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Sample records for coronavirus rna synthesis

  1. 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. PMID:25451065

  2. Synthesis of coronavirus mRNAs: kinetics of inactivation of infectious bronchitis virus RNA synthesis by UV light. [Chickens

    SciTech Connect

    Stern, D.F.; Sefton, B.M.

    1982-05-01

    Infection of cells with the avian coronavirus infectious bronchitis virus results in the synthesis of five major subgenomic RNAs. These RNAs and the viral genome form a 3' coterminal nested set. We found that the rates of inactivation of synthesis of the RNAs by UV light were different and increased with the length of the transcript. These results show that each RNA is transcribed from a unique promoter and that extensive processing of the primary transcripts probably does not occur.

  3. 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. PMID:26847650

  4. RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

    PubMed Central

    Liao, C L; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur

  5. 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

  6. Integrity of the Early Secretory Pathway Promotes, but Is Not Required for, Severe Acute Respiratory Syndrome Coronavirus RNA Synthesis and Virus-Induced Remodeling of Endoplasmic Reticulum Membranes▿ †

    PubMed Central

    Knoops, Kèvin; Swett-Tapia, Cindy; van den Worm, Sjoerd H. E.; te Velthuis, Aartjan J. W.; Koster, Abraham J.; Mommaas, A. Mieke; Snijder, Eric J.; Kikkert, Marjolein

    2010-01-01

    To accommodate its RNA synthesis in the infected cell, severe acute respiratory syndrome coronavirus (SARS-CoV) induces a cytoplasmic reticulovesicular network (RVN) that is derived from endoplasmic reticulum (ER) membranes. We set out to investigate how the early secretory pathway interacts with the RVN and the viral replication/transcription complex (RTC) that is anchored to it. When the secretory pathway was disrupted by brefeldin A (BFA) treatment at the start of infection, RVN formation and viral RTC activity were not blocked and continued up to 11 h postinfection, although RNA synthesis was reduced by ca. 80%. In vitro RTC assays, using membrane fractions from infected cells, demonstrated that BFA does not directly interfere with the activity of the viral RNA-synthesizing enzymes. Confocal microscopy studies showed that early secretory pathway components are not associated with SARS-CoV-induced replication sites, although our studies revealed that infection induces a remarkable redistribution of the translocon subunit Sec61α. Ultrastructural studies, including electron tomography, revealed that the formation of the RVN and all its previously documented features can occur in the presence of BFA, despite differences in the volume and morphology of the network. We therefore conclude that early secretory pathway proteins do not play a direct role in RVN morphogenesis or the functionality of the SARS-CoV RTC. The BFA-induced disruption of ER integrity and functionality probably affects the overall quality of the membrane scaffold that is needed to support the viral RTC and/or the availability of specific host factors, which in turn compromises viral RNA synthesis. PMID:19889777

  7. 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. PMID:25197083

  8. 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

  9. 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

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

    PubMed Central

    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

    2013-01-01

    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. PMID:23275571

  11. Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

    PubMed Central

    Siddell, Stuart G.; Wege, Helmut; Barthel, Andrea; ter Meulen, Volker

    1980-01-01

    Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA. Images PMID:7365865

  12. Coronavirus Multiplication Strategy I. Identification and Characterization of Virus-Specified RNA

    PubMed Central

    Stern, David F.; Kennedy, S. Ian T.

    1980-01-01

    We examined the synthesis of intracellular RNA in primary chicken embryo kidney cells infected with the avian coronavirus infectious bronchitis virus. Infected cells were labeled with 32Pi in the presence of actinomycin D for the duration of the viral multiplication cycle, and nucleic acids were extracted, denatured, and analyzed on agarose slab gels. Six major RNA species were found. None of these RNAs was found in extracts of mock-infected cells. All six of the virus-specified RNAs (designated species A through F) were single stranded, and RNA species F had the same electrophoretic mobility as purified viral genome RNA. The molecular weights of the five subgenomic RNAs were estimated to be 0.8 × 106, 0.9 × 106, 1.3 × 106, 1.5 × 106, and 2.6 × 106 for species A through E, respectively. All of the RNAs were polyadenylated and are therefore likely to be viral mRNA's. The RNAs were synthesized in approximately constant proportions throughout the viral multiplication cycle. Intracellular RNA species A, B, C, D, and F and the purified viral genome were analyzed by RNase T1 fingerprinting. The results confirmed the identification of RNA species F as the intracellular genome and the derivation of the four smaller RNAs from the genome. Fingerprinting also showed that the intracellular RNAs constitute a nested set such that the nucleotide sequence of each RNA is contained within all larger RNAs and each larger RNA contains an additional sequence congruent with its greater size. Finally, the possible modes of transcription and translation of the infectious bronchitis virus RNAs are discussed. Images PMID:6247505

  13. A cis-acting function for the coronavirus leader in defective interfering RNA replication.

    PubMed Central

    Chang, R Y; Hofmann, M A; Sethna, P B; Brian, D A

    1994-01-01

    To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that, in contrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements originating from discontinuous downstream regions within the polymerase gene for replication or for packaging. Images PMID:7966615

  14. 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

  15. 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

  16. 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

  17. A Three-Stemmed mRNA Pseudoknot in the SARS Coronavirus Frameshift Signal

    PubMed Central

    2005-01-01

    A wide range of RNA viruses use programmed −1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed −1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics. PMID:15884978

  18. 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

  19. 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.

  20. 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. PMID:23719724

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

    PubMed Central

    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. PMID:22619553

  2. Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.

    PubMed Central

    Liao, C L; Lai, M M

    1994-01-01

    We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The

  3. 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

  4. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    PubMed

    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

  5. 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. PMID

  6. SARS/avian coronaviruses.

    PubMed

    Monceyron Jonassen, C

    2006-01-01

    In the hunt for the aetiology of the SARS outbreak in 2003, a newly developed virus DNA micro-array was successfully used to hybridise PCR products obtained by random amplification of nucleic acids extracted from a cell culture infected with material from a SARS patient. The SARS agent was found to hybridise with micro-array probes from both coronaviruses and astroviruses, but one of the coronavirus probes and the four astrovirus probes contained redundant sequences, spanning a highly conserved motif, named s2m, found at the 3' end of the genomes of almost all astroviruses, one picornavirus, and the poultry coronaviruses. The three other coronavirus probes, that hybridised with the SARS agent, were located in the replicase gene, and it could be concluded that the SARS agent was a novel coronavirus, harbouring s2m. The presence of this motif in different virus families is probably the result of recombinations between unrelated viruses, but its presence in both poultry and SARS coronaviruses could suggest a bird involvement in the history of the SARS coronavirus. A recent screening of wild birds for the presence of coronaviruses, using a pan-coronavirus RT-PCR, led to the identification of novel coronaviruses in the three species studied. Phylogenetic analyses performed on both replicase gene and nucleocapsid protein could not add support to a close relationship between avian and SARS coronaviruses, but all the novel avian coronaviruses were found to harbour s2m. The motif is inserted at a homologous place in avian and SARS coronavirus genomes, but in a somewhat different context for the SARS coronavirus. If the presence of s2m in these viruses is a result of two separate recombination events, this suggests that its particular position in these genomes is the only one that would not be deleterious for coronaviral replication, or that it is the result of a copy-choice recombination between coronaviruses, following an ancestral introduction in the coronavirus family by

  7. Flavivirus RNA synthesis in vitro.

    PubMed

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

    2015-12-01

    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

  8. SARS Coronavirus-unique Domain (SUD): Three-domain Molecular Architecture in Solution and RNA Binding

    PubMed Central

    Johnson, Margaret A.; Chatterjee, Amarnath; Neuman, Benjamin W.; Wüthrich, Kurt

    2010-01-01

    The nonstructural protein 3 (nsp3) of the severe acute respiratory syndrome coronavirus (SARS-CoV) includes a “SARS-unique region” (SUD) consisting of three globular domains separated by short linker peptide segments. This paper reports NMR structure determinations of the C-terminal domain (SUD-C) and of a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution, and in SUD-NM there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin-like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observation of the 15N-labeled proteins further resulted in the delineation of the RNA binding sites, i.e., in SUD-M a positively charged surface area with a pronounced cavity, and in SUD-C several residues of an antiparallel β-sheet. Overall, the present data provide evidence for molecular mechanisms involving concerted actions of SUD-M and SUD-C, which result in specific RNA-binding that might be unique to the SUD, and thus to the SARS-CoV. PMID:20493876

  9. Characterization of an Essential RNA Secondary Structure in the 3′ Untranslated Region of the Murine Coronavirus Genome

    PubMed Central

    Hsue, Bilan; Hartshorne, Toinette; Masters, Paul S.

    2000-01-01

    We have previously identified a functionally essential bulged stem-loop in the 3′ untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication. PMID:10888630

  10. Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR.

    PubMed Central

    Herrewegh, A A; de Groot, R J; Cepica, A; Egberink, H F; Horzinek, M C; Rottier, P J

    1995-01-01

    A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV. PMID:7751377

  11. 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.

  12. Evidence for coronavirus discontinuous transcription.

    PubMed Central

    Jeong, Y S; Makino, S

    1994-01-01

    Coronavirus subgenomic mRNA possesses a 5'-end leader sequence which is derived from the 5' end of genomic RNA and is linked to the mRNA body sequence. This study examined whether coronavirus transcription involves a discontinuous transcription step; the possibility that a leader sequence from mouse hepatitis virus (MHV) genomic RNA could be used for MHV subgenomic defective interfering (DI) RNA transcription was examined. This was tested by using helper viruses and DI RNAs that were easily distinguishable. MHV JHM variant JHM(2), which synthesizes a subgenomic mRNA encoding the HE gene, and variant JHM(3-9), which does not synthesize this mRNA, were used. An MHV DI RNA, DI(J3-9), was constructed to contain a JHM(3-9)-derived leader sequence and an inserted intergenic region derived from the region preceding the MHV JHM HE gene. DI(J3-9) replicated efficiently in JHM(2)- or JHM(3-9)-infected cells, whereas synthesis of subgenomic DI RNAs was observed only in JHM(2)-infected cells. Sequence analyses demonstrated that the 5' regions of both helper virus genomic RNAs and genomic DI RNAs maintained their original sequences in DI RNA-replicating cells, indicating that the genomic leader sequences derived from JHM(2) functioned for subgenomic DI RNA transcription. Replication and transcription of DI(J3-9) were observed in cells infected with an MHV A59 strain whose leader sequence was similar to that of JHM(2), except for one nucleotide substitution within the leader sequence. The 5' region of the helper virus genomic RNA and that of the DI RNA were the same as their original structures in virus-infected cells, and the leader sequence of DI(J3-9) subgenomic DI RNA contained the MHV A59-derived leader sequence. The leader sequence of subgenomic DI RNA was derived from that of helper virus; therefore, the genomic leader sequence had a trans-acting property indicative of a discontinuous step in coronavirus transcription. Images PMID:8139040

  13. 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 ...

  14. Coronavirus mRNA transcription: UV light transcriptional mapping studies suggest an early requirement for a genomic-length template.

    PubMed Central

    Yokomori, K; Banner, L R; Lai, M M

    1992-01-01

    Mouse hepatitis virus (MHV) synthesizes seven to eight mRNAs, each of which contains a leader RNA derived from the 5' end of the genome. To understand the mechanism of synthesis of these mRNAs, we studied how the synthesis of each mRNA was affected by UV irradiation at different time points after infection. When MHV-infected cells were UV irradiated at a late time in infection (5 h postinfection), the syntheses of the various mRNAs were inhibited to different extents in proportion to the sizes of the mRNAs. Analysis of the UV inactivation kinetics revealed that the UV target size of each mRNA was equivalent to its own physical size. In contrast, when cells were irradiated at 2.5 or 3 h postinfection, there appeared to be two different kinetics of inhibition of mRNA synthesis: the synthesis of every mRNA was inhibited to the same extent by a small UV dose, but the remaining mRNA synthesis was inhibited by additional UV doses at different rates for different mRNAs in proportion to RNA size. The analysis of the UV inactivation kinetics indicated that the UV target sizes for the majority of mRNAs were equivalent to that of the genomic-size RNA early in the infection. These results suggest that MHV mRNA synthesis requires the presence of a genomic-length RNA template at least early in the infection. In contrast, later in the infection, the sizes of the templates used for mRNA synthesis were equivalent to the physical sizes of each mRNA. The possibility that the genomic-length RNA required early in the infection was used only for the synthesis of a polymerase rather than as a template for mRNA synthesis was ruled out by examining the UV sensitivity of a defective interfering (DI) RNA. We found that the UV target size for the DI RNA early in infection was much smaller than that for mRNAs 6 and 7, which are approximately equal to or smaller in size than the DI RNA. This result indicates that even though DI RNA and viral mRNAs are synthesized by the same polymerase, m

  15. 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. PMID:22635272

  16. 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. PMID:25596968

  17. Genotyping bovine coronaviruses.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  18. 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.

  19. A 5'-proximal RNA sequence of murine coronavirus as a potential initiation site for genomic-length mRNA transcription.

