However, the evolutionary pathways of bat CoVs remain elusive. We provide evidence for natural recombination between distantly related African bat coronaviruses associated with Triaenops afer and Hipposideros sp. bats that resulted in a NL63-like virus, an ancestor of the human pathogen HCoV-NL63. These results suggest that interspecies recombination may play an important role in CoV evolution and the emergence of novel CoVs with zoonotic potential.

  5. Interacting RNA polymerase motors on a DNA track: Effects of traffic congestion and intrinsic noise on RNA synthesis

    NASA Astrophysics Data System (ADS)

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  6. DNA-Directed Cell-Free Synthesis of Biologically Active Transfer RNA: su+III Tyrosyl-tRNA

    PubMed Central

    Zubay, Geoffrey; Cheong, Loretta; Gefter, Malcolm

    1971-01-01

    Biologically active su+III tyrosyl-tRNA has been synthesized in a DNA-directed cell-free system from Escerichia coli. Such a system should be most useful for studying the mechanism of tRNA synthesis. This tRNA is capable of suppressing amber mutations in the gene coding for β-galactosidase (EC 3.2.1.23) and, therefore, must be capable of being charged and transferring amino acids. A 4-fold stimulation in the activity of the tRNA formed de novo is obtained with isopentenyl pyrophosphate, a compound involved in the post-transcriptional acylation of an adenine base adjacent to the anticodon. It has been suggested elsewhere that formation of RNA subject to stringent control may be inhibited by guanosine tetraphosphate (ppGpp). However, guanosine tetraphosphate did not affect the synthesis of su+III tyrosyl-tRNA, even though the synthesis of this tRNA is subject to stringent control. PMID:4943792

  7. Interacting RNA polymerase motors on a DNA track: effects of traffic congestion and intrinsic noise on RNA synthesis.

    PubMed

    Tripathi, Tripti; Chowdhury, Debashish

    2008-01-01

    RNA polymerase (RNAP) is an enzyme that synthesizes a messenger RNA (mRNA) strand which is complementary to a single-stranded DNA template. From the perspective of physicists, an RNAP is a molecular motor that utilizes chemical energy input to move along the track formed by DNA. In many circumstances, which are described in this paper, a large number of RNAPs move simultaneously along the same track; we refer to such collective movements of the RNAPs as RNAP traffic. Here we develop a theoretical model for RNAP traffic by incorporating the steric interactions between RNAPs as well as the mechanochemical cycle of individual RNAPs during the elongation of the mRNA. By a combination of analytical and numerical techniques, we calculate the rates of mRNA synthesis and the average density profile of the RNAPs on the DNA track. We also introduce, and compute, two different measures of fluctuations in the synthesis of RNA. Analyzing these fluctuations, we show how the level of intrinsic noise in mRNA synthesis depends on the concentrations of the RNAPs as well as on those of some of the reactants and the products of the enzymatic reactions catalyzed by RNAP. We suggest appropriate experimental systems and techniques for testing our theoretical predictions.

  8. Mercurated nucleotides: assessment of a new tool to study RNA synthesis and processing in isolated nuclei.

    PubMed Central

    Schäfer, K P

    1977-01-01

    Mercurated pyrimidine nucleotides have been used to study RNA synthesis and processing in isolated nuclei from mouse L cells. 5-mercuridine triphosphate (5-Hg-UTP) or 5-Hg-CTP are accepted as substrates by the purified RNA polymerases (I+III) and (II) from mouse cells, respectively, as well as by the enzymes still bound to the nuclear chromatin. In nuclei, RNA synthesis in the presence of Hg-UTP is reduced to 60-70% of a control. 30-60% of RNA labeled in vitro with (3H)UTP in isolated nuclei is not retained on sulfhydryl sepharose columns. Sucrose gradient analysis reveals a size distribution of the non-bound RNA similar to non-mercurated control RNA. Hg-RNA is found in a single peak from 4-10S. Chase experiments indicate that this RNA is the original transcript. It is argued that Hg-nucleotides may cause premature chain termination. Methylation of RNA in vitro by S-adenosyl methionine ((3H)SAM) is reduced to 75% of controls in the presence of Hg-UTP. Only 6% of the methyl groups appear in Hg-RNA. Polyadenylation is reduced as well. 15% of poly(A) (+)RNA are found in control assays whereas only 1% of Hg-RNA carries a poly(A) end added in vitro. These results limit the use of mercurated nucleotides for studies of nuclear RNA synthesis and processing. PMID:600804

  9. Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia.

    PubMed

    Rihtaric, Danijela; Hostnik, Peter; Steyer, Andrej; Grom, Joze; Toplak, Ivan

    2010-04-01

    Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, such as Hendra virus, Nipah virus, Ebola virus, Marburg virus, rabies and other lyssaviruses. Recently, a large number of viruses closely related to members of the genus Coronavirus have been associated with severe acute respiratory syndrome (SARS) and detected in bat species. In this study, samples were collected from 106 live bats of seven different bat species from 27 different locations in Slovenia. Coronaviruses were detected by RT-PCR in 14 out of 36 horseshoe bat (Rhinolophus hipposideros) fecal samples, with 38.8% virus prevalence. Sequence analysis of a 405-nucleotide region of the highly conserved RNA polymerase gene (pol) showed that all coronaviruses detected in this study are genetically closely related, with 99.5-100% nucleotide identity, and belong to group 2 of the coronaviruses. The most closely related virus sequence in GenBank was SARS bat isolate Rp3/2004 (DQ071615) within the SARS-like CoV cluster, sharing 85% nucleotide identity and 95.6% amino acid identity. The potential risk of a new group of bat coronaviruses as a reservoir for human infections is highly suspected, and further molecular epidemiologic studies of these bat coronaviruses are needed.

  10. Biochemical Characterization of Middle East Respiratory Syndrome Coronavirus Helicase

    PubMed Central

    Lazarus, Hilary

    2016-01-01

    ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) helicase is a superfamily 1 helicase containing seven conserved motifs. We have cloned, expressed, and purified a Strep-fused recombinant MERS-CoV nonstructural protein 13 (M-nsp13) helicase. Characterization of its biochemical properties showed that it unwound DNA and RNA similarly to severe acute respiratory syndrome CoV nsp13 (S-nsp13) helicase. We showed that M-nsp13 unwound in a 5′-to-3′ direction and efficiently unwound the partially duplex RNA substrates with a long loading strand relative to those of the RNA substrates with a short or no loading strand. Moreover, the Km of ATP for M-nsp13 is inversely proportional to the length of the 5′ loading strand of the partially duplex RNA substrates. Finally, we also showed that the rate of unwinding (ku) of M-nsp13 is directly proportional to the length of the 5′ loading strand of the partially duplex RNA substrate. These results provide insights that enhance our understanding of the biochemical properties of M-nsp13. IMPORTANCE Coronaviruses are known to cause a wide range of diseases in humans and animals. Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel coronavirus discovered in 2012 and is responsible for acute respiratory syndrome in humans in the Middle East, Europe, North Africa, and the United States of America. Helicases are motor proteins that catalyze the processive separation of double-stranded nucleic acids into two single-stranded nucleic acids by utilizing the energy derived from ATP hydrolysis. MERS-CoV helicase is one of the most important viral replication enzymes of this coronavirus. Herein, we report the first bacterial expression, enzyme purification, and biochemical characterization of MERS-CoV helicase. The knowledge obtained from this study might be used to identify an inhibitor of MERS-CoV replication, and the helicase might be used as a therapeutic target. PMID:27631026

  11. Two conformations of a crystalline human tRNA synthetase–tRNA complex: implications for protein synthesis

    PubMed Central

    Yang, Xiang-Lei; Otero, Francella J; Ewalt, Karla L; Liu, Jianming; Swairjo, Manal A; Köhrer, Caroline; RajBhandary, Uttam L; Skene, Robert J; McRee, Duncan E; Schimmel, Paul

    2006-01-01

    Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1α, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross-dimer tRNA complex, the class I enzyme has a class II-like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species-specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF-1α, suggesting that the product of aminoacylation can be directly handed off to EF-1α for the next step of protein synthesis. PMID:16724112

  12. Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates.

    PubMed Central

    Damha, M J; Ganeshan, K; Hudson, R H; Zabarylo, S V

    1992-01-01

    The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo. Images PMID:1480476

  13. Animal models for SARS and MERS coronaviruses.

    PubMed

    Gretebeck, Lisa M; Subbarao, Kanta

    2015-08-01

    The emergence of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), two strains of animal coronaviruses that crossed the species barrier to infect and cause severe respiratory infections in humans within the last 12 years, have taught us that coronaviruses represent a global threat that does not recognize international borders. We can expect to see other novel coronaviruses emerge in the future. An ideal animal model should reflect the clinical signs, viral replication and pathology seen in humans. In this review, we present factors to consider in establishing an animal model for the study of novel coronaviruses and compare the different animal models that have been employed to study SARS-CoV and MERS-CoV.

  14. Animal models for SARS and MERS coronaviruses

    PubMed Central

    Gretebeck, Lisa M; Subbarao, Kanta

    2015-01-01

    The emergence of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), two strains of animal coronaviruses that crossed the species barrier to infect and cause severe respiratory infections in humans within the last 12 years, have taught us that coronaviruses represent a global threat that does not recognize international borders. We can expect to see other novel coronaviruses emerge in the future. An ideal animal model should reflect the clinical signs, viral replication and pathology seen in humans. In this review, we present factors to consider in establishing an animal model for the study of novel coronaviruses and compare the different animal models that have been employed to study SARS-CoV and MERS-CoV. PMID:26184451

  15. Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture.

    PubMed

    Harrison, Sally M; Tarpey, Ian; Rothwell, Lisa; Kaiser, Pete; Hiscox, Julian A

    2007-04-01

    The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry that, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination, which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross-strain immunity. Effective antiviral agents may therefore be advantageous in the treatment of IBV. Lithium chloride (LiCl) is a potent inhibitor of the DNA virus herpes simplex virus but not RNA viruses. The effect of LiCl on the replication of IBV was examined in cell culture using two model cell types; Vero cells, an African Green monkey kidney-derived epithelial cell line; and DF-1 cells, an immortalized chicken embryo fibroblast cell line. When treated with a range of LiCl concentrations, IBV RNA and protein levels and viral progeny production were reduced in a dose-dependent manner in both cell types, and the data indicated that inhibition was a cellular rather than a virucidal effect. Host cell protein synthesis still took place in LiCl-treated cells and the level of a standard cellular housekeeping protein remained unchanged, indicating that the effect of LiCl was specifically against IBV.

  16. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  17. DNA and RNA Synthesis in Animal Cells in Culture--Methods for Use in Schools

    ERIC Educational Resources Information Center

    Godsell, P. M.; Balls, M.

    1973-01-01

    Describes the experimental procedures used for detecting DNA and RNA synthesis in xenopus cells by autoradiography. The method described is suitable for senior high school laboratory classes or biology projects, if supervised by a teacher qualified to handle radioisotopes. (JR)

  18. Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

    PubMed Central

    Cruz, Jazmina L. G.; Sola, Isabel; Becares, Martina; Alberca, Berta; Plana, Joan; Enjuanes, Luis; Zuñiga, Sonia

    2011-01-01

    Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of

  19. [Synthesis in E. coli cells of short RNA encoded in plasmids].

    PubMed

    Dontsova, O A; Bogdanova, S L; Kopylov, A M

    1989-05-01

    The synthesis of 5S rRNA and 4.5S RNA in E. coli HB 101 cells harbouring plasmids pKK 5-1 and pKK 247-2 was studied. The plasmids were derived from pBK 322 and contained genes coding for 5S rRNA and 4.5S RNA with regulatory elements of an rRNA transcription operon rrn B. When the cells were grown on enriched or minimal media (2 and 0.3 duplications per hour), the synthesis of both 5S rRNA and 4.5S RNA was proportional to the gene dosage and was greater in the plasmid than in the host strain. Such RNA accumulation did not change the cell growth parameters and was thus not toxic for the cells. At high growth rates, the RNA synthesis in the cells became excessive, and the processing system was upset with the accumulation of RNA precursors. The fact confirms the hypothesis, according to which the whole rRNA operon is essential for its own feedback regulation.

  20. Synthesis and biological activity of artificial mRNA prepared with novel phosphorylating reagents

    PubMed Central

    Nagata, Seigo; Hamasaki, Tomohiro; Uetake, Koichi; Masuda, Hirofumi; Takagaki, Kazuchika; Oka, Natsuhisa; Wada, Takeshi; Ohgi, Tadaaki; Yano, Junichi

    2010-01-01

    Though medicines that target mRNA are under active investigation, there has been little or no effort to develop mRNA itself as a medicine. Here, we report the synthesis of a 130-nt mRNA sequence encoding a 33-amino-acid peptide that includes the sequence of glucagon-like peptide-1, a peptide that stimulates glucose-dependent insulin secretion from the pancreas. The synthesis method used, which had previously been developed in our laboratory, was based on the use of 2-cyanoethoxymethyl as the 2′-hydroxy protecting group. We also developed novel, highly reactive phosphotriester pyrophosphorylating reagents to pyrophosphorylate the 5′-end of the 130-mer RNA in preparation for capping. We completed the synthesis of the artificial mRNA by the enzymatic addition of a 5′-cap and a 3′-poly(A) tail to the pyrophosphorylated 130-mer and showed that the resulting mRNA supported protein synthesis in a cell-free system and in whole cells. As far as we know, this is the first time that mRNA has been prepared from a chemically synthesized RNA sequence. As well as providing a research tool for the intracellular expression of peptides, the technology described here may be used for the production of mRNA for medical applications. PMID:20660478

  1. Defective antitermination of rRNA transcription and derepression of rRNA and tRNA synthesis in the nusB5 mutant of Escherichia coli.

    PubMed Central

    Sharrock, R A; Gourse, R L; Nomura, M

    1985-01-01

    The nusB5 mutant of Escherichia coli was originally selected for reduced ability to support the antitermination of transcription that is mediated by the gene N product of bacteriophage lambda. By analyzing pulse-labeled RNA with an RNA.DNA filter hybridization technique, we have shown that, in the nusB5 mutant, the ratio of promoter-proximal rRNA transcripts to promoter-distal transcripts is increased at least by a factor of 1.6; that is, in the absence of the functional nusB gene product, premature transcription termination takes place within rRNA operons. These results demonstrate that rRNA transcription in E. coli utilizes an antitermination mechanism that has at least one factor in common with the phage lambda system, the nusB gene product. We have also observed that the transcription initiation frequency at rRNA promoters is increased in the nusB5 strain and that this strain accumulates 30S and 50S ribosomal subunits at approximately the same rate as the parent. Thus, it appears that E. coli compensates for premature termination of rRNA transcription by derepressing rRNA operon expression. The increase in rRNA promoter activity in the nusB5 mutant is accompanied by a parallel derepression of synthesis of tRNAs that are not encoded by rRNA operons. These results are consistent with a model for negative feedback regulation of rRNA and tRNA synthesis by products of rRNA operons. PMID:3161080

  2. The C-Terminal Tail of TRIM56 Dictates Antiviral Restriction of Influenza A and B Viruses by Impeding Viral RNA Synthesis

    PubMed Central

    Liu, Baoming; Li, Nan L.; Shen, Yang; Bao, Xiaoyong; Elbahesh, Husni; Webby, Richard J.

    2016-01-01

    ABSTRACT Accumulating data suggest that tripartite-motif-containing (TRIM) proteins participate in host responses to viral infections, either by acting as direct antiviral restriction factors or through regulating innate immune signaling of the host. Of >70 TRIMs, TRIM56 is a restriction factor of several positive-strand RNA viruses, including three members of the family Flaviviridae (yellow fever virus, dengue virus, and bovine viral diarrhea virus) and a human coronavirus (OC43), and this ability invariably depends upon the E3 ligase activity of TRIM56. However, the impact of TRIM56 on negative-strand RNA viruses remains unclear. Here, we show that TRIM56 puts a check on replication of influenza A and B viruses in cell culture but does not inhibit Sendai virus or human metapneumovirus, two paramyxoviruses. Interestingly, the anti-influenza virus activity was independent of the E3 ligase activity, B-box, or coiled-coil domain. Rather, deletion of a 63-residue-long C-terminal-tail portion of TRIM56 abrogated the antiviral function. Moreover, expression of this short C-terminal segment curtailed the replication of influenza viruses as effectively as that of full-length TRIM56. Mechanistically, TRIM56 was found to specifically impede intracellular influenza virus RNA synthesis. Together, these data reveal a novel antiviral activity of TRIM56 against influenza A and B viruses and provide insights into the mechanism by which TRIM56 restricts these medically important orthomyxoviruses. IMPORTANCE Options to treat influenza are limited, and drug-resistant influenza virus strains can emerge through minor genetic changes. Understanding novel virus-host interactions that alter influenza virus fitness may reveal new targets/approaches for therapeutic interventions. We show here that TRIM56, a tripartite-motif protein, is an intrinsic host restriction factor of influenza A and B viruses. Unlike its antiviral actions against positive-strand RNA viruses, the anti

  3. An intrinsically disordered peptide from Ebola virus VP35 controls viral RNA synthesis by modulating nucleoprotein-RNA interactions

    DOE PAGES

    Leung, Daisy  W.; Borek, Dominika; Luthra, Priya; ...

    2015-04-01

    During viral RNA synthesis, Ebola virus (EBOV) nucleoprotein (NP) alternates between an RNA-template-bound form and a template-free form to provide the viral polymerase access to the RNA template. In addition, newly synthesized NP must be prevented from indiscriminately binding to noncognate RNAs. Here, we investigate the molecular bases for these critical processes. We identify an intrinsically disordered peptide derived from EBOV VP35 (NPBP, residues 20–48) that binds NP with high affinity and specificity, inhibits NP oligomerization, and releases RNA from NP-RNA complexes in vitro. The structure of the NPBP/ΔNPNTD complex, solved to 3.7 Å resolution, reveals how NPBP peptide occludesmore » a large surface area that is important for NP-NP and NP-RNA interactions and for viral RNA synthesis. Together, our results identify a highly conserved viral interface that is important for EBOV replication and can be targeted for therapeutic development.« less

  4. Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth.

    PubMed

    Drygin, Denis; Lin, Amy; Bliesath, Josh; Ho, Caroline B; O'Brien, Sean E; Proffitt, Chris; Omori, Mayuko; Haddach, Mustapha; Schwaebe, Michael K; Siddiqui-Jain, Adam; Streiner, Nicole; Quin, Jaclyn E; Sanij, Elaine; Bywater, Megan J; Hannan, Ross D; Ryckman, David; Anderes, Kenna; Rice, William G

    2011-02-15

    Deregulated ribosomal RNA synthesis is associated with uncontrolled cancer cell proliferation. RNA polymerase (Pol) I, the multiprotein complex that synthesizes rRNA, is activated widely in cancer. Thus, selective inhibitors of Pol I may offer a general therapeutic strategy to block cancer cell proliferation. Coupling medicinal chemistry efforts to tandem cell- and molecular-based screening led to the design of CX-5461, a potent small-molecule inhibitor of rRNA synthesis in cancer cells. CX-5461 selectively inhibits Pol I-driven transcription relative to Pol II-driven transcription, DNA replication, and protein translation. Molecular studies demonstrate that CX-5461 inhibits the initiation stage of rRNA synthesis and induces both senescence and autophagy, but not apoptosis, through a p53-independent process in solid tumor cell lines. CX-5461 is orally bioavailable and demonstrates in vivo antitumor activity against human solid tumors in murine xenograft models. Our findings position CX-5461 for investigational clinical trials as a potent, selective, and orally administered agent for cancer treatment. ©2010 AACR.

  5. A Model for the Origin of Protein Synthesis as Coreplicational Scanning of Nascent RNA

    NASA Astrophysics Data System (ADS)

    Yakhnin, Alexander V.

    2007-12-01

    The origin of protein synthesis is one of the major riddles of molecular biology. It was proposed a decade ago that the ribosomal RNA evolved from an earlier RNA-replisome (a ribozyme fulfilling RNA replication) while transfer RNA (tRNA) evolved from a genomic replication origin. Applying these hypotheses, I suggest that protein synthesis arose for the purpose of segregating copy and template RNA during replication through the conventional formation of a complementary strand. Nascent RNA was scanned in 5' to 3' direction following the progress of replication. The base pairing of several tRNA-like molecules with nascent RNA released the replication intermediates trapped in duplex. Synthesis of random peptides evolved to fuel the turnover of tRNAs. Then the combination of replication-coupled peptide formation and the independent development of amino acid-specific tRNA aminoacylation resulted in template-based protein synthesis. Therefore, the positioning of tRNAs adjacent to each other developed for the purpose of replication rather than peptide synthesis. This hypothesis does not include either selection for useful peptides or specific recognition of amino acids at the initial evolution of translation. It does, however, explain a number of features of modern translation apparatus, such as the relative flexibility of genetic code, the number of proteins shared by the transcription and translation machines, the universal participation of an RNA subunit in co-translational protein secretion, ‘unscheduled translation’, and factor-independent translocation. Assistance of original ribosomes in keeping apart the nascent transcript from its template is still widely explored by modern bacteria and perhaps by other domains of life.

  6. On-chip synthesis of RNA aptamer microarrays for multiplexed protein biosensing with SPR imaging measurements.

    PubMed

    Chen, Yulin; Nakamoto, Kohei; Niwa, Osamu; Corn, Robert M

    2012-06-05

    Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

  7. A Recombinant Collagen-mRNA Platform for Controllable Protein Synthesis.

    PubMed

    Sun, Liping; Xiong, Yunjing; Bashan, Anat; Zimmerman, Ella; Shulman Daube, Shirley; Peleg, Yoav; Albeck, Shira; Unger, Tamar; Yonath, Hagith; Krupkin, Miri; Matzov, Donna; Yonath, Ada

    2015-07-06

    We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.

  8. The parable of the caveman and the Ferrari: protein synthesis and the RNA world.

    PubMed

    Noller, Harry F

    2017-03-19

    The basic steps of protein synthesis are carried out by the ribosome, a very large and complex ribonucleoprotein particle. In keeping with its proposed emergence from an RNA world, all three of its core mechanisms-aminoacyl-tRNA selection, catalysis of peptide bond formation and coupled translocation of mRNA and tRNA-are embodied in the properties of ribosomal RNA, while its proteins play a supportive role.This article is part of the themed issue 'Perspectives on the ribosome'.

  9. Accessory proteins of SARS-CoV and other coronaviruses.

    PubMed

    Liu, Ding Xiang; Fung, To Sing; Chong, Kelvin Kian-Long; Shukla, Aditi; Hilgenfeld, Rolf

    2014-09-01

    The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)). Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Comparative studies of effect of auxin and ethylene on permeability and synthesis of RNA and protein.

    PubMed

    Sacher, J A; Salminen, S O

    1969-10-01

    The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (alpha-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is concluded that in these tissues the auxin effect is not an ethylene effect.

  11. Gold-nanobeacons for real-time monitoring of RNA synthesis.

    PubMed

    Rosa, João; Conde, João; de la Fuente, Jesus M; Lima, João C; Baptista, Pedro V

    2012-01-01

    Measuring RNA synthesis and, when required, the level of inhibition, is crucial towards the development of practical strategies to evaluate silencing efficiency of gene silencing approaches. We developed a direct method to follow RNA synthesis in real time based on gold nanoparticles (AuNPs) functionalized with a fluorophore labeled hairpin-DNA, i.e. gold-nanobeacon (Au-nanobeacon). Under hairpin configuration, proximity to gold nanoparticles leads to fluorescence quenching; hybridization to a complementary target restores fluorescence emission due to the Au-nanobeacons' conformational reorganization that causes the fluorophore and the AuNP to part from each other, yielding a quantitative response. With this reporter Au-nanobeacon we were able to measure the rate of in vitro RNA synthesis (~10.3 fmol of RNA per minute). Then, we designed a second Au-nanobeacon targeting the promoter sequence (inhibitor) so as to inhibit transcription whilst simultaneously monitor the number of promoters being silenced. Using the two Au-nanobeacons in the same reaction mixture, we are capable of quantitatively assess in real time the synthesis of RNA and the level of inhibition. The biosensor concept can easily be extended and adapted to situations when real-time quantitative assessment of RNA synthesis and determination of the level of inhibition are required. In fact, this biosensor may assist the in vitro evaluation of silencing potential of a given sequence to be later used for in vivo gene silencing.

  12. The role of the priming loop in Influenza A virus RNA synthesis

    PubMed Central

    te Velthuis, Aartjan J.W.; Robb, Nicole C.; Kapanidis, Achillefs N.; Fodor, Ervin

    2016-01-01

    RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes1. They adopt a closed, right-handed fold with conserved subdomains called palm, fingers, and thumb1,2. Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs, and magnesium ions to facilitate nucleotide condensation1. For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism3. The Influenza A virus RdRp in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication4. To understand how the Influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation, and single-molecule FRET assays. Our data shows that this β-hairpin is essential for terminal initiation during replication, but auxiliary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of Influenza A virus RNA synthesis and identifies the initiation platform of viral replication. PMID:27274864

  13. Towards a Coronavirus-Based HIV Multigene Vaccine

    PubMed Central

    Eriksson, Klara K.; Makia, Divine; Maier, Reinhard; Ludewig, Burkhard; Thiel, Volker

    2006-01-01

    Human immunodeficiency virus (HIV) infection represents one of the major health threats in the developing world. The costly treatment of infected individuals with multiple highly efficient anti-HIV drugs is only affordable in industrialized countries. Thus, an efficient vaccination strategy is required to prevent the further spread of the infection. The molecular biology of coronaviruses and particular features of the human coronavirus 229E (HCoV 229E) indicate that HCoV 229E-based vaccine vectors can become a new class of highly efficient vaccines. First, the receptor of HCoV 229E, human aminopeptidase N (hAPN or CD13) is expressed mainly on human dendritic cells (DCs) and macrophages indicating that targeting of HCoV 229E-based vectors to professional antigen presenting cells can be achieved by receptor-mediated transduction. Second, HCoV 229E structural genes can be replaced by multiple transcriptional units encoding various antigens. These virus-like particles (VLPs) containing HCoV 229E-based vector RNA have the ability to transduce human DCs and to mediate heterologous gene expression in these cells. Finally, coronavirus infections are associated with mainly respiratory and enteric diseases, and natural transmission of coronaviruses occurs via mucosal surfaces. In humans, HCoV 229E causes common cold by infecting the upper respiratory tract. HCoV 229E infections are mainly encountered in children and re-infection occurs frequently in adults. It is thus most likely that pre-existing immunity against HCoV 229E will not significantly impact on the vaccination efficiency if HCoV 229E-based vectors are used in humans. PMID:17162377

  14. Reformation of nucleolus-like bodies in the absence of postmitotic RNA synthesis.

    PubMed

    Stevens, A R; Prescott, D M

    1971-03-01

    The dependence of nucleolar reformation on RNA synthesis that resumes in late anaphase or early telophase has been investigated in synchronously dividing Amoeba proteus. RNA synthesis was completely inhibited throughout all stages of mitosis and the early hours of interphase with high concentrations of actinomycin D. In such cells, nucleolus-like bodies that bind azure B and pyronin were apparent in the reformed nuclei. The bodies appear as dense, fibrous masses with loosely associated, finely fibrillar material. There are no characteristic granular regions in the reformed structures. It is suggested that the bodies probably represent mainly nucleolar protein and residual RNA which can bring about the reorganization of nucleoli in the absence of postmitotic RNA synthesis.

  15. Induction of vitellogenin synthesis by estrogen in avian liver: relationship between level of vitellogenin mRNA and vitellogenin synthesis.

    PubMed Central

    Mullinix, K P; Wetekam, W; Deeley, R G; Gordon, J I; Meyers, M; Kent, K A; Goldberger, R F

    1976-01-01

    We have investigated the estrogen-mediated induction of vitellogenin synthesis in rooster liver. We compared the concentrations of vitellogenin messenger RNA (mRNA) in the liver with the concentrations of vitellogenin in the sera of roosters that had recieved various treatments with estrogen. We found no vitellogenin mRNA in the livers of the unstimulated roosters. An initial injection of estrogen was attended by de novo synthesis of vitellogenin mRNA in the liver and accumulation of vitellogenin in the serum. When vitellogenin was no longer present in the serum or liver (the "post-estrogen-serum-negative" state), the liver was found to contain appreciable amounts of vitellogenin mRNA. This mRNA was of the same size as that found in the liver of the rooster actively synthesizing vitellogenin in response to estrogen. Whereas vitellogenin mRNA was in large polysomes in the livers of the roosters actively synthesizing vitellogenin, the vitellogenin mRNA in the liver of the post-estrogen-serum-negative rooster was not associated with polysomes. The possible relevance of these findings to the fact that the rooster responds differently to a primary stimulation with estrogen than to subsequent stimulations is discussed. PMID:1064017

  16. Control of rRNA Synthesis in Escherichia coli: a Systems Biology Approach†

    PubMed Central

    Dennis, Patrick P.; Ehrenberg, Mans; Bremer, Hans

    2004-01-01

    The first part of this review contains an overview of the various contributions and models relating to the control of rRNA synthesis reported over the last 45 years. The second part describes a systems biology approach to identify the factors and effectors that control the interactions between RNA polymerase and rRNA (rrn) promoters of Escherichia coli bacteria during exponential growth in different media. This analysis is based on measurements of absolute rrn promoter activities as transcripts per minute per promoter in bacterial strains either deficient or proficient in the synthesis of the factor Fis and/or the effector ppGpp. These absolute promoter activities are evaluated in terms of rrn promoter strength (Vmax/Km) and free RNA polymerase concentrations. Three major conclusions emerge from this evaluation. First, the rrn promoters are not saturated with RNA polymerase. As a consequence, changes in the concentration of free RNA polymerase contribute to changes in rrn promoter activities. Second, rrn P2 promoter strength is not specifically regulated during exponential growth at different rates; its activity changes only when the concentration of free RNA polymerase changes. Third, the effector ppGpp reduces the strength of the rrn P1 promoter both directly and indirectly by reducing synthesis of the stimulating factor Fis. This control of rrn P1 promoter strength forms part of a larger feedback loop that adjusts the synthesis of ribosomes to the availability of amino acids via amino acid-dependent control of ppGpp accumulation. PMID:15590778

  17. Coronaviruses in poultry and other birds.

    PubMed

    Cavanagh, Dave

    2005-12-01

    The number of avian species in which coronaviruses have been detected has doubled in the past couple of years. While the coronaviruses in these species have all been in coronavirus Group 3, as for the better known coronaviruses of the domestic fowl (infectious bronchitis virus [IBV], in Gallus gallus), turkey (Meleagris gallopavo) and pheasant (Phasianus colchicus), there is experimental evidence to suggest that birds are not limited to infection with Group 3 coronaviruses. In China coronaviruses have been isolated from peafowl (Pavo), guinea fowl (Numida meleagris; also isolated in Brazil), partridge (Alectoris) and also from a non-gallinaceous bird, the teal (Anas), all of which were being reared in the vicinity of domestic fowl. These viruses were closely related in genome organization and in gene sequences to IBV. Indeed, gene sequencing and experimental infection of chickens indicated that the peafowl isolate was the H120 IB vaccine strain, while the teal isolate was possibly a field strain of a nephropathogenic IBV. Thus the host range of IBV does extend beyond the chicken. Most recently, Group 3 coronaviruses have been detected in greylag goose (Anser anser), mallard duck (Anas platyrhynchos) and pigeon (Columbia livia). It is clear from the partial genome sequencing of these viruses that they are not IBV, as they have two additional small genes near the 3' end of the genome. Twenty years ago a coronavirus was isolated after inoculation of mice with tissue from the coastal shearwater (Puffinus puffinus). While it is not certain whether the virus was actually from the shearwater or from the mice, recent experiments have shown that bovine coronavirus (a Group 2 coronavirus) can infect and also cause enteric disease in turkeys. Experiments with some Group 1 coronaviruses (all from mammals, to date) have shown that they are not limited to replicating or causing disease in a single host. SARS-coronavirus has a wide host range. Clearly there is the potential for

  18. Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

    PubMed Central

    Cavanagh, Amy T.; Sperger, Jamie M.; Wassarman, Karen M.