    PubMed Central

    Zhang, X; Lai, M M

    1996-01-01

    Coronavirus transcription is a discontinuous process, involving interactions between a trans-acting leader and the intergenic transcription initiation sequences. A 9-nucleotide (nt) sequence (UUUAUAAAC), which is located immediately downstream of the leader at the 5' terminus of the mouse hepatitis virus (MHV) genomic RNA, contains a sequence resembling the consensus intergenic sequence (UCUAAAC). It has been shown previously that the presence of the 9-nt sequence facilitates leader RNA switching and may enhance subgenomic mRNA transcription. It is unclear how the 9-nt sequence exerts these functions. In this study, we inserted the 9-nt sequence into a defective interfering (DI) RNA reporter system and demonstrated that mRNA transcription could be initiated from the 9-nt sequence almost as efficiently as from the intergenic sequence between genes 6 and 7. Sequence analysis of the mRNAs showed that the 9-nt sequence served as a site of fusion between the leaders and mRNA. The transcription initiation function of the 9-nt sequence could not be substituted by other 5'-terminal sequences. When the entire 5'-terminal sequence, including four copies of the UCUAA sequence plus the 9-nt sequence, was present, transcription could be initiated from any of the UCUAA copies or the 9-nt sequence, resulting in different copy numbers of the UCUAA sequence and the deletion of the 9-nt sequence in some mRNAs. All of these heterogeneous RNA species were also detected from the 5'-terminal region of the viral genomic-length RNA in MHV-infected cells. These results thus suggest tha the heterogeneity of the copy number of UCUAA sequences at the 5' end, the deletion of the 9-nt sequence in viral and DI RNAs, and the leader RNA switching are the results of transcriptional initiation from the 9-nt site. They also show that an mRNA species (mRNA 1) that lacks the 9-nt sequence can be synthesized during MHV infection. Therefore, MHV genomic RNA replication and mRNA 1 transcription may be

  20. 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

    2014-01-01

    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. PMID:24957745

  1. Yeast-based assays for the high-throughput screening of inhibitors of coronavirus RNA cap guanine-N7-methyltransferase.

    PubMed

    Sun, Ying; Wang, Zidao; Tao, Jiali; Wang, Yi; Wu, Andong; Yang, Ziwen; Wang, Kaimei; Shi, Liqiao; Chen, Yu; Guo, Deyin

    2014-04-01

    The 5'-cap structure is a distinct feature of eukaryotic mRNAs and is important for RNA stability and protein translation by providing a molecular signature for the distinction of self or non-self mRNA. Eukaryotic viruses generally modify the 5'-end of their RNAs to mimic the cellular mRNA structure, thereby facilitating viral replication in host cells. However, the molecular organization and biochemical mechanisms of the viral capping apparatus typically differ from its cellular counterpart, which makes viral capping enzymes attractive targets for drug discovery. Our previous work showed that SARS coronavirus (SARS-CoV) non-structural protein 14 represents a structurally novel and unique guanine-N7-methyltransferase (N7-MTase) that is able to functionally complement yeast cellular N7-MTase. In the present study, we developed a yeast-based system for identifying and screening inhibitors against coronavirus N7-MTase using both 96-well and 384-well microtiter plates. The MTase inhibitors previously identified by in vitro biochemical assays were tested, and some, such as sinefungin, effectively suppressed N7-MTase in the yeast system. However, other compounds, such as ATA and AdoHcy, did not exert an inhibitory effect within a cellular context. These results validated the yeast assay system for inhibitor screening yet also demonstrated the difference between cell-based and in vitro biochemical assays. The yeast system was applied to the screening of 3000 natural product extracts, and three were observed to more potently inhibit the activity of coronavirus than human N7-MTase. PMID:24530452

  2. 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

  3. Regulation of Flavivirus RNA synthesis and replication.

    PubMed

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-12-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  4. Regulation of Flavivirus RNA synthesis and replication

    PubMed Central

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-01-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  5. Enhancement of RNA Synthesis, Protein Synthesis, and Abscission by Ethylene

    PubMed Central

    Abeles, F. B.; Holm, R. E.

    1966-01-01

    Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed. PMID:16656405

  6. Nonstructural Proteins 7 and 8 of Feline Coronavirus Form a 2:1 Heterotrimer That Exhibits Primer-Independent RNA Polymerase Activity

    PubMed Central

    Xiao, Yibei; Ma, Qingjun; Restle, Tobias; Shang, Weifeng; Svergun, Dmitri I.; Ponnusamy, Rajesh; Sczakiel, Georg

    2012-01-01

    Nonstructural proteins 7 and 8 of severe acute respiratory syndrome coronavirus (SARS-CoV) have previously been shown by X-ray crystallography to form an 8:8 hexadecamer. In addition, it has been demonstrated that N-terminally His6-tagged SARS-CoV Nsp8 is a primase able to synthesize RNA oligonucleotides with a length of up to 6 nucleotides. We present here the 2.6-Å crystal structure of the feline coronavirus (FCoV) Nsp7:Nsp8 complex, which is a 2:1 heterotrimer containing two copies of the α-helical Nsp7 with conformational differences between them, and one copy of Nsp8 that consists of an α/β domain and a long-α-helix domain. The same stoichiometry is found for the Nsp7:Nsp8 complex in solution, as demonstrated by chemical cross-linking, size exclusion chromatography, and small-angle X-ray scattering. Furthermore, we show that FCoV Nsp8, like its SARS-CoV counterpart, is able to synthesize short oligoribonucleotides of up to 6 nucleotides in length when carrying an N-terminal His6 tag. Remarkably, the same protein harboring the sequence GPLG instead of the His6 tag at its N terminus exhibits a substantially increased, primer-independent RNA polymerase activity. Upon addition of Nsp7, the RNA polymerase activity is further enhanced so that RNA up to template length (67 nucleotides) can be synthesized. Further, we show that the unprocessed intermediate polyprotein Nsp7-10 of human coronavirus (HCoV) 229E is also capable of synthesizing oligoribonucleotides up to a chain length of six. These results indicate that in case of FCoV as well as of HCoV 229E, the formation of a hexadecameric Nsp7:Nsp8 complex is not necessary for RNA polymerase activity. Further, the FCoV Nsp7:Nsp8 complex functions as a noncanonical RNA polymerase capable of synthesizing RNA of up to template length. PMID:22318142

  7. 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

  8. About Coronavirus

    MedlinePlus

    ... or surfaces then touching your mouth, nose, or eyes. Also see MERS-CoV Transmission and How SARS Spreads . Q: When can I get infected? A: In the United States, people usually get infected with common human coronaviruses in the fall and winter. However, you ...

  9. Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus

    PubMed Central

    Weiss, Susan R.; Navas-Martin, Sonia

    2005-01-01

    Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV. PMID:16339739

  10. Preparation of His-Tagged Armored RNA Phage Particles as a Control for Real-Time Reverse Transcription-PCR Detection of Severe Acute Respiratory Syndrome Coronavirus

    PubMed Central

    Cheng, Yangjian; Niu, Jianjun; Zhang, Yongyou; Huang, Jianwei; Li, Qingge

    2006-01-01

    Armored RNA has been increasingly used as both an external and internal positive control in nucleic acid-based assays for RNA virus. In order to facilitate armored RNA purification, a His6 tag was introduced into the loop region of the MS2 coat protein, which allows the exposure of multiple His tags on the surface during armored RNA assembly. The His-tagged armored RNA particles were purified to homogeneity and verified to be free of DNA contamination in a single run of affinity chromatography. A fragment of severe acute respiratory syndrome coronavirus (SARS-CoV) genome targeted for SARS-CoV detection was chosen for an external positive control preparation. A plant-specific gene sequence was chosen for a universal noncompetitive internal positive control preparation. Both controls were purified by Co2+ affinity chromatography and were included in a real-time reverse transcription-PCR assay for SARS-CoV. The noncompetitive internal positive control can be added to clinical samples before RNA extraction and enables the identification of potential inhibitive effects without interfering with target amplification. The external control could be used for the quantification of viral loads in clinical samples. PMID:17021082

  11. 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.

  12. 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

  13. Initiator RNA in Discontinuous Polyoma DNA Synthesis*

    PubMed Central

    Reichard, Peter; Eliasson, Rolf; Söderman, Gunilla

    1974-01-01

    During replication of polyoma DNA in isolated nuclei, RNA was found attached to the 5′ ends of growing progeny strands. This RNA starts with either ATP or GTP and can be labeled at its 5′ end with 32P from β-labeled nucleotides. Digestion of progeny strands with pancreatic DNase released 32P-labeled RNA that, on gel electrophoresis, gave a distinct peak in the position expected for a decanucleotide. We believe that this short RNA is involved in the initiation of the discontinuous synthesis of DNA and propose the name “initiator RNA” for it. The covalent linkage of initiator RNA to 5′ ends of growing DNA chains was substantiated by the finding that 32P was transferred to ribonucleotides by alkaline hydrolysis of purified initiator RNA obtained by DNase digestion of polyoma progeny strands synthesized from [α-32P]dTTP. While initiator RNA was quite homogeneous in size, it had no unique base sequence since digestion with pancreatic RNase of initiator RNA labeled at its 5′ end with 32P released a variety of different [32P]oligonucleotides. The switch from RNA to DNA synthesis during strand elongation may thus depend on the size of initiator RNA rather than on a specific base sequence. PMID:4373733

  14. 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.

  15. The effect of two closely inserted transcription consensus sequences on coronavirus transcription.

    PubMed Central

    Joo, M; Makino, S

    1995-01-01

    Insertion of an intergenic region from the murine coronavirus mouse hepatitis virus into a mouse hepatitis virus defective interfering (DI) RNA led to transcription of subgenomic DI RNA in helper virus-infected cells. Using this system, we studied how two intergenic regions in close proximity affected subgenomic RNA synthesis. When two intergenic regions were separated by more than 100 nucleotides, slightly less of the larger subgenomic DI RNA (synthesized from the upstream intergenic region) was made; this difference was significant when the intergenic region separation was less than about 35 nucleotides. Deletion of sequences flanking the two intergenic regions inserted in close proximity did not affect transcription. No significant change in the ratio of the two subgenomic DI RNAs was observed when the sequence between the two intergenic regions was altered. Removal of the downstream intergenic region restored transcription of the larger subgenomic DI RNA. The UCUAAAC consensus sequence was needed for efficient suppression of the larger subgenomic DI RNA synthesis. These results demonstrated that the downstream intergenic sequence was suppressing subgenomic DI RNA synthesis from the upstream intergenic region. We discuss possible mechanisms to account for the regulation of this suppression of subgenomic DI RNA synthesis and the ways in which they relate to the general regulation of coronavirus transcription. PMID:7983719

  16. 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

  17. Identification of a Novel Coronavirus in Bats

    PubMed Central

    Poon, L. L. M.; Chu, D. K. W.; Chan, K. H.; Wong, O. K.; Ellis, T. M.; Leung, Y. H. C.; Lau, S. K. P.; Woo, P. C. Y.; Suen, K. Y.; Yuen, K. Y.; Guan, Y.; Peiris, J. S. M.

    2005-01-01

    Exotic wildlife can act as reservoirs of diseases that are endemic in the area or can be the source of new emerging diseases through interspecies transmission. The recent emergence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) highlights the importance of virus surveillance in wild animals. Here, we report the identification of a novel bat coronavirus through surveillance of coronaviruses in wildlife. Analyses of the RNA sequence from the ORF1b and S-gene regions indicated that the virus is a group 1 coronavirus. The virus was detected in fecal and respiratory samples from three bat species (Miniopterus spp.). In particular, 63% (12 of 19) of fecal samples from Miniopterus pusillus were positive for the virus. These findings suggest that this virus might be commonly circulating in M. pusillus in Hong Kong. PMID:15681402

  18. 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. PMID:23212740

  19. 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.

  20. The Structure and Functions of Coronavirus Genomic 3’ and 5’ Ends

    PubMed Central

    Yang, Dong; Leibowitz, Julian L.

    2015-01-01

    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 thirty-five 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. PMID:25736566

  1. RNA template-directed RNA synthesis by T7 RNA polymerase.

    PubMed Central

    Cazenave, C; Uhlenbeck, O C

    1994-01-01

    In an attempt to synthesize an oligoribonucleotide by run-off transcription by bacteriophage T7 RNA polymerase, a major transcript was produced that was much longer than expected. Analysis of the reaction indicated that the product resulted from initial DNA-directed run-off transcription followed by RNA template-directed RNA synthesis. This reaction occurred because the RNA made from the DNA template displayed self-complementarity at its 3' end and therefore could form an intra- or intermolecular primed template. In reactions containing only an RNA template, the rate of incorporation of NTPs was quite comparable to DNA-dependent transcription. RNA template-directed RNA synthesis has been found to occur with a great number of oligoribonucleotides, even with primed templates that are only marginally stable. In one instance, we observed a multistep extension reaction converting the oligonucleotide into a final product longer than twice its original length. Presumably, such a process could have generated some of the RNAs found to be efficiently replicated by T7 RNA polymerase. Images PMID:7518923

  2. Identification of a Gamma Interferon-Activated Inhibitor of Translation-Like RNA Motif at the 3′ End of the Transmissible Gastroenteritis Coronavirus Genome Modulating Innate Immune Response

    PubMed Central

    Marquez-Jurado, Silvia; Nogales, Aitor; Zuñiga, Sonia; Almazán, Fernando

    2015-01-01

    ABSTRACT A 32-nucleotide (nt) RNA motif located at the 3′ end of the transmissible gastroenteritis coronavirus (TGEV) genome was found to specifically interact with the host proteins glutamyl-prolyl-tRNA synthetase (EPRS) and arginyl-tRNA synthetase (RRS). This RNA motif has high homology in sequence and secondary structure with the gamma interferon-activated inhibitor of translation (GAIT) element, which is located at the 3′ end of several mRNAs encoding proinflammatory proteins. The GAIT element is involved in the translation silencing of these mRNAs through its interaction with the GAIT complex (EPRS, heterogeneous nuclear ribonucleoprotein Q, ribosomal protein L13a, and glyceraldehyde 3-phosphate dehydrogenase) to favor the resolution of inflammation. Interestingly, we showed that the viral RNA motif bound the GAIT complex and inhibited the in vitro translation of a chimeric mRNA containing this RNA motif. To our knowledge, this is the first GAIT-like motif described in a positive RNA virus. To test the functional role of the GAIT-like RNA motif during TGEV infection, a recombinant coronavirus harboring mutations in this motif was engineered and characterized. Mutations of the GAIT-like RNA motif did not affect virus growth in cell cultures. However, an exacerbated innate immune response, mediated by the melanoma differentiation-associated gene 5 (MDA5) pathway, was observed in cells infected with the mutant virus compared with the response observed in cells infected with the parental virus. Furthermore, the mutant virus was more sensitive to beta interferon than the parental virus. All together, these data strongly suggested that the viral GAIT-like RNA motif modulates the host innate immune response. PMID:25759500

  3. 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. PMID:15288921

  4. Drug Targets for Rational Design against Emerging Coronaviruses.

    PubMed

    Zhao, Qi; Weber, Erin; Yang, Haitao

    2013-07-26

    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. PMID:23885693

  5. 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. PMID:23895136

  6. 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

  7. Initiation of minus-strand RNA synthesis by the brome mosaicvirus RNA-dependent RNA polymerase: use of oligoribonucleotide primers.