    2012-01-01

    6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ70 in Escherichia coli, EσA in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ70 during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ70 to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from EσA prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well. PMID:22102588

  19. Purified coronavirus spike protein nanoparticles induce coronavirus neutralizing antibodies in mice.

    PubMed

    Coleman, Christopher M; Liu, Ye V; Mu, Haiyan; Taylor, Justin K; Massare, Michael; Flyer, David C; Glenn, Gregory M; Smith, Gale E; Frieman, Matthew B

    2014-05-30

    Development of vaccination strategies for emerging pathogens are particularly challenging because of the sudden nature of their emergence and the long process needed for traditional vaccine development. Therefore, there is a need for development of a rapid method of vaccine development that can respond to emerging pathogens in a short time frame. The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003 and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in late 2012 demonstrate the importance of coronaviruses as emerging pathogens. The spike glycoproteins of coronaviruses reside on the surface of the virion and are responsible for virus entry. The spike glycoprotein is the major immunodominant antigen of coronaviruses and has proven to be an excellent target for vaccine designs that seek to block coronavirus entry and promote antibody targeting of infected cells. Vaccination strategies for coronaviruses have involved live attenuated virus, recombinant viruses, non-replicative virus-like particles expressing coronavirus proteins or DNA plasmids expressing coronavirus genes. None of these strategies has progressed to an approved human coronavirus vaccine in the ten years since SARS-CoV emerged. Here we describe a novel method for generating MERS-CoV and SARS-CoV full-length spike nanoparticles, which in combination with adjuvants are able to produce high titer antibodies in mice.

  20. The murine norovirus core subgenomic RNA promoter consists of a stable stem-loop that can direct accurate initiation of RNA synthesis.

    PubMed

    Yunus, Muhammad Amir; Lin, Xiaoyan; Bailey, Dalan; Karakasiliotis, Ioannis; Chaudhry, Yasmin; Vashist, Surender; Zhang, Guo; Thorne, Lucy; Kao, C Cheng; Goodfellow, Ian

    2015-01-15

    All members of the Caliciviridae family of viruses produce a subgenomic RNA during infection. The subgenomic RNA typically encodes only the major and minor capsid proteins, but in murine norovirus (MNV), the subgenomic RNA also encodes the VF1 protein, which functions to suppress host innate immune responses. To date, the mechanism of norovirus subgenomic RNA synthesis has not been characterized. We have previously described the presence of an evolutionarily conserved RNA stem-loop structure on the negative-sense RNA, the complementary sequence of which codes for the viral RNA-dependent RNA polymerase (NS7). The conserved stem-loop is positioned 6 nucleotides 3' of the start site of the subgenomic RNA in all caliciviruses. We demonstrate that the conserved stem-loop is essential for MNV viability. Mutant MNV RNAs with substitutions in the stem-loop replicated poorly until they accumulated mutations that revert to restore the stem-loop sequence and/or structure. The stem-loop sequence functions in a noncoding context, as it was possible to restore the replication of an MNV mutant by introducing an additional copy of the stem-loop between the NS7- and VP1-coding regions. Finally, in vitro biochemical data suggest that the stem-loop sequence is sufficient for the initiation of viral RNA synthesis by the recombinant MNV RNA-dependent RNA polymerase, confirming that the stem-loop forms the core of the norovirus subgenomic promoter. Noroviruses are a significant cause of viral gastroenteritis, and it is important to understand the mechanism of norovirus RNA synthesis. Here we describe the identification of an RNA stem-loop structure that functions as the core of the norovirus subgenomic RNA promoter in cells and in vitro. This work provides new insights into the molecular mechanisms of norovirus RNA synthesis and the sequences that determine the recognition of viral RNA by the RNA-dependent RNA polymerase. Copyright © 2015, American Society for Microbiology. All Rights

  1. Activation of ribosomal and messenger RNA synthesis in excised Jerusalem artichoke tuber slices.

    PubMed

    Byrne, H; Setterfield, G

    1977-01-01

    RNA synthesis was studied in Jerusalem artichoke (Helianthus tuberosus L.) tuber slices immediately following excision and during the early period of aging in water. Incorporation of [(3)H]adenosine into RNA was detected as early as 20 min after excision. Measurement of the specific activities of RNA (cpm/μg) and of ATP showed that RNA synthesis proceeded at a constant rate for the first several hours of aging and then increased moderately. [(3)H]adenosine was incorporated into polysomes throughout the aging period examined. Sucrose gradient fractionation of EDTA-dissociated polysomes showed that during the first 2 h of aging most of this incorporation was not into ribosome subunits but into presumed mRNA. Autoradiographic analysis of [(3)H]adenosine labelled nuclei showed that this was caused, at least in part, by a delay in the onset of rRNA synthesis synthesized during this time chromatographed as poly(A)-RNA on oligo(dT)-cellulose, indicating that a large part of the mRNA was not polyadenylated.

  2. Depletion of ribosomal protein S19 causes a reduction of rRNA synthesis

    PubMed Central

    Juli, Giada; Gismondi, Angelo; Monteleone, Valentina; Caldarola, Sara; Iadevaia, Valentina; Aspesi, Anna; Dianzani, Irma; Proud, Christopher G.; Loreni, Fabrizio

    2016-01-01

    Ribosome biogenesis plays key roles in cell growth by providing increased capacity for protein synthesis. It requires coordinated production of ribosomal proteins (RP) and ribosomal RNA (rRNA), including the processing of the latter. Here, we show that, the depletion of RPS19 causes a reduction of rRNA synthesis in cell lines of both erythroid and non-erythroid origin. A similar effect is observed upon depletion of RPS6 or RPL11. The deficiency of RPS19 does not alter the stability of rRNA, but instead leads to an inhibition of RNA Polymerase I (Pol I) activity. In fact, results of nuclear run-on assays and ChIP experiments show that association of Pol I with the rRNA gene is reduced in RPS19-depleted cells. The phosphorylation of three known regulators of Pol I, CDK2, AKT and AMPK, is altered during ribosomal stress and could be involved in the observed downregulation. Finally, RNA from patients with Diamond Blackfan Anemia (DBA), shows, on average, a lower level of 47S precursor. This indicates that inhibition of rRNA synthesis could be one of the molecular alterations at the basis of DBA. PMID:27734913

  3. Gene 5 of the Avian Coronavirus Infectious Bronchitis Virus Is Not Essential for Replication

    PubMed Central

    Casais, Rosa; Davies, Marc; Cavanagh, David; Britton, Paul

    2005-01-01

    The avian coronavirus Infectious bronchitis virus (IBV), like other coronaviruses, expresses several small nonstructural (ns) proteins in addition to those from gene 1 (replicase) and the structural proteins. These coronavirus ns genes differ both in number and in amino acid similarity between the coronavirus groups but show some concordance within a group or subgroup. The functions and requirements of the small ns gene products remain to be elucidated. With the advent of reverse genetics for coronaviruses, the first steps in elucidating their role can be investigated. We have used our reverse genetics system for IBV (R. Casais, V. Thiel, S. G. Siddell, D. Cavanagh, and P. Britton, J. Virol. 75:12359-12369, 2001) to investigate the requirement of IBV gene 5 for replication in vivo, in ovo, and ex vivo. We produced a series of recombinant viruses, with an isogenic background, in which complete expression of gene 5 products was prevented by the inactivation of gene 5 following scrambling of the transcription-associated sequence, thereby preventing the expression of IBV subgenomic mRNA 5, or scrambling either separately or together of the translation initiation codons for the two gene 5 products. As all of the recombinant viruses replicated very similarly to the wild-type virus, Beau-R, we conclude that the IBV gene 5 products are not essential for IBV replication per se and that they are accessory proteins. PMID:15956552

  4. Structure of the C-terminal domain of nsp4 from feline coronavirus

    SciTech Connect

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  5. Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China.

    PubMed

    Zhuang, Qing-Ye; Wang, Kai-Cheng; Liu, Shuo; Hou, Guang-Yu; Jiang, Wen-Ming; Wang, Su-Chun; Li, Jin-Ping; Yu, Jian-Min; Chen, Ji-Ming

    2015-01-01

    The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV), and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV). The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks.

  6. Stability of bovine coronavirus on lettuce surfaces under household refrigeration conditions.

    PubMed

    Mullis, Lisa; Saif, Linda J; Zhang, Yongbin; Zhang, Xuming; Azevedo, Marli S P

    2012-05-01

    Fecal suspensions with an aerosol route of transmission were responsible for a cluster of severe acute respiratory syndrome (SARS) cases in 2003 in Hong Kong. Based on that event, the World Health Organization recommended that research be implemented to define modes of transmission of SARS coronavirus through sewage, feces, food and water. Environmental studies have shown that animal coronaviruses remain infectious in water and sewage for up to a year depending on the temperature and humidity. In this study, we examined coronavirus stability on lettuce surfaces. A cell culture adapted bovine coronavirus, diluted in growth media or in bovine fecal suspensions to simulate fecal contamination was used to spike romaine lettuce. qRT-PCR detected viral RNA copy number ranging from 6.6 × 10⁴ to 1.7 × 10⁶ throughout the experimental period of 30 days. Whereas infectious viruses were detected for at least 14 days, the amount of infectious virus varied, depending upon the diluent used for spiking the lettuce. UV and confocal microscopic observation indicated attachment of residual labeled virions to the lettuce surface after the elution procedure, suggesting that rates of inactivation or detection of the virus may be underestimated. Thus, it is possible that contaminated vegetables may be potential vehicles for coronavirus zoonotic transmission to humans.

  7. Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China

    PubMed Central

    Liu, Shuo; Hou, Guang-Yu; Jiang, Wen-Ming; Wang, Su-Chun; Li, Jin-Ping; Yu, Jian-Min; Chen, Ji-Ming

    2015-01-01

    The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV), and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV). The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks. PMID:26053682

  8. Synthesis of ribosomal RNA during the mitotic cycle on the slime mould Physarum polycephalum.

    PubMed

    Hall, L; Turnock, G

    1976-03-01

    1. An isotope dilution technique has been used to analyze the synthesis of metabolically stable nucleic acids during the mitotic cycle in surface plasmodia of the slime mould Physarum polycephalum. Microplasmodia that had been labelled with [3H]uridine were used to prepare a surface culture, after a period of growth long enough to ensure that radioactivity was present only in tRNA, rRNA and DNA. The synthesis of rRNA or nuclear DNA during the growth of the surface plasmodium was then followed by measuring the specific activity of the nucleic acid. 2. Synthesis of rRNA during the mitotic cycle shows the following characteristics: (a) it is low during the immediate period of nuclear division, (b) synthesis is then continuous throughout interphase and (c)the rate of synthesis increases 5-6-fold between the beginning and end of interphase. These results are discussed in relation to the pattern of replication of the genes for rRNA. 3. Approximately 80% of the nuclear DNA replicates during the first 90 min of the mitotic cycle; completion of replication, however, occupies the remainder of interphase.

  9. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV.

    PubMed

    Tashjian, Tommy F; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M; Godoy, Veronica G

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB's fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability.

  10. RNA Primer Extension Hinders DNA Synthesis by Escherichia coli Mutagenic DNA Polymerase IV

    PubMed Central

    Tashjian, Tommy F.; Lin, Ida; Belt, Verena; Cafarelli, Tiziana M.; Godoy, Veronica G.

    2017-01-01

    In Escherichia coli the highly conserved DNA damage regulated dinB gene encodes DNA Polymerase IV (DinB), an error prone specialized DNA polymerase with a central role in stress-induced mutagenesis. Since DinB is the DNA polymerase with the highest intracellular concentrations upon induction of the SOS response, further regulation must exist to maintain genomic stability. Remarkably, we find that DinB DNA synthesis is inherently poor when using an RNA primer compared to a DNA primer, while high fidelity DNA polymerases are known to have no primer preference. Moreover, we show that the poor DNA synthesis from an RNA primer is conserved in DNA polymerase Kappa, the human DinB homolog. The activity of DinB is modulated by interactions with several other proteins, one of which is the equally evolutionarily conserved recombinase RecA. This interaction is known to positively affect DinB’s fidelity on damaged templates. We find that upon interaction with RecA, DinB shows a significant reduction in DNA synthesis when using an RNA primer. Furthermore, with DinB or DinB:RecA a robust pause, sequence and lesion independent, occurs only when RNA is used as a primer. The robust pause is likely to result in abortive DNA synthesis when RNA is the primer. These data suggest a novel mechanism to prevent DinB synthesis when it is not needed despite its high concentrations, thus protecting genome stability. PMID:28298904

  11. [The first steps of chlorophyll synthesis: RNA involvement and regulation]. Progress report, January 1990--June 1992

    SciTech Connect

    Soell, D.

    1992-12-31

    Glu-tRNA{sup Glu} is synthesized from glutamate and tRNA{sup Glu} by glutamyl-tRNA synthetase (GluRS). Recent work has demonstrated that Glu-tRNA{sup Glu} has dual functions and is a precursor for protein and 5-aminolevulinate (ALA) synthesis. Current data does not provide compelling evidence for the notion that GluRS is regulated by chlorophyll precursors or in concert with the other enzymes of ALA synthesis. We have redefined the C5-pathway as a two-step route to ALA starting with Glu-tRNA{sup Glu}. Only two enzymes, Glu-tRNA reductase (GluTR) and GSA-2,1-amino-mutase (GSA-AM), are specifically involved in ALA synthesis. We have purified these enzymatic activities from Chlamydomonas and demonstrated that the two purified proteins in the presence of their cofactors NADPH and pyridoxal phosphate are sufficient for the in vitro Glu-tRNA {yields} ALA conversion. We have cloned the genes encoding GluTR. The sequences of the GluTR proteins deduced from these genes share highly conserved regions with those of bacterial origin. We havealso cloned and analyzed the gene encoding GSA-AM from Arabidopsis. As in Salmonella typhimurium, there are indications of the existence of an additional pathway for ALA formation in E. coli. To shed light on the recognition of the single tRNA{sup Glu} by the chloroplast enzymes GluTR, GluRS we characterized a chlorophyll-deficient mutant of Euglena having tRNA{sup Glu} with a point mutation in the T{Psi}C-loop. The altered tRNA supports protein but not ALA synthesis.

  12. Rotavirus Replication: Plus-Sense Templates for Double-Stranded RNA Synthesis Are Made in Viroplasms

    PubMed Central

    Silvestri, Lynn S.; Taraporewala, Zenobia F.; Patton, John T.

    2004-01-01

    Rotavirus plus-strand RNAs not only direct protein synthesis but also serve as templates for the synthesis of the segmented double-stranded RNA (dsRNA) genome. In this study, we identified short-interfering RNAs (siRNAs) for viral genes 5, 8, and 9 that suppressed the expression of NSP1, a nonessential protein; NSP2, a component of viral replication factories (viroplasms); and VP7, an outer capsid protein, respectively. The loss of NSP2 expression inhibited viroplasm formation, genome replication, virion assembly, and synthesis of the other viral proteins. In contrast, the loss of VP7 expression had no effect on genome replication; instead, it inhibited only outer-capsid morphogenesis. Similarly, neither genome replication nor any other event of the viral life cycle was affected by the loss of NSP1. The data indicate that plus-strand RNAs templating dsRNA synthesis within viroplasms are not susceptible to siRNA-induced RNase degradation. In contrast, plus-strand RNAs templating protein synthesis in the cytosol are susceptible to degradation and thus are not the likely source of plus-strand RNAs for dsRNA synthesis in viroplasms. Indeed, immunofluorescence analysis of bromouridine (BrU)-labeled RNA made in infected cells provided evidence that plus-strand RNAs are synthesized within viroplasms. Furthermore, transfection of BrU-labeled viral plus-strand RNA into infected cells suggested that plus-strand RNAs introduced into the cytosol do not localize to viroplasms. From these results, we propose that plus-strand RNAs synthesized within viroplasms are the primary source of templates for genome replication and that trafficking pathways do not exist within the cytosol that transport plus-strand RNAs to viroplasms. The lack of such pathways confounds the development of reverse genetics systems for rotavirus. PMID:15220450

  13. Synthesis of capped RNA using a DMT group as a purification handle.

    PubMed

    Veliath, Elizabeth; Gaffney, Barbara L; Jones, Roger A

    2014-01-01

    We report a new method for synthesis of capped RNA or 2'-OMe RNA that uses a N(2-)4,4'-dimethoxytrityl (DMT) group as a lipophilic purification handle to allow convenient isolation and purification of the capped RNA. The DMT group is easily removed under mild conditions without degradation of the cap. We have used this approach to prepare capped 10- and 20-mers. This method is compatible with the many condensation reactions that have been reported for preparation of capped RNA or cap analogues.

  14. Overview of methods in RNA nanotechnology: synthesis, purification, and characterization of RNA nanoparticles.

    PubMed

    Haque, Farzin; Guo, Peixuan

    2015-01-01

    RNA nanotechnology encompasses the use of RNA as a construction material to build homogeneous nanostructures by bottom-up self-assembly with defined size, structure, and stoichiometry; this pioneering concept demonstrated in 1998 (Guo et al., Molecular Cell 2:149-155, 1998; featured in Cell) has emerged as a new field that also involves materials engineering and synthetic structural biology (Guo, Nature Nanotechnology 5:833-842, 2010). The field of RNA nanotechnology has skyrocketed over the last few years, as evidenced by the burst of publications in prominent journals on RNA nanostructures and their applications in nanomedicine and nanotechnology. Rapid advances in RNA chemistry, RNA biophysics, and RNA biology have created new opportunities for translating basic science into clinical practice. RNA nanotechnology holds considerable promise in this regard. Increased evidence also suggests that substantial part of the 98.5 % of human genome (Lander et al. Nature 409:860-921, 2001) that used to be called "junk DNA" actually codes for noncoding RNA. As we understand more on how RNA structures are related to function, we can fabricate synthetic RNA nanoparticles for the diagnosis and treatment of diseases. This chapter provides a brief overview of the field regarding the design, construction, purification, and characterization of RNA nanoparticles for diverse applications in nanotechnology and nanomedicince.

  15. Severe Acute Respiratory Syndrome-Coronavirus Papain-Like Novel Protease Inhibitors: Design, Synthesis, Protein-Ligand X-ray Structure and Biological Evaluation

    SciTech Connect

    Ghosh, Arun K.; Takayama, Jun; Rao, Kalapala Venkateswar; Ratia, Kiira; Chaudhuri, Rima; Mulhearn, Debbie C.; Lee, Hyun; Nichols, Daniel B.; Baliji, Surendranath; Baker, Susan C.; Johnson, Michael E.; Mesecar, Andrew D.

    2012-02-21

    The design, synthesis, X-ray crystal structure, molecular modeling, and biological evaluation of a series of new generation SARS-CoV PLpro inhibitors are described. A new lead compound 3 (6577871) was identified via high-throughput screening of a diverse chemical library. Subsequently, we carried out lead optimization and structure-activity studies to provide a series of improved inhibitors that show potent PLpro inhibition and antiviral activity against SARS-CoV infected Vero E6 cells. Interestingly, the (S)-Me inhibitor 15h (enzyme IC{sub 50} = 0.56 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) and the corresponding (R)-Me 15g (IC{sub 50} = 0.32 {mu}M; antiviral EC{sub 50} = 9.1 {mu}M) are the most potent compounds in this series, with nearly equivalent enzymatic inhibition and antiviral activity. A protein-ligand X-ray structure of 15g-bound SARS-CoV PLpro and a corresponding model of 15h docked to PLpro provide intriguing molecular insight into the ligand-binding site interactions.

  16. Synthesis of RNA containing 5-hydroxymethyl-, 5-formyl-, and 5-carboxycytidine.

    PubMed

    Michaelides, Iacovos N; Tago, Nobuhiro; Viverge, Bastien; Carell, Thomas

    2017-09-14

    5-Hydroxymethyl-, 5-formyl-, and 5-carboxycytidine are new epigenetic bases (hmdC, fdC, cadC) that were recently discovered in the DNA of higher eukaryotes. The same bases (hmC, fC and caC) have now also been detected in mammalian RNA with a high abundance in mRNA. While for the DNA bases phosphoramidites (PAs) are available that allow the synthesis of xdC-containing oligonucleotides for deeper biological studies, the corresponding RNA PAs for fC and caC have not yet been disclosed. Here we report novel RNA PAs for hmC, fC and caC that can be used in routine RNA synthesis. The new building blocks are compatible with the canonical PAs and also with themselves, which enables even the synthesis of RNA strands containing all three of these bases. The study will pave the way for detailed physical, biochemical and biological studies to unravel the function of these non-canonical modifications in RNA. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Novel phenotype of RNA synthesis expressed by vesicular stomatitis virus isolated from persistent infection.

    PubMed Central

    Frey, T K; Youngner, J S

    1982-01-01

    Vesicular stomatitis virus (VSV) stocks isolated from two persistently infected mouse L-cell lines (designated VSV-PI stocks) express an altered phenotype of RNA synthesis. This phenotype is different from the RNA synthesis phenotype expressed by the viruses used to initiate the persistently infected lines, wild-type VSV and VSV ts-0-23 (a group III, ts-, RNA+ mutant). At 34 and 37 degrees C in L cells productively infected with VSV-PI stocks derived from the two cell lines, transcription of virus mRNA was significantly reduced, whereas replication of the 40S genomic RNA species was enhanced compared with wild-type VSV or ts-0-23. At 34 and 37 degrees C, both VSV-PI stocks replicated with equal or greater efficiency than wild-type VSV; 37 degrees C was the temperature at which the persistently infected cultures were maintained. At 40 degrees C, both VSV-PI stocks were temperature sensitive, and clonal VSV-PI isolates from both cell lines belong to complementation group I (RNA-). Standard ts- mutants (derived by mutagenesis of wild-type VSV) belonging to RNA- complementation groups I, II, and IV do not express the VSV-PI RNA synthesis phenotype at the permissive temperature, making this phenotype distinctive to persistent infection. Since the two VSV-PI populations from persistently infected cell lines initiated with different viruses both evolved this unique phenotype of RNA synthesis, the expression of this phenotype may play an important role in the maintenance of persistence. Images PMID:6292483

  18. Reliable semi-synthesis of hydrolysis-resistant 3'-peptidyl-tRNA conjugates containing genuine tRNA modifications.

    PubMed

    Graber, Dagmar; Moroder, Holger; Steger, Jessica; Trappl, Krista; Polacek, Norbert; Micura, Ronald

    2010-10-01

    The 3'-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3'-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3'-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5'-fragments carrying the modifications. After dephosphorylation of the 2',3'-cyclophosphate moieties from these fragments, they are ligated to the respective 3'-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3'-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner.

  19. A fast click-slow release strategy towards the HPLC-free synthesis of RNA.

    PubMed

    Agustin, E; Asare Okai, P N; Khan, I; Miller, M R; Wang, R; Sheng, J; Royzen, M

    2016-01-25

    A general strategy for purification of oligonucleotides synthesized by solid phase synthesis is described. It is based on a recently developed concept involving a bio-orthogonal inverse electron demand Diels-Alder reaction between trans-cyclooctene and tetrazine, termed 'click-to-release'. The strategy has been applied towards the synthesis and purification of a model hairpin RNA strand, as well as a 34 nt long aptamer.

  20. Inhibition of RNA synthesis by bradykinin involves both the B1 and B2 receptor subtypes.

    PubMed

    Yau, L; Pinsk, M; Zahradka, P

    1996-04-01

    The efficacy of angiotensin converting enzyme inhibitors in the treatment of heart disease is due in part to the accumulation of bradykinin (BK). Since BK can exert its effect by influencing cell proliferation, we chose to study the effect of BK on the growth of A10 vascular smooth muscle cells. Ligand binding studies to determine which BK receptor subtypes are present on A10 cells showed that both B1 and B2 receptors were present in approximately equal numbers. Examination of RNA synthesis demonstrated that BK inhibits uridine incorporation in a dose-dependent manner. This decrease in RNA synthesis was blocked by both B1 and B2 receptor antagonists, as well as by addition of indomethacin, a cyclooxygenase inhibitor. The latter result suggested that prostaglandins mediate the biological actions of BK. Consequently, we examined the direct effect of two prostaglandins, PGE2 and PGI2 (prostacyclin), on A10 cells. PGE2 caused a decrease in RNA synthesis, thus mimicking the effect of BK, while PGI2 did not. Therefore, the inhibition of RNA synthesis in A10 vascular smooth muscle cells by BK requires both B1 and B2 receptor subtypes and this action of BK is apparently mediated by de novo synthesis of prostaglandins.

  1. Reformation of functional liver polyribosomes from ribosome monomers in the absence of RNA synthesis.

    PubMed

    Stewart, G A; Farber, E

    1967-07-07

    The administration to rats of the ethyl analog of methionine, ethionine, results in the rapid decrease in the hepatic concentration of adenosine triphosphate followed by an extensive disaggregation of polysomes to ribosome monomers and a concomitant inhibition of protein synthesis. These effects are readily reversed by the injection of methionine or precursors of adenine nucleotides such as adenine. The reformation of liver polyribosomes in such animals following the administration of adenine plus methionine was found to occur under conditions in which new RNA synthesis was markedly inhibited. Free messenger RNA without attached ribosomes must be capable of remaining functionally active in the liver cytoplasm for many hours.

  2. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    PubMed

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan

    2014-08-01

    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  3. RT-PCR detection of avian coronaviruses of galliform birds (chicken, turkey, pheasant) and in a parrot.

    PubMed

    Culver, Francesca Anne; Britton, Paul; Cavanagh, Dave

    2008-01-01

    Of the many primer combinations that we have investigated for the detection of avian coronaviruses, two have worked better than any of the others: they worked with the largest number of strains/samples of a given coronavirus and the most species of avian coronavirus, and they also produced the most sensitive detection tests. The primer combinations were: oligonucleotide pair 2Bp/4Bm, which is in a region of gene 1 that is moderately conserved among all species of coronavirus (1); and UTR11-/UTR41+, which are in a highly conserved part of the 3' untranslated region of avian coronaviruses related to infectious bronchitis virus (2). The gene 1 primer pair enabled the detection of a new coronavirus in a green-checked Amazon parrot (Amazon viridigenalis Cassin). In this chapter we describe the use of these oligonucleotides in a one-step (single-tube) RT-PCR, and describe the procedure that we used to extract RNA from turkey feces.

  4. Synthesis of Specifically Modified Oligonucleotides for Application in Structural and Functional Analysis of RNA

    PubMed Central

    Rublack, Nico; Nguyen, Hien; Appel, Bettina; Springstubbe, Danilo; Strohbach, Denise; Müller, Sabine

    2011-01-01

    Nowadays, RNA synthesis has become an essential tool not only in the field of molecular biology and medicine, but also in areas like molecular diagnostics and material sciences. Beyond synthetic RNAs for antisense, aptamer, ribozyme, and siRNA technologies, oligoribonucleotides carrying site-specific modifications for structure and function studies are needed. This often requires labeling of the RNA with a suitable spectroscopic reporter group. Herein, we describe the synthesis of functionalized monomer building blocks that upon incorporation in RNA allow for selective reaction with a specific reporter or functional entity. In particular, we report on the synthesis of 5′-O-dimethoxytrityl-2′-O-tert-butyldimethylsilyl protected 3′-O-phosphoramidites of nucleosides that carry amino linkers of different lengths and flexibility at the heterocyclic base, their incorporation in a variety of RNAs, and postsynthetic conjugation with fluorescent dyes and nitroxide spin labels. Further, we show the synthesis of a flavine mononucleotide-N-hydroxy-succinimidyl ester and its conjugation to amino functionalized RNA. PMID:22013508

  5. Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl-uridine-Modified RNA

    PubMed Central

    Riml, Christian; Micura, Ronald

    2016-01-01

    We report on the syntheses of 5-hydroxymethyl-uridine [5hm(rU)] and -cytidine [5hm(rC)] phosphoramidites and their incorporation into RNA by solid-phase synthesis. Deprotection of the oligonucleotides is accomplished in a straightforward manner using standard conditions, confirming the appropriateness of the acetyl protection used for the pseudobenzylic alcohol moieties. The approach provides robust access to 5hm(rC/U)-modified RNAs that await applications in pull-down experiments to identify potential modification enzymes. They will also serve as synthetic probes for the development of high-throughput-sequencing methods in native RNAs. 1Introduction2Protection Strategies Reported for the Synthesis of 5hm(dC)-Modified DNA3Synthesis of 5-Hydroxymethylpyrimidine-Modified RNA3.1Synthesis of 5hm(rC) Phosphoramidite3.2Synthesis of 5hm(rU) Phosphoramidite3.3Synthesis of 5hm(rC)- and 5hm(rU)-Modified RNA4Conclusions PMID:27413246

  6. Influenza virus RNA polymerase: insights into the mechanisms of viral RNA synthesis

    PubMed Central

    te Velthuis, Aartjan J.W.; Fodor, Ervin

    2016-01-01

    The genome of influenza viruses consists of multiple segments of single stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, forming viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, how it carries out transcription and replication, and how its activities are modulated by viral and host factors. Furthermore, we discuss how advances in our understanding of polymerase function could help identifying new antiviral targets. PMID:27396566

  7. The emerging complexity of the tRNA world: mammalian tRNAs beyond protein synthesis.

    PubMed

    Schimmel, Paul

    2017-09-06

    The discovery of the genetic code and tRNAs as decoders of the code transformed life science. However, after establishing the role of tRNAs in protein synthesis, the field moved to other parts of the RNA world. Now, tRNA research is blooming again, with demonstration of the involvement of tRNAs in various other pathways beyond translation and in adapting translation to environmental cues. These roles are linked to the presence of tRNA sequence variants known as isoacceptors and isodecoders, various tRNA base modifications, the versatility of protein binding partners and tRNA fragmentation events, all of which collectively create an incalculable complexity. This complexity provides a vast repertoire of tRNA species that can serve various functions in cellular homeostasis and in adaptation of cellular functions to changing environments, and it likely arose from the fundamental role of RNAs in early evolution.