    PubMed Central

    Kao, C C; Sun, J H

    1996-01-01

    Various DNA- and RNA-dependent RNA polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. For the brome mosaic virus RNA-dependent RNA polymerase (RdRp), we determined that in reactions performed with limited GTP concentrations, minus-strand RNA synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. Furthermore, guanylyl-3',5'-guanosine (GpG) was incorporated into minus-strand RNA and increased the rate of minus-strand RNA synthesis. In the presence of GpG, RdRp's Km for GTP decreased from 50 microM to approximately 3 microM while the Kms for other nucleotides were unaffected. These results have implications for the mechanism of initiation by RdRp. PMID:8794323

  8. Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis.

    PubMed

    Stefanovsky, Victor Y; Moss, Tom

    2016-01-01

    RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates. PMID:27576716

  9. 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

  10. 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

  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. Regulation of Stress Responses and Translational Control by Coronavirus.

    PubMed

    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

  13. Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

    PubMed Central

    Millet, Jean Kaoru; Whittaker, Gary R.

    2015-01-01

    Coronaviruses are a large group of enveloped, single-stranded positive-sense RNA viruses that infect a wide range of avian and mammalian species, including humans. The emergence of deadly human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) have bolstered research in these viral and often zoonotic pathogens. While coronavirus cell and tissue tropism, host range, and pathogenesis are initially controlled by interactions between the spike envelope glycoprotein and host cell receptor, it is becoming increasingly apparent that proteolytic activation of spike by host cell proteases also plays a critical role. Coronavirus spike proteins are the main determinant of entry as they possess both receptor binding and fusion functions. Whereas binding to the host cell receptor is an essential first step in establishing infection, the proteolytic activation step is often critical for the fusion function of spike, as it allows for controlled release of the fusion peptide into target cellular membranes. Coronaviruses have evolved multiple strategies for proteolytic activation of spike, and a large number of host proteases have been shown to proteolytically process the spike protein. These include, but are not limited to, endosomal cathepsins, cell surface transmembrane protease/serine (TMPRSS) proteases, furin, and trypsin. This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity. PMID:25445340

  14. Synthesis of Amplified DNA That Codes for Ribosomal RNA

    PubMed Central

    Crippa, Marco; Tocchini-Valentini, Glauco P.

    1971-01-01

    During the amplification stage in ovaries, the complete repetitive unit of the DNA that codes for ribosomal RNA in Xenopus appears to be transcribed. This large RNA transcript is found in a complex with DNA. Substitution experiments with 5-bromodeoxyuridine do not show any evidence that a complete amplified cistron is used as a template for further amplification. A derivative of rifampicin, 2′,5′-dimethyl-N(4′)benzyl-N(4′)[desmethyl] rifampicin, preferentially inhibits the DNA synthesis responsible for ribosomal gene amplification. These results are consistent with the hypothesis that RNA-dependent DNA synthesis is involved in gene amplification. PMID:5288254

  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

    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. 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

  18. The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

    PubMed Central

    Graham, Rachel L.; Sims, Amy C.; Brockway, Sarah M.; Baric, Ralph S.; Denison, Mark R.

    2005-01-01

    The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function. PMID:16227261

  19. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication.

    PubMed

    Graham, Rachel L; Sims, Amy C; Brockway, Sarah M; Baric, Ralph S; Denison, Mark R

    2005-11-01

    The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function. PMID:16227261

  20. [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.

  1. 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

  2. In vitro RNA synthesis by infectious pancreatic necrosis virus-associated RNA polymerase.

    PubMed

    Mertens, P P; Jamieson, P B; Dobos, P

    1982-03-01

    The presence of an RNA-dependent RNA polymerase was demonstrated in purified infectious pancreatic necrosis virus (IPNV). The enzyme was active in vitro without any pretreatment of the virus. Optimum activity was shown at 30 degrees C, pH 8 and in the presence of 6 mM-magnesium ions. Approx. 50% of the polymerase product remained associated with the dsRNA template of the virions. The remainder was found as extravirion ssRNA broken down to 5S to 7S fragments by virus-associated RNase(s). Although the addition of bentonite considerably reduced the amount of RNA synthesized, it protected the ssRNA product from degradation. This, in turn, permitted the synthesis of small amounts of ssRNA, which when analysed by sucrose gradient centrifugation or polyacrylamide gel electrophoresis behaved identically to the 24S single-stranded virus mRNA produced in infected cells. The virion polymerase was not stimulated by S-adenosyl-L-methionine or the addition of cellular or capped reovirus ssRNA. Several other modifications of the assay system were tried in an attempt to increase 24S RNA synthesis, but with little success. When [3H]uridine-labelled virus was used in the polymerase reaction, some labelled 24S ssRNA was obtained, indicating that in vitro transcription may proceed by a semi-conservative (displacement) mechanism. PMID:6175731

  3. Coronavirus Infection Modulates the Unfolded Protein Response and Mediates Sustained Translational Repression▿

    PubMed Central

    Bechill, John; Chen, Zhongbin; Brewer, Joseph W.; Baker, Susan C.

    2008-01-01

    During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6α. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing α-mannosidase-like protein, and p58IPK, or expression of UPR reporter constructs. Translation initiation factor eIF2α is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2α during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed. PMID:18305036

  4. 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

  5. [Effect of gibberellic acid on RNA synthesis in dwarf peas].

    PubMed

    Kilev, S N; Kholodar', A V; Chekurov, V M; Mertvetsov, N P

    1982-04-01

    The effect of gibberellic acid (GA) on total RNA and polysomal poly-[A]+-RNA synthesis in epicotylia and embryos of dwarf pea of two varieties differing in their physiological sensitivity to GA was studied. It was found that incubation with GA increases the accumulation of total RNA in pea epicotylia, var. "Pioner" and "Polzunok". The maximal stimulation of RNA accumulation makes up to 40% for the low sensitivity variety "Polzunok" and 150% for the highly sensitive variety "Pioner". GA increases the synthesis of polysomal poly (A)+-mRNA in 5-year-old pea sprouts and that of newly synthesized poly (A)+-mRNA in epicotylian polysomes of both varieties 5, 24, 48 and 72 hrs after incubation with GA. GA at concentrations of 10(-6) and 10(-5) stimulates the incorporation of [3H]uridine into polysomal mRNA during the first 1--3 hours after treatment and enhances the accumulation of newly synthesized mRNA in pea embryonic polyribosomes. The stimulating effect is directly proportional to the dose of the hormone. The mechanisms of GA effect on the transcription and translation in pea plant cells are discussed. PMID:6177351

  6. 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. PMID:7539631

  7. A novel human coronavirus: Middle East respiratory syndrome human coronavirus.

    PubMed

    Geng, HeYuan; Tan, WenJie

    2013-08-01

    In 2012, a novel coronavirus, initially named as human coronavirus EMC (HCoV-EMC) but recently renamed as Middle East respiratory syndrome human coronavirus (MERS-CoV), was identified in patients who suffered severe acute respiratory infection and subsequent renal failure that resulted in death. Ongoing epidemiological investigations together with retrospective studies have found 61 laboratory-confirmed cases of infection with this novel coronavirus, including 34 deaths to date. This novel coronavirus is culturable and two complete genome sequences are now available. Furthermore, molecular detection and indirect immunofluorescence assay have been developed. The present paper summarises the limited recent advances of this novel human coronavirus, including its discovery, genomic characterisation and detection. PMID:23917839

  8. Montmorillonite Clay-Catalyzed Synthesis of RNA Oligomers

    NASA Astrophysics Data System (ADS)

    Ferris, J. P.; Miyakawa, S.; Huang, W.; Joshi, P.

    2005-12-01

    It is proposed that catalysis had a central role in the origins of life. This will be illustrated using the montmorillonite clay-catalyzed synthesis of oligomers of RNA from activated monomers, (Ferris and Ertem, 1993) a possible step in the origin of the RNA world (Ferris, 2005). Structural analysis of oligomers formed in the reaction of the activated monomer of 5'-AMP with that of 5'-CMP demonstrated that the oligomers formed were not produced by random synthesis but rather the sequences observed were directed by the montmorillonite catalyst (Miyakawa and Ferris, 2003). RNA oligomers containing up to 40 mers have been synthesized in reactions performed in water at 25 oC in the presence of montmorillonite (Huang and Ferris, 2003). Analysis of the structure elements in these oligomers from the 7 to 39 mers showed that they did not vary. Reaction of D, L-mixtures of the activated monomers of A and U resulted in the formation of greater amounts of the homochiral amounts of dimers and trimers of A than would be expected if there was no selectivity in the reaction. A limited number of the dimers and trimers of U were also formed but here the selectivity was for the formation of an excess of heterochiral products (Joshi et al., 2000). A postulate that explains why homochiral trimers of U are not formed and the significance of catalysis in prebiotic synthesis will be discussed. Ferris, J.P. (2005) Origins of life, molecular basis of. In R.A. Meyers, Ed. Encyclopedia of Molecular Cell Biology and Molecular Medicine, 10. Wiley-VCH Verlag, Weinheim, Germany. Ferris, J.P., and Ertem, G. (1993) Montmorillonite catalysis of RNA oligomer formation in aqueous solution. A model for the prebiotic formation of RNA. J. Am. Chem. Soc., 115, 12270-12275. Huang, W., and Ferris, J.P. (2003) Synthesis of 35-40 mers of RNA oligomers from unblocked monomers. A simple approach to the RNA world. Chem. Commun., 1458-1459. Joshi, P.C., Pitsch, S., and Ferris, J.P. (2000) Homochiral selection

  9. Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNA(Cys).

    PubMed

    Zhang, Chun-Mei; Liu, Cuiping; Slater, Simon; Hou, Ya-Ming

    2008-05-01

    Cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNA(Cys) by an indirect pathway, whereby O-phosphoseryl-tRNA synthetase (SepRS) acylates tRNA(Cys) with phosphoserine (Sep), and Sep-tRNA-Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNA(Cys) tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1alpha or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNA(Cys). PMID:18425141

  10. 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. PMID:26879854

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

    PubMed

    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

    2016-03-01

    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

  12. 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

  13. Detection of bat coronaviruses from Miniopterus fuliginosus in Japan.

    PubMed

    Shirato, Kazuya; Maeda, Ken; Tsuda, Shumpei; Suzuki, Kazuo; Watanabe, Shumpei; Shimoda, Hiroshi; Ueda, Naoya; Iha, Koichiro; Taniguchi, Satoshi; Kyuwa, Shigeru; Endoh, Daiji; Matsuyama, Shutoku; Kurane, Ichiro; Saijo, Masayuki; Morikawa, Shigeru; Yoshikawa, Yasuhiro; Akashi, Hiroomi; Mizutani, Tetsuya

    2012-02-01

    Bats have great potential as reservoirs for emerging viruses such as severe acute respiratory syndrome-coronavirus. In this study, bat coronaviruses (BtCoVs) were detected by RT-PCR from intestinal and fecal specimens of Miniopterus fuliginosus breeding colonies in Wakayama Prefecture caves, where we previously identified bat betaherpesvirus 2. Two primer sets were used for the detection of BtCoV: one was for the RNA-dependent RNA polymerase (RdRp) region and the other was for the spike (S) protein region. Eleven and 73% of intestinal and fecal specimens, respectively, were positive for RdRp region, and 2 and 40% of those were positive for S protein region. Sequencing and phylogenetic analysis showed that the detected BtCoV belonged to the group 1 (alpha) coronaviruses. These data suggest that BtCoV is endemic in M. fuliginosus in Japan. PMID:21877208

  14. 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…

  15. The 5' untranslated region of alfalfa mosaic virus RNA 1 is involved in negative-strand RNA synthesis.

    PubMed

    Vlot, A Corina; Bol, John F

    2003-10-01

    The three genomic RNAs of alfalfa mosaic virus each contain a unique 5' untranslated region (5' UTR). Replacement of the 5' UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5' stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5' stem-loop structure is present in RNA 2 but not in RNA 3. PMID:14512577

  16. 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

  17. 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. PMID:16595304

  18. Nuclear Localization of Flavivirus RNA Synthesis in Infected Cells

    PubMed Central

    Uchil, Pradeep Devappa; Kumar, Anil V. A.; Satchidanandam, Vijaya

    2006-01-01

    Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex. PMID:16699025

  19. Inhibition of poliovirus RNA synthesis by brefeldin A.

    PubMed Central

    Maynell, L A; Kirkegaard, K; Klymkowsky, M W

    1992-01-01

    Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles. Images PMID:1312615

  20. Primer-Dependent and Primer-Independent Initiation of Double Stranded RNA Synthesis by Purified Arabidopsis RNA-Dependent RNA Polymerases RDR2 and RDR6

    PubMed Central

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  1. Primer-dependent and primer-independent initiation of double stranded RNA synthesis by purified Arabidopsis RNA-dependent RNA polymerases RDR2 and RDR6.

    PubMed

    Devert, Anthony; Fabre, Nicolas; Floris, Maïna; Canard, Bruno; Robaglia, Christophe; Crété, Patrice

    2015-01-01

    Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants. PMID:25793874

  2. RNA synthesis in isolated rat osteoclasts: inhibitory effect of calcitonin.

    PubMed

    Zheng, M H; Papadimitriou, J M; Nicholson, G C

    1991-01-01

    The metabolism of RNA has not been studied in the osteoclast (OC) because these bone-resorbing cells are only available in small numbers and cultures are always contaminated with other cells. Using two single-cell assay techniques, tritiated uridine (3H-UdR) autoradiography and gallocyanin quantitative cytophotometry, we have examined RNA synthesis in OCs isolated from neonatal rats. Oligo-nuclear OCs showed greater nuclear uptake of 3H-UdR than cells with many nuclei, and the variance of nuclear labeling within polykarya was greater in the latter, possibly because they contain nuclei of various ages. Salmon calcitonin (sCT) was a potent (ED50 approximately 5 x 10(-12) M) and rapid (40% reduction in 2 h, 75% reduction in 6 h) inhibitor of 3H-UdR uptake, and also reduced cytochemical total cellular RNA by 22% within 4 h. Forskolin (10(-5) M) inhibited nuclear uptake of 3H-UdR, suggesting that the sCT response may be mediated by cyclic AMP. Following a short (30 min) exposure to sCT, there was a progressive decline in labeling, followed by complete recovery by 4.5 h, a response possibly related to the phenomenon of calcitonin-induced persistent activation of adenylate cyclase. Inhibition of OC RNA synthesis may be an important component of its anti-resorptive action. PMID:1723609

  3. 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

  4. 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

  5. Bromovirus RNA replication and transcription require compatibility between the polymerase- and helicase-like viral RNA synthesis proteins.

    PubMed Central

    Dinant, S; Janda, M; Kroner, P A; Ahlquist, P

    1993-01-01

    The positive-strand RNA bromoviruses encode two nonstructural proteins, 1a and 2a, involved in RNA-dependent RNA replication. These proteins have extensive sequence similarities with methyltransferase, helicase, and polymerase proteins of other plant and animal viruses. 1a and 2a can also form a complex in vitro. To explore whether 1a-2a interaction is required for RNA replication in vivo, we reassorted the 1a and 2a genes from two different bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). 1a and 2a were expressed independently of viral replication by using RNA- or DNA-based transient expression, and their in vivo RNA replication activities were tested in protoplasts with BMV and CCMV RNA3 templates. RNA-based transient expression confirmed prior indications that bromovirus RNA replication is more sensitive to reductions in 1a expression than to reductions in 2a expression. DNA-based expression of the homologous combinations of 1a and 2a supported high levels of RNA synthesis, but both 1a-2a heterologous combinations exhibited RNA synthesis defects. The combination of CCMV 1a and BMV 2a did not support detectable synthesis of negative-strand, positive-strand, or subgenomic RNA. The converse combination of BMV 1a and CCMV 2a was preferentially defective in positive-strand and subgenomic RNA accumulation, showing that 1a-2a interaction is involved in these processes in ways distinct from negative-strand RNA synthesis, which was only slightly affected. These results indicate that at least some functions of 1a and 2a operate in a mutually dependent manner in vivo and that the mechanisms of positive- and negative-strand RNA synthesis are differentiated in part by features of such interactions. Images PMID:8230440

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

    PubMed

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

    2016-07-27

    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 tRNA(Glu) UUC 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

  7. Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos.

    PubMed Central

    Gong, Z Y; Brandhorst, B P

    1988-01-01

    An increased level of unpolymerized tubulin caused by depolymerization of microtubules in sea urchin larvae resulted in a rapid loss of tubulin mRNA, which was prevented by nearly complete inhibition of protein synthesis. Results of an RNA run-on assay indicated that inhibition of protein synthesis does not alter tubulin gene transcription. Analysis of the decay of tubulin mRNA in embryos in which RNA synthesis was inhibited by actinomycin D indicated that inhibition of protein synthesis prevents the destabilization of tubulin mRNA. The effect was similar whether mRNA was maintained on polysomes in the presence of emetine or anisomycin or displaced from the polysomes in the presence of puromycin or pactamycin; thus, the stabilization of tubulin mRNA is not dependent on the state of the polysomes after inhibition of protein synthesis. Even after tubulin mRNA declined to a low level after depolymerization of microtubules, it could be rescued by treatment of embryos with inhibitors of protein synthesis. Tubulin mRNA could be induced to accumulate prematurely in gastrulae but not in plutei if protein synthesis was inhibited, an observation that is indicative of the importance of the autogenous regulation of tubulin mRNA stability during embryogenesis. Possible explanations for the role of protein synthesis in the control of mRNA stability are discussed. Images PMID:3211150

  8. From SARS coronavirus to novel animal and human coronaviruses

    PubMed Central

    To, Kelvin K. W.; Hung, Ivan F. N.; Chan, Jasper F. W.

    2013-01-01

    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. PMID:23977429

  9. 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.

  10. Stochastic mRNA synthesis in mammalian cells.

    PubMed

    Raj, Arjun; Peskin, Charles S; Tranchina, Daniel; Vargas, Diana Y; Tyagi, Sanjay

    2006-10-01

    Individual cells in genetically homogeneous populations have been found to express different numbers of molecules of specific proteins. We investigated the origins of these variations in mammalian cells by counting individual molecules of mRNA produced from a reporter gene that was stably integrated into the cell's genome. We found that there are massive variations in the number of mRNA molecules present in each cell. These variations occur because mRNAs are synthesized in short but intense bursts of transcription beginning when the gene transitions from an inactive to an active state and ending when they transition back to the inactive state. We show that these transitions are intrinsically random and not due to global, extrinsic factors such as the levels of transcriptional activators. Moreover, the gene activation causes burst-like expression of all genes within a wider genomic locus. We further found that bursts are also exhibited in the synthesis of natural genes. The bursts of mRNA expression can be buffered at the protein level by slow protein degradation rates. A stochastic model of gene activation and inactivation was developed to explain the statistical properties of the bursts. The model showed that increasing the level of transcription factors increases the average size of the bursts rather than their frequency. These results demonstrate that gene expression in mammalian cells is subject to large, intrinsically random fluctuations and raise questions about how cells are able to function in the face of such noise. PMID:17048983

  11. 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. PMID:26920708

  12. The Evolutionary Processes of Canine Coronaviruses

    PubMed Central

    Pratelli, Annamaria

    2011-01-01

    Since the first identification of the virus in 1971, the disease caused by canine coronavirus (CCoV) has not been adequately investigated, and the role that the virus plays in canine enteric illness has not been well established. Only after the emergence in 2002 of SARS in human has new attention been focused on coronaviruses. As a consequence of the relatively high mutation frequency of RNA-positive stranded viruses, CCoV has evolved and, with the biomolecular techniques developed over the last two decades, new virus strains, serotypes, and subtypes have been identified in infected dogs. Considering the widespread nature of CCoV infections among dog populations, several studies have been carried out, focusing upon the epidemiological relevance of these viruses and underlining the need for further investigation into the biology of CCoVs and into the pathogenetic role of the infections. This paper reports the evolutionary processes of CCoVs with a note onto recent diagnostic methods. PMID:22315601

  13. Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.

    PubMed Central

    Wittekind, M; Kolb, J M; Dodd, J; Yamagishi, M; Mémet, S; Buhler, J M; Nomura, M

    1990-01-01

    The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986). Images PMID:2183018

  14. A MicroRNA precursor surveillance system in quality control of MicroRNA synthesis.

    PubMed

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

    2014-09-18

    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 (Ago)-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

  15. 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

  16. Myelin basic protein synthesis is regulated by small non-coding RNA 715.

    PubMed

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-09-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein. PMID:22744314

  17. Myelin Basic Protein synthesis is regulated by small non-coding RNA 715

    PubMed Central

    Bauer, Nina M; Moos, Christina; van Horssen, Jack; Witte, Maarten; van der Valk, Paul; Altenhein, Benjamin; Luhmann, Heiko J; White, Robin

    2012-01-01

    Oligodendroglial Myelin Basic Protein (MBP) synthesis is essential for myelin formation in the central nervous system. During oligodendrocyte differentiation, MBP mRNA is kept in a translationally silenced state while intracellularly transported, until neuron-derived signals initiate localized MBP translation. Here we identify the small non-coding RNA 715 (sncRNA715) as an inhibitor of MBP translation. SncRNA715 localizes to cytoplasmic granular structures and associates with MBP mRNA transport granule components. We also detect increased levels of sncRNA715 in demyelinated chronic human multiple sclerosis lesions, which contain MBP mRNA but lack MBP protein. PMID:22744314

  18. Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

    PubMed Central

    Fogg, Mark H.; Teterina, Natalya L.; Ehrenfeld, Ellie

    2003-01-01

    Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation. PMID:14557626

  19. 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. PMID:18351890

  20. 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. PMID:20217155

  1. 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.

  2. Detection of Coronaviruses in Bats of Various Species in Italy

    PubMed Central

    Lelli, Davide; Papetti, Alice; Sabelli, Cristiano; Rosti, Enrica; Moreno, Ana; Boniotti, Maria B.

    2013-01-01

    Bats are natural reservoirs for many mammalian coronaviruses, which have received renewed interest after the discovery of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) CoV in humans. This study describes the identification and molecular characterization of alphacoronaviruses and betacoronaviruses in bats in Italy, from 2010 to 2012. Sixty-nine faecal samples and 126 carcasses were tested using pan-coronavirus RT-PCR. Coronavirus RNAs were detected in seven faecal samples and nine carcasses. A phylogenetic analysis of RNA-dependent RNA polymerase sequence fragments aided in identifying two alphacoronaviruses from Kuhl’s pipistrelle (Pipistrellus kuhlii), three clade 2b betacoronaviruses from lesser horseshoe bats (Rhinolophus hipposideros), and 10 clade 2c betacoronaviruses from Kuhl’s pipistrelle, common noctule (Nyctalus noctula), and Savi’s pipistrelle (Hypsugo savii). This study fills a substantive gap in the knowledge on bat-CoV ecology in Italy, and extends the current knowledge on clade 2c betacoronaviruses with new sequences obtained from bats that have not been previously described as hosts of these viruses. PMID:24184965

  3. Detection of coronaviruses in bats of various species in Italy.

    PubMed

    Lelli, Davide; Papetti, Alice; Sabelli, Cristiano; Rosti, Enrica; Moreno, Ana; Boniotti, Maria B

    2013-11-01

    Bats are natural reservoirs for many mammalian coronaviruses, which have received renewed interest after the discovery of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS) CoV in humans. This study describes the identification and molecular characterization of alphacoronaviruses and betacoronaviruses in bats in Italy, from 2010 to 2012. Sixty-nine faecal samples and 126 carcasses were tested using pan-coronavirus RT-PCR. Coronavirus RNAs were detected in seven faecal samples and nine carcasses. A phylogenetic analysis of RNA-dependent RNA polymerase sequence fragments aided in identifying two alphacoronaviruses from Kuhl's pipistrelle (Pipistrellus kuhlii), three clade 2b betacoronaviruses from lesser horseshoe bats (Rhinolophus hipposideros), and 10 clade 2c betacoronaviruses from Kuhl's pipistrelle, common noctule (Nyctalus noctula), and Savi's pipistrelle (Hypsugo savii). This study fills a substantive gap in the knowledge on bat-CoV ecology in Italy, and extends the current knowledge on clade 2c betacoronaviruses with new sequences obtained from bats that have not been previously described as hosts of these viruses. PMID:24184965

  4. 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

  5. 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)

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

    DOE PAGESBeta

    Leung, Daisy  W.; Borek, Dominika; Luthra, Priya; Binning, Jennifer  M.; Anantpadma, Manu; Liu, Gai; Harvey, Ian B.; Su, Zhaoming; Endlich-Frazier, Ariel; Pan, Juanli; et al

    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

  7. Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis.

    PubMed

    Komoda, Keisuke; Ishibashi, Kazuhiro; Kawamura-Nagaya, Kazue; Ishikawa, Masayuki

    2014-11-01

    Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV. Based on activity-guided fractionation of the cell-free extract, we identified eukaryotic elongation factor (eEF) 1A as a possible host factor facilitating TSWV transcription and replication. The RNA synthesis-supporting activity decreased in the presence of an eEF1A inhibitor, suggesting that eEF1A plays an important role in RNA synthesis of TSWV. PMID:25151062

  8. Highly diversified coronaviruses in neotropical bats.

    PubMed

    Corman, Victor Max; Rasche, Andrea; Diallo, Thierno Diawo; Cottontail, Veronika M; Stöcker, Andreas; Souza, Breno Frederico de Carvalho Dominguez; Corrêa, Jefferson Ivan; Carneiro, Aroldo José Borges; Franke, Carlos Roberto; Nagy, Martina; Metz, Markus; Knörnschild, Mirjam; Kalko, Elisabeth K V; Ghanem, Simon J; Morales, Karen D Sibaja; Salsamendi, Egoitz; Spínola, Manuel; Herrler, Georg; Voigt, Christian C; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

    2013-09-01

    Bats host a broad diversity of coronaviruses (CoVs), including close relatives of human pathogens. There is only limited data on neotropical bat CoVs. We analysed faecal, blood and intestine specimens from 1562 bats sampled in Costa Rica, Panama, Ecuador and Brazil for CoVs by broad-range PCR. CoV RNA was detected in 50 bats representing nine different species, both frugivorous and insectivorous. These bat CoVs were unrelated to known human or animal pathogens, indicating an absence of recent zoonotic spill-over events. Based on RNA-dependent RNA polymerase (RdRp)-based grouping units (RGUs) as a surrogate for CoV species identification, the 50 viruses represented five different alphacoronavirus RGUs and two betacoronavirus RGUs. Closely related alphacoronaviruses were detected in Carollia perspicillata and C. brevicauda across a geographical distance exceeding 5600 km. Our study expands the knowledge on CoV diversity in neotropical bats and emphasizes the association of distinct CoVs and bat host genera. PMID:23761408

  9. 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.