  8. Structural Characterization of Human Coronavirus NL63 N Protein.

    PubMed

    Szelazek, Bozena; Kabala, Wojciech; Kus, Krzysztof; Zdzalik, Michal; Twarda-Clapa, Aleksandra; Golik, Przemyslaw; Burmistrz, Michal; Florek, Dominik; Wladyka, Benedykt; Pyrc, Krzysztof; Dubin, Grzegorz

    2017-06-01

    Coronaviruses are responsible for upper and lower respiratory tract infections in humans. It is estimated that 1 to 10% of the population suffers annually from cold-like symptoms related to infection with human coronavirus NL63 (HCoV-NL63), an alphacoronavirus. The nucleocapsid (N) protein, the major structural component of the capsid, facilitates RNA packing, links the capsid to the envelope, and is also involved in multiple other processes, including viral replication and evasion of the immune system. Although the role of N protein in viral replication is relatively well described, no structural data are currently available regarding the N proteins of alphacoronaviruses. Moreover, our understanding of the mechanisms of RNA binding and nucleocapsid formation remains incomplete. In this study, we solved the crystal structures of the N- and C-terminal domains (NTD, residues 10 to 140, and CTD, residues 221 to 340, respectively) of the N protein of HCoV-NL63, both at a 1.5-Å resolution. Based on our structure of NTD solved here, we proposed and experimentally evaluated a model of RNA binding. The structure of the CTD reveals the mode of N protein dimerization. Overall, this study expands our understanding of the initial steps of N protein-nucleic acid interaction and may facilitate future efforts to control the associated infections.IMPORTANCE Coronaviruses are responsible for the common cold and other respiratory tract infections in humans. According to multiple studies, 1 to 10% of the population is infected each year with HCoV-NL63. Viruses are relatively simple organisms composed of a few proteins and the nucleic acids that carry the information determining their composition. The nucleocapsid (N) protein studied in this work protects the nucleic acid from the environmental factors during virus transmission. This study investigated the structural arrangement of N protein, explaining the first steps of its interaction with nucleic acid at the initial stages of

  9. MicroRNA and AU-rich element regulation of prostaglandin synthesis

    PubMed Central

    Moore, Ashleigh E.; Young, Lisa E.

    2012-01-01

    Many liness of evidence demonstrate that prostaglandins play an important role in cancer, and enhanced synthesis of prostaglandin E2 (PGE2) is often observed in various human malignancies often associated with poor prognosis. PGE2 synthesis is initiated with the release of arachidonic acid by phospholipase enzymes, where it is then converted into the intermediate prostaglandin prostaglandin H2 (PGH2) by members of the cyclooxygenase family. The synthesis of PGE2 from PGH2 is facilitated by three different PGE synthases, and functional PGE2 can promote tumor growth by binding to four EP receptors to activate signaling pathways that control cell proliferation, migration, apoptosis, and angiogenesis. An integral method of controlling gene expression is by posttranscriptional mechanisms that regulate mRNA stability and protein translation. Messenger RNA regulatory elements typically reside within the 3′ untranslated region (3′UTR) of the transcript and play a critical role in targeting specific mRNAs for posttranscriptional regulation through micro-RNA (miRNA) binding and adenylate- and uridylate-rich element RNA-binding proteins. In this review, we highlight the current advances in our understanding of the impact these RNA sequence elements have upon regulating PGE2 levels. We also identify various RNA sequence elements consistently observed within the 3′UTRs of the genes involved in the PGE2 pathway, indicating these binding sites for miRNAs and RNA-binding proteins to be central regulators of PGE2 synthesis and function. These findings may provide a rationale for the development of new therapeutic approaches to control tumor growth and metastasis promoted by elevated PGE2 levels. PMID:22005950

  10. AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

    PubMed Central

    Ueda, Yuko; Ooshio, Ikumi; Fusamae, Yasuyuki; Kitae, Kaori; Kawaguchi, Megumi; Jingushi, Kentaro; Hase, Hiroaki; Harada, Kazuo; Hirata, Kazumasa; Tsujikawa, Kazutake

    2017-01-01

    The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion. PMID:28205560

  11. Lipid-assisted Synthesis of RNA-like Polymers from Mononucleotides

    NASA Astrophysics Data System (ADS)

    Rajamani, Sudha; Vlassov, Alexander; Benner, Seico; Coombs, Amy; Olasagasti, Felix; Deamer, David

    2008-02-01

    A fundamental problem in research on the origin of life is the process by which polymers capable of catalysis and replication were produced on the early Earth. Here we show that RNA-like polymers can be synthesized non-enzymatically from mononucleotides in lipid environments. The RNA-like polymers were initially identified by nanopore analysis, a technique with single molecule sensitivity. To our knowledge, this is the first such application of a nanopore instrument to detect RNA synthesis under simulated prebiotic conditions. The synthesis of the RNA-like polymers was confirmed by standard methods of enzymatic end labeling followed by gel electrophoresis. Chemical activation of the mononucleotides is not required. Instead, synthesis of phosphodiester bonds is driven by the chemical potential of fluctuating anhydrous and hydrated conditions, with heat providing activation energy during dehydration. In the final hydration step, the RNA-like polymer is encapsulated within lipid vesicles. This process provides a laboratory model of an early stage of evolution toward an RNA World.

  12. Alteration of PFK subunit protein, synthesis, and mRNA during neonatal brain development.

    PubMed

    Mhaskar, Y; Dunaway, G A

    1995-03-16

    During neonatal maturation of rat brain, a similar biphasic relationship exists between the previously reported pattern of glucose utilization and levels of each type of 6-phosphofructo-1-kinase (PFK) subunit protein, relative synthesis, and mRNA. The increasing amounts of each subunit isoform generally correlated with elevated protein synthesis which was promoted by greater amounts of each type of subunit mRNA. For each parameter, the early phase, 1 to 10 days after birth, was characterized by small increases, and the subsequent period from ten to thirty days postpartum was characterized by a much greater rate of increase. By 30 days after birth, adult values were observed. The apparent efficiency of translation of each type of PFK subunit mRNA in brain suggests that the M-type subunit mRNA is the most efficient and that the L-type subunit mRNA is the least. The greatest relative increases in subunit protein, mRNA, and synthesis were observed for the C-type subunit. Since enhanced translation apparently makes little, if any, contribution, a possible explanation of these phenomena could be increased transcription of the PFK genes. These neonatal changes could involve age-dependent alteration of methylation of the PFK gene promotor(s) and/or activity of effectors of the transcription of the PFK genes.

  13. Isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14, from domestic rabbits.

    PubMed

    Lau, Susanna K P; Woo, Patrick C Y; Yip, Cyril C Y; Fan, Rachel Y Y; Huang, Yi; Wang, Ming; Guo, Rongtong; Lam, Carol S F; Tsang, Alan K L; Lai, Kenneth K Y; Chan, Kwok-Hung; Che, Xiao-Yan; Zheng, Bo-Jian; Yuen, Kwok-Yung

    2012-05-01

    We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 10(8) copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5'-UCUAAAC-3'. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits.

  14. Isolation and Characterization of a Novel Betacoronavirus Subgroup A Coronavirus, Rabbit Coronavirus HKU14, from Domestic Rabbits

    PubMed Central

    Lau, Susanna K. P.; Woo, Patrick C. Y.; Yip, Cyril C. Y.; Fan, Rachel Y. Y.; Huang, Yi; Wang, Ming; Guo, Rongtong; Lam, Carol S. F.; Tsang, Alan K. L.; Lai, Kenneth K. Y.; Chan, Kwok-Hung; Che, Xiao-Yan; Zheng, Bo-Jian

    2012-01-01

    We describe the isolation and characterization of a novel Betacoronavirus subgroup A coronavirus, rabbit coronavirus HKU14 (RbCoV HKU14), from domestic rabbits. The virus was detected in 11 (8.1%) of 136 rabbit fecal samples by reverse transcriptase PCR (RT-PCR), with a viral load of up to 108 copies/ml. RbCoV HKU14 was able to replicate in HRT-18G and RK13 cells with cytopathic effects. Northern blotting confirmed the production of subgenomic mRNAs coding for the HE, S, NS5a, E, M, and N proteins. Subgenomic mRNA analysis revealed a transcription regulatory sequence, 5′-UCUAAAC-3′. Phylogenetic analysis showed that RbCoV HKU14 formed a distinct branch among Betacoronavirus subgroup A coronaviruses, being most closely related to but separate from the species Betacoronavirus 1. A comparison of the conserved replicase domains showed that RbCoV HKU14 possessed <90% amino acid identities to most members of Betacoronavirus 1 in ADP-ribose 1″-phosphatase (ADRP) and nidoviral uridylate-specific endoribonuclease (NendoU), indicating that RbCoV HKU14 should represent a separate species. RbCoV HKU14 also possessed genomic features distinct from those of other Betacoronavirus subgroup A coronaviruses, including a unique NS2a region with a variable number of small open reading frames (ORFs). Recombination analysis revealed possible recombination events during the evolution of RbCoV HKU14 and members of Betacoronavirus 1, which may have occurred during cross-species transmission. Molecular clock analysis using RNA-dependent RNA polymerase (RdRp) genes dated the most recent common ancestor of RbCoV HKU14 to around 2002, suggesting that this virus has emerged relatively recently. Antibody against RbCoV was detected in 20 (67%) of 30 rabbit sera tested by an N-protein-based Western blot assay, whereas neutralizing antibody was detected in 1 of these 20 rabbits. PMID:22398294

  15. Reduced secreted mu mRNA synthesis in selective IgM deficiency of Bloom's syndrome.

    PubMed Central

    Kondo, N; Ozawa, T; Kato, Y; Motoyoshi, F; Kasahara, K; Kameyama, T; Orii, T

    1992-01-01

    Serum IgM concentrations were low although serum IgG and IgA concentrations were normal in both our patients with Bloom's syndrome. Although the percentages of surface IgM-bearing cells were not reduced, the numbers of IgM-secreting cells were markedly reduced. The membrane-bound mu (microns) and secreted mu (microseconds) mRNAs are produced from transcripts of a single immunoglobulin mu gene by alternative RNA processing pathways. The control of microseconds mRNA synthesis depends on the addition of poly(A) to microseconds C-terminal segment. In both patients, mu mRNA was well detected but microseconds C-terminal mRNA was scarcely detected, suggesting that microns mRNA was well transcribed but microseconds mRNA was not. There was, at least, no mutation or deletion in the microseconds C-terminal coding sequence, the RNA splice site (GG/TAAAC) at the 5' end of microseconds C-terminal segment and the AATAAA poly(A) signal sequence in both patients. Our results suggest that selective IgM deficiency in Bloom's syndrome is due to an abnormality in the maturation of surface IgM-bearing B cells into IgM-secreting cells and a failure of microseconds mRNA synthesis. Moreover, reduced microseconds mRNA synthesis may be due to the defect on developmental regulation of the site at which poly(A) is added to transcripts of the mu gene. Images Fig. 2 PMID:1563106

  16. [Interrelationship between protein synthesis and mRNA metabolism in rat liver cells].

    PubMed

    Arbuzov, V A

    1976-08-01

    It is demonstrated that RNA isolated from polyribosomes and postmitochondrial fraction of rat liver cells and bound to nitrocellulose filters (Milliport) represent mRNA. RNA taken from the nitrocellulose filters sedimented in sucrose concentration gradient with a wide peak within the range of 18--6S, attaining a maximum at 12S. The (A+U)/(G+C) ratio of this RNA was equal to 1.04. On the other hand, the same ratio for rRNA was 0.64. Specific radioactivity of polysomal mRNA containing poly-A sequences, was significantly lower at 14-hour labelling with 14C-orotate than at 4-hour labelling (control). Inhibitors (cycloheximide, puromycin, ethionine, actinomycin D) stabilized polysomal mRNA. Specific radioactivity of postmitochondrial fraction mRNA was higher at 14-hour labelling than at 4-hour labelling. Specific radioactivity of postmitochondrial fraction mRNA during protein synthesis blocking by different inhibitors was comparable to those of control animals. It is hypothesized that active translation is necessary for the initiation of rat liver mRNA degradation.

  17. Requirements for Assembly of Poliovirus Replication Complexes and Negative-Strand RNA Synthesis

    PubMed Central

    Teterina, Natalya L.; Egger, Denise; Bienz, Kurt; Brown, David M.; Semler, Bert L.; Ehrenfeld, Ellie

    2001-01-01

    HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5′ noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5′-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5′-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3′ end. PMID:11264373

  18. Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

    PubMed Central

    de la Cruz, J; Vioque, A

    2001-01-01

    tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628

  19. In vitro synthesis of vertebrate U1 snRNA.

    PubMed Central

    Lund, E; Dahlberg, J E

    1989-01-01

    We have developed a DNA-dependent in vitro transcription system for vertebrate snRNA genes. By isolating the nuclei (germinal vesicles, GVs) of Xenopus laevis oocytes under oil to maintain the in vivo composition of their internal milieu, we are able to prepare nuclei that retain their ability to synthesize snRNAs efficiently. Homogenates of these GVs synthesize correctly initiated and terminated U1 snRNA using exogenous X.laevis U1 genes as templates. The templates may be either injected into the nucleus prior to its isolation or added to the nuclear homogenate. Images PMID:2714253

  20. The SARS coronavirus nucleocapsid protein--forms and functions.

    PubMed

    Chang, Chung-ke; Hou, Ming-Hon; Chang, Chi-Fon; Hsiao, Chwan-Deng; Huang, Tai-huang

    2014-03-01

    The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. It is a protein with multifarious activities. In this article we will review our current understanding of the N protein structure and its interaction with nucleic acid. Highlights of the progresses include uncovering the modular organization, determining the structures of the structural domains, realizing the roles of protein disorder in protein-protein and protein-nucleic acid interactions, and visualizing the ribonucleoprotein (RNP) structure inside the virions. It was also demonstrated that N-protein binds to nucleic acid at multiple sites with a coupled-allostery manner. We propose a SARS-CoV RNP model that conforms to existing data and bears resemblance to the existing RNP structures of RNA viruses. The model highlights the critical role of modular organization and intrinsic disorder of the N protein in the formation and functions of the dynamic RNP capsid in RNA viruses. This paper forms part of a symposium in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses." Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Anti-SARS coronavirus agents: a patent review (2008 - present).

    PubMed

    Kumar, Vathan; Jung, Young-Sik; Liang, Po-Huang

    2013-10-01

    A novel coronavirus (CoV), unlike previous typical human coronaviruses (HCoVs), was identified as causative agent for severe acute respiratory syndrome (SARS). SARS first surfaced as a pandemic in late 2002 and originated in southern China. SARS-CoV rapidly spread to > 30 countries by 2003, infecting nearly 8,000 people and causing around 800 fatalities. After 10 years of silence, a 2012 report alarmed researchers about the emergence of a new strain of CoV causing SARS-like disease. To combat SARS, scientists applied for patents on various therapeutic agents, including small-molecule inhibitors targeting the essential proteases, helicase and other proteins of the virus, natural products, approved drugs, molecules binding to the virus, neutralizing antibodies, vaccines, anti-sense RNA, siRNA and ribozyme against SARS-CoV. In this article, the patents published from 2008 to the present for the new therapeutics that could potentially be used in the prophylaxis and treatment of SARS are reviewed. The therapeutic interventions or prophylaxis discussed in this review seems to offer promising solutions to tackle SARS. Rather than being complacent about the results, we should envisage how to transform them into drug candidates that may be useful in combating SARS and related viral infections in the future.

  2. First-In-Class Inhibitor of Ribosomal RNA Synthesis with Antimicrobial Activity against Staphylococcus aureus.

    PubMed

    Yang, Xiao; Luo, Ming Jing; Yeung, Apple C M; Lewis, Peter J; Chan, Paul K S; Ip, Margaret; Ma, Cong

    2017-09-26

    We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

  3. Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

    SciTech Connect

    Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre

    2006-04-01

    Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4{sub 1}32 or P4{sub 3}32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.

  4. HTCC: Broad Range Inhibitor of Coronavirus Entry

    PubMed Central

    Milewska, Aleksandra; Kaminski, Kamil; Ciejka, Justyna; Kosowicz, Katarzyna; Zeglen, Slawomir; Wojarski, Jacek; Nowakowska, Maria; Szczubiałka, Krzysztof; Pyrc, Krzysztof

    2016-01-01

    To date, six human coronaviruses have been known, all of which are associated with respiratory infections in humans. With the exception of the highly pathogenic SARS and MERS coronaviruses, human coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-229E, and HCoV-HKU1) circulate worldwide and typically cause the common cold. In most cases, infection with these viruses does not lead to severe disease, although acute infections in infants, the elderly, and immunocompromised patients may progress to severe disease requiring hospitalization. Importantly, no drugs against human coronaviruses exist, and only supportive therapy is available. Previously, we proposed the cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and its hydrophobically-modified derivative (HM-HTCC) as potent inhibitors of the coronavirus HCoV-NL63. Here, we show that HTCC inhibits interaction of a virus with its receptor and thus blocks the entry. Further, we demonstrate that HTCC polymers with different degrees of substitution act as effective inhibitors of all low-pathogenic human coronaviruses. PMID:27249425

  5. Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

    PubMed Central

    Osman, T. A. M.; Hemenway, C. L.; Buck, K. W.

    2000-01-01

    A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity. PMID:11090166

  6. Crystallization and diffraction analysis of the SARS coronavirus nsp10–nsp16 complex

    PubMed Central

    Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

    2011-01-01

    To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2′-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress. PMID:21393853

  7. Crystallization and diffraction analysis of the SARS coronavirus nsp10-nsp16 complex.

    PubMed

    Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

    2011-03-01

    To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1-16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2'-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress.

  8. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis.

    PubMed

    Fei, Jingyi; Richard, Arianne C; Bronson, Jonathan E; Gonzalez, Ruben L

    2011-08-21

    Translocation of tRNAs through the ribosome during protein synthesis involves large-scale structural rearrangement of the ribosome and ribosome-bound tRNAs that is accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. How the rearrangement of individual tRNA-ribosome interactions influences tRNA movement during translocation, however, remains largely unknown. To address this question, we used single-molecule FRET to characterize the dynamics of ribosomal pretranslocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate of PRE complex rearrangement into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. Notably, our results suggest that the conformational flexibility of the tRNA molecule has a crucial role in directing the structural dynamics of the PRE complex during translocation.

  9. Role of RNA Synthesis in the Estrogen Induction of a Specific Uterine Protein*

    PubMed Central

    DeAngelo, Anthony B.; Gorski, Jack

    1970-01-01

    The rate of amino acid incorporation into a specific uterine protein (induced protein band) isolated by gel electrophoresis has been shown to be markedly stimulated within an hour after estrogen administration. Injection of actinomycin D (8 mg/kg) prior to estrogen blocks the synthesis of induced protein. The accumulation of the product of the actinomycin D-sensitive step (induced protein band RNA) is significant 15 minutes after estrogen, and its synthesis would appear to be initiated as soon as the estrogen-receptor complex reaches the nucleus. Blocking protein synthesis with puromycin or cycloheximide did not affect the accumulation of induced protein band RNA, indicating that this is one of the earliest macromolecular synthetic events to occur after estrogen administration. PMID:5269235

  10. The “Speedy” Synthesis of Atom-Specific 15N Imino/Amido-Labeled RNA

    PubMed Central

    Kreutz, Christoph; Micura, Ronald

    2016-01-01

    Although numerous reports on the synthesis of atom-specific 15N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of 15N(1) adenosine and 15N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve 15N(3) uridine and 15N(3) cytidine amidites in order to tap full potential of 1H/15N/15N-COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered. PMID:26237536

  11. Codon-specific and general inhibition of protein synthesis by the tRNA-sequestering minigenes.

    PubMed

    Delgado-Olivares, Luis; Zamora-Romo, Efraín; Guarneros, Gabriel; Hernandez-Sanchez, Javier

    2006-07-01

    The expression of minigenes in bacteria inhibits protein synthesis and cell growth. Presumably, the translating ribosomes, harboring the peptides as peptidyl-tRNAs, pause at the last sense codon of the minigene directed mRNAs. Eventually, the peptidyl-tRNAs drop off and, under limiting activity of peptidyl-tRNA hydrolase, accumulate in the cells reducing the concentration of specific aminoacylable tRNA. Therefore, the extent of inhibition is associated with the rate of starvation for a specific tRNA. Here, we used minigenes harboring various last sense codons that sequester specific tRNAs with different efficiency, to inhibit the translation of reporter genes containing, or not, these codons. A prompt inhibition of the protein synthesis directed by genes containing the codons starved for their cognate tRNA (hungry codons) was observed. However, a non-specific in vitro inhibition of protein synthesis, irrespective of the codon composition of the gene, was also evident. The degree of inhibition correlated directly with the number of hungry codons in the gene. Furthermore, a tRNA(Arg4)-sequestering minigene promoted the production of an incomplete beta-galactosidase polypeptide interrupted, during bacterial polypeptide chain elongation at sites where AGA codons were inserted in the lacZ gene suggesting ribosome pausing at the hungry codons.

  12. Akt activation enhances ribosomal RNA synthesis through casein kinase II and TIF-IA.

    PubMed

    Nguyen, Le Xuan Truong; Mitchell, Beverly S

    2013-12-17

    Transcription initiation factor I (TIF-IA) plays an essential role in regulating ribosomal RNA (rRNA) synthesis by tethering RNA polymerase I (Pol I) to the rDNA promoter. We have found that activated Akt enhances rRNA synthesis through the phosphorylation of casein kinase IIα (CK2α) on a threonine residue near its N terminus. CK2 in turn phosphorylates TIF-IA, thereby increasing rDNA transcription. Activated Akt also stabilizes TIF-IA, induces its translocation to the nucleolus, and enhances its interaction with Pol I. Treatment with AZD8055, an inhibitor of both Akt and mammalian target of rapamycin phosphorylation, but not with rapamycin, disrupts Akt-mediated TIF-IA stability, translocation, and activity. These data support a model in which activated Akt enhances rRNA synthesis both by preventing TIF-IA degradation and phosphorylating CK2α, which in turn phosphorylates TIF-IA. This model provides an explanation for the ability of activated Akt to promote cell proliferation and, potentially, transformation.

  13. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2005-07-05

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

  14. Catalytic RNA and synthesis of the peptide bond

    NASA Technical Reports Server (NTRS)

    Usher, D. A.; Kozlowski, M.; Zou, X.

    1991-01-01

    We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested.

  15. The relationship of severe acute respiratory syndrome coronavirus with avian and other coronaviruses.

    PubMed

    Jackwood, Mark W

    2006-09-01

    In February 2003, a severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in humans in Guangdong Province, China, and caused an epidemic that had severe impact on public health, travel, and economic trade. Coronaviruses are worldwide in distribution, highly infectious, and extremely difficult to control because they have extensive genetic diversity, a short generation time, and a high mutation rate. They can cause respiratory, enteric, and in some cases hepatic and neurological diseases in a wide variety of animals and humans. An enormous, previously unrecognized reservoir of coronaviruses exists among animals. Because coronaviruses have been shown, both experimentally and in nature, to undergo genetic mutations and recombination at a rate similar to that of influenza viruses, it is not surprising that zoonosis and host switching that leads to epidemic diseases have occurred among coronaviruses. Analysis of coronavirus genomic sequence data indicates that SARS-CoV emerged from an animal reservoir. Scientists examining coronavirus isolates from a variety of animals in and around Guangdong Province reported that SARS-CoV has similarities with many different coronaviruses including avian coronaviruses and SARS-CoV-like viruses from a variety of mammals found in live-animal markets. Although a SARS-like coronavirus isolated from a bat is thought to be the progenitor of SARS-CoV, a lack of genomic sequences for the animal coronaviruses has prevented elucidation of the true origin of SARS-CoV. Sequence analysis of SARS-CoV shows that the 5' polymerase gene has a mammalian ancestry; whereas the 3' end structural genes (excluding the spike glycoprotein) have an avian origin. Spike glycoprotein, the host cell attachment viral surface protein, was shown to be a mosaic of feline coronavirus and avian coronavirus sequences resulting from a recombination event. Based on phylogenetic analysis designed to elucidate evolutionary links among viruses, SARS-CoV is believed

  16. Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis

    PubMed Central

    Politz, Joan C.; Lewandowski, Laura B.; Pederson, Thoru

    2002-01-01

    The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I–specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions. PMID:12427865

  17. Human coronavirus NL63, France.

    PubMed

    Vabret, Astrid; Mourez, Thomas; Dina, Julia; van der Hoek, Lia; Gouarin, Stéphanie; Petitjean, Joëlle; Brouard, Jacques; Freymuth, François

    2005-08-01

    The human coronavirus NL63 (HCoV-NL63) was first identified in The Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory specimens for HCoV-NL63. Of the 300 samples, 28 (9.3%) were positive for HCoV-NL63. The highest prevalence was found in February (18%). The main symptoms were fever (61%), rhinitis (39%), bronchiolitis (39%), digestive problems (33%), otitis (28%), pharyngitis (22%), and conjunctivitis (17%). A fragment of the spike protein gene was sequenced to determine the variety of circulating HCoV-NL63. Phylogenetic analysis indicated that strains with different genetic markers cocirculate in France.

  18. In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor[C][W][OPEN

    PubMed Central

    Okano, Yukari; Senshu, Hiroko; Hashimoto, Masayoshi; Neriya, Yutaro; Netsu, Osamu; Minato, Nami; Yoshida, Tetsuya; Maejima, Kensaku; Oshima, Kenro; Komatsu, Ken; Yamaji, Yasuyuki; Namba, Shigetou

    2014-01-01

    RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs. PMID:24879427

  19. Why should cancer biologists care about tRNAs? tRNA synthesis, mRNA translation and the control of growth.

    PubMed

    Grewal, Savraj S

    2015-07-01

    Transfer RNAs (tRNAs) are essential for mRNA translation. They are transcribed in the nucleus by RNA polymerase III and undergo many modifications before contributing to cytoplasmic protein synthesis. In this review I highlight our understanding of how tRNA biology may be linked to the regulation of mRNA translation, growth and tumorigenesis. First, I review how oncogenes and tumour suppressor signalling pathways, such as the PI3 kinase/TORC1, Ras/ERK, Myc, p53 and Rb pathways, regulate Pol III and tRNA synthesis. In several cases, this regulation contributes to cell, tissue and body growth, and has implications for our understanding of tumorigenesis. Second, I highlight some recent work, particularly in model organisms such as yeast and Drosophila, that shows how alterations in tRNA synthesis may be not only necessary, but also sufficient to drive changes in mRNA translation and growth. These effects may arise due to both absolute increases in total tRNA levels, but also changes in the relative levels of tRNAs in the overall pool. Finally, I review some recent studies that have revealed how tRNA modifications (amino acid acylation, base modifications, subcellular shuttling, and cleavage) can be regulated by growth and stress cues to selectively influence mRNA translation. Together these studies emphasize the importance of the regulation of tRNA synthesis and modification as critical control points in protein synthesis and growth. This article is part of a Special Issue entitled: Translation and Cancer.

  20. Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5.

    PubMed

    Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul; Wu, Jianguo; He, Biao

    2015-12-01

    Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of

  1. Asphalt, water, and the prebiotic synthesis of ribose, ribonucleosides, and RNA.

    PubMed

    Benner, Steven A; Kim, Hyo-Joong; Carrigan, Matthew A

    2012-12-18

    RNA has been called a "prebiotic chemist's nightmare" because of its combination of large size, carbohydrate building blocks, bonds that are thermodynamically unstable in water, and overall intrinsic instability. However, a discontinuous synthesis model is well-supported by experimental work that might produce RNA from atmospheric CO(2), H(2)O, and N(2). For example, electrical discharge in such atmospheres gives formaldehyde (HCHO) in large amounts and glycolaldehyde (HOCH(2)CHO) in small amounts. When rained into alkaline aquifers generated by serpentinizing rocks, these substances were undoubtedly converted to carbohydrates including ribose. Likewise, atmospherically generated HCN was undoubtedly converted in these aquifers to formamide and ammonium formate, precursors for RNA nucleobases. Finally, high reduction potentials maintained by mantle-derived rocks and minerals would allow phosphite to be present in equilibrium with phosphate, mobilizing otherwise insoluble phosphorus for the prebiotic synthesis of phosphite and phosphate esters after oxidation. So why does the community not view this discontinuous synthesis model as compelling evidence for the RNA-first hypothesis for the origin of life? In part, the model is deficient because no experiments have joined together those steps without human intervention. Further, many steps in the model have problems. Some are successful only if reactive compounds are presented in a specific order in large amounts. Failing controlled addition, the result produces complex mixtures that are inauspicious precursors for biology, a situation described as the "asphalt problem". Many bonds in RNA are thermodynamically unstable with respect to hydrolysis in water, creating a "water problem". Finally, some bonds in RNA appear to be "impossible" to form under any conditions considered plausible for early Earth. To get a community-acceptable "RNA first" model for the origin of life, the discontinuous synthesis model must be

  2. Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

    PubMed Central

    Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

    2015-01-01

    ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the

  3. Evolutionary Relationships between Bat Coronaviruses and Their Hosts

    PubMed Central

    Cui, Jie; Han, Naijian; Streicker, Daniel; Li, Gang; Tang, Xianchun; Shi, Zhengli; Hu, Zhihong; Zhao, Guoping; Fontanet, Arnaud; Guan, Yi; Wang, Linfa; Jones, Gareth; Field, Hume E.

    2007-01-01

    Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (CoVs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (SARS) CoV, which caused an outbreak of respiratory illness in humans during 2002–2003. We examined the evolutionary relationships between bat CoVs and their hosts by using sequence data of the virus RNA-dependent RNA polymerase gene and the bat cytochrome b gene. Phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their CoVs, which suggested that host shifts have occurred in the recent evolutionary history of this group. These shifts may be due to either virus biologic traits or host behavioral traits. This finding has implications for the emergence of SARS and for the potential future emergence of SARS-CoVs or related viruses. PMID:18258002

  4. Surveillance of avian coronaviruses in wild bird populations of Korea.

    PubMed

    Kim, Hye-Ryoung; Oem, Jae-Ku

    2014-10-01

    We examined the role of wild birds in the epidemiology of avian coronaviruses by studying oropharyngeal swabs from 32 wild bird species. The 14 avian coronaviruses detected belonged to the gamma-coronaviruses and shared high nucleotide sequence identity with some previously identified strains in wild waterfowl, but not with infectious bronchitis viruses.

  5. Interaction of TIF-90 and filamin A in the regulation of rRNA synthesis in leukemic cells.

    PubMed

    Nguyen, Le Xuan Truong; Chan, Steven M; Ngo, Tri Duc; Raval, Aparna; Kim, Kyeong Kyu; Majeti, Ravindra; Mitchell, Beverly S

    2014-07-24

    The transcription initiation factor I (TIF-IA) is an important regulator of the synthesis of ribosomal RNA (rRNA) through its facilitation of the recruitment of RNA polymerase I (Pol I) to the ribosomal DNA promoter. Activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway, which occurs commonly in acute myelogenous leukemia, enhances rRNA synthesis through TIF-IA stabilization and phosphorylation. We have discovered that TIF-IA coexists with a splicing isoform, TIF-90, which is expressed preferentially in the nucleolus and at higher levels in proliferating and transformed hematopoietic cells. TIF-90 interacts directly with Pol I to increase rRNA synthesis as a consequence of Akt activation. Furthermore, TIF-90 binds preferentially to a 90-kDa cleavage product of the actin binding protein filamin A (FLNA) that inhibits rRNA synthesis. Increased expression of TIF-90 overcomes the inhibitory effect of this cleavage product and stimulates rRNA synthesis. Because activated Akt also reduces FLNA cleavage, these results indicate that activated Akt and TIF-90 function in parallel to increase rRNA synthesis and, as a consequence, cell proliferation in leukemic cells. These results provide evidence that the direct targeting of Akt would be an effective therapy in acute leukemias in which Akt is activated. © 2014 by The American Society of Hematology.

  6. May Cyclic Nucleotides Be a Source for Abiotic RNA Synthesis?

    NASA Astrophysics Data System (ADS)

    Costanzo, Giovanna; Pino, Samanta; Botta, Giorgia; Saladino, Raffaele; di Mauro, Ernesto

    2011-12-01

    Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

  7. Clustered Bottlenecks in mRNA Translation and Protein Synthesis

    NASA Astrophysics Data System (ADS)

    Chou, Tom; Lakatos, Greg

    2004-11-01

    Using a model based on the totally asymmetric exclusion process, we investigate the effects of slow codons along messenger RNA. Ribosome density profiles near neighboring clusters of slow codons interact, enhancing suppression of ribosome throughput when such bottlenecks are closely spaced. Increasing the slow codon cluster size beyond ˜3 4 codons does not significantly reduce the ribosome current. Our results are verified by both extensive MonteCarlo simulations and numerical calculation, and provide a biologically motivated explanation for the experimentally observed clustering of low-usage codons.

  8. RNA synthesis and turnover in the molluscan nervous system studied by Click-iT method.

    PubMed

    Ierusalimsky, Victor N; Balaban, Pavel M

    2016-02-15

    RNA synthesis can be detected by means of the in vivo incorporation of 5-ethynyluridine (EU) in newly-synthesized RNA with the relatively simple Click-iT method. We used this method to study the RNA synthesis in the CNS tissue of adult and juvenile terrestrial snails Helix lucorum L. Temporally, first labeled neurons were detected in the adult CNS after 4-h of isolated CNS incubation in EU solution, while 12-h of incubation led to extensive labeling of most CNS neurons. The EU labeling was present as the nuclear and nucleolar staining. The cytoplasm staining was observed after 2-3 days of CNS washout following the EU exposure for 16 h. In juvenile CNS, the first staining reaction was apparent as the staining of apical region in the procerebral lobe of cerebral ganglia after 1h of CNS incubation in EU, while the maximum pattern of staining was obtained after 4h of CNS incubation. Thus, age-related differences in RNA synthesis are present. Activation of neurons elicited by serotonin and caffeine applications noticeably increased the intensity of staining. EU readily penetrates into the bodies of juvenile snails immersed in the EU solution. When the intact juvenile animals were immersed in the EU solution for 1h, the procerebrum staining, similar to the one detected in the incubated juvenile CNS, was observed.

  9. Meteorites and the RNA World: A Thermodynamic Model of Nucleobase Synthesis within Planetesimals.

    PubMed

    Pearce, Ben K D; Pudritz, Ralph E

    2016-11-01

    The possible meteorite parent body origin of Earth's pregenetic nucleobases is substantiated by the guanine (G), adenine (A), and uracil (U) measured in various meteorites. Cytosine (C) and thymine (T), however, are absent in meteorites, making the emergence of an RNA and later RNA/DNA/protein world problematic. We investigated the meteorite parent body (planetesimal) origin of all nucleobases by computationally modeling 18 reactions that potentially contribute to nucleobase formation in such environments. Out of this list, we identified the two most important reactions for each nucleobase and found that these involve small molecules such as HCN, CO, NH3, and water that ultimately arise from the protoplanetary disks in which planetesimals are built. The primary result of this study is that cytosine is unlikely to persist within meteorite parent bodies due to aqueous deamination. Thymine has a thermodynamically favorable reaction pathway from uracil, formaldehyde, and formic acid but likely did not persist within planetesimals containing H2O2 due to an oxidation reaction with this molecule. Finally, while Fischer-Tropsch (FT) synthesis is found to be the dominant source of nucleobases within our model planetesimal, non-catalytic (NC) synthesis may still be significant under certain chemical conditions (e.g., within CR2 parent bodies). We discuss several major consequences of our results for the origin of the RNA world. Key Words: Astrobiology-Cosmochemistry-Meteorites-RNA world-Abiotic organic synthesis. Astrobiology 16, 853-872.

  10. AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

    PubMed

    Xu, Kaibiao; Yang, Lele; Zhao, Danyun; Wu, Yaoyao; Qi, Huayu

    2014-06-01

    Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells

  11. RNA synthesis in isolated brian nuclei after administration of d-lysergic acid diethylamide (LSD) in vivo.

    PubMed Central

    Brown, I R

    1975-01-01

    RNA synthesis in isolated brain nuclei was analyzed 2.5 hr after the intravenous administration of d-lysergic acid diethylamide (LSD) to young rabbits. The drug stimulated transcription by 54% in brain stem nuclei and by 13% in cerebral hemisphere nuclei expressed over saline controls. Both nucleoplasmic and nucleolar RNA synthesis were increased. The main activity in the isolated nuclei assay was due to nucleoplasmic RNA polymerase, since alpha-amanitin reduced synthesis by over 70% in either drug or control treatments. PMID:1055380

  12. RNA and protein synthesis is required for Ancylostoma caninum larval activation.

    PubMed

    Dryanovski, Dilyan I; Dowling, Camille; Gelmedin, Verena; Hawdon, John M

    2011-06-30

    The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 μM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 μM), cycloheximide (IC(50)=50 μM), and anisomycin (IC(50)=200 μM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h. None of the RNA or protein synthesis inhibitors prevented secretion of the activation-associated protein ASP-1, despite nearly complete inhibition of feeding. The results indicate that unlike dauer recovery in C. elegans, de novo gene expression is required for hookworm larval activation, and the critical genes are expressed within 12h of exposure to activating stimuli. However, secretion of infection-associated proteins is independent of gene expression, indicating that the proteins are pre-synthesized and stored for rapid release during the initial stages of infection. The genes that are inhibited

  13. Discovery, diversity and evolution of novel coronaviruses sampled from rodents in China.

    PubMed

    Wang, Wen; Lin, Xian-Dan; Guo, Wen-Ping; Zhou, Run-Hong; Wang, Miao-Ruo; Wang, Cai-Qiao; Ge, Shuang; Mei, Sheng-Hua; Li, Ming-Hui; Shi, Mang; Holmes, Edward C; Zhang, Yong-Zhen

    2015-01-01

    Although rodents are important reservoirs for RNA viruses, to date only one species of rodent coronavirus (CoV) has been identified. Herein, we describe a new CoV, denoted Lucheng Rn rat coronavirus (LRNV), and novel variants of two Betacoronavirus species termed Longquan Aa mouse coronavirus (LAMV) and Longquan Rl rat coronavirus (LRLV), that were identified in a survey of 1465 rodents sampled in China during 2011-2013. Phylogenetic analysis revealed that LAMV and LRLV fell into lineage A of the genus Betacoronavirus, which included CoVs discovered in humans and domestic and wild animals. In contrast, LRNV harbored by Rattus norvegicus formed a distinct lineage within the genus Alphacoronavirus in the 3CL(pro), RdRp, and Hel gene trees, but formed a more divergent lineage in the N and S gene trees, indicative of a recombinant origin. Additional recombination events were identified in LRLV. Together, these data suggest that rodents may carry additional unrecognized CoVs.

  14. Structure of the C-terminal domain of nsp4 from feline coronavirus

    PubMed Central

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-01-01

    Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. PMID:19622868

  15. Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CL(pro)

    SciTech Connect

    Bacha,U.; Barilla, J.; Gabelli, S.; Kiso, Y.; Amzel, L.; Freire, E.

    2008-01-01

    Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CLpro (Mpro), which is highly conserved among all coronaviruses. Here 3CLpro derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CLpro with Ki's as low as 300 nm. The crystal structure of the complex of one of the compounds with 3CLpro indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

  16. Switchable Protecting Strategy for Solid Phase Synthesis of DNA and RNA Interacting Nucleopeptides.

    PubMed

    Mercurio, Maria Emilia; Tomassi, Stefano; Gaglione, Maria; Russo, Rosita; Chambery, Angela; Lama, Stefania; Stiuso, Paola; Cosconati, Sandro; Novellino, Ettore; Di Maro, Salvatore; Messere, Anna

    2016-12-02

    Nucleopeptides are promising nucleic acid mimetics in which the peptide backbone bears nucleobases. They can recognize DNA and RNA targets modulating their biological functions. To date, the lack of an effective strategy for the synthesis of nucleopeptides prevents their evaluation for biological and biomedical applications. Herein, we describe an unprecedented approach that enables the synthesis of cationic both homo and heterosequence nucleopeptides wholly on solid support with high yield and purity. Spectroscopic studies indicate advantageous properties of the nucleopeptides in terms of binding, thermodynamic stability and sequence specific recognition. Biostability assay and laser scanning confocal microscopy analyses reveal that the nucleopeptides feature acceptable serum stability and ability to cross the cell membrane.

  17. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

    PubMed Central

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

    2012-01-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

  18. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    PubMed

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C; Chang, Kyeong-Ok

    2013-02-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  19. Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis

    PubMed Central

    El Hage, Aziz; French, Sarah L.; Beyer, Ann L.; Tollervey, David

    2010-01-01

    Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5–Air1/Air2–Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5′ region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5′ region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5′ region of the rDNA, imposing persistent transcription blocks when RNase H is limiting. PMID:20634320

  20. The cellular and molecular pathogenesis of coronaviruses.

    PubMed

    Compton, S R; Barthold, S W; Smith, A L

    1993-02-01

    Coronaviruses cause a wide spectrum of diseases in humans and animals but generally fall into two classes, with respiratory or enteric tropisms. Mouse hepatitis virus (MHV) and rat coronaviruses are the viruses most frequently encountered in the laboratory animal setting. This review focuses primarily on the cellular and molecular aspects of MHV pathogenesis. The high mutation and recombination rates of coronaviruses lead to a diverse, ever-changing population of MHV strains. The spike (S) protein is the most variable coronavirus protein and is responsible for binding to cell surface receptors, inducing cell fusion and humoral and cellular immunity. Differences within the S protein of different MHV strains have been linked to their variable tropisms. Since immunity to MHV is strain-specific, seropositive mice can be reinfected with different strains of MHV. Natural infections with MHV are acute, with persistence occurring at the population level, not within an individual mouse, unless it is immunocompromised. Age, genotype, immunologic status of the mouse, and MHV strain influence the type and severity of disease caused by MHV. Interference with research by MHV has been reported primarily in the fields of immunology and tumor biology and may be a reflection of MHV's capacity to grow in several types of immune cells. While many methods are available to diagnose coronavirus infection, serologic tests, primarily ELISA and IFA, are the most commonly used. MHV is best managed on a preventive basis. Elimination of MHV from a population requires cessation of breeding and halting the introduction of naive mice into the population.

  1. Pathogenic characteristics of persistent feline enteric coronavirus infection in cats.

    PubMed

    Vogel, Liesbeth; Van der Lubben, Mariken; te Lintelo, Eddie G; Bekker, Cornelis P J; Geerts, Tamara; Schuijff, Leontine S; Grinwis, Guy C M; Egberink, Herman F; Rottier, Peter J M

    2010-01-01

    Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.

  2. Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

    PubMed Central

    Vogel, Liesbeth; Van der Lubben, Mariken; Te Lintelo, Eddie G.; Bekker, Cornelis P.J.; Geerts, Tamara; Schuijff, Leontine S.; Grinwis, Guy C.M.; Egberink, Herman F.; Rottier, Peter J.M.

    2010-01-01

    Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed. PMID:20663472

  3. Clustered bottlenecks in mRNA translation and protein synthesis

    NASA Astrophysics Data System (ADS)

    Chou, Tom; Lakatos, Greg

    2004-03-01

    We construct an algorithm that generates large, band-diagonal transition matrices for a totally asymmetric exclusion process (TASEP) with local hopping rate inhomogeneities. The matrices are diagonalized numerically to find steady-state currents of TASEPs with local variations in hopping rate. The results are then used to investigate clustering of slow codons along mRNA. Ribosome density profiles near neighboring clusters of slow codons interact, enhancing suppression of ribosome throughput when such bottlenecks are closely spaced. Increasing the slow codon cluster size, beyond ˜ 3-4, does not significantly reduce ribosome current. Our results are verified by extensive Monte-Carlo simulations and provide a biologically-motivated explanation for the experimentally-observed clustering of low-usage codons.

  4. Coronavirus Spike Protein and Tropism Changes.

    PubMed

    Hulswit, R J G; de Haan, C A M; Bosch, B-J

    2016-01-01

    Coronaviruses (CoVs) have a remarkable potential to change tropism. This is particularly illustrated over the last 15 years by the emergence of two zoonotic CoVs, the severe acute respiratory syndrome (SARS)- and Middle East respiratory syndrome (MERS)-CoV. Due to their inherent genetic variability, it is inevitable that new cross-species transmission events of these enveloped, positive-stranded RNA viruses will occur. Research into these medical and veterinary important pathogens-sparked by the SARS and MERS outbreaks-revealed important principles of inter- and intraspecies tropism changes. The primary determinant of CoV tropism is the viral spike (S) entry protein. Trimers of the S glycoproteins on the virion surface accommodate binding to a cell surface receptor and fusion of the viral and cellular membrane. Recently, high-resolution structures of two CoV S proteins have been elucidated by single-particle cryo-electron microscopy. Using this new structural insight, we review the changes in the S protein that relate to changes in virus tropism. Different concepts underlie these tropism changes at the cellular, tissue, and host species level, including the promiscuity or adaptability of S proteins to orthologous receptors, alterations in the proteolytic cleavage activation as well as changes in the S protein metastability. A thorough understanding of the key role of the S protein in CoV entry is critical to further our understanding of virus cross-species transmission and pathogenesis and for development of intervention strategies. © 2016 Elsevier Inc. All rights reserved.

  5. An efficient ribosomal frame-shifting signal in the polymerase-encoding region of the coronavirus IBV.

    PubMed Central

    Brierley, I; Boursnell, M E; Binns, M M; Bilimoria, B; Blok, V C; Brown, T D; Inglis, S C

    1987-01-01

    The polymerase-encoding region of the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains two very large, briefly overlapping open reading frames (ORF), F1 and F2, and it has been suggested on the basis of sequence analysis that expression of the downstream ORF, F2, might be mediated through ribosomal frame-shifting. To examine this possibility a cDNA fragment containing the F1/F2 overlap region was cloned within a marker gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Messenger RNA transcribed from this plasmid, when translated in cell-free systems, specified the synthesis of polypeptides whose size was entirely consistent with the products predicted by an efficient ribosomal frame-shifting event within the overlap region. The nature of the products was confirmed by their reactivity with antisera raised against defined portions of the flanking marker gene. This is the first non-retroviral example of ribosomal frame-shifting in higher eukaryotes. Images Fig. 4. Fig. 6. PMID:3428275

  6. Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

    SciTech Connect

    Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

    1985-07-01

    To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

  7. RNA synthesis in whole cells and protoplasts of centaurea: a comparison.

    PubMed

    Kulikowski, R R; Mascarenhas, J P

    1978-04-01

    Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [(3)H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.

  8. Synthesis, Thermodynamic Properties, and Crystal Structure of RNA Oligonucleotides Containing 5-Hydroxymethylcytosine.

    PubMed

    Riml, Christian; Lusser, Alexandra; Ennifar, Eric; Micura, Ronald

    2017-08-04

    5-Hydroxymethylcytosine (hm(5)C) is an RNA modification that has attracted significant interest because of the finding that RNA hydroxymethylation can favor mRNA translation. For insight into the mechanistic details of hm(5)C function to be obtained, the availability of RNAs containing this modification at defined positions that can be used for in vitro studies is highly desirable. In this work, we present an eight-step route to 5-hydroxymethylcytidine (hm(5)rC) phosphoramidite for solid-phase synthesis of modified RNA oligonucleotides. Furthermore, we examined the effects of hm(5)rC on RNA duplex stability and its impact on structure formation using the sarcin-ricin loop (SRL) motif. Thermal denaturation experiments revealed that hm(5)rC increases RNA duplex stability. By contrast, when cytosine within an UNCG tetraloop motif was replaced by hm(5)rC, the thermodynamic stability of the corresponding hairpin fold was attenuated. Importantly, incorporation of hm(5)rC into the SRL motif resulted in an RNA crystal structure at 0.85 Å resolution. Besides changes in the hydration pattern at the site of modification, a slight opening of the hm(5)rC-G pair compared to the unmodified C-G in the native structure was revealed.

  9. Heat shock represses rRNA synthesis by inactivation of TIF-IA and lncRNA-dependent changes in nucleosome positioning

    PubMed Central

    Zhao, Zhongliang; Dammert, Marcel A.; Hoppe, Sven; Bierhoff, Holger; Grummt, Ingrid

    2016-01-01

    Attenuation of ribosome biogenesis in suboptimal growth environments is crucial for cellular homeostasis and genetic integrity. Here, we show that shutdown of rRNA synthesis in response to elevated temperature is brought about by mechanisms that target both the RNA polymerase I (Pol I) transcription machinery and the epigenetic signature of the rDNA promoter. Upon heat shock, the basal transcription factor TIF-IA is inactivated by inhibition of CK2-dependent phosphorylations at Ser170/172. Attenuation of pre-rRNA synthesis in response to heat stress is accompanied by upregulation of PAPAS, a long non-coding RNA (lncRNA) that is transcribed in antisense orientation to pre-rRNA. PAPAS interacts with CHD4, the adenosine triphosphatase subunit of NuRD, leading to deacetylation of histones and movement of the promoter-bound nucleosome into a position that is refractory to transcription initiation. The results exemplify how stress-induced inactivation of TIF-IA and lncRNA-dependent changes of chromatin structure ensure repression of rRNA synthesis in response to thermo-stress. PMID:27257073

  10. DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

    PubMed Central

    Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

    2016-01-01

    Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

  11. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains.

  12. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  13. Osteoblast fibronectin mRNA, protein synthesis, and matrix are unchanged after exposure to microgravity

    NASA Technical Reports Server (NTRS)

    Hughes-Fulford, M.; Gilbertson, V.

    1999-01-01

    The well-defined osteoblast line, MC3T3-E1 was used to examine fibronectin (FN) mRNA levels, protein synthesis, and extracellular FN matrix accumulation after growth activation in spaceflight. These osteoblasts produce FN extracellular matrix (ECM) known to regulate adhesion, differentiation, and function in adherent cells. Changes in bone ECM and osteoblast cell shape occur in spaceflight. To determine whether altered FN matrix is a factor in causing these changes in spaceflight, quiescent osteoblasts were launched into microgravity and were then sera activated with and without a 1-gravity field. Synthesis of FN mRNA, protein, and matrix were measured after activation in microgravity. FN mRNA synthesis is significantly reduced in microgravity (0-G) when compared to ground (GR) osteoblasts flown in a centrifuge simulating earth's gravity (1-G) field 2.5 h after activation. However, 27.5 h after activation there were no significant differences in mRNA synthesis. A small but significant reduction of FN protein was found in the 0-G samples 2.5 h after activation. Total FN protein 27.5 h after activation showed no significant difference between any of the gravity conditions, however, there was a fourfold increase in absolute amount of protein synthesized during the incubation. Using immunofluorescence, we found no significant differences in the amount or in the orientation of the FN matrix after 27.5 h in microgravity. These results demonstrate that FN is made by sera-activated osteoblasts even during exposure to microgravity. These data also suggest that after a total period of 43 h of spaceflight FN transcription, translation, or altered matrix assembly is not responsible for the altered cell shape or altered matrix formation of osteoblasts.

  14. Detection of feline coronavirus using microcantilever sensors

    NASA Astrophysics Data System (ADS)

    Velanki, Sreepriya; Ji, Hai-Feng

    2006-11-01

    This work demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (SARS-CoV) using microcantilever technology by showing that the feline coronavirus (FIP) type I virus can be detected by a microcantilever modified by feline coronavirus (FIP) type I anti-viral antiserum. A microcantilever modified by FIP type I anti-viral antiserum was developed for the detection of FIP type I virus. When the FIP type I virus positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the FIP type I virus by the antiserum on the surface of the microcantilever. A negative control sample that does not contain FIP type I virus did not cause any bending of the microcantilever. The detection limit of the sensor was 0.1 µg ml-1 when the assay time was <1 h.

  15. Feline and Canine Coronaviruses: Common Genetic and Pathobiological Features

    PubMed Central

    Le Poder, Sophie

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP) and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV) will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses. PMID:22312347

  16. Feline and canine coronaviruses: common genetic and pathobiological features.

    PubMed

    Le Poder, Sophie

    2011-01-01

    A new human coronavirus responsible for severe acute respiratory syndrome (SARS) was identified in 2003, which raised concern about coronaviruses as agents of serious infectious disease. Nevertheless, coronaviruses have been known for about 50 years to be major agents of respiratory, enteric, or systemic infections of domestic and companion animals. Feline and canine coronaviruses are widespread among dog and cat populations, sometimes leading to the fatal diseases known as feline infectious peritonitis (FIP) and pantropic canine coronavirus infection in cats and dogs, respectively. In this paper, different aspects of the genetics, host cell tropism, and pathogenesis of the feline and canine coronaviruses (FCoV and CCoV) will be discussed, with a view to illustrating how study of FCoVs and CCoVs can improve our general understanding of the pathobiology of coronaviruses.

  17. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

    PubMed Central

    Jewett, Michael C; Fritz, Brian R; Timmerman, Laura E; Church, George M

    2013-01-01

    Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties. PMID:23799452

  18. In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation.

    PubMed

    Jewett, Michael C; Fritz, Brian R; Timmerman, Laura E; Church, George M

    2013-06-25

    Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties.

  19. Enteral glutamine stimulates protein synthesis and decreases ubiquitin mRNA level in human gut mucosa.

    PubMed

    Coëffier, Moïse; Claeyssens, Sophie; Hecketsweiler, Bernadette; Lavoinne, Alain; Ducrotté, Philippe; Déchelotte, Pierre

    2003-08-01

    Effects of glutamine on whole body and intestinal protein synthesis and on intestinal proteolysis were assessed in humans. Two groups of healthy volunteers received in a random order enteral glutamine (0.8 mmol.kg body wt(-1)x h(-1)) compared either to saline or isonitrogenous amino acids. Intravenous [2H5]phenylalanine and [13C]leucine were simultaneously infused. After gas chromatography-mass spectrometry analysis, whole body protein turnover was estimated from traced plasma amino acid fluxes and the fractional synthesis rate (FSR) of gut mucosal protein was calculated from protein and intracellular phenylalanine and leucine enrichments in duodenal biopsies. mRNA levels for ubiquitin, cathepsin D, and m-calpain were analyzed in biopsies by RT-PCR. Glutamine significantly increased mucosal protein FSR compared with saline. Glutamine and amino acids had similar effects on FSR. The mRNA level for ubiquitin was significantly decreased after glutamine infusion compared with saline and amino acids, whereas cathepsin D and m-calpain mRNA levels were not affected. Enteral glutamine stimulates mucosal protein synthesis and may attenuate ubiquitin-dependent proteolysis and thus improve protein balance in human gut.

  20. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis.

    PubMed Central

    Ornelles, D A; Fey, E G; Penman, S

    1986-01-01

    Cytochalasin D was shown to be a reversible inhibitor of protein synthesis in HeLa cells. The inhibition was detectable at drug levels typically used to perturb cell structure and increased in a dose-dependent manner. The drug also released mRNA from the cytoskeletal framework in direct proportion to the inhibition of protein synthesis. The released mRNA was unaltered in its translatability as measured in vitro but was no longer translated in the cytochalasin-treated HeLa cells. The residual protein synthesis occurred on polyribosomes that were reduced in amount but displayed a normal sedimentation distribution. The results support the hypothesis that mRNA binding to the cytoskeletal framework is necessary although not sufficient for translation. Analysis of the cytoskeletal framework, which binds the polyribosomes, revealed no alterations in composition or amount of protein as a result of treatment with cytochalasin D. Electron microscopy with embedment-free sections shows the framework in great detail. The micrographs revealed the profound reorganization effected by the drug but did not indicate substantial disaggregation of the cytoskeletal elements. Images PMID:3785175

  1. Coronavirus avian infectious bronchitis virus.

    PubMed

    Cavanagh, Dave

    2007-01-01

    Infectious bronchitis virus (IBV), the coronavirus of the chicken (Gallus gallus), is one of the foremost causes of economic loss within the poultry industry, affecting the performance of both meat-type and egg-laying birds. The virus replicates not only in the epithelium of upper and lower respiratory tract tissues, but also in many tissues along the alimentary tract and elsewhere e.g. kidney, oviduct and testes. It can be detected in both respiratory and faecal material. There is increasing evidence that IBV can infect species of bird other than the chicken. Interestingly breeds of chicken vary with respect to the severity of infection with IBV, which may be related to the immune response. Probably the major reason for the high profile of IBV is the existence of a very large number of serotypes. Both live and inactivated IB vaccines are used extensively, the latter requiring priming by the former. Their effectiveness is diminished by poor cross-protection. The nature of the protective immune response to IBV is poorly understood. What is known is that the surface spike protein, indeed the amino-terminal S1 half, is sufficient to induce good protective immunity. There is increasing evidence that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection. Experimental vector IB vaccines and genetically manipulated IBVs--with heterologous spike protein genes--have produced promising results, including in the context of in ovo vaccination.

  2. Inhibition of human coronavirus NL63 infection at early stages of the replication cycle.

    PubMed

    Pyrc, Krzysztof; Bosch, Berend Jan; Berkhout, Ben; Jebbink, Maarten F; Dijkman, Ronald; Rottier, Peter; van der Hoek, Lia

    2006-06-01

    Human coronavirus NL63 (HCoV-NL63), a recently discovered member of the Coronaviridae family, has spread worldwide and is associated with acute respiratory illness in young children and elderly and immunocompromised persons. Further analysis of HCoV-NL63 pathogenicity seems warranted, in particular because the virus uses the same cellular receptor as severe acute respiratory syndrome-associated coronavirus. As there is currently no HCoV-NL63-specific and effective vaccine or drug therapy available, we evaluated several existing antiviral drugs and new synthetic compounds as inhibitors of HCoV-NL63, targeting multiple stages of the replication cycle. Of the 28 compounds that we tested, 6 potently inhibited HCoV-NL63 at early steps of the replication cycle. Intravenous immunoglobulins, heptad repeat 2 peptide, small interfering RNA1 (siRNA1), siRNA2, beta-D-N(4)-hydroxycytidine, and 6-azauridine showed 50% inhibitory concentrations of 125 microg/ml, 2 microM, 5 nM, 3 nM, 400 nM, and 32 nM, respectively, and low 50% cytotoxicity concentrations (>10 mg/ml, >40 microM, >200 nM, >200 nM, >100 microM, and 80 microM, respectively). These agents may be investigated further for the treatment of coronavirus infections.

  3. Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses.

    PubMed

    Wang, Jingjing; Deng, Feng; Ye, Gang; Dong, Wanyu; Zheng, Anjun; He, Qigai; Peng, Guiqing

    2016-02-01

    The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

  4. Poliovirus 5′-Terminal Cloverleaf RNA Is Required in cis for VPg Uridylylation and the Initiation of Negative-Strand RNA Synthesis

    PubMed Central

    Lyons, Traci; Murray, Kenneth E.; Roberts, Allan W.; Barton, David J.

    2001-01-01

    Chimeric poliovirus RNAs, possessing the 5′ nontranslated region (NTR) of hepatitis C virus in place of the 5′ NTR of poliovirus, were used to examine the role of the poliovirus 5′ NTR in viral replication. The chimeric viral RNAs were incubated in cell-free reaction mixtures capable of supporting the sequential translation and replication of poliovirus RNA. Using preinitiation RNA replication complexes formed in these reactions, we demonstrated that the 3′ NTR of poliovirus RNA was insufficient, by itself, to recruit the viral replication proteins required for negative-strand RNA synthesis. The 5′-terminal cloverleaf of poliovirus RNA was required in cis to form functional preinitiation RNA replication complexes capable of uridylylating VPg and initiating the synthesis of negative-strand RNA. These results are consistent with a model in which the 5′-terminal cloverleaf and 3′ NTRs of poliovirus RNA interact via temporally dynamic ribonucleoprotein complexes to coordinately mediate and regulate the sequential translation and replication of poliovirus RNA. PMID:11602711

  5. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

    SciTech Connect

    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.; Saphire, Erica Ollmann; Schnell, Matthias Johannes

    2016-10-18

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis.

  6. Analysis of global RNA synthesis at the single cell level following hypoxia.

    PubMed

    Biddlestone, John; Druker, Jimena; Shmakova, Alena; Ferguson, Gus; Swedlow, Jason R; Rocha, Sonia

    2014-05-13

    Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.  Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

  7. Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine.