  10. A cis-acting mutation in the Sindbis virus junction region which affects subgenomic RNA synthesis.

    PubMed Central

    Grakoui, A; Levis, R; Raju, R; Huang, H V; Rice, C M

    1989-01-01

    The synthesis of Sindbis virus minus-strand and genomic and subgenomic RNAs is believed to require specific cis-acting sequences or structures in the template RNAs and a combination of virus-specific proteins and host components which act in trans. A conserved sequence of about 21 nucleotides in the junction region and encompassing the start site for the subgenomic RNA has been proposed to function as the promoter on the minus-strand template for synthesis of the subgenomic RNA (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982). We introduced a three-base insertion in this sequence, which also inserts a single amino acid near the COOH terminus of nsP4, in a cDNA clone of Sindbis virus from which infectious RNA transcripts can be generated. The phenotype of this mutant, called Toto1100CR4.1, was studied after RNA transfection of chicken embryo fibroblasts or BHK cells. The mutation leads to a drastic reduction in the level of the subgenomic RNA but does not alter the start site of the RNA. Probably as a consequence of depressed structural-protein synthesis, very few progeny virions are released and the mutant makes tiny or indistinct plaques even after prolonged incubation. The cis-acting effect of this mutation was demonstrated by incorporating either a wild-type or mutant junction region into a defective-interfering RNA and examining the relative synthesis of defective-interfering RNA-derived subgenomic RNA in vivo in the presence of wild-type helper virus. These results show that the junction region is recognized by yet unidentified viral trans-acting components for subgenomic RNA synthesis. When the Toto1100CR4.1 mutant was passaged in culture, plaque morphology variants readily arose. A total of 24 independent revertants were isolated, and 16 were characterized in detail. All revertants analyzed showed an increase in the level of subgenomic RNA synthesis. Sequence analysis of the junction region

  11. A molecular nanodevice for targeted degradation of mRNA during protein synthesis

    PubMed Central

    Lee, Kyung-Ho; Min, Seung-Eui; Kim, Haseong; Lee, Seung-Goo; Kim, Dong-Myung

    2016-01-01

    RNase H is an endonuclease that catalyzes the cleavage of RNA. Because it only acts on RNA in RNA:DNA hybrids, RNase H can be used for targeted degradation of RNA when used in combination with antisense oligodeoxyribonucleotides (ASODNs) designed against a specific sequence of the target RNA. In this study, ASODN and RNase H were co-conjugated on magnetic nanoparticles. The resulting nanoparticles, having integrated functions of probing and processing target RNA, were able to remove target mRNA sequences more effectively than free ASODNs. The paramagnetic property of the nanoparticles also enabled timed engagement and disengagement of the RNA-degrading components in a given system, and these nanoparticles were able to be used for ON/OFF control of gene expression during cell-free protein synthesis reactions. PMID:26857021

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

    PubMed

    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 genomes(1). They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb(1,2). Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation(1). For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism(3). 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 replication(4). 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 Förster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary 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:27572643

  13. Synthesis of ¹⁸O-labeled RNA for application to kinetic studies and imaging.

    PubMed

    Hamasaki, Tomohiro; Matsumoto, Takahiro; Sakamoto, Naoya; Shimahara, Akiko; Kato, Shiori; Yoshitake, Ayumi; Utsunomiya, Ayumi; Yurimoto, Hisayoshi; Gabazza, Esteban C; Ohgi, Tadaaki

    2013-07-01

    Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of (18)O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging (18)O atom into the phosphate group during the oxidation step of the synthetic cycle by using (18)O water as the oxygen donor. The (18)O label in the RNA was stable at pH 3-8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The (18)O/(16)O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of (18)O-labeled RNA, and this technique was used to determine the blood concentration of (18)O-labeled RNA after administration to mice. (18)O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies. PMID:23632164

  14. PATTERNS OF RNA SYNTHESIS IN T5-INFECTED CELLS I. AS STUDIED BY THE TECHNIQUE OF DNA-RNA HYBRIDIZATION-COMPETITION*

    PubMed Central

    Moyer, Richard W.; Buchanan, John M.

    1969-01-01

    The RNA-labeling patterns obtained after T5 infection of Escherichia coli F agree with the patterns of protein labeling published by McCorquodale and Buchanan.1 Three distinct classes of RNA formed sequentially during the period of viral development can be recognized by the DNA-RNA hybridization-competition technique. Class I RNA is formed within 5 minutes after the beginning of viral metabolism and corresponds to the RNA synthesized in response to infection with the 8 per cent segment of T5 DNA. Protein synthesis directed by this 8 per cent segment is required in some capacity for the cessation of class I synthesis and the beginning of the synthesis of class II at 4 to 5 min after infection. Class III RNA synthesis begins between 9 and 12 minutes. Its appearance is prevented when chloramphenicol is added immediately after complete expression of class I functions. PMID:4916923

  15. Microinjection of purified ornithine decarboxylase into Xenopus oocytes selectively stimulates ribosomal RNA synthesis.

    PubMed Central

    Russell, D H

    1983-01-01

    This study has utilized stage VI oocytes of Xenopus laevis which have amplified the rDNA gene 1,000-fold to assess whether the microinjection of ornithine decarboxylase (OrnDCase) would stimulate [alpha-32P]guanosine incorporation into 45S and 18S/28S RNA selectively. The injection of purified OrnDCase into individual oocytes resulted in a greater than 2-fold increase in the incorporation of [32P]guanosine into 45S RNA and 18S/28S RNA with no increased incorporation into low molecular weight RNA. Further, an irreversible inhibitor of OrnDCase, alpha-difluoromethylornithine (CHF2-Orn), rapidly inhibited the endogenous activity of OrnDCase when added to the buffered Hepes solution bathing the oocytes and also inhibited the incorporation of [32P]guanosine into rRNA. The inhibitory effect of CHF2-Orn could not be reversed totally by addition of 10 microM putrescine to the oocytes. OrnDCase injected into oocytes in the presence of CHF2-Orn in the media did not stimulate incorporation of [32P]guanosine label into rRNA. However, when CHF2-Orn was removed from the buffered medium at the time of the injection of label and enzyme, a 3-fold increase of 32P incorporation into 18S/28S RNA occurred. Therefore, in an in vivo model in which amplified extrachromosomal rDNA gene copies are present, the microinjection of OrnDCase was capable of specifically stimulating rRNA synthesis. CHF2-Orn, a suicide enzyme inactivator of OrnDCase, was able to inhibit rRNA synthesis and, after washout, there was a more marked stimulation of rRNA synthesis than occurred after only the injection of OrnDCase alone. These data suggest further that OrnDCase is the labile protein that regulates the initiation of RNA synthesis. PMID:6402779

  16. The Murine Norovirus Core Subgenomic RNA Promoter Consists of a Stable Stem-Loop That Can Direct Accurate Initiation of RNA Synthesis

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. IMPORTANCE 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. PMID:25392209

  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. Influenza virion transcriptase: synthesis in vitro of large, polyadenylic acid-containing complementary RNA.

    PubMed Central

    Plotch, S J; Krug, R M

    1977-01-01

    The influenza virion transcriptase is capable of synthesizing in vitro complementary RNA (cRNA) that is similar in several characteristics to the cRNA synthesized in the infected cell, which is the viral mRNA. Most of the in vitro cRNA is large (approximately 2.5 X 10(5) to 10(6) daltons), similar in size to in vivo cRNA. The in vitro transcripts initiate in adenosine (A) or guanosine (G) at the 5' end, as also appears to be the case with in vivo cRNA (R.M. Krug et al., 1976). The in vitro transcripts contain covalently linked polyadenylate [poly(A)] sequences, which are longer and more heterogeneous than the poly(A) sequences found on in vivo cRNA. The synthesis in vitro of cRNA with these characteristics requires both the proper divalent cation, Mg2+, and a specific dinulceside monophosphage (DNMP), ApG or GpG. These DNMPs stimulate cRNA synthesis about 100-fold in the presence of Mg2+ and act as primers to initiate RNA chains, as demonstrated by the fact that the 5'-phosphorylated derivatives of these DNMP's, 32pApG or 32pGpG, are incroporated at the 5' end of the product RNA. The RNA synthesized in vitro differs from in vivo cRNA in that neither capping nor methylation of the in vitro transcripts has been detected. The virion does contain a methylase activity, as shown by its ability to methylate exogenous methyl-deficient Escherichia coli tRNA. PMID:833924

  19. SARS: lessons learned from other coronaviruses.

    PubMed

    Navas-Martin, Sonia; Weiss, Susan R

    2003-01-01

    The identification of a new coronavirus as the etiological agent of severe acute respiratory syndrome (SARS) has evoked much new interest in the molecular biology and pathogenesis of coronaviruses. This review summarizes present knowledge on coronavirus molecular biology and pathogenesis with particular emphasis on mouse hepatitis virus (MHV). MHV, a member of coronavirus group 2, is a natural pathogen of the mouse; MHV infection of the mouse is considered one of the best models for the study of demyelinating disease, such as multiple sclerosis, in humans. As a result of the SARS epidemic, coronaviruses can now be considered as emerging pathogens. Future research on SARS needs to be based on all the knowledge that coronavirologists have generated over more than 30 years of research. PMID:14733734

  20. The hepatitis C virus core protein can modulate RNA-dependent RNA synthesis by the 2a polymerase

    PubMed Central

    Wen, Y.; Cheng Kao, C.

    2014-01-01

    RNA replication enzymes are multi-subunit protein complexes whose activity can be modulated by other viral and cellular factors. For genotype 1b Hepatitis C virus (HCV), the RNA-dependent RNA polymerase (RdRp) subunit of the replicase, NS5B, has been reported to interact with the HCV Core protein to decrease RNA synthesis (Kang et al., 2009). Here we used a cell-based assay for RNA synthesis to examine the Core–NS5B interaction of genotype 2a HCV. Unlike the 1b NS5B, the activity of the 2a NS5B was stimulated by the Core protein. Using the bimolecular fluorescence complementation assay, the 2a Core co-localized with 2a NS5B when they were transiently expressed in cells. The two proteins can form a coimmunoprecipitable complex. Deletion analysis showed that the N-terminal 75 residues of 2a Core were required to contact 2a NS5B to modulate its activity. The C-terminal transmembrane helix of 2a NS5B also contributes to the interaction with the 2a Core. To determine the basis for the differential effects of the Core–RdRp interaction, we found that the 2a RdRp activity was enhanced by both the 1b Core and 2a Core. However, the 1b NS5B activity was slightly inhibited by either Core protein. The replication of the 2a JFH-1 replicon was increased by co-expressed 2a Core while the genotype 1b Con1 replicon was not significantly affected by the corresponding Core. Mutations in 2a NS5B that affected the closed RdRp structure were found to be less responsive to 2a Core. Finally, we determined that RNA synthesis by the RdRps from genotypes 2a, 3a and 4a HCV were increased by the Core proteins from HCV of genotypes 1–4. These results reveal another difference between RNA syntheses by the different genotype RdRps and add additional examples of a viral structural protein regulating viral RNA synthesis. PMID:24874198

  1. The yin and yang of hepatitis C: synthesis and decay of hepatitis C virus RNA.

    PubMed

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

    2015-09-01

    Hepatitis C virus (HCV) is an unusual RNA virus that has a striking capacity to persist for the remaining life of the host 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 the host RNA-binding proteins and microRNAs, and describe how these interactions influence the competing processes of viral RNA synthesis and decay to achieve stable, long-term persistence of the viral genome. Furthermore, we discuss how these processes affect hepatitis C pathogenesis and therapeutic strategies against HCV. PMID:26256788

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

    PubMed Central

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

    2016-01-01

    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. PMID:22265299

  3. 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.

  4. 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. PMID:22265299

  5. 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

  6. Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes.

    PubMed Central

    Sethna, P B; Brian, D A

    1997-01-01

    The majority of porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%). They therefore are probably free molecules in solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-to-minus-strand molar ratio of approximately 8:1 and viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs. PMID:9311859

  7. [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.

  8. The synthesis and stability of cytoplasmic messenger RNA during myoblast differentiation in culture.

    PubMed

    Buckingham, M E; Caput, D; Cohen, A; Whalen, R G; Gros, F

    1974-04-01

    The synthesis of poly(A)-containing cytoplasmic RNA was examined in primary myoblast cultures prepared from skeletal muscle of fetal calves. After a period of cell division, these cells undergo fusion, with concomitant appearance of acetylcholine receptor and subsequent myosin synthesis. In the dividing myoblast there is a high level of messenger RNA synthesis, including a 26S RNA, the size of a putative messenger for the large subunit of myosin. In the transition period prior to fusion, there are quantitative changes in RNA synthesis. At this time, there is a pronounced production of 26S RNA, which diminishes during fusion. The possibility that 26S RNA is accumulated in the dividing myoblast was investigated by chase experiments. At fusion, there is a marked increase in the half-lives of a number of messenger RNA species, including 26 S, which increases from about 10 hr in the dividing cell to a value of more than 50 hr. The identity of the more rapidly turning over 26 S in the myoblasts, compared to that of the 26 S at fusion, was examined in terms of polysomal distribution, migration on gels, and hybridization with complementary DNA for the myosin message. The results of these analyses suggest that the 26S species are identical. Thus, it would appear that in a predetermined cell like the myoblast, the transition to the differentiated state of myotube that is synthesizing muscle specific proteins is effected by the stabilization of messenger already being actively transcribed: terminal differentiation, with respect to myosin synthesis, is preceded by the stabilization of 26S RNA. PMID:4524649

  9. Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation

    PubMed Central

    Thornbrough, Joshua M.; Jha, Babal K.; Yount, Boyd; Goldstein, Stephen A.; Li, Yize; Elliott, Ruth; Sims, Amy C.; Baric, Ralph S.; Silverman, Robert H.

    2016-01-01

    ABSTRACT Middle East respiratory syndrome coronavirus (MERS-CoV) is the first highly pathogenic human coronavirus to emerge since severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002. Like many coronaviruses, MERS-CoV carries genes that encode multiple accessory proteins that are not required for replication of the genome but are likely involved in pathogenesis. Evasion of host innate immunity through interferon (IFN) antagonism is a critical component of viral pathogenesis. The IFN-inducible oligoadenylate synthetase (OAS)-RNase L pathway activates upon sensing of viral double-stranded RNA (dsRNA). Activated RNase L cleaves viral and host single-stranded RNA (ssRNA), which leads to translational arrest and subsequent cell death, preventing viral replication and spread. Here we report that MERS-CoV, a lineage C Betacoronavirus, and related bat CoV NS4b accessory proteins have phosphodiesterase (PDE) activity and antagonize OAS-RNase L by enzymatically degrading 2′,5′-oligoadenylate (2-5A), activators of RNase L. This is a novel function for NS4b, which has previously been reported to antagonize IFN signaling. NS4b proteins are distinct from lineage A Betacoronavirus PDEs and rotavirus gene-encoded PDEs, in having an amino-terminal nuclear localization signal (NLS) and are localized mostly to the nucleus. However, the expression level of cytoplasmic MERS-CoV NS4b protein is sufficient to prevent activation of RNase L. Finally, this is the first report of an RNase L antagonist expressed by a human or bat coronavirus and provides a specific mechanism by which this occurs. Our findings provide a potential mechanism for evasion of innate immunity by MERS-CoV while also identifying a potential target for therapeutic intervention. PMID:27025250

  10. Light-regulated protein and poly(A)+ mRNA synthesis in Neurospora crassa.

    PubMed Central

    Chambers, J A; Hinkelammert, K; Russo, V E

    1985-01-01

    We have examined the effect of illumination upon the patterns of protein synthesis in the filamentous ascomycete Neurospora crassa by pulse labelling and two-dimensional gel electrophoresis. Light did not affect overall rates of protein synthesis but did induce the synthesis of six novel polypeptides whose appearance followed a temporally regulated pattern. When translation products of mRNA from illuminated cultures and dark control cultures were compared it was found that the synthesis of five out of six of the polypeptides specific to illuminated cultures could be seen in vitro. We believe that this is consistent with the hypothesis that light regulates the transcription of some genes in N. crassa, although we cannot exclude effects on mRNA stability or the control of precursor splicing. Images Fig. 2. Fig. 3. PMID:2868891

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

    PubMed

    Te Velthuis, Aartjan J W; Fodor, Ervin

    2016-08-01

    The genomes of influenza viruses consist 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, which form 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, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets. PMID:27396566

  12. L Protein Requirement for In Vitro RNA Synthesis by Vesicular Stomatitis Virus

    PubMed Central

    Emerson, Suzanne U.; Wagner, Robert R.

    1973-01-01

    The endogenous transcriptase present in purified vesicular stomatitis (VS) virions was solubilized with a Triton X-100 high-salt solution. The polymerase activity was purified on glycerol gradients and by phosphocellulose column chromatography; the viral proteins present in the active enzyme fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was demonstrated that L protein, but not NS protein, was required for in vitro RNA synthesis on the VS viral nucleocapsid template. Solubilized L protein rebinds to the ribonucleoprotein template when the transcription complex is reconstituted, and the RNA synthesized in vitro by purified L protein hybridizes to virion RNA. Cyanogen bromide peptide fingerprints indicate that the large L protein is a unique polypeptide chain. It is concluded that the L protein functions as the transcriptase, and the nucleocapsid NS protein is not essential for in vitro RNA synthesis. PMID:4357510

  13. 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

  14. 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

  15. Nonenzymatic template-directed RNA synthesis inside model protocells.

    PubMed

    Adamala, Katarzyna; Szostak, Jack W

    2013-11-29

    Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg(2+) on fatty acid membranes. Here we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg(2+) ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg(2+)-catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides. PMID:24288333

  16. De novo synthesis of minus strand RNA by the rotavirus RNA polymerase in a cell-free system involves a novel mechanism of initiation.

    PubMed Central

    Chen, D; Patton, J T

    2000-01-01

    The replicase activity of rotavirus open cores has been used to study the synthesis of (-) strand RNA from viral (+) strand RNA in a cell-free replication system. The last 7 nt of the (+) strand RNA, 5'-UGUGACC-3', are highly conserved and are necessary for efficient (-) strand synthesis in vitro. Characterization of the cell-free replication system revealed that the addition of NaCl inhibited (-) strand synthesis. By preincubating open cores with (+) strand RNA and ATP, CTP, and GTP prior to the addition of NaCl and UTP, the salt-sensitive step was overcome. Thus, (-) strand initiation, but not elongation, was a salt-sensitive process in the cell-free system. Further analysis of the requirements for initiation showed that preincubating open cores and the (+) strand RNA with GTP or UTP, but not with ATP or CTP, allowed (-) strand synthesis to occur in the presence of NaCl. Mutagenesis suggested that in the presence of GTP, (-) strand synthesis initiated at the 3'-terminal C residue of the (+) strand template, whereas in the absence of GTP, an aberrant initiation event occurred at the third residue upstream from the 3' end of the (+) strand RNA. During preincubation with GTP, formation of the dinucleotides pGpG and ppGpG was detected; however, no such products were made during preincubation with ATP, CTP, or UTP. Replication assays showed that pGpG, but not GpG, pApG, or ApG, served as a specific primer for (-) strand synthesis and that the synthesis of pGpG may occur by a template-independent process. From these data, we conclude that initiation of rotavirus (-) strand synthesis involves the formation of a ternary complex consisting of the viral RNA-dependent RNA polymerase, viral (+) strand RNA, and possibly a 5'-phosphorylated dinucleotide, that is, pGpG or ppGpG. PMID:11073221

  17. 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.

  18. Mammalian transcription in support of hybrid mRNA and protein synthesis in testis and lung.

    PubMed

    Fitzgerald, Carolyn; Sikora, Curtis; Lawson, Vannice; Dong, Karen; Cheng, Min; Oko, Richard; van der Hoorn, Frans A

    2006-12-15

    Post-transcriptional mechanisms including differential splicing expand the protein repertoire beyond that provided by the one gene-one protein model. Trans-splicing has been observed in mammalian systems but is low level (sometimes referred to as noise), and a contribution to hybrid protein expression is unclear. In the study of rat sperm tail proteins a cDNA, called 1038, was isolated representing a hybrid mRNA derived in part from the ornithine decarboxylase antizyme 3 (Oaz3) gene located on rat chromosome 2 fused to sequences encoded by a novel gene on chromosome 4. Cytoplasmic Oaz3 mRNA is completely testis specific. However, in several tissues Oaz3 is transcribed and contributes to hybrid 1038 mRNA synthesis, without concurrent Oaz3 mRNA synthesis. 1038 mRNA directs synthesis of a hybrid 14-kDa protein, part chromosome 2- and part chromosome 4-derived as shown in vitro and in transfected cells. Antisera that recognize a chromosome 4-encoded C-terminal peptide confirm the hybrid character of endogenous 14-kDa protein and its presence in sperm tail structures and 1038-positive tissue. Our data suggest that the testis-specific OAZ3 gene may be an example of a mammalian gene that in several tissues is transcribed to contribute to a hybrid mRNA and protein. This finding expands the repertoire of known mechanisms available to cells to generate proteome diversity. PMID:17040916

  19. Enhancement of Polyribosome Formation and RNA Synthesis of Gibberellic Acid in Wounded Potato Tuber Tissue 1

    PubMed Central

    Wielgat, Bernard; Kahl, Günter

    1979-01-01

    As part of a more detailed study on plant tumorigenesis, the action of gibberellic acid (GA3) in wounded potato tuber tissues as a model system has been evaluated. GA3 stimulates total RNA synthesis in wounded tissues, the optimal concentration being 0.1 micromolar. The responsiveness of the tissue toward the hormone develops with time after wounding. Whereas freshly wounded tissue does not respond at all to the hormone, it becomes competent after about 6 hours, the competence being maximal after 1 day of wound healing. GA3 enhances the formation of polyribosomes in wounded tissues and stimulates the synthesis of both ribosomal RNAs, transfer RNAs, 5S RNA, and a fraction, which in sucrose density gradients sediments between 18S rRNA and 5S RNA. This fraction contains presumptive mRNA. The hormone, then, is somehow recognized by wounded potato tissue in a time-specific way; the signal is transferred to the genome and triggers the synthesis of various RNA species. PMID:16661070

  20. 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

  1. Systems approaches to coronavirus pathogenesis

    PubMed Central

    Schäfer, Alexandra; Baric, Ralph S.; Ferris, Martin T.

    2014-01-01

    Coronaviruses comprise a large group of emergent human and animal pathogens, including the highly pathogenic SARS-CoV and MERS-CoV strains that cause significant morbidity and mortality in infected individuals, especially the elderly. As emergent viruses may cause episodic outbreaks of disease over time, human samples are limited. Systems biology and genetic technologies maximize opportunities for identifying critical host and viral genetic factors that regulate susceptibility and virus-induced disease severity. These approaches provide discovery platforms that highlight and allow targeted confirmation of critical targets for prophylactics and therapeutics, especially critical in an outbreak setting. Although poorly understood, it has long been recognized that host regulation of virus-associated disease severity is multigenic. The advent of systems genetic and biology resources provide new opportunities for deconvoluting the complex genetic interactions and expression networks that regulate pathogenic or protective host response patterns following virus infection. Using SARS-CoV as a model, dynamic transcriptional network changes and disease-associated phenotypes have been identified in different genetic backgrounds, leading to the promise of population-wide discovery of the underpinnings of Coronavirus pathogenesis. PMID:24842079

  2. Studies on the Role of RNA Synthesis in Auxin Induction of Cell Enlargement 1

    PubMed Central

    Nooden, Larry D.

    1968-01-01

    Selective inhibitors were used to study the connection between nucleic acid synthesis and indoleacetic acid (IAA) induction of cell enlargement. Actinomycin D (act D) and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber disks when they are added simultaneously with IAA. In contrast, when they are added 24 hours after the hormone, these inhibitors have little or no effect on the induced growth which continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably supporting metabolism are still essential. In corn coleoptile sections auxin-induced growth did not show any pronounced tendency to become less sensitive to act D as the IAA treatment progressed. Act D did not completely inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth when added with the IAA. Although IAA has a very large and very rapid stimulatory effect (within 10 min) on incorporation of 32P-orthophosphate into RNA in disks, it did not cause a detectable change in the base composition of the RNA synthesized. Furthermore, the promotive effect could be accounted for through increased uptake of the 32P. That much of the RNA synthesis in these tissues is not necessary for auxin action is indicated by the results with fluorouracil (FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections. FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion of respiration by auxin likewise may not be entirely necessary for growth. At least in the artichoke disks, RNA synthesis is required for auxin induction of cell enlargement and not for cell enlargement itself. The possible

  3. 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

  4. Synthesis of Poly Linear shRNA Expression Cassettes Through Branch-PCR.

    PubMed

    Liu, Jianbing; Xi, Zhen

    2016-01-01

    A facile and universal strategy to construct the poly linear small hairpin RNA (shRNA) expression cassettes with multiple shRNA transcription templates through polymerase chain reaction with flexible branched primers (branch-PCR) is described in this protocol. Double-stranded RNA (dsRNA) is not stable enough for the study of RNA interference (RNAi) delivery in mammalian cells. Therefore, the more stable shRNA transcription template is employed to produce the endogenous transcribed dsRNA. Then, the covalent crosslinked linear shRNA expression cassettes are constructed through the branch-PCR for the long-lasting RNAi effect in this protocol. The branched primer pair is efficiently synthesized through classic click chemistry. In one step of PCR, the much more stable poly linear shRNA expression cassettes can be produced in large scale. This strategy of efficient synthesis of the poly linear gene expression cassettes can also be applied in the field for other target gene delivery. © 2016 by John Wiley & Sons, Inc. PMID:27584702

  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. Genomic and serological detection of bat coronavirus from bats in the Philippines.

    PubMed

    Tsuda, Shumpei; Watanabe, Shumpei; Masangkay, Joseph S; Mizutani, Tetsuya; Alviola, Phillip; Ueda, Naoya; Iha, Koichiro; Taniguchi, Satoshi; Fujii, Hikaru; Kato, Kentaro; Horimoto, Taisuke; Kyuwa, Shigeru; Yoshikawa, Yasuhiro; Akashi, Hiroomi

    2012-12-01

    Bat coronavirus (BtCoV) is assumed to be a progenitor of severe acute respiratory syndrome (SARS)-related coronaviruses. To explore the distribution of BtCoVs in the Philippines, we collected 179 bats and detected viral RNA from intestinal or fecal samples by RT-PCR. The overall prevalence of BtCoVs among bats was 29.6 %. Phylogenetic analysis of the partial RNA-dependent RNA polymerase gene suggested that one of the detected BtCoVs was a novel alphacoronavirus, while the others belonged to the genus Betacoronavirus. Western blotting revealed that 66.5 % of bat sera had antibodies to BtCoV. These surveys suggested the endemic presence of BtCoVs in the Philippines. PMID:22833101

  7. 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.

  8. SARS coronavirus protein 7a interacts with human Ap4A-hydrolase

    PubMed Central

    2010-01-01

    The SARS coronavirus (SARS-CoV) open reading frame 7a (ORF 7a) encodes a 122 amino acid accessory protein. It has no significant sequence homology with any other known proteins. The 7a protein is present in the virus particle and has been shown to interact with several host proteins; thereby implicating it as being involved in several pathogenic processes including apoptosis, inhibition of cellular protein synthesis, and activation of p38 mitogen activated protein kinase. In this study we present data demonstrating that the SARS-CoV 7a protein interacts with human Ap4A-hydrolase (asymmetrical diadenosine tetraphosphate hydrolase, EC 3.6.1.17). Ap4A-hydrolase is responsible for metabolizing the "allarmone" nucleotide Ap4A and therefore likely involved in regulation of cell proliferation, DNA replication, RNA processing, apoptosis and DNA repair. The interaction between 7a and Ap4A-hydrolase was identified using yeast two-hybrid screening. The interaction was confirmed by co-immunoprecipitation from cultured human cells transiently expressing V5-His tagged 7a and HA tagged Ap4A-hydrolase. Human tissue culture cells transiently expressing 7a and Ap4A-hydrolase tagged with EGFP and Ds-Red2 respectively show these proteins co-localize in the cytoplasm. PMID:20144233

  9. 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

  10. Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice.