    PubMed

    Cui, Song; Kim, Yong-Hoon; Jin, Cheng-Hao; Kim, Sang Kook; Rhee, Man-Hee; Kwon, Oh-Shin; Moon, Byung Jo

    2009-06-30

    5-Hydroxyuridine (5-OHU) is a major lesion of uridine and cytosine produced in RNA by various chemical oxidants. To elucidate its biochemical and biophysical effects on RNA replication, the site-specifically modified oligoribonucleotides containing 5-OHU were synthesized with C5-hydroxy-5'-O-DMTr-2'-TBDMS-uridine phosphoramidite using automated solid phase synthesis. The base-pairing properties of nucleotides opposite 5-OHU in 24 mer oligoribonulcleotides with dNTP were studied using three reverse transcriptases (Super-Script(TM)II-, AMV-, MMLV-RT) in cDNA synthesis. Adenine as well as guanine was incorporated preferentially by all reverse transcriptases. In the UV-melting temperature experiment, the results from the relative stabilities of the base pairs were A : 5-OHU > G : 5-OHU > T : 5-OHU approximately C : 5-OHU. Circular Dichroism (CD) studies showed that DNA-RNA containing 5-OHU heteroduplexes exhibit a similar conformation between the A-type RNA and B-type DNA. These results suggest that 5-OHU from oxidative damage was mainly influenced by adenine mismatch.

  8. The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

    PubMed Central

    Kirchdoerfer, Robert N.; Moyer, Crystal L.; Abelson, Dafna M.; Saphire, Erica Ollmann

    2016-01-01

    Filoviruses are capable of causing deadly hemorrhagic fevers. All nonsegmented negative-sense RNA-virus nucleocapsids are composed of a nucleoprotein (NP), a phosphoprotein (VP35) and a polymerase (L). However, the VP30 RNA-synthesis co-factor is unique to the filoviruses. The assembly, structure, and function of the filovirus RNA replication complex remain unclear. Here, we have characterized the interactions of Ebola, Sudan and Marburg virus VP30 with NP using in vitro biochemistry, structural biology and cell-based mini-replicon assays. We have found that the VP30 C-terminal domain interacts with a short peptide in the C-terminal region of NP. Further, we have solved crystal structures of the VP30-NP complex for both Ebola and Marburg viruses. These structures reveal that a conserved, proline-rich NP peptide binds a shallow hydrophobic cleft on the VP30 C-terminal domain. Structure-guided Ebola virus VP30 mutants have altered affinities for the NP peptide. Correlation of these VP30-NP affinities with the activity for each of these mutants in a cell-based mini-replicon assay suggests that the VP30-NP interaction plays both essential and inhibitory roles in Ebola virus RNA synthesis. PMID:27755595

  9. Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

    PubMed Central

    Kuo, Lili; Hurst-Hess, Kelley R.; Koetzner, Cheri A.

    2016-01-01

    ABSTRACT The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its

  10. Human Coronavirus 229E Remains Infectious on Common Touch Surface Materials.

    PubMed

    Warnes, Sarah L; Little, Zoë R; Keevil, C William

    2015-11-10

    severe infections with high mortality, such as severe acute respiratory syndrome (SARS) and, more recently, Middle East respiratory syndrome (MERS). We show here that a closely related human coronavirus, 229E, which causes upper respiratory tract infection in healthy individuals and serious disease in patients with comorbidities, remained infectious on surface materials common to public and domestic areas for several days. The low infectious dose means that this is a significant infection risk to anyone touching a contaminated surface. However, rapid inactivation, irreversible destruction of viral RNA, and massive structural damage were observed in coronavirus exposed to copper and copper alloy surfaces. Incorporation of copper alloy surfaces in conjunction with effective cleaning regimens and good clinical practice could help to control transmission of respiratory coronaviruses, including MERS and SARS. Copyright © 2015 Warnes et al.

  11. Synthesis and tau RNA binding evaluation of ametantrone-containing ligands.

    PubMed

    Artigas, Gerard; Marchán, Vicente

    2015-02-20

    We describe the synthesis and characterization of ametantrone-containing RNA ligands based on the derivatization of this intercalator with two neamine moieties (Amt-Nea,Nea) or with one azaquinolone heterocycle and one neamine (Amt-Nea,Azq) as well as its combination with guanidinoneamine (Amt-NeaG4). Biophysical studies revealed that guanidinylation of the parent ligand (Amt-Nea) had a positive effect on the binding of the resulting compound for Tau pre-mRNA target as well as on the stabilization upon complexation of some of the mutated RNA sequences associated with the development of tauopathies. Further studies by NMR revealed the existence of a preferred binding site in the stem-loop structure, in which ametantrone intercalates in the characteristic bulged region. Regarding doubly-functionalized ligands, binding affinity and stabilizing ability of Amt-Nea,Nea were similar to those of the guanidinylated ligand, but the two aminoglycoside fragments seem to interfere with its accommodation in a single binding site. However, Amt-Nea,Azq binds at the bulged region in a similar way than Amt-NeaG4. Overall, these results provide new insights on fine-tuning RNA binding properties of ametantrone by single or double derivatization with other RNA recognition motifs, which could help in the future design of new ligands with improved selectivity for disease-causing RNA molecules.

  12. Synthesis and processing of tRNA-related SINE transcripts in Arabidopsis thaliana

    PubMed Central

    Pélissier, Thierry; Bousquet-Antonelli, Cécile; Lavie, Laurence; Deragon, Jean-Marc

    2004-01-01

    Despite the ubiquitous distribution of tRNA-related short interspersed elements (SINEs) in eukaryotic species, very little is known about the synthesis and processing of their RNAs. In this work, we have characterized in detail the different RNA populations resulting from the expression of a tRNA-related SINE S1 founder copy in Arabidopsis thaliana. The main population is composed of poly(A)-ending (pa) SINE RNAs, while two minor populations correspond to full-length (fl) or poly(A) minus [small cytoplasmic (sc)] SINE RNAs. Part of the poly(A) minus RNAs is modified by 3′-terminal addition of C or CA nucleotides. All three RNA populations accumulate in the cytoplasm. Using a mutagenesis approach, we show that the poly(A) region and the 3′ end unique region, present at the founder locus, are both important for the maturation and the steady-state accumulation of the different S1 RNA populations. The observation that primary SINE transcripts can be post-transcriptionally processed in vivo into a poly(A)-ending species introduces the possibility that this paRNA is used as a retroposition intermediate. PMID:15282328

  13. Chemical synthesis of a biologically active natural tRNA with its minor bases.

    PubMed Central

    Gasparutto, D; Livache, T; Bazin, H; Duplaa, A M; Guy, A; Khorlin, A; Molko, D; Roget, A; Téoule, R

    1992-01-01

    The complete chemical synthesis of an E. coli tRNA(Ala) with its specific minor nucleosides, dihydrouridine, ribothymidine and pseudouridine, is reported. The method makes use of protected 2'-O-tertiobutyldimethylsilyl-ribonucleoside-3'-O-(2-cyanoethyl-N- ethyl-N- methyl)phosphoramidites. The exocyclic amino functions of the bases were protected by the phenoxyacetyl group for purines and acetyl for cytosine. The assembling has been performed on a silica support with coupling yield better than 98% within 2 min of condensation. Triethylamine tris-hydrofluoride allowed a clean and complete deprotection of the tBDMS groups. The synthetic tRNA(Ala) has been transcribed into cDNA by reverse transcriptase and sequenced. With E. coli alanyl-tRNA synthetase the alanyl acceptance activity and kcat/Km were 672 pmol/A260 and 6 x 10(4)M-1s-1, respectively. Images PMID:1383941

  14. Differential Effects of Actinomycin D and Cordycepin in Lettuce Seed Germination and RNA Synthesis 1

    PubMed Central

    Tao, Kar-Ling; Khan, Anwar A.

    1976-01-01

    Intact lettuce seed germination was inhibited by cordycepin but not by actinomycin D; however, when seeds were clipped at the cotyledonary end, actinomycin D partially inhibited germination. Uptake studies with intact seeds using 3H-actinomycin D showed that it was unable to reach the embryo prior to radical protrusion. 3H-Cordycepin uptake studies using intact seeds showed that cordycepin was able to reach the embryo during the first 3 hours of incubation and at subsequent times. The pericarp and endosperm offered resistance to penetration of cordycepin into the embryo. In contrast to actinomycin D, cordycepin markedly inhibited 3H-uridine incorporation into RNA of intact seeds during the first 10 and 12 hours of incubation. About 60% of 3H-adenosine incorporation into poly A-RNA was inhibited by cordycepin during 12 hours of incubation, whereas actinomycin D had little effect. RNA synthesis appears to be essential for seed germination. PMID:16659763

  15. The prebiotic synthesis of modified purines and their potential role in the RNA world

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for these compounds in the evolution of life. These include N6-methyladenine, 1-methyladenine, N6,N6-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N2-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional groups in ribozymes, especially for the construction of hydrophobic binding pockets.

  16. The prebiotic synthesis of modified purines and their potential role in the RNA world

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Bada, J. L. (Principal Investigator)

    1999-01-01

    Modified purines are found in all organisms in the tRNA, rRNA, and even DNA, raising the possibility of an early role for these compounds in the evolution of life. These include N6-methyladenine, 1-methyladenine, N6,N6-dimethyladenine, 1-methylhypoxanthine, 1-methylguanine, and N2-methylguanine. We find that these bases as well as a number of nonbiological modified purines can be synthesized from adenine and guanine by the simple reaction of an amine or an amino group with adenine and guanine under the concentrated conditions of the drying-lagoon or drying-beach model of prebiotic synthesis with yields as high as 50%. These compounds are therefore as prebiotic as adenine and guanine and could have played an important role in the RNA world by providing additional functional groups in ribozymes, especially for the construction of hydrophobic binding pockets.

  17. Regiospecific solid-phase synthesis of branched oligoribonucleotides that mimic intronic lariat RNA intermediates.

    PubMed

    Katolik, Adam; Johnsson, Richard; Montemayor, Eric; Lackey, Jeremy G; Hart, P John; Damha, Masad J

    2014-02-07

    We have developed new solid phase methods for the synthesis of branched RNAs that mimic intronic lariat RNA intermediates. These methods produce branched oligoribonucleotide sequences of arbitrary length, base composition, and regiochemistry at the branchpoint junction. The methods utilize branching monomers that allow for the growth of each branch regioselectively from any of the hydroxyl positions (5′, 3′, or 2′) at the branch-point junction. The integrity and branchpoint connectivity of the synthetic products have been confirmed by HPLC and MS analysis, and cleavage of the 2′,5′ linkage by recombinant debranching enzyme. Nonhydrolyzable branched RNA analogues containing arabinose instead of ribose at the branchpoint junction were shown to inhibit debranching activity and, hence, represent “decoys” for sequestering RNA binding proteins thought to drive amyotrophic lateral sclerosis (ALS).

  18. Coronaviruses in bats from Mexico

    PubMed Central

    Ojeda-Flores, R.; Rico-Chávez, O.; Navarrete-Macias, I.; Zambrana-Torrelio, C. M.; Rostal, M. K.; Epstein, J. H.; Tipps, T.; Liang, E.; Sanchez-Leon, M.; Sotomayor-Bonilla, J.; Aguirre, A. A.; Ávila-Flores, R.; Medellín, R. A.; Goldstein, T.; Suzán, G.; Daszak, P.

    2013-01-01

    Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (β)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were β-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A β-CoV with 96.5 % amino acid identity to the β-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health. PMID:23364191

  19. Coronaviruses in bats from Mexico.

    PubMed

    Anthony, S J; Ojeda-Flores, R; Rico-Chávez, O; Navarrete-Macias, I; Zambrana-Torrelio, C M; Rostal, M K; Epstein, J H; Tipps, T; Liang, E; Sanchez-Leon, M; Sotomayor-Bonilla, J; Aguirre, A A; Ávila-Flores, R; Medellín, R A; Goldstein, T; Suzán, G; Daszak, P; Lipkin, W I

    2013-05-01

    Bats are reservoirs for a wide range of human pathogens including Nipah, Hendra, rabies, Ebola, Marburg and severe acute respiratory syndrome coronavirus (CoV). The recent implication of a novel beta (β)-CoV as the cause of fatal respiratory disease in the Middle East emphasizes the importance of surveillance for CoVs that have potential to move from bats into the human population. In a screen of 606 bats from 42 different species in Campeche, Chiapas and Mexico City we identified 13 distinct CoVs. Nine were alpha (α)-CoVs; four were β-CoVs. Twelve were novel. Analyses of these viruses in the context of their hosts and ecological habitat indicated that host species is a strong selective driver in CoV evolution, even in allopatric populations separated by significant geographical distance; and that a single species/genus of bat can contain multiple CoVs. A β-CoV with 96.5 % amino acid identity to the β-CoV associated with human disease in the Middle East was found in a Nyctinomops laticaudatus bat, suggesting that efforts to identify the viral reservoir should include surveillance of the bat families Molossidae/Vespertilionidae, or the closely related Nycteridae/Emballonuridae. While it is important to investigate unknown viral diversity in bats, it is also important to remember that the majority of viruses they carry will not pose any clinical risk, and bats should not be stigmatized ubiquitously as significant threats to public health.

  20. Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

    NASA Technical Reports Server (NTRS)

    Raghavan, V.

    1991-01-01

    Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

  1. Gene activity during germination of spores of the fern, Onoclea sensibilis: RNA and protein synthesis and the role of stored mRNA

    NASA Technical Reports Server (NTRS)

    Raghavan, V.

    1991-01-01

    Pattern of 3H-uridine incorporation into RNA of spores of Onoclea sensibilis imbibed in complete darkness (non-germinating conditions) and induced to germinate in red light was followed by oligo-dT cellulose chromatography, gel electrophoresis coupled with fluorography and autoradiography. In dark-imbibed spores, RNA synthesis was initiated about 24 h after sowing, with most of the label accumulating in the high mol. wt. poly(A) -RNA fraction. There was no incorporation of the label into poly(A) +RNA until 48 h after sowing. In contrast, photo-induced spores began to synthesize all fractions of RNA within 12 h after sowing and by 24 h, incorporation of 3H-uridine into RNA of irradiated spores was nearly 70-fold higher than that into dark-imbibed spores. Protein synthesis, as monitored by 3H-arginine incorporation into the acid-insoluble fraction and by autoradiography, was initiated in spores within 1-2 h after sowing under both conditions. Autoradiographic experiments also showed that onset of protein synthesis in the cytoplasm of the germinating spore is independent of the transport of newly synthesized nuclear RNA. One-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis of 35S-methionine-labelled proteins revealed a good correspondence between proteins synthesized in a cell-free translation system directed by poly(A) +RNA of dormant spores and those synthesized in vivo by dark-imbibed and photo-induced spores. These results indicate that stored mRNAs of O. sensibilis spores are functionally competent and provide templates for the synthesis of proteins during dark-imbibition and germination.

  2. Suppression of coronavirus replication by cyclophilin inhibitors.

    PubMed

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2013-05-22

    Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS), feline infectious peritonitis (FIP), mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA), could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

  3. Comparative Studies of Effect of Auxin and Ethylene on Permeability and Synthesis of RNA and Protein 1

    PubMed Central

    Sacher, Joseph A.; Salminen, Seppo O.

    1969-01-01

    The effects of ethylene on permeability and RNA and protein synthesis were assayed over a 6 to 26 hr period in tissue sections from avocado (Persea gratissima Gaertn. F., var. Fuerte), both pulp and peel of banana (Musa sapientum L., var. Gros Michel), bean endocarp (Phaseolus vulgaris L., var. Kentucky Wonder Pole beans) and leaves of Rhoeo discolor. Ethylene had no effect on permeability in 4 of the 5 tissues, but sometimes enhanced solute uptake in banana peel; it had either no effect or an inhibitory effect on synthesis of RNA and protein in sections from fruits of avocado and banana. Auxin (α-naphthalene acetic acid) stimulated synthesis of RNA and protein in bean endocarp and Rhoeo leaf sections, whereas ethylene inhibited both basal and auxin-induced synthesis. It is concluded that in these tissues the auxin effect is not an ethylene effect. PMID:16657212

  4. Transfer RNA-mediated regulation of ribosome dynamics during protein synthesis

    PubMed Central

    Fei, Jingyi; Richard, Arianne C.; Bronson, Jonathan E.; Gonzalez, & Ruben L.

    2011-01-01

    Translocation of transfer RNAs (tRNAs) through the ribosome during protein synthesis involves large-scale structural rearrangements of the ribosome and the ribosome-bound tRNAs that are accompanied by extensive and dynamic remodeling of tRNA-ribosome interactions. The contributions that rearranging individual tRNA-ribosome interactions make to directing tRNA movements during translocation, however, remain largely unknown. To address this question, we have used single-molecule fluorescence resonance energy transfer to characterize the dynamics of ribosomal pre-translocation (PRE) complex analogs carrying either wild-type or systematically mutagenized tRNAs. Our data reveal how specific tRNA-ribosome interactions regulate the rate with which the PRE complex rearranges into a critical, on-pathway translocation intermediate and how these interactions control the stability of the resulting configuration. More interestingly, our results suggest that the conformational flexibility of the tRNA molecule itself plays a crucial role in directing the structural dynamics of the PRE complex during translocation. PMID:21857664

  5. Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

    PubMed Central

    Ogilvie, K K; Usman, N; Nicoghosian, K; Cedergren, R J

    1988-01-01

    Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47. Images PMID:3413059

  6. Cell cycle regulation of tubulin RNA level, tubulin protein synthesis, and assembly of microtubules in Physarum.

    PubMed

    Schedl, T; Burland, T G; Gull, K; Dove, W F

    1984-07-01

    The temporal relationship between tubulin expression and the assembly of the mitotic spindle microtubules has been investigated during the naturally synchronous cell cycle of the Physarum plasmodium. The cell cycle behavior of the tubulin isoforms was examined by two-dimensional gel electrophoresis of proteins labeled in vivo and by translation of RNA in vitro. alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin synthesis increases coordinately until metaphase, and then falls, with beta 2 falling more rapidly than beta 1. Nucleic acid hybridization demonstrated that alpha- and beta-tubulin RNAs accumulate coordinately during G2, peaking at metaphase. Quantitative analysis demonstrated that alpha-tubulin RNA increases with apparent exponential kinetics, peaking with an increase over the basal level of greater than 40-fold. After metaphase, tubulin RNA levels fall exponentially, with a short half-life (19 min). Electron microscopic analysis of the plasmodium showed that the accumulation of tubulin RNA begins long before the polymerization of mitotic spindle microtubules. By contrast, the decay of tubulin RNA after metaphase coincides with the depolymerization of the spindle microtubules.

  7. RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

    PubMed Central

    1975-01-01

    A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs. PMID:1171872

  8. Synthesis of Biotin-Labeled RNA for Gene Expression Measurements Using Oligonucleotide Arrays

    PubMed Central

    Vázquez, Ana E.; Nie, Liping; Yamoah, Ebenezer N.

    2010-01-01

    Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ~ 500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests. PMID:18839339

  9. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  10. In low protein diets, microRNA-19b regulates urea synthesis by targeting SIRT5

    PubMed Central

    Sun, Rui-Ping; Xi, Qian-Yun; Sun, Jia-Jie; Cheng, Xiao; Zhu, Yan-Ling; Ye, Ding-Ze; Chen, Ting; Wei, Li-Min; Ye, Rui-Song; Jiang, Qing-Yan; Zhang, Yong-Liang

    2016-01-01

    Ammonia detoxification, which takes place via the hepatic urea cycle, is essential for nitrogen homeostasis and physiological well-being. It has been reported that a reduction in dietary protein reduces urea nitrogen. MicroRNAs (miRNAs) are major regulatory non-coding RNAs that have significant effects on several metabolic pathways; however, little is known on whether miRNAs regulate hepatic urea synthesis. The objective of this study was to assess the miRNA expression profile in a low protein diet and identify miRNAs involved in the regulation of the hepatic urea cycle using a porcine model. Weaned 28-days old piglets were fed a corn-soybean normal protein diet (NP) or a corn-soybean low protein diet (LP) for 30 d. Hepatic and blood samples were collected, and the miRNA expression profile was assessed by sequencing and qRT-PCR. Furthermore, we evaluated the possible role of miR-19b in urea synthesis regulation. There were 25 differentially expressed miRNAs between the NP and LP groups. Six of these miRNAs were predicted to be involved in urea cycle metabolism. MiR-19b negatively regulated urea synthesis by targeting SIRT5, which is a positive regulator of CPS1, the rate limiting enzyme in the urea cycle. Our study presented a novel explanation of ureagenesis regulation by miRNAs. PMID:27686746

  11. Abiotic synthesis of RNA in water: a common goal of prebiotic chemistry and bottom-up synthetic biology.

    PubMed

    Cafferty, Brian J; Hud, Nicholas V

    2014-10-01

    For more than half a century chemists have searched for a plausible prebiotic synthesis of RNA. The initial advances of the 1960s and 1970s were followed by decades of measured progress and a growing pessimism about overcoming remaining challenges. Fortunately, the past few years have provided a number of important advances, including new abiotic routes for the synthesis of nucleobases, nucleosides, and nucleotides. Recent discoveries also provide additional support for the hypothesis that RNA is the product of evolution, being preceded by ancestral genetic polymers, or pre-RNAs, that are synthesized more easily than RNA. In some cases, parallel searches for plausible prebiotic routes to RNA and pre-RNAs have provided more than one experimentally verified synthesis of RNA substructures and possible predecessors. Just as the synthesis of a contemporary biological molecule cannot be understood without knowledge of cellular metabolism, it is likely that an integrated approach that takes into account both plausible prebiotic reactions and plausible prebiotic environments will ultimately provide the most satisfactory and unifying chemical scenarios for the origin of nucleic acids. In this context, recent advances towards the abiotic synthesis of RNA and candidates for pre-RNAs are beginning to suggest that some molecules (e.g., urea) were multi-faceted contributors to the origin of nucleic acids, and the origin of life. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. IFITM Proteins Inhibit Entry Driven by the MERS-Coronavirus Spike Protein: Evidence for Cholesterol-Independent Mechanisms

    PubMed Central

    Wrensch, Florian; Winkler, Michael; Pöhlmann, Stefan

    2014-01-01

    The interferon-inducible transmembrane (IFITM) proteins 1, 2 and 3 inhibit the host cell entry of several enveloped viruses, potentially by promoting the accumulation of cholesterol in endosomal compartments. IFITM3 is essential for control of influenza virus infection in mice and humans. In contrast, the role of IFITM proteins in coronavirus infection is less well defined. Employing a retroviral vector system for analysis of coronavirus entry, we investigated the susceptibility of human-adapted and emerging coronaviruses to inhibition by IFITM proteins. We found that entry of the recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is sensitive to inhibition by IFITM proteins. In 293T cells, IFITM-mediated inhibition of cellular entry of the emerging MERS- and SARS-CoV was less efficient than blockade of entry of the globally circulating human coronaviruses 229E and NL63. Similar differences were not observed in A549 cells, suggesting that cellular context and/or IFITM expression levels can impact inhibition efficiency. The differential IFITM-sensitivity of coronaviruses observed in 293T cells afforded the opportunity to investigate whether efficiency of entry inhibition by IFITMs and endosomal cholesterol accumulation correlate. No such correlation was observed. Furthermore, entry mediated by the influenza virus hemagglutinin was robustly inhibited by IFITM3 but was insensitive to accumulation of endosomal cholesterol, indicating that modulation of cholesterol synthesis/transport did not account for the antiviral activity of IFITM3. Collectively, these results show that the emerging MERS-CoV is a target of the antiviral activity of IFITM proteins and demonstrate that mechanisms other than accumulation of endosomal cholesterol can contribute to viral entry inhibition by IFITMs. PMID:25256397

  13. Existence of two forms of rat liver arginyl-tRNA synthetase suggests channeling of aminoacyl-tRNA for protein synthesis.

    PubMed Central

    Sivaram, P; Deutscher, M P

    1990-01-01

    Arginyl-tRNA synthetase (arginine-tRNA ligase, EC 6.1.1.19) is found in extracts of mammalian cells both as a free protein (Mr = 60,000) and as a component (Mr approximately 72,000) of the high molecular weight aminoacyl-tRNA synthetase complex (Mr greater than 10(6). Several pieces of evidence indicate that the low molecular weight free form is not a proteolytic degradation product of the complex-bound enzyme but that it preexists in vivo: (i) the endogenous free form differs in size from the active proteolytic fragment generated in vitro, (ii) conditions expected to increase or decrease the amount of proteolysis do not alter the ratio of the two forms of the enzyme, and (iii) the free form contains an NH2-terminal methionine residue. A model is presented that provides a rationale for the existence of two forms of arginyl-tRNA synthetase in cells. In this model the complexed enzyme supplies arginyl-tRNA for protein synthesis, whereas the free enzyme provides arginyl-tRNA for the NH2-terminal arginine modification of proteins by arginyl-tRNA:protein arginyltransferase. This latter process targets certain proteins for removal by the ubiquitin-dependent protein degradation pathway. The necessity for an additional pool of arginyl-tRNA for the modification reaction leads to the conclusion that the arginyl-tRNA destined for protein synthesis (and/or protein modification) is channeled and unavailable for other processes. Other evidence supporting channeling in protein synthesis is discussed. Images PMID:2187187

  14. Full genome analysis of a novel type II feline coronavirus NTU156.

    PubMed

    Lin, Chao-Nan; Chang, Ruey-Yi; Su, Bi-Ling; Chueh, Ling-Ling

    2013-04-01

    Infections by type II feline coronaviruses (FCoVs) have been shown to be significantly correlated with fatal feline infectious peritonitis (FIP). Despite nearly six decades having passed since its first emergence, different studies have shown that type II FCoV represents only a small portion of the total FCoV seropositivity in cats; hence, there is very limited knowledge of the evolution of type II FCoV. To elucidate the correlation between viral emergence and FIP, a local isolate (NTU156) that was derived from a FIP cat was analyzed along with other worldwide strains. Containing an in-frame deletion of 442 nucleotides in open reading frame 3c, the complete genome size of NTU156 (28,897 nucleotides) appears to be the smallest among the known type II feline coronaviruses. Bootscan analysis revealed that NTU156 evolved from two crossover events between type I FCoV and canine coronavirus, with recombination sites located in the RNA-dependent RNA polymerase and M genes. With an exchange of nearly one-third of the genome with other members of alphacoronaviruses, the new emerging virus could gain new antigenicity, posing a threat to cats that either have been infected with a type I virus before or never have been infected with FCoV.

  15. Reverse transcription recombinase polymerase amplification assay for the detection of middle East respiratory syndrome coronavirus.

    PubMed

    Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T; Weidmann, Manfred

    2013-12-12

    The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) in the eastern Mediterranean and imported cases to Europe has alerted public health authorities. Currently, detection of MERS-CoV in patient samples is done by real-time RT-PCR. Samples collected from suspected cases are sent to highly-equipped centralized laboratories for screening. A rapid point-of-care test is needed to allow more widespread mobile detection of the virus directly from patient material. In this study, we describe the development of a reverse transcription isothermal Recombinase Polymerase Amplification (RT-RPA) assay for the identification of MERS-CoV. A partial nucleocapsid gene RNA molecular standard of MERS-coronavirus was used to determine the assay sensitivity. The isothermal (42°C) MERS-CoV RT-RPA was as sensitive as real-time RT-PCR (10 RNA molecules), rapid (3-7 minutes) and mobile (using tubescanner weighing 1kg). The MERS-CoV RT-RPA showed cross-detection neither of any of the RNAs of several coronaviruses and respiratory viruses affecting humans nor of the human genome. The developed isothermal real-time RT-RPA is ideal for rapid mobile molecular MERS-CoV monitoring in acute patients and may also facilitate the search for the animal reservoir of MERS-CoV.

  16. [Visual Detection of Human Coronavirus NL63 by Reverse Transcription Loop-Mediated Isothermal Amplification].

    PubMed

    Geng, Heyuan; Wang, Shengqiang; Xie, Xiaoqian; Xiao, Yu; Zhang, Ting; Tan, Wenjie; Su, Chuan

    2016-01-01

    A simple and sensitive assay for rapid detection of human coronavirus NL63 (HCoV-NL63) was developed by colorimetic reverse transcription loop-mediated isothermal amplification (RT-LAMP). The method employed six specially designed primers that recognized eight distinct regions of the HCoV-NL63 nucleocapsid protein gene for amplification of target sequences under isothermal conditions at 63 degrees C for 1 h Amplification of RT-LAMP was monitored by addition of calcein before amplification. A positive reaction was confirmed by change from light-brown to yellow-green under visual detection. Specificity of the RT-LAMP assay was validated by cross-reaction with different human coronaviruses, norovirus, influenza A virus, and influenza B virus. Sensitivity was evaluated by serial dilution of HCoV-NL63 RNA from 1.6 x 10(9) to 1.6 x 10(1) per reaction. The RT-LAMP assay could achieve 1,600 RNA copies per reaction with high specificity. Hence, our colorimetric RT-LAMP assay could be used for rapid detection of human coronavirus NL63.

  17. Meteorites and the RNA World: A Thermodynamic Model of Nucleobase Synthesis within Planetesimals

    NASA Astrophysics Data System (ADS)

    Pearce, Ben K. D.; Pudritz, Ralph E.

    2016-11-01

    The possible meteorite parent body origin of Earth's pregenetic nucleobases is substantiated by the guanine (G), adenine (A), and uracil (U) measured in various meteorites. Cytosine (C) and thymine (T), however, are absent in meteorites, making the emergence of an RNA and later RNA/DNA/protein world problematic. We investigated the meteorite parent body (planetesimal) origin of all nucleobases by computationally modeling 18 reactions that potentially contribute to nucleobase formation in such environments. Out of this list, we identified the two most important reactions for each nucleobase and found that these involve small molecules such as HCN, CO, NH3, and water that ultimately arise from the protoplanetary disks in which planetesimals are built. The primary result of this study is that cytosine is unlikely to persist within meteorite parent bodies due to aqueous deamination. Thymine has a thermodynamically favorable reaction pathway from uracil, formaldehyde, and formic acid but likely did not persist within planetesimals containing H2O2 due to an oxidation reaction with this molecule. Finally, while Fischer-Tropsch (FT) synthesis is found to be the dominant source of nucleobases within our model planetesimal, non-catalytic (NC) synthesis may still be significant under certain chemical conditions (e.g., within CR2 parent bodies). We discuss several major consequences of our results for the origin of the RNA world.

  18. [Synthesis of specific RNA at the early S-stage of the cell cycle of the myxomycete Physarum polycephalum].

    PubMed

    Bukhman, V L; Cherveniak, Iu

    1982-01-01

    Incorporation of [3H]uridine into polyribosomes and polyribosomal RNA at different stages of the cell cycle of myxomycete Physarum polycephalum was investigated. It has been shown that at the early S-stage (during the first hour after mitosis) there is no rRNA synthesis. About 10--12% of polyribosomal RNA, synthesized during this period is poly(A)+RNA. Approximately 50% of poly(A)-RNA sediments as 4--6S and 20--25% as 7--14S in sucrose density gradient. It has been found that labeled RNA is associated with the light polyribosome fractions. The bulk of RNA synthesized during other periods of the cell cycle consists of rRNA, which is evenly distributed between different polysome fractions. We suppose that light polyribosomes are involved in the protein synthesis at the early S-stage of the cell cycle. 4--6S and 7--14S polyribosomal poly(A)-RNA was investigated by electrophoresis in denaturing conditions. By comparing with other organisms we may suppose that the 7--14S poly(A)-RNA, synthesized during the beginning of the S-stage is histone messenger RNA.

  19. A 3′-end structure in RNA2 of a crinivirus is essential for viral RNA synthesis and contributes to replication-associated translation activity

    PubMed Central

    Mongkolsiriwattana, Chawin; Zhou, Jaclyn S.; Ng, James C. K.

    2016-01-01

    The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3′ terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5′ and YSS-containing 3′ terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons. PMID:27694962

  20. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions.

    PubMed

    Liu, Fakeng; Jin, Rui; Liu, Xiuju; Huang, Henry; Wilkinson, Scott C; Zhong, Diansheng; Khuri, Fadlo R; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-19

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells.

  1. The relation of RNA synthesis to chondroitin sulphate biosynthesis in cultured bovine cartilage.

    PubMed Central

    McQuillan, D J; Handley, C J; Robinson, H C; Ng, K; Tzaicos, C

    1986-01-01

    Addition of actinomycin D (or cordycepin, an alternative inhibitor of RNA synthesis) to cartilage cultures resulted in a first-order decrease in the rate of incorporation of [35S]sulphate into proteoglycan (half-life = 7.5 +/- 1.1 h). Addition of 1.0 mM-benzyl beta-D-xyloside relieved the initial inhibition of glycosaminoglycan synthesis induced by actinomycin D; however, after a lag of about 10 h the rate of xyloside-initiated glycosaminoglycan synthesis also decreased with apparent first-order kinetics (half-life = 7.1 +/- 1.8 h), which paralleled the decrease in the rate of core-protein-initiated glycosaminoglycan synthesis. The hydrodynamic size of the proteoglycans formed in the presence of actinomycin D remained essentially constant (Kav. 0.21-0.23), whereas the constituent glycosaminoglycan chains were larger than those formed by control cultures, which suggested that the core protein was substituted with fewer but larger glycosaminoglycan chains. Proteoglycans formed in the presence of beta-D-xyloside were significantly smaller (Kav. approximately 0.33) than those synthesized by control cultures, and were further diminished in size after exposure of cultures to actinomycin D. Glycosaminoglycan chains synthesized by these same cultures on to both core-protein and xyloside acceptors were also smaller than those of control cultures. The decrease in synthesis observed after exposure to actinomycin D was not reflected by any significant decrease in the activities of several glycosyltransferases involved in chondroitin sulphate synthesis (galactosyltransferase-I, galactosyltransferase-II, N-acetylgalactosaminyltransferase and glucuronosyltransferase-II). PMID:2427073

  2. Properties of Native High-Density Lipoproteins Inspire Synthesis of Actively Targeted In Vivo siRNA Delivery Vehicles.

    PubMed

    McMahon, Kaylin M; Plebanek, Michael P; Thaxton, C Shad

    2016-11-15

    Efficient systemic administration of therapeutic short interfering RNA (siRNA) is challenging. High-density lipoproteins (HDL) are natural in vivo RNA delivery vehicles. Specifically, native HDLs: 1) Load single-stranded RNA; 2) Are anionic, which requires charge reconciliation between the RNA and HDL, and 3) Actively target scavenger receptor type B-1 (SR-B1) to deliver RNA. Emphasizing these particular parameters, we employed templated lipoprotein particles (TLP), mimics of spherical HDLs, and self-assembled them with single-stranded complements of, presumably, any highly unmodified siRNA duplex pair after formulation with a cationic lipid. Resulting siRNA templated lipoprotein particles (siRNA-TLP) are anionic and tunable with regard to RNA assembly and function. Data demonstrate that the siRNA-TLPs actively target SR-B1 to potently reduce androgen receptor (AR) and enhancer of zeste homolog 2 (EZH2) proteins in multiple cancer cell lines. Systemic administration of siRNA-TLPs demonstrated no off-target toxicity and significantly reduced the growth of prostate cancer xenografts. Thus, native HDLs inspired the synthesis of a hybrid siRNA delivery vehicle that can modularly load single-stranded RNA complements after charge reconciliation with a cationic lipid, and that function due to active targeting of SR-B1.

  3. Coronavirus entry and release in polarized epithelial cells: a review.

    PubMed

    Cong, Yingying; Ren, Xiaofeng

    2014-09-01

    Most coronaviruses cause respiratory or intestinal infections in their animal or human host. Hence, their interaction with polarized epithelial cells plays a critical role in the onset and outcome of infection. In this paper, we review the knowledge regarding the entry and release of coronaviruses, with particular emphasis on the severe acute respiratory syndrome and Middle East respiratory syndrome coronaviruses. As these viruses approach the epithelial surfaces from the apical side, it is not surprising that coronavirus cell receptors are exposed primarily on the apical domain of polarized epithelial cells. With respect to release, all possibilities appear to occur. Thus, most coronaviruses exit through the apical surface, several through the basolateral one, although the Middle East respiratory syndrome coronavirus appears to use both sides. These observations help us understand the local or systematic spread of the infection within its host as well as the spread of the virus within the host population.

  4. Solid-supported synthesis of 5'-mRNA CAP-4 from Trypanosomatids.

    PubMed

    Lewdorowicz, Magdalena; Jemielity, Jacek; Kierzek, Ryszard; Shapira, Michal; Stepinski, Janusz; Darzynkiewicz, Edward

    2007-01-01

    The unique structure of 5' mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5'-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn(2+) as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl(2).

  5. Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

    SciTech Connect

    Luthra, Priya; Jordan, David S.; Leung, Daisy W.; Amarasinghe, Gaya K.; Basler, Christopher F.; Lyles, D. S.

    2015-03-04

    Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

  6. Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells▿ †

    PubMed Central

    Bellecave, Pantxika; Cazenave, Christian; Rumi, Julie; Staedel, Cathy; Cosnefroy, Ophélie; Andreola, Marie-Line; Ventura, Michel; Tarrago-Litvak, Laura; Astier-Gin, Thérèse

    2008-01-01

    We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection. PMID:18347106

  7. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: evidence for differential gene expression.

    PubMed Central

    Kim, S Y; Byrn, R; Groopman, J; Baltimore, D

    1989-01-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. We have established a single-cycle growth condition for HIV in H9 cells, a human CD4+ lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes. Images PMID:2760980

  8. Synthesis of acetylene-substituted probes with benzene-phosphate backbones for RNA labeling.

    PubMed

    Kitamura, Yoshiaki; Ueno, Yoshihito; Kitade, Yukio

    2014-06-24

    Conversion of dimethyl 5-aminoisophthalate into the iodoarene via the corresponding diazonium intermediate, followed by Sonogashira coupling with trimethylsilylacetylene afford the alkynylarene, which is reduced with LiAlH4 to give 5-ethynyl-1,3-benzenedimethanol (B(E)). One hydroxyl group is protected with a 4,4'-dimethoxytrityl (DMTr) group and subsequently another hydroxyl group is phosphitylated to produce the phosphoramidite. The mono-DMTr compound is also modified to afford the corresponding succinate, which is then reacted with controlled pore glass (CPG) to provide the solid support. Either the phosphoramidite or the solid support is employed in solid-phase synthesis of RNA containing B(E). RNA oligomers bearing B(E) rapidly react with 4-fluorobenzylazide to produce the cycloaddition products in good to excellent yield.

  9. Receptor recognition mechanisms of coronaviruses: a decade of structural studies.

    PubMed

    Li, Fang

    2015-02-01

    Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general.

  10. Receptor Recognition Mechanisms of Coronaviruses: a Decade of Structural Studies

    PubMed Central

    2014-01-01

    Receptor recognition by viruses is the first and essential step of viral infections of host cells. It is an important determinant of viral host range and cross-species infection and a primary target for antiviral intervention. Coronaviruses recognize a variety of host receptors, infect many hosts, and are health threats to humans and animals. The receptor-binding S1 subunit of coronavirus spike proteins contains two distinctive domains, the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), both of which can function as receptor-binding domains (RBDs). S1-NTDs and S1-CTDs from three major coronavirus genera recognize at least four protein receptors and three sugar receptors and demonstrate a complex receptor recognition pattern. For example, highly similar coronavirus S1-CTDs within the same genus can recognize different receptors, whereas very different coronavirus S1-CTDs from different genera can recognize the same receptor. Moreover, coronavirus S1-NTDs can recognize either protein or sugar receptors. Structural studies in the past decade have elucidated many of the puzzles associated with coronavirus-receptor interactions. This article reviews the latest knowledge on the receptor recognition mechanisms of coronaviruses and discusses how coronaviruses have evolved their complex receptor recognition pattern. It also summarizes important principles that govern receptor recognition by viruses in general. PMID:25428871

  11. Alteration of 6-phosphofructo-1-kinase subunit protein, synthesis rates, and mRNA during rat neonatal development.

    PubMed

    Mhaskar, Y; Dunaway, G A

    1996-03-29

    For the three 6-phosphofructo-1-kinase (PFK) subunits in heart, skeletal muscle, liver and kidney, developmentally-associated changes in protein, mRNA and apparent synthesis rates were observed. During neonatal maturation, all three phenomena for the M-type in heart and skeletal muscle exhibited large increases. Also, during neonatal development, the L-type and C-type subunits were unaffected in heart but disappeared from skeletal muscle. In the newborn liver and kidney, the amounts of each type of PFK subunit protein were nearly identical. During neonatal development, the levels of all three PFK subunit proteins in kidney increased more than twofold; and this was associated with a similar increase in apparent subunit synthesis rates and mRNA levels. During liver neonatal development, the L-type subunit protein, synthesis and mRNA levels also increased more than twofold. However, during hepatic maturation, M-type subunit protein, synthesis and mRNA levels were unchanged and apparently unaffected. The C-type subunit protein during neonatal liver development decreased approximately 80% as did its apparent synthesis rate. These data suggest that regulation of the alteration of the PFK subunit proteins during neonatal maturation can vary among these tissues and is not the same for each subunit type. Different mechanisms, such as transcription, translation, and mRNA stability could be involved.

  12. Significance of mineral salts in prebiotic RNA synthesis catalyzed by montmorillonite.

    PubMed

    Joshi, Prakash C; Aldersley, Michael F

    2013-06-01

    The montmorillonite-catalyzed reactions of the 5'-phosphorimidazolide of adenosine used as a model generated RNA type oligomers. These reactions were found to be dependent on the presence of mineral salts. Whereas montmorillonite (pH 7) produced only dimers and traces of trimer in water, addition of sodium chloride (0.1-2.0 M) enhanced the chain length of oligomers to 10-mers as detected by HPLC. Maximum catalytic activity was observed with sodium chloride at a concentration between 0.8 and 1.2 M. This concentration of sodium chloride resembled its abundance in the ancient oceans (0.9-1.2 M). Magnesium chloride produced a similar effect but its joint action with sodium chloride did not produce any difference in the oligomer chain length. Therefore, Mg(2+) was not deemed necessary for generating longer oligomers. The effect of monovalent cations upon RNA chain length was: Li(+) > Na(+) > K(+). A similar effect was observed with the anions with enhanced oligomer length in the following order: Cl(-) > Br(-) > I(-). Thus, the smaller ions facilitated the formation of the longest oligomers. Inorganic salts that tend to salt out organic compounds from water and salts which show salt-in effects had no influence on the oligomerization process indicating that the montmorillonite-catalyzed RNA synthesis is not affected by either of these hydrophobic or hydrophilic interactions. A 2.3-fold decrease in the yield of cyclic dimer was observed upon increasing the sodium chloride concentration from 0.2 to 2.0 M. Inhibition of cyclic dimer formation is vital for increasing the yield of linear dimers and longer oligomers. In summary, sodium chloride is likely to have played an essential role in any clay mineral-catalyzed prebiotic RNA synthesis.

  13. Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes

    SciTech Connect

    Suh, D.; Reddy, R. ); Wright, D. )

    1991-06-04

    Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes (reviewed in Dahlberg and Lund (1988)). The authors studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the {minus}1,200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in Hela cells.

  14. Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination.

    PubMed

    Barr, John N

    2007-05-01

    Messenger RNA transcription by Bunyaviridae family members is unique within the group of negative-strand RNA viruses as it requires on-going protein synthesis. The long-standing model explaining this phenomenon proposes that the translational requirement is not for a protein product, but instead is for ribosomal translocation along nascent mRNAs. This movement is proposed to disrupt spurious transcription termination signals that otherwise cause premature mRNA truncation leading to a fatal loss of gene expression. This model was tested by introducing translational stop codons into model RNA genomes of Bunyamwera virus, the prototypic member of the Bunyaviridae family. This directly showed that translation of nascent mRNAs prevents transcription termination. While such coupling of transcription and translation is common in prokaryotic systems, these results represent the first report of such obligatory coupling in a eukaryotic cell environment. The results also provide insight into the bunyavirus termination mechanism and suggest it is mechanistically similar to prokaryotic intrinsic termination.

  15. Catalytic effects of Murchison material: prebiotic synthesis and degradation of RNA precursors.

    PubMed

    Saladino, Raffaele; Crestini, Claudia; Cossetti, Cristina; Di Mauro, Ernesto; Deamer, David

    2011-10-01

    Mineral components of the Murchison meteorite were investigated in terms of potential catalytic effects on synthetic and hydrolytic reactions related to ribonucleic acid. We found that the mineral surfaces catalyzed condensation reactions of formamide to form carboxylic acids, amino acids, nucleobases and sugar precursors. These results suggest that formamide condensation reactions in the parent bodies of carbonaceous meteorites could give rise to multiple organic compounds thought to be required for the emergence of life. Previous studies have demonstrated similar catalytic effects for mineral assemblies likely to have been present in the early Earth environment. The minerals had little or no effect in promoting hydrolysis of RNA (24mer of polyadenylic acid) at 80°C over a pH range from 4.2 to 9.3. RNA was most stable in the neutral pH range, with a half-life ~5 h, but at higher and lower pH ranges the half-life decreased to ~1 h. These results suggest that if RNA was somehow incorporated into a primitive form of RNA-based thermophilic life, either it must be protected from random hydrolytic events, or the rate of synthesis must exceed the rate of hydrolysis.

  16. Wild animal surveillance for coronavirus HKU1 and potential variants of other coronaviruses.

    PubMed

    Yuen, K Y; Lau, S K P; Woo, P C Y

    2012-02-01

    1. Although CoV-HKU1 was not identified in any of the studied animals, a coronavirus closely related to SARS-CoV (bat-SARS-CoV) was identified in 23 (19%) of 118 wild Chinese horseshoe bats by reverse transcriptase polymerase chain reaction (RT-PCR). 2. Complete genome sequencing and phylogenetic analysis showed that bat-SARS-CoV formed a distinct cluster with SARS-CoV as group 2b coronaviruses, distantly related to known group 2 coronaviruses. 3. Most differences between the bat-SARS-CoV and SARS-CoV genomes were observed in the spike gene. The presence of a29-bp insertion in ORF 8 of bat-SARS-CoV genome, not in most human SARS-CoV genomes, suggests that it has a common ancestor with civet SARS-CoV. 4. Antibody against recombinant bat-SARS-CoV nucleocapsid protein was detected in 84% of Chinese horseshoe bats using an enzyme immunoassay.Neutralising antibody to human SARS-CoV was also detected in those with lower viral loads.5. This study also revealed a previously unknown diversity of coronaviruses in bats, which are important natural reservoir for coronaviruses including SARS-CoV-like viruses.

  17. Understanding bat SARS-like coronaviruses for the preparation of future coronavirus outbreaks - Implications for coronavirus vaccine development.

    PubMed

    Ng, Oi-Wing; Tan, Yee-Joo

    2017-01-02

    The severe acute respiratory syndrome coronavirus (SARS-CoV) first emerged in 2003, causing the SARS epidemic which resulted in a 10% fatality rate. The advancements in metagenomic techniques have allowed the identification of SARS-like coronaviruses (SL-CoVs) sequences that share high homology to the human SARS-CoV epidemic strains from wildlife bats, presenting concrete evidence that bats are the origin and natural reservoir of SARS-CoV. The application of reverse genetics further enabled that characterization of these bat CoVs and the prediction of their potential to cause disease in humans. The knowledge gained from such studies is valuable in the surveillance and preparation of a possible future outbreak caused by a spill-over of these bat SL-CoVs.

  18. Coronavirus genome: prediction of putative functional domains in the non-structural polyprotein by comparative amino acid sequence analysis.

    PubMed Central

    Gorbalenya, A E; Koonin, E V; Donchenko, A P; Blinov, V M

    1989-01-01

    Amino acid sequences of 2 giant non-structural polyproteins (F1 and F2) of infectious bronchitis virus (IBV), a member of Coronaviridae, were compared, by computer-assisted methods, to sequences of a number of other positive strand RNA viral and cellular proteins. By this approach, juxtaposed putative RNA-dependent RNA polymerase, nucleic acid binding ("finger"-like) and RNA helicase domains were identified in F2. Together, these domains might constitute the core of the protein complex involved in the primer-dependent transcription, replication and recombination of coronaviruses. In F1, two cysteine protease-like domains and a growth factor-like one were revealed. One of the putative proteases of IBV is similar to 3C proteases of picornaviruses and related enzymes of como- nepo- and potyviruses. Search of IBV F1 and F2 sequences for sites similar to those cleaved by the latter proteases and intercomparison of the surrounding sequence stretches revealed 13 dipeptides Q/S(G) which are probably cleaved by the coronavirus 3C-like protease. Based on these observations, a partial tentative scheme for the functional organization and expression strategy of the non-structural polyproteins of IBV was proposed. It implies that, despite the general similarity to other positive strand RNA viruses, and particularly to potyviruses, coronaviruses possess a number of unique structural and functional features. PMID:2526320

  19. A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells.

    PubMed

    Castorena, Kathryn M; Weeks, Spencer A; Stapleford, Kenneth A; Cadwallader, Amy M; Miller, David J

    2007-08-01

    The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.

  20. [Development and comparison of real-time and conventional RT-PCR assay for detection of human coronavirus NL63 and HKU1].

    PubMed

    Lu, Rou-jian; Zhang, Ling-lin; Tan, Wen-jie; Zhou, Wei-min; Wang, Zhong; Peng, Kun; Ruan, Li

    2008-07-01

    We designed specific primers and fluorescence-labeled probes to develop real-time and conventional RT-PCR assays for detection of human coronavirus NL63 or HKU1. Subsequently, experiments were undertaken to assess diagnostic criteria such as specificity, sensitivity and reproducibility. The detection limit of the real-time RT-PCR assays was 10 RNA copies per reaction mixture. No cross-reactivity was observed between RNA samples derived from designed HCoV and other HCoV or human metapneumovirus. A total of 158 nasopharyngeal swab specimens collected from adult patients with acute respiratory tract infection in Beijing were screened for the presence of human coronavirus NL63 and HKU1 by using real-time RT-PCR and conventional RT-PCR method. The fluorescence quantitative RT-PCR method detected six specimens positive for human coronavirus NL63, five specimens positive for human coronavirus HKU1; and conventional RT-PCR method detected three HCoV-NL63 positive and three HCoV-HKU1 positive, respectively. The convention RT-PCR products of positive samples were obtained and sequence analysis confirmed the reliability of the above methods. In summary, the real-time RT-PCR assay for HCoV- NL63 or HKU1 was more sensitive than conventional RT-PCR and with less time (less than 4 hours) for completion. It may be suitable for molecular epidemiological surveillance and clinical diagnosis for human coronavirus NL63 and HKU1.

  1. Human coronaviruses: insights into environmental resistance and its influence on the development of new antiseptic strategies.

    PubMed

    Geller, Chloé; Varbanov, Mihayl; Duval, Raphaël E

    2012-11-12

    The Coronaviridae family, an enveloped RNA virus family, and, more particularly, human coronaviruses (HCoV), were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002-2003, the outbreak of severe acute respiratory syndrome (SARS), due to a newly discovered coronavirus, the SARS-associated coronavirus (SARS-CoV); led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection, and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1), NL63, HKU1 or SARS-CoV) to survive in different environmental conditions (e.g. temperature and humidity), on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biological fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections), the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance) make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to survive in the

  2. Human Coronaviruses: Insights into Environmental Resistance and Its Influence on the Development of New Antiseptic Strategies

    PubMed Central

    Geller, Chloé; Varbanov, Mihayl; Duval, Raphaël E.

    2012-01-01

    The Coronaviridae family, an enveloped RNA virus family, and, more particularly, human coronaviruses (HCoV), were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002–2003, the outbreak of severe acute respiratory syndrome (SARS), due to a newly discovered coronavirus, the SARS-associated coronavirus (SARS-CoV); led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection, and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1), NL63, HKU1 or SARS-CoV) to survive in different environmental conditions (e.g. temperature and humidity), on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biological fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections), the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance) make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to survive in the

  3. Methyldeficient mammalian 4S RNA: evidence for L-ethionine-induced inhibition of N6-dimethyladenosine synthesis in rat liver tRNA

    PubMed Central

    Wildenauer, Dieter; Gross, Hans J.

    1974-01-01

    The nucleotide composition of 4s RNA from livers of rats fed with a diet containing 0.3% D-ethionine was found to be identical with that from untreated animals. In contrast, one single modified nucleotide was absent in 4s RNA from livers of rats fed with a 0.3% L-ethionine diet. The minor nucleo=tide was also absent in liver 4s RNA from rats fed with a 0.3% L-ethionine diet followed by ten days of normal food. It was identified after dephosphorylation by ultraviolet absorption spectra, cochromatography with authentic material and mass spectra as N6-dimethyladenosine. It is concluded that S-adenosylethionine, the primary product of L-ethionine in the liver, causes strong and selective inhibition of the specific RNA-methylase responsible for adenosine to N6-dimethyl=adenosine methylation in rat liver 4s RNA. Compared to the strong inhibition of N6-dimethyladenosine formation described here, L-ethionine-dependent ethylation of liver 4s RNA is far less efficient. The quantitation of l-methyladenosine, ribothymidine and 3′-terminal adenosine in this 4s RNA as well as its aminoacid acceptor activity is typical for tRNA; hence it may be concluded that N6-dimethyladenosine is a component of rat liver tRNA. This may demonstrate the first evidence for the existence of specifically methyl-deficient mammalian tRNA. A possible correlation between the activity of L-ethionine as a liver carcinogen and its ability to induce the formation of methyl-deficient tRNA by selectively inhibiting the synthesis of N6-dimethyladenosine on the tRNA level in the same organ is discussed. PMID:4414662

  4. Cell cycle specific distribution of killin: evidence for negative regulation of both DNA and RNA synthesis.

    PubMed

    Qiao, Man; Luo, Dan; Kuang, Yi; Feng, Haiyan; Luo, Guangping; Liang, Peng

    2015-01-01

    p53 tumor-suppressor gene is a master transcription factor which controls cell cycle progression and apoptosis. killin was discovered as one of the p53 target genes implicated in S-phase control coupled to cell death. Due to its extreme proximity to pten tumor-suppressor gene on human chromosome 10, changes in epigenetic modification of killin have also been linked to Cowden syndrome as well as other human cancers. Previous studies revealed that Killin is a high-affinity DNA-binding protein with preference to single-stranded DNA, and it inhibits DNA synthesis in vitro and in vivo. Here, co-localization studies of RFP-Killin with either GFP-PCNA or endogenous single-stranded DNA binding protein RPA during S-phase show that Killin always adopts a mutually exclusive punctuated nuclear expression pattern with the 2 accessory proteins in DNA replication. In contrast, when cells are not in S-phase, RFP-Killin largely congregates in the nucleolus where rRNA transcription normally occurs. Both of these cell cycle specific localization patterns of RFP-Killin are stable under high salt condition, consistent with Killin being tightly associated with nucleic acids within cell nuclei. Together, these cell biological results provide a molecular basis for Killin in competitively inhibiting the formation of DNA replication forks during S-phase, as well as potentially negatively regulate RNA synthesis during other cell cycle phases.

  5. Targeting of Arenavirus RNA Synthesis by a Carboxamide-Derivatized Aromatic Disulfide with Virucidal Activity

    PubMed Central

    Sepúlveda, Claudia S.; García, Cybele C.; Levingston Macleod, Jesica M.

    2013-01-01

    Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37°C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed. PMID:24278404

  6. Targeting of arenavirus RNA synthesis by a carboxamide-derivatized aromatic disulfide with virucidal activity.

    PubMed

    Sepúlveda, Claudia S; García, Cybele C; Levingston Macleod, Jesica M; López, Nora; Damonte, Elsa B

    2013-01-01

    Several arenaviruses can cause severe hemorrhagic fever (HF) in humans, representing a public health threat in endemic areas of Africa and South America. The present study characterizes the potent virucidal activity of the carboxamide-derivatized aromatic disulfide NSC4492, an antiretroviral zinc finger-reactive compound, against Junín virus (JUNV), the causative agent of Argentine HF. The compound was able to inactivate JUNV in a time and temperature-dependent manner, producing more than 99 % reduction in virus titer upon incubation with virions at 37 °C for 90 min. The ability of NSC4492-treated JUNV to go through different steps of the multiplication cycle was then evaluated. Inactivated virions were able to bind and enter into the host cell with similar efficiency as control infectious particles. In contrast, treatment with NSC4492 impaired the capacity of JUNV to drive viral RNA synthesis, as measured by quantitative RT-PCR, and blocked viral protein expression, as determined by indirect immunofluorescence. These results suggest that the disulfide NSC4492 targets on the arenavirus replication complex leading to impairment in viral RNA synthesis. Additionally, analysis of VLP produced in NSC4492-treated cells expressing JUNV matrix Z protein revealed that the compound may interact with Z resulting in an altered aggregation behavior of this protein, but without affecting its intrinsic self-budding properties. The potential perspectives of NSC4492 as an inactivating vaccinal compound for pathogenic arenaviruses are discussed.

  7. Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

    PubMed Central

    Deshmukh, Abhijit S.; Mitra, Pallabi; Maruthi, Mulaka

    2016-01-01

    Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5′-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis. PMID:27759017

  8. The SARS-like coronaviruses: the role of bats and evolutionary relationships with SARS coronavirus.

    PubMed

    Balboni, Andrea; Battilani, Mara; Prosperi, Santino

    2012-01-01

    Bats represent an order of great evolutionary success, with elevated geographical diffusion and species diversity. This order harbors viruses of high variability which have a great possibility of acquiring the capacity of infecting other animals,including humans. Bats are the natural reservoir for several viruses genetically closely related to the SARScoronavirus which is the etiological agent of severe acute respiratory syndrome (SARS), a human epidemic which emerged in China in 2002-2003. In the last few years, it has been discovered that the association between coronaviruses and bats is a worldwide phenomenon, and it has been hypothesised that all mammalian coronaviruses were derived from ancestral viruses residing in bats. This review analyzes the role of bats as a reservoir of zoonotic viruses focusing more extensively on SARS-related coronaviruses and taking into account the role of African and European strains in the evolutionary history of these viruses.

  9. NUCLEOLAR AND NUCLEAR RNA SYNTHESIS DURING THE CELL LIFE CYCLE IN MONKEY AND PIG KIDNEY CELLS IN VITRO

    PubMed Central

    Showacre, Jane L.; Cooper, W. G.; Prescott, D. M.

    1967-01-01

    The incorporation of 5-3H-uridine and 5-3H-cytidine into nucleolar and nonnucleolar RNA in the nucleus of monkey and pig kidney cells was measured in vitro during the cell life cycle. Time-lapse cinematographic records were made of cells during asynchronous exponential proliferation, in order to identify the temporal position of individual cells in relation to the preceding mitosis. Immediately following cinematography, cells were labeled with uridine-3H and cytidine-3H for a short period, fixed, and analyzed by radioautography. Since the data permit correlation of the rate of RNA labeling with the position of a cell within the cycle, curves could be constructed describing the rate of RNA synthesis over the average cell cycle. RNA synthesis was absent in early telophase, and rose very abruptly in rate in late telophase and in very early G1 in both the nucleus and the reconstituting nucleolus. Thereafter, through the G1 and S periods the rate of nuclear RNA synthesis rose gradually. When we used a 10-min pulse, there was no detectable change in the rate for nucleolar RNA labeling in monkey kidney cells during G1 or S. When we used a 30-min labeling time, the rate of nucleolar RNA labeling rose gradually in pig kidney cells. With increasing time after mitosis, the data became more variable, which may, in part, be related to the variation in generation times for individual cells. PMID:6039371

  10. A Functional Interface at the rDNA Connects rRNA Synthesis, Pre-rRNA Processing and Nucleolar Surveillance in Budding Yeast

    PubMed Central

    Leporé, Nathalie; Lafontaine, Denis L. J.

    2011-01-01

    Ribogenesis is a multistep error-prone process that is actively monitored by quality control mechanisms. How ribosomal RNA synthesis, pre-rRNA processing and nucleolar surveillance are integrated is unclear. Nor is it understood how defective ribosomes are recognized. We report in budding yeast that, in vivo, the interaction between the transcription elongation factor Spt5 and Rpa190, the largest subunit of RNA polymerase (Pol) I, requires the Spt5 C-terminal region (CTR), a conserved and highly repetitive domain that is reminiscent of the RNA Pol II C-terminal domain (CTD). We show that this sequence is also required for the interaction between Spt5 and Nrd1, an RNA specific binding protein, and an exosome cofactor. Both the Spt4-Spt5, and the Nrd1-Nab3 complexes interact functionally with Rrp6, and colocalize at the rDNA. Mutations in the RNA binding domain of Nrd1, but not in its RNA Pol II CTD-interacting domain, and mutations in the RRM of Nab3 led to the accumulation of normal and aberrant polyadenylated pre-rRNAs. Altogether these results indicate that Nrd1-Nab3 contributes to recruiting the nucleolar surveillance to elongating polymerases to survey nascent rRNA transcripts. PMID:21949810

  11. Control of synthesis of mRNA's for T4 bacteriophage-specific dihydrofolate reductase and deoxycytidylate hydroxymethylase.