    PubMed

    Yount, Boyd; Roberts, Rhonda S; Sims, Amy C; Deming, Damon; Frieman, Matthew B; Sparks, Jennifer; Denison, Mark R; Davis, Nancy; Baric, Ralph S

    2005-12-01

    SARS coronavirus (SARS-CoV) encodes several unique group-specific open reading frames (ORFs) relative to other known coronaviruses. To determine the significance of the SARS-CoV group-specific ORFs in virus replication in vitro and in mice, we systematically deleted five of the eight group-specific ORFs, ORF3a, OF3b, ORF6, ORF7a, and ORF7b, and characterized recombinant virus replication and gene expression in vitro. Deletion of the group-specific ORFs of SARS-CoV, either alone or in various combinations, did not dramatically influence replication efficiency in cell culture or in the levels of viral RNA synthesis. The greatest reduction in virus growth was noted following ORF3a deletion. SARS-CoV spike (S) glycoprotein does not encode a rough endoplasmic reticulum (rER)/Golgi retention signal, and it has been suggested that ORF3a interacts with and targets S glycoprotein retention in the rER/Golgi apparatus. Deletion of ORF3a did not alter subcellular localization of the S glycoprotein from distinct punctuate localization in the rER/Golgi apparatus. These data suggest that ORF3a plays little role in the targeting of S localization in the rER/Golgi apparatus. In addition, insertion of the 29-bp deletion fusing ORF8a/b into the single ORF8, noted in early-stage SARS-CoV human and civet cat isolates, had little if any impact on in vitro growth or RNA synthesis. All recombinant viruses replicated to wild-type levels in the murine model, suggesting that either the group-specific ORFs play little role in in vivo replication efficiency or that the mouse model is not of sufficient quality for discerning the role of the group-specific ORFs in disease origin and development. PMID:16282490

  11. Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1.

    PubMed

    Song, Yunke; Liu, Kelvin J; Wang, Tza-Huei

    2015-01-01

    MicroRNA profiling methods have become increasingly important due to the rapid rise of microRNA in both basic and translational sciences. A critical step in many microRNA profiling assays is adapter ligation using pre-adenylated adapters. While pre-adenylated adapters can be chemically or enzymatically prepared, enzymatic adenylation is preferred due to its ease and high yield. However, previously reported enzymatic methods either require tedious purification steps or use thermostable ligases that can generate side products during the subsequent ligation step. We have developed a highly efficient, template- and purification-free, adapter adenylation method using T4 RNA ligase 1. This method is capable of adenylating large amounts of adapter at ~100% efficiency and can efficiently adenylate both DNA and RNA bases. We find that the adenylation reaction speed can differ between DNA and RNA and between terminal nucleotides, leading to bias if reactions are not allowed to run to completion. We further find that the addition of high PEG levels can effectively suppress these differences. PMID:26500066

  12. The Riia Gene of Bacteriophage T4. II. Regulation of Its Messenger RNA Synthesis

    PubMed Central

    Daegelen, P.; Brody, E.

    1990-01-01

    When the rII genes are first introduced into cells which had been previously infected by T4 phage deleted for these genes, the kinetics of synthesis of rIIA and rIIB RNA are rapid and identical. We show that this rapid synthesis depends on a functional motA gene for rIIB, but not for rIIA, RNA synthesis. By primer-extension mapping of T4 messenger RNA, we find three promoters close to the rIIA gene. One of them is an early promoter just before the rIIA.1 gene; it is used under all conditions tested. Another is in the coding portion of the rIIA.1 gene; it is weak, primarily because of a 19-bp spacing between the -10 and -35 elements, and its use is stimulated by T4 functions. The third is a motA-dependent (middle) promoter which has an unusual CCCGCTT box at -33. We present results which suggest that none of these promoters is likely to be the site at which the motB and motC gene products exercise their major influence on rIIA RNA synthesis. PMID:2379818

  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. 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

  15. Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response

    PubMed Central

    Paulsen, Michelle T.; Veloso, Artur; Prasad, Jayendra; Bedi, Karan; Ljungman, Emily A.; Tsan, Ya-Chun; Chang, Ching-Wei; Tarrier, Brendan; Washburn, Joseph G.; Lyons, Robert; Robinson, Daniel R.; Kumar-Sinha, Chandan; Wilson, Thomas E.; Ljungman, Mats

    2013-01-01

    Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings. PMID:23345452

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

    PubMed Central

    Nguyen, Le Xuan Truong; Mitchell, Beverly S.

    2013-01-01

    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. PMID:24297901

  17. Fidaxomicin Is an Inhibitor of the Initiation of Bacterial RNA Synthesis

    PubMed Central

    Artsimovitch, Irina; Seddon, Jaime; Sears, Pamela

    2012-01-01

    Fidaxomicin was recently approved for the treatment of Clostridium difficile infection. It inhibits transcription by bacterial RNA polymerase. Because transcription is a multistep process, experiments were conducted in which fidaxomicin was added at different stages of transcriptional initiation to identify the blocked step. DNA footprinting experiments were also conducted to further elucidate the stage inhibited. Fidaxomicin blocks initiation only if added before the formation of the “open promoter complex,” in which the template DNA strands have separated but RNA synthesis has not yet begun. Binding of fidaxomicin precludes the initial separation of DNA strands that is prerequisite to RNA synthesis. These studies show that it has a mechanism distinct from that of elongation inhibitors, such as streptolydigin, and from the transcription initiation inhibitors myxopyronin and the rifamycins. PMID:22752861

  18. 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.

  19. 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

  20. Guanine nucleotide metabolism in a mutant strain of Escherichia coli with a temperature sensitive lesion in rRNA synthesis.

    PubMed

    Harris, J S; Chaney, S G

    1978-12-21

    We have described a mutant of Escherichia coli (designated 2S142) which shows specific inhibition of rRNA synthesis at 42 degrees C. ppGpp levels increase at the restrictive temperature, as expected. However, when the cells are returned to 30 degrees C, rRNA synthesis resumes before ppGpp levels have returned to normal. Furthermore, when ppGpp levels are decreased by the addition of tetracycline or choramphenicol, rRNA synthesis does not resume at 42 degrees C. Also, a derivative of 2S142 with a temperature-sensitive G factor (which cannot synthesize either protein or ppGpp at 42 degrees C) shows identical kinetics of rRNA shut-off at 42 degrees C as 2S142. Thus, the elevated ppGpp levels in this mutant do not appear to be directly responsible for the cessation of rRNA synthesis at 42 degrees C. PMID:367439

  1. Synthesis of double-stranded RNA in a virus-enriched fraction from Agaricus bisporus

    SciTech Connect

    Sriskantha, A.; Wach, P.; Schlagnhaufer, B.; Romaine, C.P.

    1986-03-01

    Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from health sporophores. Enzyme activity was dependent upon the presence of Mg/sup 2 +/ and the four nucleoside triphosphates and was insensitive to actinomycin D, ..cap alpha..-amanitin, and rifampin. The /sup 3/H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 /times/ 10/sup 6/ and 1.4 /times/ 10/sup 6/. Cs/sub 2/SO/sub 4/ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 /times/ 10/sup 6/ and 1.4 /times/ 10/sup 6/. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.

  2. A novel in vitro replication system for Dengue virus. Initiation of RNA synthesis at the 3'-end of exogenous viral RNA templates requires 5'- and 3'-terminal complementary sequence motifs of the viral RNA.

    PubMed

    You, S; Padmanabhan, R

    1999-11-19

    Positive strand viral replicases are membrane-bound complexes of viral and host proteins. The mechanism of viral replication and the role of host proteins are not well understood. To understand this mechanism, a viral replicase assay that utilizes extracts from dengue virus-infected mosquito (C6/36) cells and exogenous viral RNA templates is reported in this study. The 5'- and 3'-terminal regions (TR) of the template RNAs contain the conserved elements including the complementary (cyclization) motifs and stem-loop structures. RNA synthesis in vitro requires both 5'- and 3'-TR present in the same template molecule or when the 5'-TR RNA was added in trans to the 3'-untranslated region (UTR) RNA. However, the 3'-UTR RNA alone is not active. RNA synthesis occurs by elongation of the 3'-end of the template RNA to yield predominantly a double-stranded hairpin-like RNA product, twice the size of the template RNA. These results suggest that an interaction between 5'- and 3'-TR of the viral RNA that modulates the 3'-UTR RNA structure is required for RNA synthesis by the viral replicase. The complementary cyclization motifs of the viral genome also seem to play an important role in this interaction. PMID:10559263

  3. 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.

  4. 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.

  5. 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

  6. Preferential synthesis of low-molecular-weight RNA in uv-irradiated plasma of Physarum polycephalum

    SciTech Connect

    Kumari, P.A.V.; Nair, V.R.

    1981-10-01

    Mitotically synchronous surface plasmodia of Physarum polycephalum were irradiated during the G2 phase with a Philips 15-W germicidal lamp. At different intervals after irradiation, the plasmodia were pulse-labeled with (/sup 3/H)uridine, and RNA was extracted and analyzed on linear sucrose gradients. The radioactivity profiles of the RNA showed that irradiated plasmodia synthesize preferentially low-molecular-weight RNA types, including 4 SRNA, during the delay period prior to the first postirradiation mitosis and during the following short mitotic cycle. Double-labeling experiments, employing (/sup 14/C)uridine-prelabeled plasmodia which were pulse-labeled with (/sup 3/H)uridine after irradiation, confirmed this finding. It is also seen that there is an overall reduction in the rate of synthesis of rRNA in the irradiated plasmodia.

  7. An antisense RNA controls synthesis of an SOS-induced toxin evolved from an antitoxin

    PubMed Central

    Kawano, Mitsuoki; Aravind, L; Storz, Gisela

    2007-01-01

    Only few small, regulatory RNAs encoded opposite another gene have been identified in bacteria. Here, we report the characterization of a locus where a small RNA (SymR) is encoded in cis to an SOS-induced gene whose product shows homology to the antitoxin MazE (SymE). Synthesis of the SymE protein is tightly repressed at multiple levels by the LexA repressor, the SymR RNA and the Lon protease. SymE co-purifies with ribosomes and overproduction of the protein leads to cell growth inhibition, decreased protein synthesis and increased RNA degradation. These properties are shared with several RNA endonuclease toxins of the toxin-antitoxin modules, and we show that the SymE protein represents evolution of a toxin from the AbrB fold, whose representatives are typically antitoxins. We suggest that SymE promotion of RNA cleavage may be important for the recycling of RNAs damaged under SOS-inducing conditions. PMID:17462020

  8. Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery

    PubMed Central

    Srinivasan, Selvi; Shubin, Andrew D.; Stayton, Patrick S.

    2011-01-01

    Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-copropylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior.1,2 These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here, we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system, as the folate receptor is an attractive target for tumor-selective therapies due to its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer. PMID:21634800

  9. 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.

  10. Synthesis of oligodiaminomannoses and analysis of their RNA duplex binding properties and their potential application as siRNA-based drugs.

    PubMed

    Iwata, Rintaro; Doi, Akiko; Maeda, Yusuke; Wada, Takeshi

    2015-09-28

    The synthesis of artificial cationic oligodiaminosaccharides, α-(1 → 4)-linked-2,6-diamino-2,6-dideoxy-d-mannopyranose oligomers (ODAMans), and their interactions with RNA duplexes are described. The monomer through the pentamer, all of which bear unnatural 2,6-diaminomannose moieties, were successfully prepared. UV melting and fluorescence anisotropy analyses revealed that the ODAMans bound and thermodynamically stabilized both 12mer RNA duplexes and an siRNA. Furthermore, it was clearly shown that the siRNA acquired substantial RNase A resistance due to its binding to the ODAMan 4mer. PMID:26256756

  11. 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.

  12. [Electron-microscopic autoradiography of RNA synthesis in the myocardium after damage to it].

    PubMed

    Galankin, V N; Pal'tsyn, A A; Badikova, A K

    1977-06-01

    Thermic burn of the wall of the left cardiac ventricle was inflicted to new born rats. Twenty-four hours after the injury the RNA synthesis of the myocardial cells remote from the site of burn were investigated by electron-microscopic autoradiography. Tissue samples were fixed 2 and 6 hours after the 3H-uridine injections. As compared with the control, experimental animals displayed a reduction of silver grains density over the nucleus and the cytoplasm of cardiomyocytes. PMID:884310

  13. Membrane ectopeptidases targeted by human coronaviruses

    PubMed Central

    Bosch, Berend Jan; Smits, Saskia L.; Haagmans, Bart L.

    2014-01-01

    Six coronaviruses, including the recently identified Middle East respiratory syndrome coronavirus, are known to target the human respiratory tract causing mild to severe disease. Their interaction with receptors expressed on cells located in the respiratory tract is an essential first step in the infection. Thus far three membrane ectopeptidases, dipeptidyl peptidase 4 (DPP4), angiotensin-converting enzyme 2 (ACE2) and aminopeptidase N (APN), have been identified as entry receptors for four human-infecting coronaviruses. Although the catalytic activity of the ACE2, APN, and DPP4 peptidases is not required for virus entry, co-expression of other host proteases allows efficient viral entry. In addition, evolutionary conservation of these receptors may permit interspecies transmissions. Because of the physiological function of these peptidase systems, pathogenic host responses may be potentially amplified and cause acute respiratory distress. PMID:24762977

  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. Guanine nucleotide depletion inhibits pre-ribosomal RNA synthesis and causes nucleolar disruption.