    PubMed Central

    Witmer, H; Baros, A; Ende, D; Dosmar, M

    1976-01-01

    A 30 degrees C, functional messengers for dCMP hydroxymethylase first appeared 3 to 6 min postinfection and reached their maximum levels at 12 min. Chloramphenicol, added before the phage, reduced the rate of mRNA accumulation. When the antibiotic was added 6 min postinfection, mRNA levels increased at their normal rate but there was no obvious repression of messenger accumulation. Delaying the addition of drug until 8 or 12 min had progressively less effect on the pattern of hydroxymethylase mRNA metabolism. When chloramphenicol was present from preinfection times or from 6 min postinfection, all hydroxymethylase mRNA's synthesized were stable; at later times, however, the ability of the drug to stabilize mRNA decreased with its ability to delay the turnoff of mRNA production. An overaccumulation of hydroxymethylase mRNA was also seen when phage-specific DNA synthesis was inhibited either by mutational lesion in an essential viral gene or by 5-fluorodeoxyuridine. By min 20 of a DNA-negative program, hydroxymethylase mRNA synthesis was repressed to the point where it no longer compensated for decay. However, a finite level of hydroxymethylase mRNA synthesis was maintained at later times of a DNA-negative infection. Such results indicate that replication of the phage chromosome is necessary but not sufficient for a complete turnoff of hydroxymethylase mRNA production. Functions controlled by the maturation-defective proteins (the products of genes 55 and 33) played only a minor role in the regulation of hydroxymethylase mRNA, metabolism. Thus, we favor the hypothesis that a complete turnoff of hydroxymethylase messenger production requires one or more new proteins as well as an interval of DNA replication. The absence of DNA synthesis had no particular effect upon dihydrofolate reductase messenger production. The preinfection addition of chloramphenicol likewise had little effect on dihydrofolate reductase messenger metabolism. These latter data imply that prior

  12. Actin mRNA levels and actin synthesis during the encystation of Entamoeba invadens.

    PubMed

    Manning-Cela, R; Meraz, M A; Hernandez, J M; Meza, I

    1994-01-01

    Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate the encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeletion occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.

  13. A trans-spliced telomerase RNA dictates telomere synthesis in Trypanosoma brucei

    PubMed Central

    Sandhu, Ranjodh; Sanford, Samantha; Basu, Shrabani; Park, MinA; Pandya, Unnati M; Li, Bibo; Chakrabarti, Kausik

    2013-01-01

    Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation. Although both telomerase activity and the telomerase catalytic protein component, TbTERT, have been identified in the eukaryotic pathogen Trypanosoma brucei, the RNA molecule that dictates telomere synthesis remains unknown. Here, we identify the RNA component of Trypanosoma brucei telomerase, TbTR, and provide phylogenetic and in vivo evidence for TbTR's native folding and activity. We show that TbTR is processed through trans-splicing, and is a capped transcript that interacts and copurifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD), which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences, demonstrating that telomere synthesis in T. brucei is dependent on TbTR. We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments, thus defining TbTR domains that may have important functional implications in telomere synthesis. Identification and characterization of TbTR not only provide important insights into T. brucei telomere functions, which have been shown to play important roles in T. brucei pathogenesis, but also offer T. brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes. PMID:23478302

  14. Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein.

    PubMed

    Lee, Wai-Ming; Ahlquist, Paul

    2003-12-01

    Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive

  15. Antibacterial activity of lichen secondary metabolite usnic acid is primarily caused by inhibition of RNA and DNA synthesis.

    PubMed

    Maciąg-Dorszyńska, Monika; Węgrzyn, Grzegorz; Guzow-Krzemińska, Beata

    2014-04-01

    Usnic acid, a compound produced by various lichen species, has been demonstrated previously to inhibit growth of different bacteria and fungi; however, mechanism of its antimicrobial activity remained unknown. In this report, we demonstrate that usnic acid causes rapid and strong inhibition of RNA and DNA synthesis in Gram-positive bacteria, represented by Bacillus subtilis and Staphylococcus aureus, while it does not inhibit production of macromolecules (DNA, RNA, and proteins) in Escherichia coli, which is resistant to even high doses of this compound. However, we also observed slight inhibition of RNA synthesis in a Gram-negative bacterium, Vibrio harveyi. Inhibition of protein synthesis in B. subtilis and S. aureus was delayed, which suggest indirect action (possibly through impairment of transcription) of usnic acid on translation. Interestingly, DNA synthesis was halted rapidly in B. subtilis and S. aureus, suggesting interference of usnic acid with elongation of DNA replication. We propose that inhibition of RNA synthesis may be a general mechanism of antibacterial action of usnic acid, with additional direct mechanisms, such as impairment of DNA replication in B. subtilis and S. aureus.

  16. Elimination and utilization of oxidized guanine nucleotides in the synthesis of RNA and its precursors.

    PubMed

    Sekiguchi, Takeshi; Ito, Riyoko; Hayakawa, Hiroshi; Sekiguchi, Mutsuo

    2013-03-22

    Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

  17. Nop9 is an RNA binding protein present in pre-40S ribosomes and required for 18S rRNA synthesis in yeast

    PubMed Central

    Thomson, Emma; Rappsilber, Juri; Tollervey, David

    2007-01-01

    Proteomic analyses in yeast have identified a large number of proteins that are associated with preribosomal particles. However, the product of the yeast ORF YJL010C, herein designated as Nop9, failed to be identified in any previous physical or genetic analysis of preribosomes. Here we report that Nop9 is a nucleolar protein, which is associated with 90S and 40S preribosomes. In cells depleted of Nop9p, early cleavages of the 35S pre-rRNA are inhibited, resulting in the nucleolar retention of accumulated precursors and a failure to synthesize 18S rRNA. Nop9 contains multiple pumilio-like putative RNA binding repeats and displays robust in vitro RNA binding activity. The identification of Nop9p as a novel, essential factor in the nuclear maturation of 90S and pre-40S ribosomal subunits shows that the complement of ribosome synthesis factors remains incomplete. PMID:17956976

  18. Recent developments in anti-severe acute respiratory syndrome coronavirus chemotherapy

    PubMed Central

    Barnard, Dale L; Kumaki, Yohichi

    2011-01-01

    Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in early 2003 to cause a very severe acute respiratory syndrome, which eventually resulted in a 10% case-fatality rate. Owing to excellent public health measures that isolated focus cases and their contacts, and the use of supportive therapies, the epidemic was suppressed to the point that further cases have not appeared since 2005. However, despite intensive research since then (over 3500 publications), it remains an untreatable disease. The potential for re-emergence of the SARS-CoV or a similar virus with unknown but potentially serious consequences remains high. This is due in part to the extreme genetic variability of RNA viruses such as the coronaviruses, the many animal reservoirs that seem to be able host the SARS-CoV in which reassortment or recombination events could occur and the ability coronaviruses have to transmit relatively rapidly from species to species in a short period of time. Thus, it seems prudent to continue to explore and develop antiviral chemotherapies to treat SARS-CoV infections. To this end, the various efficacious anti-SARS-CoV therapies recently published from 2007 to 2010 are reviewed in this article. In addition, compounds that have been tested in various animal models and were found to reduce virus lung titers and/or were protective against death in lethal models of disease, or otherwise have been shown to ameliorate the effects of viral infection, are also reported. PMID:21765859

  19. Physiological effects of the fructose-1,6-diphosphate aldolase ts8 mutation on stable RNA synthesis in Escherichia coli.

    PubMed Central

    Singer, M; Walter, W A; Cali, B M; Rouviere, P; Liebke, H H; Gourse, R L; Gross, C A

    1991-01-01

    The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown. PMID:1717436

  20. Synthesis and multiple incorporations of 2'-O-methyl-5-hydroxymethyl-, 5-hydroxymethyl- and 5-formylcytidine monomers into RNA oligonucleotides.

    PubMed

    Balasubramanian, Shankar; Tanpure, Arun Ankush

    2017-09-13

    The synthesis of 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm), 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C) phosphoramidite monomers has been developed. Optimization of mild post-synthetic deprotection conditions enabled the synthesis of RNA containing all four naturally occurring cytosine-modifications (hm5Cm, hm5C, f5C plus 5-methylcytosine). Given the considerable interest in RNA modifications and epitranscriptomics, the availability of synthetic monomers and RNAs containing these modifications will be valuable to elucidate their biological function(s). © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. [Protein and RNA synthesis in larvae of the yellow mealworm beetle (Tenebrio molitor) during cooling and cold acclimatization].

    PubMed

    Gulevskiĭ, A K; Riazantsev, V V; Grishchenkova, E A; Relina, L I

    1996-01-01

    Functioning of protein-synthesis system freeze-avoiding larvae of Tenebrio molitor. (Tenebrinidae family) has been investigated at temperature lowering to 6-8 degrees. It has been shown that mealworms maintain the protein and RNA biosynthesis under cold acclimation (6-8 degrees C). Judging by the angle of inclination of Arrenius of plot reorganization of protein synthesis system takes place in the temperature range 0-10 degrees C. Biosynthesis of proteins at the room and low temperatures is inhibited by cycloheximide while biosynthesis of RNA--by alpha-amanitine.

  2. Enzymatic synthesis of RNAs capped with nucleotide analogues reveals the molecular basis for substrate selectivity of RNA capping enzyme: impacts on RNA metabolism.

    PubMed

    Issur, Moheshwarnath; Bougie, Isabelle; Despins, Simon; Bisaillon, Martin

    2013-01-01

    RNA cap binding proteins have evolved to specifically bind to the N7-methyl guanosine cap structure found at the 5' ends of eukaryotic mRNAs. The specificity of RNA capping enzymes towards GTP for the synthesis of this structure is therefore crucial for mRNA metabolism. The fact that ribavirin triphosphate was described as a substrate of a viral RNA capping enzyme, raised the possibility that RNAs capped with nucleotide analogues could be generated in cellulo. Owing to the fact that this prospect potentially has wide pharmacological implications, we decided to investigate whether the active site of the model Paramecium bursaria Chlorella virus-1 RNA capping enzyme was flexible enough to accommodate various purine analogues. Using this approach, we identified several key structural determinants at each step of the RNA capping reaction and generated RNAs harboring various different cap analogues. Moreover, we monitored the binding affinity of these novel capped RNAs to the eIF4E protein and evaluated their translational properties in cellulo. Overall, this study establishes a molecular rationale for the specific selection of GTP over other NTPs by RNA capping enzyme It also demonstrates that RNAs can be enzymatically capped with certain purine nucleotide analogs, and it also describes the impacts of modified RNA caps on specific steps involved in mRNA metabolism. For instance, our results indicate that the N7-methyl group of the classical N7-methyl guanosine cap is not always indispensable for binding to eIF4E and subsequently for translation when compensatory modifications are present on the capped residue. Overall, these findings have important implications for our understanding of the molecular determinants involved in both RNA capping and RNA metabolism.

  3. Synthesis of (6-(13)C)pyrimidine nucleotides as spin-labels for RNA dynamics.

    PubMed

    Wunderlich, Christoph H; Spitzer, Romana; Santner, Tobias; Fauster, Katja; Tollinger, Martin; Kreutz, Christoph

    2012-05-02

    We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed. © 2012 American Chemical Society

  4. Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.

    PubMed

    Baranovskiy, Andrey G; Babayeva, Nigar D; Zhang, Yinbo; Gu, Jianyou; Suwa, Yoshiaki; Pavlov, Youri I; Tahirov, Tahir H

    2016-05-06

    The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Local synthesis of axonal and presynaptic RNA in squid model systems.

    PubMed

    Eyman, Maria; Cefaliello, Carolina; Ferrara, Eugenia; De Stefano, Rosanna; Lavina, Zeno Scotto; Crispino, Marianna; Squillace, Angela; van Minnen, Jan; Kaplan, Barry B; Giuditta, Antonio

    2007-01-01

    The presence of active systems of protein synthesis in axons and nerve endings raises the question of the cellular origin of the corresponding RNAs. Our present experiments demonstrate that, besides a possible derivation from neuronal cell bodies, axoplasmic RNAs originate in periaxonal glial cells and presynaptic RNAs derive from nearby cells, presumably glial cells. Indeed, in perfused squid giant axons, delivery of newly synthesized RNA to the axon perfusate is strongly stimulated by axonal depolarization or agonists of glial glutamate and acetylcholine receptors. Likewise, incubation of squid optic lobe slices with [3H]uridine leads to a marked accumulation of [3H]RNA in the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons. As the cell bodies of these neurons lie outside the optic lobe, the data demonstrate that presynaptic RNA is locally synthesized, presumably by perisynaptic glial cells. Overall, our results support the view that axons and presynaptic regions are endowed with local systems of gene expression which may prove essential for the maintenance and plasticity of these extrasomatic neuronal domains.

  6. A non-isotopic assay uses bromouridine and RNA synthesis to detect DNA damage responses.

    PubMed

    Hasegawa, Mayu; Iwai, Shigenori; Kuraoka, Isao

    2010-06-17

    Individuals with inherited xeroderma pigmentosum (XP) disorder and Cockayne syndrome (CS) are deficient in nucleotide excision repair and experience hypersensitivity to sunlight. Although there are several diagnostic assays for these disorders, the recovery of RNA synthesis (RRS) assay that can discriminate between XP cells and CS cells is very laborious. Here, we report on a novel non-radioisotope RRS assay that uses bromouridine (a uridine analog) as an alternative to (3)H-uridine. This assay can easily detect RNA polymerase I transcription in nucleoli and RNA polymerase II transcription in nuclei. The non-RI RSS assay also can rapidly detect normal RRS activity in HeLa cells. Thus, this assay is useful as a novel and easy technique for CS diagnosis. Because RRS is thought to be related to transcription-coupled DNA repair, which is triggered by the blockage of transcriptional machinery by DNA lesions, this assay may be of use for analysis of DNA repair, transcription, and/or genetic toxicity. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Biological and genetic analysis of a bovine-like coronavirus isolated from water buffalo (Bubalus bubalis) calves.

    PubMed

    Decaro, Nicola; Martella, Vito; Elia, Gabriella; Campolo, Marco; Mari, Viviana; Desario, Costantina; Lucente, Maria Stella; Lorusso, Alessio; Greco, Grazia; Corrente, Marialaura; Tempesta, Maria; Buonavoglia, Canio

    2008-01-05

    We describe the isolation, biological and genetic characterization of a host-range variant of bovine coronavirus (BCoV) detected in water buffalo (Bubalus bubalis). By conventional and real-time RT-PCR assays, the virus was demonstrated in the intestinal contents of two 20-day-old buffalo calves dead of a severe form of enteritis and in the feces of additional 17 buffalo calves with diarrhea. Virus isolation, hemagglutination and receptor-destroying enzyme activity showed that the buffalo coronavirus (BuCoV) is closely related to BCoV but possesses some different biological properties. Sequence and phylogenetic analyses of the 3' end (9.6 kb) of the BuCoV RNA revealed a genomic organization typical of group 2 coronaviruses. Moreover, the genetic distance between BuCoV and BCoV was proven to be the same or even higher than the distance between other ruminant coronaviruses and BCoV. In conclusion, our data support the existence of a host-range variant of BCoV associated with enteritis in buffaloes.

  8. Novel avian coronavirus and fulminating disease in guinea fowl, France.

    PubMed

    Liais, Etienne; Croville, Guillaume; Mariette, Jérôme; Delverdier, Maxence; Lucas, Marie-Noëlle; Klopp, Christophe; Lluch, Jérôme; Donnadieu, Cécile; Guy, James S; Corrand, Léni; Ducatez, Mariette F; Guérin, Jean-Luc

    2014-01-01

    For decades, French guinea fowl have been affected by fulminating enteritis of unclear origin. By using metagenomics, we identified a novel avian gammacoronavirus associated with this disease that is distantly related to turkey coronaviruses. Fatal respiratory diseases in humans have recently been caused by coronaviruses of animal origin.

  9. Novel Avian Coronavirus and Fulminating Disease in Guinea Fowl, France

    PubMed Central

    Liais, Etienne; Croville, Guillaume; Mariette, Jérôme; Delverdier, Maxence; Lucas, Marie-Noëlle; Klopp, Christophe; Lluch, Jérôme; Donnadieu, Cécile; Guy, James S.; Corrand, Léni; Ducatez, Mariette F.

    2014-01-01

    For decades, French guinea fowl have been affected by fulminating enteritis of unclear origin. By using metagenomics, we identified a novel avian gammacoronavirus associated with this disease that is distantly related to turkey coronaviruses. Fatal respiratory diseases in humans have recently been caused by coronaviruses of animal origin. PMID:24377831

  10. Genetic characterization of coronaviruses from domestic ferrets, Japan.

    PubMed

    Terada, Yutaka; Minami, Shohei; Noguchi, Keita; Mahmoud, Hassan Y A H; Shimoda, Hiroshi; Mochizuki, Masami; Une, Yumi; Maeda, Ken

    2014-02-01

    We detected ferret coronaviruses in 44 (55.7%) of 79 pet ferrets tested in Japan and classified the viruses into 2 genotypes on the basis of genotype-specific PCR. Our results show that 2 ferret coronaviruses that cause feline infectious peritonitis-like disease and epizootic catarrhal enteritis are enzootic among ferrets in Japan.

  11. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain.

    PubMed

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-10-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. The methyltransferase domain of dengue virus protein NS5 ensures efficient RNA synthesis initiation and elongation by the polymerase domain

    PubMed Central

    Potisopon, Supanee; Priet, Stéphane; Collet, Axelle; Decroly, Etienne; Canard, Bruno; Selisko, Barbara

    2014-01-01

    Viral RNA-dependent RNA polymerases (RdRps) responsible for the replication of single-strand RNA virus genomes exert their function in the context of complex replication machineries. Within these replication complexes the polymerase activity is often highly regulated by RNA elements, proteins or other domains of multi-domain polymerases. Here, we present data of the influence of the methyltransferase domain (NS5-MTase) of dengue virus (DENV) protein NS5 on the RdRp activity of the polymerase domain (NS5-Pol). The steady-state polymerase activities of DENV-2 recombinant NS5 and NS5-Pol are compared using different biochemical assays allowing the dissection of the de novo initiation, transition and elongation steps of RNA synthesis. We show that NS5-MTase ensures efficient RdRp activity by stimulating the de novo initiation and the elongation phase. This stimulation is related to a higher affinity of NS5 toward the single-strand RNA template indicating NS5-MTase either completes a high-affinity RNA binding site and/or promotes the correct formation of the template tunnel. Furthermore, the NS5-MTase increases the affinity of the priming nucleotide ATP upon de novo initiation and causes a higher catalytic efficiency of the polymerase upon elongation. The complex stimulation pattern is discussed under the perspective that NS5 adopts several conformations during RNA synthesis. PMID:25209234

  13. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  14. Biodiversity impact of host interferon-stimulated-gene-product 15 on the coronavirus Papain-like protease deISGylase functions

    USDA-ARS?s Scientific Manuscript database

    Coronaviruses are single-stranded, positive sense RNA viruses whose members have severe impact on human health and cause significant economic hardships. Some pertinent examples include severe acute and Middle East respiratory syndromes (SARS-CoV; MERS-CoV), porcine epidemic diarrhea virus (PEDV), an...

  15. Structure, Function, and Evolution of Coronavirus Spike Proteins

    PubMed Central

    Li, Fang

    2017-01-01

    The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding functions from other viruses and host cells. PMID:27578435

  16. The large genome segment of dsRNA bacteriophage phi6 is the key regulator in the in vitro minus and plus strand synthesis.

    PubMed Central

    Frilander, M; Poranen, M; Bamford, D H

    1995-01-01

    Bacteriophage phi6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (L, M, S). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4, and P7. Empty preformed procapsids are able to package the plus-sense single-stranded RNA (ssRNA) of each genome segment, to synthesize the corresponding minus strands ("replication") to form dsRNA segments, and to continue to the plus strand synthesis ("transcription") in which the dsRNA segments are used as templates in production of plus-sense ssRNA. In this study, we have investigated the requirements for the switch-on of minus and plus strand syntheses. We show that there exists an inverse relationship between regulation of the ssRNA packaging and minus strand synthesis. The packaging of single-stranded l, which has previously been shown to be packaged as the last, is the necessary signal for the onset of the minus strand synthesis. The absolute requirement for plus strand synthesis is minus strand synthesis of l, but in addition, the minus strand synthesis of m and the packaging of s segment are needed for efficient plus strand synthesis. Furthermore, the second nucleotide at the 5'-end of each segment regulates the extent of the transcription. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:7489512

  17. Development of animal models against emerging coronaviruses: From SARS to MERS coronavirus.

    PubMed

    Sutton, Troy C; Subbarao, Kanta

    2015-05-01

    Two novel coronaviruses have emerged to cause severe disease in humans. While bats may be the primary reservoir for both viruses, SARS coronavirus (SARS-CoV) likely crossed into humans from civets in China, and MERS coronavirus (MERS-CoV) has been transmitted from camels in the Middle East. Unlike SARS-CoV that resolved within a year, continued introductions of MERS-CoV present an on-going public health threat. Animal models are needed to evaluate countermeasures against emerging viruses. With SARS-CoV, several animal species were permissive to infection. In contrast, most laboratory animals are refractory or only semi-permissive to infection with MERS-CoV. This host-range restriction is largely determined by sequence heterogeneity in the MERS-CoV receptor. We describe animal models developed to study coronaviruses, with a focus on host-range restriction at the level of the viral receptor and discuss approaches to consider in developing a model to evaluate countermeasures against MERS-CoV. Copyright © 2015. Published by Elsevier Inc.

  18. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-05

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA.

  19. LKB1 promotes cell survival by modulating TIF-IA-mediated pre-ribosomal RNA synthesis under uridine downregulated conditions

    PubMed Central

    Liu, Xiuju; Huang, Henry; Wilkinson, Scott C.; Zhong, Diansheng; Khuri, Fadlo R.; Fu, Haian; Marcus, Adam; He, Yulong; Zhou, Wei

    2016-01-01

    We analyzed the mechanism underlying 5-aminoimidazole-4-carboxamide riboside (AICAR) mediated apoptosis in LKB1-null non-small cell lung cancer (NSCLC) cells. Metabolic profile analysis revealed depletion of the intracellular pyrimidine pool after AICAR treatment, but uridine was the only nucleotide precursor capable of rescuing this apoptosis, suggesting the involvement of RNA metabolism. Because half of RNA transcription in cancer is for pre-ribosomal RNA (rRNA) synthesis, which is suppressed by over 90% after AICAR treatment, we evaluated the role of TIF-IA-mediated rRNA synthesis. While the depletion of TIF-IA by RNAi alone promoted apoptosis in LKB1-null cells, the overexpression of a wild-type or a S636A TIF-IA mutant, but not a S636D mutant, attenuated AICAR-induced apoptosis. In LKB1-null H157 cells, pre-rRNA synthesis was not suppressed by AICAR when wild-type LKB1 was present, and cellular fractionation analysis indicated that TIF-IA quickly accumulated in the nucleus in the presence of a wild-type LKB1 but not a kinase-dead mutant. Furthermore, ectopic expression of LKB1 was capable of attenuating AICAR-induced death in AMPK-null cells. Because LKB1 promotes cell survival by modulating TIF-IA-mediated pre-rRNA synthesis, this discovery suggested that targeted depletion of uridine related metabolites may be exploited in the clinic to eliminate LKB1-null cancer cells. PMID:26506235

  20. An RNA sequence of hundreds of nucleotides at the 5' end of poliovirus RNA is involved in allowing viral protein synthesis.

    PubMed Central

    Trono, D; Andino, R; Baltimore, D

    1988-01-01

    Twenty-one mutations were engineered in the 5' noncoding region of poliovirus type 1 RNA, using an infectious cDNA copy of the viral genome. RNA was made from these constructs and used to transfect HeLa cells. Viable virus was recovered from 12 of these transfection experiments, including six strains with a recognizable phenotype, mapping in four different regions. One mutant of each site was studied in more detail. Mutant 5NC-11, having a 4-base insertion at nucleotide 70, was dramatically deficient in RNA synthesis, suggesting that the far 5' end of the genome is primarily involved in one or more steps of RNA replication. Mutants 5NC-13, 5NC-114, and 5NC-116, mapping at nucleotides 224, 270, and 392, respectively, showed a similar behavior; they made very little viral protein, they did not inhibit host cell translation, and they synthesized a significant amount of viral RNA, although with some delay compared with wild type. These three mutants were efficiently complemented by all other poliovirus mutants tested, except those with lesions in protein 2A. Our results imply that these three mutants map in a region (region P) primarily involved in viral protein synthesis and that their inability to shut off host cell translation is secondary to a quantitative defect in protein 2A. The exact function of region P is still to be determined, but our data supports the hypothesis of a single functional module allowing viral protein synthesis and extending over several hundred nucleotides. Images PMID:2836612

  1. Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety.

    PubMed

    Shanmugasundaram, Muthian; Charles, Irudaya; Kore, Anilkumar R

    2016-03-15

    The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-O-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. De Novo mRNA Synthesis Is Required for Both Consolidation and Reconsolidation of Fear Memories in the Amygdala

    ERIC Educational Resources Information Center

    Duvarci, Sevil; Nader, Karim; LeDoux, Joseph E.

    2008-01-01

    Memory consolidation is the process by which newly learned information is stabilized into long-term memory (LTM). Considerable evidence indicates that retrieval of a consolidated memory returns it to a labile state that requires it to be restabilized. Consolidation of new fear memories has been shown to require de novo RNA and protein synthesis in…

  3. Serum concentration and increased temperature alter Mayaro virus RNA and protein synthesis in Aedes albopictus (mosquito)-infected cells.

    PubMed

    Motta, M C; Fournier, M V; Carvalho, M G

    1995-01-01

    We have previously shown the inhibition of Mayaro virus multiplication in Aedes albopictus-infected cells maintained at a supraoptimal temperature for growth (37 degrees C) and a stimulation of virus production in response to high serum concentrations in the incubation medium. In the present study, we addressed the question of how the effect of continuous heat stress and high serum concentration soon after infection interfere with virus macromolecule synthesis. Cells maintained at 28 degrees C in the presence of 2% serum synthesized a viral genomic RNA of 12 kb and a subgenomic RNA of 5.2 kb 6 h postinfection. Analysis of the protein profile showed the presence of the viral nucleocapsid protein of 34 kDa (P34). However, if infected cells were maintained at 37 degrees C, a smear starting immediately below the 5.2-kb RNA was noticed and the viral P34 was not detected by SDS-PAGE. Addition of 10% serum to the growth medium of infected cells maintained at 37 degrees C results in a viral RNA profile and protein synthesis similar to those observed in cultures kept at 28 degrees C, i.e., the smear was not observed and the P34 protein was detected. The results suggest that the inhibition of virus multiplication by temperature may be related to the inhibition of viral nonstructural protein synthesis early during infection. The presence of high serum levels in the incubation medium protects macromolecule synthesis against heat stress.

  4. De Novo mRNA Synthesis Is Required for Both Consolidation and Reconsolidation of Fear Memories in the Amygdala

    ERIC Educational Resources Information Center

    Duvarci, Sevil; Nader, Karim; LeDoux, Joseph E.

    2008-01-01

    Memory consolidation is the process by which newly learned information is stabilized into long-term memory (LTM). Considerable evidence indicates that retrieval of a consolidated memory returns it to a labile state that requires it to be restabilized. Consolidation of new fear memories has been shown to require de novo RNA and protein synthesis in…

  5. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed

    Menninger, J R; Otto, D P

    1982-05-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation.

  6. Erythromycin, carbomycin, and spiramycin inhibit protein synthesis by stimulating the dissociation of peptidyl-tRNA from ribosomes.

    PubMed Central

    Menninger, J R; Otto, D P

    1982-01-01

    In mutant Escherichia coli with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase; EC 3.1.1.29), peptidyl-tRNA accumulates at the nonpermissive temperature (40 degrees C), and the cells die. These consequences of high temperature were enhanced if the cells were first treated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30 degrees C). Since peptidyl-tRNA hydrolase in he mutant cells is inactivated rapidly and irreversibly at 40 degrees C, the enhanced accumulation of peptidyl-tRNA and killing were the result of enhanced dissociation, stimulated by the antibiotics, of peptidyl-tRNA from ribosomes. The implications of these findings for inhibition of cell growth and protein synthesis are discussed. Certain alternative interpretations are shown to be inconsistent with the relevant data. Previous conflicting observations on the effects of macrolide antibiotics are explained in terms of our observations. We conclude that erythromycin, carbomycin, and spiramycin (and probably all macrolides) have as a primary mechanism of action the stimulation of dissociation of peptidyl-tRNA from ribosomes, probably during translocation. PMID:6179465

  7. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells

    PubMed Central

    Maddocks, Oliver D.K.; Labuschagne, Christiaan F.; Adams, Peter D.; Vousden, Karen H.

    2016-01-01

    Summary Crosstalk between cellular metabolism and the epigenome regulates epigenetic and metabolic homeostasis and normal cell behavior. Changes in cancer cell metabolism can directly impact epigenetic regulation and promote transformation. Here we analyzed the contribution of methionine and serine metabolism to methylation of DNA and RNA. Serine can contribute to this pathway by providing one-carbon units to regenerate methionine from homocysteine. While we observed this contribution under methionine-depleted conditions, unexpectedly, we found that serine supported the methionine cycle in the presence and absence of methionine through de novo ATP synthesis. Serine starvation increased the methionine/S-adenosyl methionine ratio, decreasing the transfer of methyl groups to DNA and RNA. While serine starvation dramatically decreased ATP levels, this was accompanied by lower AMP and did not activate AMPK. This work highlights the difference between ATP turnover and new ATP synthesis and defines a vital function of nucleotide synthesis beyond making nucleic acids. PMID:26774282

  8. Reliable semi-synthesis of hydrolysis-resistant 3′-peptidyl-tRNA conjugates containing genuine tRNA modifications

    PubMed Central

    Graber, Dagmar; Moroder, Holger; Steger, Jessica; Trappl, Krista; Polacek, Norbert; Micura, Ronald

    2010-01-01

    The 3′-peptidyl-tRNA conjugates that possess a hydrolysis-resistant ribose-3′-amide linkage instead of the natural ester linkage would represent valuable substrates for ribosomal studies. Up to date, access to these derivatives is severely limited. Here, we present a novel approach for the reliable synthesis of non-hydrolyzable 3′-peptidyl-tRNAs that contain all the respective genuine nucleoside modifications. In short, the approach is based on tRNAs from natural sources that are site-specifically cleaved within the TΨC loop by using DNA enzymes to obtain defined tRNA 5′-fragments carrying the modifications. After dephosphorylation of the 2′,3′-cyclophosphate moieties from these fragments, they are ligated to the respective 3′-peptidylamino-tRNA termini that were prepared following the lines of a recently reported solid-phase synthesis. By this novel concept, non-hydrolyzable 3′-peptidyl-tRNA conjugates possessing all natural nucleoside modifications are accessible in highly efficient manner. PMID:20525967

  9. Have organic interstellar grains redox-catalyzed RNA and other synthesis from cometary precursors ?

    NASA Astrophysics Data System (ADS)

    Krueger, F. R.; Kissel, J.; Werther, W.; Schmid, E. R.

    The recent in-situ investigations of cosmic dust clearly show, that the only building blocks of life therein are nucleobases. All the other building blocks are just found as precursors in cometary dust. They themselves must be formed from precursors by hydrolysis in liquid water, like phosphates from phosphides, sugars (stabilized at mineralic surfaces) may be from polyines, and amino and lipidic acids from nitriles. Nevertheless, the self-organized synthesis of polymers like RNA's and peptides needs, i.a., additional redox-catalysis. These catalysts act in stabilizing and transmitting single electrons and/or holes in chemical reactions. At mineralic surfaces transition metals like FeII/III may take the task. However, once life has emancipated itself from minerals redox catalysts should be furtheron present yet organically without prior encoding for synthesis - as the code itself must have been evolved already before the encoded enzymes can act. However, there may be another solution for this enigma: Except archeae, all stems of life make use of quinone-type co-enzymes of the PQQ (pyrrolo-quinoline-quinone) type of redox catalysts. Interstellar dust reaching the interior of the solar system consists at least of homologuous polymers which may convert to PQQ types in liquid water, after being decelerated in the upper atmosphere. Due to their enormous radiation stability, these types are the natural end-products after long interstellar trips. They can withstand the collision processes with atmospheric molecules, then being washed out.

  10. Editing of the Sendai virus P/C mRNA by G insertion occurs during mRNA synthesis via a virus-encoded activity.

    PubMed Central

    Vidal, S; Curran, J; Kolakofsky, D

    1990-01-01

    Two forms of the Sendai virus P/C mRNA have been predicted: one an exact copy of the viral genome, and the other with a single G insertion within a run of three G's. We directly cloned the mRNA or portions of it containing the insertion site and screened the resulting colonies with oligonucleotides that could distinguish the presence of three or four G's at this position. We found that 31% of the mRNAs did in fact contain the predicted insertion, whereas the viral genomes contained no heterogeneity at this position. A smaller fraction (7%) of the mRNA contained two to eight G's inserted at this position. The insertions also took place during RNA synthesis in vitro with purified virions but were not detected when the mRNA was expressed in vivo via a vaccinia virus recombinant. When the Sendai virus- and vaccinia virus-derived P/C mRNAs were coexpressed in the same cells under conditions in which each could be distinguished, those from the Sendai genome were altered as before, but those from the vaccinia virus genome remained unaltered. The activity that alters the mRNA is therefore likely to be coded for by the virus and cannot function in trans. Images PMID:1688384

  11. Middle East Respiratory Syndrome Coronavirus Accessory Protein 4a Is a Type I Interferon Antagonist

    PubMed Central

    Niemeyer, Daniela; Zillinger, Thomas; Muth, Doreen; Zielecki, Florian; Horvath, Gabor; Suliman, Tasnim; Barchet, Winfried; Weber, Friedemann

    2013-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory infection with as yet unclear epidemiology. We previously showed that MERS-CoV counteracts parts of the innate immune response in human bronchiolar cells. Here we analyzed accessory proteins 3, 4a, 4b, and 5 for their abilities to inhibit the type I interferon response. Accessory protein 4a was found to block interferon induction at the level of melanoma differentiation-associated protein 5 (MDA5) activation presumably by direct interaction with double-stranded RNA. PMID:24027320

  12. Clay catalyzed RNA synthesis under Martian conditions: Application for Mars return samples.

    PubMed

    Joshi, Prakash C; Dubey, Krishna; Aldersley, Michael F; Sausville, Meaghen

    2015-06-26

    Catalysis by montmorillonites clay minerals is regarded as a feasible mechanism for the abiotic production and polymerization of key biomolecules on early Earth. We have investigated a montmorillonite-catalyzed reaction of the 5'-phosphorimidazolide of nucleosides as a model to probe prebiotic synthesis of RNA-type oligomers. Here we show that this model is specific for the generation of RNA oligomers despite deoxy-mononucleotides adsorbing equally well onto the montmorillonite catalytic surfaces. Optimum catalytic activity was observed over a range of pH (6-9) and salinity (1 ± 0.2 M NaCl). When the weathering steps of early Earth that generated catalytic montmorillonite were modified to meet Martian soil conditions, the catalytic activity remained intact without altering the surface layer charge. Additionally, the formation of oligomers up to tetramer was detected using as little as 0.1 mg of Na⁺-montmorillonite, suggesting that the catalytic activity of a Martian clay return sample can be investigated with sub-milligram scale samples. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Synthesis and evaluation of fluorescent cap analogues for mRNA labelling

    PubMed Central

    Ziemniak, Marcin; Szabelski, Mariusz; Lukaszewicz, Maciej; Nowicka, Anna; Darzynkiewicz, Edward; Rhoads, Robert E.; Wieczorek, Zbigniew; Jemielity, Jacek

    2013-01-01

    We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-β methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins. PMID:24273643

  14. Identification and sequence of the initiation site for rat 45S ribosomal RNA synthesis.

    PubMed Central

    Harrington, C A; Chikaraishi, D M

    1983-01-01

    The transcription initiation site for rat 45S precursor ribosomal RNA synthesis was determined by nuclease protection mapping with two single-strand endonucleases. S1 and mung bean, and one single-strand exonuclease, ExoVII. These experiments were performed with end-labeled ribosomal DNA from double-stranded pBR322 recombinants and from single-stranded M13 recombinants. Results from experiments using both kinds of DNA and all three enzymes showed that the 5' end of 45S RNA mapped to a unique site 125 bases upstream from the Hind III site in the ribosomal DNA gene. The DNA surrounding this site (designated +1) was sequenced from -281 to +641. The entire sequence of this region shows extensive homology to the comparable region of mouse. This includes three stretches of T residues in the non-coding strand between +300 and +630. Two sets of direct repeats adjacent to these T-rich regions are observed. Comparison of the mouse and human ribosomal DNA transcription initiation sites with the rat sequence reported in this paper demonstrates a conserved sequence at +2 to +16, CTGACACGCTGTCCT. This suggests that this region may be important for the initiation of transcription on mammalian ribosomal DNAs. Images PMID:6304628

  15. Alternative bases in the RNA world: the prebiotic synthesis of urazole and its ribosides

    NASA Technical Reports Server (NTRS)

    Kolb, V. M.; Dworkin, J. P.; Miller, S. L.

    1994-01-01

    Urazole is a five-membered heterocyclic compound which is isosteric with uracil's hydrogen-bonding segment. Urazole reacts spontaneoulsy with ribose (and other aldoses) to give a mixture of four ribosides: alpha and beta pyranosides and furanosides. This reaction occurs in aqueous solution at mild temperatures. Thermodynamic and kinetic parameters for the reaction of urazole with ribose were determined. In contrast, uracil is completely unreactive with ribose under these conditions. Urazole's unusual reactivity is ascribed to the hydrazine portion of the molecule. Urazole can be synthesized from biuret and hydrazine under prebiotic conditions. The prebiotic synthesis of guanazole, which is isosteric in part to diaminopyrimidine and cytosine, is accomplished from dicyandiamide and hydrazine. Kinetic parameters for both prebiotic reactions were measured. Urazole and guanazole are transparent in the UV, which would be a favorable property in the absence of an ozone layer on the early Earth. Urazole makes hydrogen bonds with adenine in DMSO similar to those of uracil, as established by H NMR. All of these properties make urazole an attractive potential precursor to uracil and guanazole a potential precursor to cytosine in the RNA or pre-RNA world.

  16. Progress in demonstrating total homochiral selection in montmorillonite-catalyzed RNA synthesis.

    PubMed

    Joshi, Prakash C; Aldersley, Michael F; Ferris, James P

    2011-10-07

    The Na(+)-montmorillonite-catalyzed reactions of 5'-phosphorimidazolides of nucleosides generates RNA oligomers. The question arises as to how chiral selectivity was introduced into this biopolymer from a simple chemical system. We have demonstrated homochiral selection in quaternary reactions of a racemic mixture of D,L-ImpA and D,L-ImpU on Na(+)-montmorillonite. The dimer, trimer, tetramer and pentamer fractions were investigated for homochiral selection. The products were collected via ion exchange HPLC and their terminal 5'-phosphate was cleaved by alkaline phosphatase. These fractions were analyzed by reverse phase HPLC for the identification of homochiral and heterochiral isomers. Encouraged by favorable homochiral excesses of dimer (63.5 ± 0.8%) and trimer (74.3 ± 1.7%), the study was extended to the analysis of higher oligomers. The tetramer and pentamer of the quaternary reaction were separated into 26 and 22 isomers, respectively, on a reverse phase column. Their co-elution with those formed in the binary reactions of d-ImpA and D-ImpU on Na(+)-montmorillonite revealed 92.7 ± 2.0% and 97.2 ± 0.5% homochirality of the tetramer and pentamer, respectively. These results suggest that Na(+)-montmorillonite not only catalyzes the prebiotic synthesis of RNA but it also facilitates homochiral selection.

  17. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia

    PubMed Central

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-01-01

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3′-to-5′ and 5′-to-3′ transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism. PMID:25593325

  18. Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia.

    PubMed

    Carradec, Quentin; Götz, Ulrike; Arnaiz, Olivier; Pouch, Juliette; Simon, Martin; Meyer, Eric; Marker, Simone

    2015-02-18

    In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3'-to-5' and 5'-to-3' transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.

  19. Early gene expression in bacteriophage T7. I. In vivo synthesis, inactivation, and translational utilization of early mRNA's.

    PubMed Central

    Hercules, K; Jovanovich, S; Sauerbrier, W

    1976-01-01

    In vivo decay rates for the individual T7 early mRNA species were determined. The physical half-lives, measured at 37 C, range from 1.1 min for gene 0.7 RNA to 4.5 min for gene 0.3 RNA. Physical half-lives, as observed after rifampin inhibition of RNA synthesis and polyacylamide electrophoresis of RNAs, are approximately 30% longer than functional half-lives, as observed by 14C-labeled amino acid uptake into individual T7 early proteins. The different RNA species are synthesized at grossly different rates, 0.3 RNA at four times the rate of 1.0 RNA, 0.7 RNA at twice the rate, and 1.1 and 1.3 RNAs at about the same or a slightly lower rate than 1.0 RNA. Rho-factor-mediated termination of transcription behind genes 0.3, 0.7, and perhaps behind 1.0 is inferred from these data. The in vivo translational utilization of the individual T7 early-message species was found to vary by not more than a factor of 2. Images PMID:1255850

  20. Coronavirus Infection and Diversity in Bats in the Australasian Region.

    PubMed

    Smith, C S; de Jong, C E; Meers, J; Henning, J; Wang, L- F; Field, H E

    2016-03-01

    Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

  1. Characterization of a novel betacoronavirus related to middle East respiratory syndrome coronavirus in European hedgehogs.

    PubMed

    Corman, Victor Max; Kallies, René; Philipps, Heike; Göpner, Gertraude; Müller, Marcel Alexander; Eckerle, Isabella; Brünink, Sebastian; Drosten, Christian; Drexler, Jan Felix

    2014-01-01

    Bats are known to host viruses closely related to important human coronaviruses (HCoVs), such as HCoV-229E, severe-acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV). As RNA viruses may coevolve with their hosts, we sought to investigate the closest sister taxon to bats, the Eulipotyphla, and screened European hedgehogs (Erinaceus europaeus) from Germany for CoV by nested reverse transcriptase PCR. A novel betacoronavirus species in a phylogenetic sister relationship to MERS-CoV and clade c bat CoVs was detected and characterized on the whole-genome level. A total of 58.9% of hedgehog fecal specimens were positive for the novel CoV (EriCoV) at 7.9 log10 mean RNA copies per ml. EriCoV RNA concentrations were higher in the intestine than in other solid organs, blood, or urine. Detailed analyses of the full hedgehog intestine showed the highest EriCoV concentrations in lower gastrointestinal tract specimens, compatible with viral replication in the lower intestine and fecal-oral transmission. Thirteen of 27 (48.2%) hedgehog sera contained non-neutralizing antibodies against MERS-CoV. The animal origins of this betacoronavirus clade that includes MERS-CoV may thus include both bat and nonbat hosts.

  2. Characterization of a Novel Betacoronavirus Related to Middle East Respiratory Syndrome Coronavirus in European Hedgehogs

    PubMed Central

    Corman, Victor Max; Kallies, René; Philipps, Heike; Göpner, Gertraude; Müller, Marcel Alexander; Eckerle, Isabella; Brünink, Sebastian

    2014-01-01

    Bats are known to host viruses closely related to important human coronaviruses (HCoVs), such as HCoV-229E, severe-acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome CoV (MERS-CoV). As RNA viruses may coevolve with their hosts, we sought to investigate the closest sister taxon to bats, the Eulipotyphla, and screened European hedgehogs (Erinaceus europaeus) from Germany for CoV by nested reverse transcriptase PCR. A novel betacoronavirus species in a phylogenetic sister relationship to MERS-CoV and clade c bat CoVs was detected and characterized on the whole-genome level. A total of 58.9% of hedgehog fecal specimens were positive for the novel CoV (EriCoV) at 7.9 log10 mean RNA copies per ml. EriCoV RNA concentrations were higher in the intestine than in other solid organs, blood, or urine. Detailed analyses of the full hedgehog intestine showed the highest EriCoV concentrations in lower gastrointestinal tract specimens, compatible with viral replication in the lower intestine and fecal-oral transmission. Thirteen of 27 (48.2%) hedgehog sera contained non-neutralizing antibodies against MERS-CoV. The animal origins of this betacoronavirus clade that includes MERS-CoV may thus include both bat and nonbat hosts. PMID:24131722

  3. Respiratory disease associated with bovine coronavirus infection in cattle herds in Southern Italy.

    PubMed

    Decaro, Nicola; Campolo, Marco; Desario, Costantina; Cirone, Francesco; D'Abramo, Maria; Lorusso, Eleonora; Greco, Grazia; Mari, Viviana; Colaianni, Maria Loredana; Elia, Gabriella; Martella, Vito; Buonavoglia, Canio

    2008-01-01

    Four outbreaks of bovine respiratory disease (BRD) associated with bovine coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2-3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 x 10(2) and 7.50 x 10(7) RNA copies/microl of template. Bovine coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

  4. Open reading frames 1a and 1b of the porcine reproductive and respiratory syndrome virus (PRRSV) collaboratively initiate viral minus-strand RNA synthesis.

    PubMed

    Tang, Yan-Dong; Fang, Qiong-Qiong; Liu, Ji-Ting; Wang, Tong-Yun; Wang, Yu; Tao, Ye; Liu, Yong-Gang; Cai, Xue-Hui

    2016-09-02

    The porcine reproductive and respiratory syndrome virus (PRRSV) causes a persistent threat to the swine industry, especially when highly pathogenic PRRSV (HP-PRRSV) emerges. Previous studies have indicated that PRRSV RNA synthesis was correlated with HP-PRRSV virulence. PRRSV RNA synthesis includes genomic RNA and sub-genomic mRNA, and these processes require minus-strand RNA as a template. However, the mechanisms involved in PRRSV minus-strand RNA synthesis are not fully understood. A mini-genome system can be used to assess viral replication mechanisms and to evaluate the effects of potential antiviral drugs on viral replicase activities. In this study, we developed a mini-genome system that uses firefly luciferase as a reporter. Based on this system, we found that PRRSV RNA-dependent RNA polymerase nsp9 alone failed to activate virus minus-strand RNA synthesis. We also demonstrated that combinations of open reading frames 1a (ORF1a) and ORF1b are necessary for viral minus-strand RNA synthesis.

  5. Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus.

    PubMed

    Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Lau, Candy C Y; Tsang, Alan K L; Lau, John H N; Bai, Ru; Teng, Jade L L; Tsang, Chris C C; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung; Yuen, Kwok-Yung

    2012-04-01

    Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination.

  6. Discovery of Seven Novel Mammalian and Avian Coronaviruses in the Genus Deltacoronavirus Supports Bat Coronaviruses as the Gene Source of Alphacoronavirus and Betacoronavirus and Avian Coronaviruses as the Gene Source of Gammacoronavirus and Deltacoronavirus

    PubMed Central

    Woo, Patrick C. Y.; Lau, Susanna K. P.; Lam, Carol S. F.; Lau, Candy C. Y.; Tsang, Alan K. L.; Lau, John H. N.; Bai, Ru; Teng, Jade L. L.; Tsang, Chris C. C.; Wang, Ming; Zheng, Bo-Jian; Chan, Kwok-Hung

    2012-01-01

    Recently, we reported the discovery of three novel coronaviruses, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13, which were identified as representatives of a novel genus, Deltacoronavirus, in the subfamily Coronavirinae. In this territory-wide molecular epidemiology study involving 3,137 mammals and 3,298 birds, we discovered seven additional novel deltacoronaviruses in pigs and birds, which we named porcine coronavirus HKU15, white-eye coronavirus HKU16, sparrow coronavirus HKU17, magpie robin coronavirus HKU18, night heron coronavirus HKU19, wigeon coronavirus HKU20, and common moorhen coronavirus HKU21. Complete genome sequencing and comparative genome analysis showed that the avian and mammalian deltacoronaviruses have similar genome characteristics and structures. They all have relatively small genomes (25.421 to 26.674 kb), the smallest among all coronaviruses. They all have a single papain-like protease domain in the nsp3 gene; an accessory gene, NS6 open reading frame (ORF), located between the M and N genes; and a variable number of accessory genes (up to four) downstream of the N gene. Moreover, they all have the same putative transcription regulatory sequence of ACACCA. Molecular clock analysis showed that the most recent common ancestor of all coronaviruses was estimated at approximately 8100 BC, and those of Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus were at approximately 2400 BC, 3300 BC, 2800 BC, and 3000 BC, respectively. From our studies, it appears that bats and birds, the warm blooded flying vertebrates, are ideal hosts for the coronavirus gene source, bats for Alphacoronavirus and Betacoronavirus and birds for Gammacoronavirus and Deltacoronavirus, to fuel coronavirus evolution and dissemination. PMID:22278237

  7. Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

    SciTech Connect

    Takeda, N.; Kuhn, R.J.; Yang, C.F.; Takegami, T.; Wimmer, E.

    1986-10-01

    An in vitro poliovirus RNA-synthesizing system derived from a crude membrance fraction of infected HeLa cells was used to analyze the mechanism of initiation of poliovirus plus-strand RNA synthesis. This system contains an activity that synthesizes the nucleotidyl proteins VPg-pU and VPg-pUpU. These molecules represent the 5'-terminal structure of nascent RNA molecules and of virion RNA. The membranous replication complex is also capable of synthesizing mucleotidyl proteins containing nine or more of the poliovirus 5'-proximal nucleotides as assayed by the formation of the RNase T/sub 1/-resistant oligonucleotide VPg-pUUAAAACAGp or by fingerprint analysis of the in vitro-synthesized /sup 32/P-RNA. Incubation of preformed VPg-pUpU with unlabeled nucleoside triphosphates resulted in the formation of VPg-pUUAAAACAGp. This reaction, which appeared to be an elongation of VPg-pUpU, was stimulated by the addition of a soluble fraction (S-10) obtained from uninfected HeLa cells. Preformed VPg-pU could be chased into VPg-pUpU in the presence of UTP. The data are consistent with a model that VPg-pU can function as a primer for poliovirus plus-strand RNA synthesis in the membranous replication complex and that the elongation reaction may be stimulated by a host cellular factor.

  8. Avian Coronavirus in Wild Aquatic Birds▿†‡

    PubMed Central

    Chu, Daniel K. W.; Leung, Connie Y. H.; Gilbert, Martin; Joyner, Priscilla H.; Ng, Erica M.; Tse, Tsemay M.; Guan, Yi; Peiris, Joseph S. M.; Poon, Leo L. M.

    2011-01-01

    We detected a high prevalence (12.5%) of novel avian coronaviruses in aquatic wild birds. Phylogenetic analyses of these coronaviruses suggest that there is a diversity of gammacoronaviruses and deltacoronaviruses circulating in birds. Gammacoronaviruses were found predominantly in Anseriformes birds, whereas deltacoronaviruses could be detected in Ciconiiformes, Pelecaniformes, and Anseriformes birds in this study. We observed that there are frequent interspecies transmissions of gammacoronaviruses between duck species. In contrast, deltacoronaviruses may have more stringent host specificities. Our analysis of these avian viral and host mitochondrial DNA sequences also suggests that some, but not all, coronaviruses may have coevolved with birds from the same order. PMID:21957308

  9. M-phase-specific protein kinase from mitotic sea urchin eggs: cyclic activation depends on protein synthesis and phosphorylation but does not require DNA or RNA synthesis.

    PubMed

    Arion, D; Meijer, L

    1989-08-01

    Histone H1 kinase (H1K) undergoes a transient activation at each early M phase of both meiotic and mitotic cell cycles. The mechanisms underlying the transient activation of this protein kinase were investigated in mitotic sea urchin eggs. Translocation of active H1K from particulate to soluble fraction does not seem to be responsible for this activation. H1K activation cannot be accounted for by the transient disappearance of a putative H1K inhibitor present in soluble fractions of homogenates. Aphidicolin, an inhibitor of DNA synthesis, and actinomycin D, an inhibitor of RNA synthesis, do not impede the transient appearance of H1K activity. H1K activation therefore does not require DNA or RNA synthesis. Fertilization triggers a rise in intracellular pH responsible for the increase of protein synthesis. H1K activation is highly dependent on the intracellular pH. Ammonia triggers an increase of intracellular pH and stimulates protein synthesis and H1K activation. Acetate lowers the intracellular pH, decreases protein synthesis, and blocks H1K activation. Protein synthesis is an absolute requirement for H1K activation as demonstrated by their identical sensitivities to emetine concentration and to time of emetine addition. About 60 min after fertilization, H1K activation and cleavage become independent of protein synthesis. The concentration of p34, a homolog of the yeast cdc2 gene product which has been recently shown to be a subunit of H1K, does not vary during the cell cycle and remains constant in emetine-treated cells. H1K activation thus requires the synthesis of either a p34 postranslational modifying enzyme or another subunit. Finally, phosphatase inhibitors and ATP slow down in the in vitro inactivation rate of H1K. These results suggest that a subunit or an activator of H1K is stored as an mRNA in the egg before mitosis and that full activation of H1K requires a phosphorylation.

  10. Identification and Survey of a Novel Avian Coronavirus in Ducks

    PubMed Central

    Chen, Gui-Qian; Zhuang, Qing-Ye; Wang, Kai-Cheng; Liu, Shuo; Shao, Jian-Zhong; Jiang, Wen-Ming; Hou, Guang-Yu; Li, Jin-Ping; Yu, Jian-Min; Li, Yi-Ping; Chen, Ji-Ming

    2013-01-01

    The rapid discovery of novel viruses using next generation sequencing (NGS) technologies including DNA-Seq and RNA-Seq, has greatly expanded our understanding of viral diversity in recent years. The timely identification of novel viruses using NGS technologies is also important for us to control emerging infectious diseases caused by novel viruses. In this study, we identified a novel duck coronavirus (CoV), distinct with chicken infectious bronchitis virus (IBV), using RNA-Seq. The novel duck-specific CoV was a potential novel species within the genus Gammacoronavirus, as indicated by sequences of three regions in the viral 1b gene. We also performed a survey of CoVs in domestic fowls in China using reverse-transcription polymerase chain reaction (RT-PCR), targeting the viral nucleocapsid (N) gene. A total of 102 CoV positives were identified through the survey. Phylogenetic analysis of the viral N sequences suggested that CoVs in domestic fowls have diverged into several region-specific or host-specific clades or subclades in the world, and IBVs can infect ducks, geese and pigeons, although they mainly circulate in chickens. Moreover, this study provided novel data supporting the notion that some host-specific CoVs other than IBVs circulate in ducks, geese and pigeons, and indicated that the novel duck-specific CoV identified through RNA-Seq in this study is genetically closer to some CoVs circulating in wild water fowls. Taken together, this study shed new insight into the diversity, distribution, evolution and control of avian CoVs. PMID:24023656

  11. Identification and survey of a novel avian coronavirus in ducks.

    PubMed

    Chen, Gui-Qian; Zhuang, Qing-Ye; Wang, Kai-Cheng; Liu, Shuo; Shao, Jian-Zhong; Jiang, Wen-Ming; Hou, Guang-Yu; Li, Jin-Ping; Yu, Jian-Min; Li, Yi-Ping; Chen, Ji-Ming

    2013-01-01

    The rapid discovery of novel viruses using next generation sequencing (NGS) technologies including DNA-Seq and RNA-Seq, has greatly expanded our understanding of viral diversity in recent years. The timely identification of novel viruses using NGS technologies is also important for us to control emerging infectious diseases caused by novel viruses. In this study, we identified a novel duck coronavirus (CoV), distinct with chicken infectious bronchitis virus (IBV), using RNA-Seq. The novel duck-specific CoV was a potential novel species within the genus Gammacoronavirus, as indicated by sequences of three regions in the viral 1b gene. We also performed a survey of CoVs in domestic fowls in China using reverse-transcription polymerase chain reaction (RT-PCR), targeting the viral nucleocapsid (N) gene. A total of 102 CoV positives were identified through the survey. Phylogenetic analysis of the viral N sequences suggested that CoVs in domestic fowls have diverged into several region-specific or host-specific clades or subclades in the world, and IBVs can infect ducks, geese and pigeons, although they mainly circulate in chickens. Moreover, this study provided novel data supporting the notion that some host-specific CoVs other than IBVs circulate in ducks, geese and pigeons, and indicated that the novel duck-specific CoV identified through RNA-Seq in this study is genetically closer to some CoVs circulating in wild water fowls. Taken together, this study shed new insight into the diversity, distribution, evolution and control of avian CoVs.

  12. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

    PubMed Central

    Kim, Yunjeong; Liu, Hongwei; Galasiti Kankanamalage, Anushka C.; Weerasekara, Sahani; Hua, Duy H.; Groutas, William C.; Chang, Kyeong-Ok; Pedersen, Niels C.

    2016-01-01

    Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further

  13. Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor.

    PubMed

    Kim, Yunjeong; Liu, Hongwei; Galasiti Kankanamalage, Anushka C; Weerasekara, Sahani; Hua, Duy H; Groutas, William C; Chang, Kyeong-Ok; Pedersen, Niels C

    2016-03-01

    Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further

  14. Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems

    PubMed Central

    Failmezger, Jurek; Nitschel, Robert; Sánchez-Kopper, Andrés; Kraml, Michael; Siemann-Herzberg, Martin

    2016-01-01

    Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems. PMID:27992588

  15. Dual Mechanisms of Translation Initiation of the Full-Length HIV-1 mRNA Contribute to Gag Synthesis

    PubMed Central

    Rivero, Matias; Cohen, Éric A.; Lopez-Lastra, Marcelo; Mouland, Andrew J.

    2013-01-01

    The precursor group-specific antigen (pr55Gag) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55Gag synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55Gag expression, the synthesis of other HIV-1 proteins, including that of pr160Gag/Pol, Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55Gag for virus assembly and production. PMID:23861855

  16. Rapid inactivation of SARS-like coronaviruses.

    SciTech Connect

    Kapil, Sanjay; Oberst, R. D.; Bieker, Jill Marie; Tucker, Mark David; Souza, Caroline Ann; Williams, Cecelia Victoria

    2004-03-01

    Chemical disinfection and inactivation of viruses is largely understudied, but is very important especially in the case of highly infectious viruses. The purpose of this LDRD was to determine the efficacy of the Sandia National Laboratories developed decontamination formulations against Bovine Coronavirus (BCV) as a surrogate for the coronavirus that causes Severe Acute Respiratory Syndrome (SARS) in humans. The outbreak of SARS in late 2002 resulted from a highly infectious virus that was able to survive and remain infectious for extended periods. For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conducted to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition. The MS-2 and T4 were slightly more resistant than BCV and required a 2 minute exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.

  17. Rapid identification of emerging pathogens: coronavirus.

    PubMed

    Sampath, Rangarajan; Hofstadler, Steven A; Blyn, Lawrence B; Eshoo, Mark W; Hall, Thomas A; Massire, Christian; Levene, Harold M; Hannis, James C; Harrell, Patina M; Neuman, Benjamin; Buchmeier, Michael J; Jiang, Yun; Ranken, Raymond; Drader, Jared J; Samant, Vivek; Griffey, Richard H; McNeil, John A; Crooke, Stanley T; Ecker, David J

    2005-03-01

    We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome-associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was approximate, equals1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day.

  18. Rapid Identification of Emerging Pathogens: Coronavirus

    PubMed Central

    Hofstadler, Steven A.; Blyn, Lawrence B.; Eshoo, Mark W.; Hall, Thomas A.; Massire, Christian; Levene, Harold M.; Hannis, James C.; Harrell, Patina M.; Neuman, Benjamin; Buchmeier, Michael J.; Jiang, Yun; Ranken, Raymond; Drader, Jared J.; Samant, Vivek; Griffey, Richard H.; McNeil, John A.; Crooke, Stanley T.; Ecker, David J.

    2005-01-01

    We describe a new approach for infectious disease surveillance that facilitates rapid identification of known and emerging pathogens. The process uses broad-range polymerase chain reaction (PCR) to amplify nucleic acid targets from large groupings of organisms, electrospray ionization mass spectrometry for accurate mass measurements of PCR products, and base composition signature analysis to identify organisms in a sample. We demonstrate this principle by using 14 isolates of 9 diverse Coronavirus spp., including the severe acute respiratory syndrome–associated coronavirus (SARS-CoV). We show that this method could identify and distinguish between SARS and other known CoV, including the human CoV 229E and OC43, individually and in a mixture of all 3 human viruses. The sensitivity of detection, measured by using titered SARS-CoV spiked into human serum, was ≈1 PFU/mL. This approach, applicable to the surveillance of bacterial, viral, fungal, or protozoal pathogens, is capable of automated analysis of >900 PCR reactions per day. PMID:15757550

  19. Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

    PubMed

    Tago, Nobuhiro; Katolik, Adam; Clark, Nathaniel E; Montemayor, Eric J; Seio, Kohji; Sekine, Mitsuo; Hart, P John; Damha, Masad J

    2015-10-16

    Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.

  20. Hyaluronan tetrasaccharides stimulate ceramide production through upregulated mRNA expression of ceramide synthesis-associated enzymes.

    PubMed

    Kage, Madoka; Tokudome, Yoshihiro

    2016-03-01

    It has been reported that hyaluronan has different physiological functions as suggested by variation in molecular weight. In addition, it has also been reported that CD44, the major hyaluronan receptor, was demonstrated to induce keratinocyte differentiation and lipid synthesis of cholesterol. We focus attention on the hyaluronan tetrasaccharides (HA4) which is the smallest unit of hyaluronan. We previously reported that HA4 induced keratinocyte differentiation and that CD44 may be involved. For the purpose of clarifying the influence of HA4 on ceramide synthesis, we evaluated