    PubMed

    Huang, Min; Ji, Yanshan; Itahana, Koji; Zhang, Yanping; Mitchell, Beverly

    2008-01-01

    Inosine monophosphate dehydrogenase (IMPDH) is a pivotal enzyme in the de novo pathway of guanine nucleotide biosynthesis. Inhibitors of this enzyme decrease intracellular guanine nucleotide levels by 50-80% and have potential as anti-neoplastic agents. Both mycophenolic acid (MPA) and AVN-944 are highly specific inhibitors of IMPDH that cause cell cycle arrest or apoptosis in lymphocytes and leukemic cell lines. We have examined the mechanisms by which these two agents cause cytotoxicity. Both MPA and AVN-944 inhibit the growth of K562 cells, and induce apoptosis in Raji B and CCRF-CEM T cells. Both compounds strikingly inhibit RNA synthesis within 2 h of exposure. Depletion of guanine nucleotides by MPA and AVN-944 also causes an early and near-complete reduction in levels of the 45S precursor rRNA synthesis and the concomitant translocation of nucleolar proteins including nucleolin, nucleophosmin, and nucleostemin from the nucleolus to the nucleoplasm. This efflux correlates temporally with the sustained induction of p53 in cell lines with wild-type p53. We conclude that inhibition of IMPDH causes a primary reduction in rRNA synthesis and secondary nucleolar disruption and efflux of nucleolar proteins that most likely mediate cell cycle arrest or apoptosis. The ability of AVN-944 to induce apoptosis in a number of leukemic cell lines supports its potential utility in the treatment of hematologic malignancies. PMID:17462731

  16. Impaired rRNA synthesis triggers homeostatic responses in hippocampal neurons

    PubMed Central

    Kiryk, Anna; Sowodniok, Katharina; Kreiner, Grzegorz; Rodriguez-Parkitna, Jan; Sönmez, Aynur; Górkiewicz, Tomasz; Bierhoff, Holger; Wawrzyniak, Marcin; Janusz, Artur K.; Liss, Birgit; Konopka, Witold; Schütz, Günther; Kaczmarek, Leszek; Parlato, Rosanna

    2013-01-01

    Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimer's disease (AD) and may play a role in dementia. Moreover, aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3, and dentate gyrus (DG). Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that 36 transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF) binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the DG a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving neuronal function. PMID:24273493

  17. Action of Inhibitors of RNA and Protein Synthesis on Cell Enlargement 1

    PubMed Central

    Noodén, Larry D.; Thimann, Kenneth V.

    1966-01-01

    Further studies with inhibitors of protein synthesis are presented to support the conclusion, drawn from work with chloramphenicol, that protein synthesis is a critical limiting factor in auxin-induced cell expansion. The indoleacetic acid-induced elongation of oat coleoptile sections was strongly inhibited by dl-p-fluorophenylalanine, and the inhibition is antagonized by phenylalanine. Puromycin at 10−4 m very strongly inhibited the indoleacetic acid-induced growth of oat coleoptile and artichoke tuber sections and exerted a less powerful effect on pea stem sections. As found earlier with chloramphenicol, concentrations of puromycin effective in inhibiting the growth of coleoptile sections had quantitatively similar effects on protein synthesis, as measured by the incorporation of C14-leucine into protein of the coleoptile tissue. Several analogues of RNA bases were also tested, but while 8-azaguanine very strongly inhibited growth of artichoke tuber disks, 6-azauracil was the only one of this group clearly inhibitory to growth in coleoptile or pea stem sections. Actinomycin D actively inhibited both elongation and the incorporation of C14-leucine into protein in oat coleoptile sections. Inhibition of the 2 processes went closely parallel. Actinomycin D also powerfully inhibited growth of artichoke tuber disks. All the compounds effective in inhibiting growth generally inhibited the uptake of leucine as well. The possibility that auxin causes cell enlargement in plants by inducing the synthesis of a messenger RNA and of one or more new but unstable enzymes, is discussed. Possible but less favored alternative explanations are: A) that auxin induces synthesis of a wall protein, or B) that the continued synthesis of some other unstable protein (by a process independent of auxin) may be a prerequisite for cell enlargement. PMID:5904588

  18. De novo mRNA synthesis is required for both consolidation and reconsolidation of fear memories in the amygdala

    PubMed Central

    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 the lateral nucleus of the amygdala (LA). We have previously shown that de novo protein synthesis in the LA is required for reconsolidation of auditory fear memories. One key question is whether protein synthesis during reconsolidation depends on already existing mRNAs or on synthesis of new mRNAs in the amygdala. In the present study, we examined the effect of mRNA synthesis inhibition during consolidation and reconsolidation of auditory fear memories. We first show that intra-LA infusion of two different mRNA inhibitors dose-dependently impairs long-term memory but leaves short-term memory (STM) intact. Next, we show that intra-LA infusion of the same inhibitors dose-dependently blocks post-reactivation long-term memory (PR-LTM), whereas post-reactivation short-term memory (PR-STM) is left intact. Furthermore, the same treatment in the absence of memory reactivation has no effect. Together, these results show that both consolidation and reconsolidation of auditory fear memories require de novo mRNA synthesis and are equally sensitive to disruption of de novo mRNA synthesis in the LA. PMID:18832561

  19. 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

    2016-05-01

    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. PMID:26975377

  20. 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.

  1. The effect of trichloroethylene and acrylonitrile on RNA and ribosome synthesis and ribosome content in Saccharomyces cells.

    PubMed

    Lochmann, E R; Ehrlich, W; Mangir, M

    1984-04-01

    The effects of trichloroethylene (TCE) and acrylonitrile (ACN) on growth, RNA synthesis, ribosome synthesis, and ribosome content were tested in yeast cells. TCE causes a delay of the growth of a cell culture (prolongation of the lag phase), but does not cause inhibition. Cells exposed to increasing concentrations of ACN show increasing damage, so that, at a certain point of the growth curve, cell division stops altogether. Similar results were obtained when RNA synthesis was investigated: After treatment with TCE, the maximum RNA synthesis of the cell culture was retarded, but subsequently reached the same level as the untreated control cells. In the presence of ACN, however, the rate of RNA synthesis was lowered with increasing ACN concentrations. The same effect was observed upon investigation of ribosome synthesis: Whereas TCE produces only a slight effect, treatment with increasing concentrations of ACN leads to a substantial decrease in ribosome synthesis, and finally to total inhibition. Parallel to this, the content of free and membrane-bound ribosomes is diminished. Obviously, the decrease in ribosome content is caused not only by an inhibition of ribosome synthesis, but also by a degradation of existing ribosomes, as well as by induction of a ribosome-associated RNase. PMID:6714140

  2. RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

    PubMed Central

    Ganguly, R; Banerjee, M R

    1978-01-01

    Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA. PMID:724523

  3. CORONAVIRUS REVERSE GENETIC SYSTEMS: INFECTIOUS CLONES AND REPLICONS

    PubMed Central

    Almazán, Fernando; Sola, Isabel; Zuñiga, Sonia; Marquez-Jurado, Silvia; Morales, Lucia; Becares, Martina; Enjuanes, Luis

    2016-01-01

    Coronaviruses (CoVs) infect humans and many animal species, and are associated with respiratory, enteric, hepatic, and central nervous system diseases. The large size of the CoV genome and the instability of some CoV replicase gene sequences during its propagation in bacteria, represent serious obstacles for the development of reverse genetic systems similar to those used for smaller positive sense RNA viruses. To overcome these limitations, several alternatives to more conventional plasmid-based approaches have been established in the last thirteen years. In this report, we briefly review and discuss the different reverse genetic systems developed for CoVs, paying special attention to the severe acute respiratory syndrome CoV (SARS-CoV). PMID:24930446

  4. 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.

  5. Effect of protein synthesis inhibitors on viral mRNA's synthesized early in adenovirus type 2 infection.

    PubMed Central

    Eggerding, F; Raskas, H J

    1978-01-01

    Viral mRNA species synthesized early in adenovirus type 2 infection in the presence of cycloheximide were compared with those synthesized in the absence of drug or in the presence of the DNA synthesis inhibitor 1-beta-D-arabinofuranosylcytosine. Cycloheximide caused approximately a 10-fold stimulation in the accumulation of [3H]uridine into early viral mRNA species. The only exception was a 24s mRNA transcribed from the transforming end of the genome; in the presence of cycloheximide, accumulation of this mRNA species was stimulated no more than 2-fold. Treatment with cycloheximide also resulted in the accumulation of polyadenylated RNAs transcribed from EcoRI-C that are heterogeneous and smaller than the 20S mRNA. Other translation inhibitors were shown to have similar effects, suggesting that inhibition of protein synthesis early after infection induces alterations in the metabolism of specific RNA sequences. PMID:621786

  6. Synthesis of human adenovirus early RNA species is similar in productive and abortive infections of monkey and human cells.

    PubMed Central

    Anderson, K P; Klessig, D F

    1982-01-01

    Northern (RNA) blot analysis has been used to show that synthesis of early mRNA species is similar in monkey cells productively or abortively infected with human adenovirus. mRNA species from all five major early regions (1A, 1B, 2, 3, 4) are identical in size and comparable in abundance whether isolated from monkey cells infected with adenovirus type 2 or with the host range mutant Ad2hr400 or coinfected with adenovirus type 2 plus simian virus 40. The mRNA species isolated from monkey cells are identical in size to those isolated from human cells. Production of virus-associated RNA is also identical in productive and abortive infections of monkey cells. Synthesis of virus-associated RNA is, however, significantly greater in HeLa cells than in CV1 cells at late times after infection regardless of which virus is used in the infection. Images PMID:6283181

  7. Rabies RNA synthesis, detected with cDNA probes, as a marker for virus transport in the rat nervous system.

    PubMed

    Ermine, A; Ceccaldi, P E; Masson, G; Tsiang, H

    1993-02-01

    The kinetics of viral RNA synthesis in different parts of the rat brain, infected with fixed or street rabies virus strains, is correlated with their anatomical neuronal connections with the masseter muscles, using hybridization with rabies cDNA probes. Viral RNA synthesis is first detected in the brain-stem and in the pons where the direct anatomical projection of the masseter muscle nervous arborization into the sensory and motor nuclei is located, through the trigeminus nerve. Rabies RNA detection is delayed in the other regions of the rat brain depending on the time course of virus transport from the trigeminal nuclei through multiple nervous connections. PMID:7681151

  8. Effects of induction of rRNA overproduction on ribosomal protein synthesis and ribosome subunit assembly in Escherichia coli.

    PubMed Central

    Yamagishi, M; Nomura, M

    1988-01-01

    Overproduction of rRNA was artificially induced in Escherichia coli cells to test whether the synthesis of ribosomal protein (r-protein) is normally repressed by feedback regulation. When rRNA was overproduced more than twofold from a hybrid plasmid carrying the rrnB operon fused to the lambda pL promoter (pL-rrnB), synthesis of individual r-proteins increased by an average of about 60%. This demonstrates that the synthesis of r-proteins is repressed under normal conditions. The increase of r-protein production, however, for unknown reasons, was not as great as the increase in rRNA synthesis and resulted in an imbalance between the amounts of rRNA and r-protein synthesis. Therefore, only a small (less than 20%) increase in the synthesis of complete 30S and 50S ribosome subunits was detected, and a considerable fraction of the excess rRNA was degraded. Lack of complete cooperativity in the assembly of ribosome subunits in vivo is discussed as a possible explanation for the absence of a large stimulation of ribosome synthesis observed under these conditions. In addition to the induction of intact rRNA overproduction from the pL-rrnB operon, the effects of unbalanced overproduction of each of the two large rRNAs, 16S rRNA and 23S rRNA, on r-protein synthesis were examined using pL-rrnB derivatives carrying a large deletion in either the 23S rRNA gene or the 16S rRNA gene. Operon-specific derepression after 23S or 16S rRNA overproduction correlated with the overproduction of rRNA containing the target site for the operon-specific repressor r-protein. These results are discussed to explain the apparent coupling of the assembly of one ribosomal subunit with that of the other which was observed in earlier studies on conditionally lethal mutants with defects in ribosome assembly. PMID:3053641

  9. Reverse Transcription Recombinase Polymerase Amplification Assay for the Detection of Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Abd El Wahed, Ahmed; Patel, Pranav; Heidenreich, Doris; Hufert, Frank T.; Weidmann, Manfred

    2013-01-01

    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. PMID:24459611

  10. Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

    PubMed Central

    Kuo, Lili; Godeke, Gert-Jan; Raamsman, Martin J. B.; Masters, Paul S.; Rottier, Peter J. M.

    2000-01-01

    Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells. PMID:10627550

  11. 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. PMID:23239278

  12. 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

    2013-01-01

    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. PMID:24459611

  13. [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. PMID:27295884

  14. 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. PMID:25438801

  15. 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. PMID:23364191

  16. 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

  17. Suppression of Coronavirus Replication by Cyclophilin Inhibitors

    PubMed Central

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2013-01-01

    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. PMID:23698397

  18. Suppression of coronavirus replication by cyclophilin inhibitors.

    PubMed

    Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

    2013-05-01

    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. PMID:23698397

  19. Hemagglutinating Encephalomyelitis Coronavirus Infection in Pigs, Argentina

    PubMed Central

    Cappuccio, Javier; Piñeyro, Pablo; Basso, Walter; Moré, Gastón; Kienast, Mariana; Schonfeld, Sergio; Cáncer, José L.; Arauz, Sandra; Pintos, María E.; Nanni, Mariana; Machuca, Mariana; Hirano, Norio; Perfumo, Carlos J.

    2008-01-01

    We describe an outbreak of vomiting, wasting, and encephalomyelitis syndrome in piglets in Argentina, caused by porcine hemagglutinating encephalomyelitis coronavirus (PHE-CoV) infection. Diagnosis was made by epidemiologic factors, pathologic features, immunohistochemistry, reverse transcription–PCR, and genomic sequencing. This study documents PHE-CoV infection in South America. PMID:18325268

  20. 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

  1. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites.

    PubMed

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A; Li, Jianhui; Randall, Glenn; Belov, George A; Wang, Xiaofeng

    2016-02-23

    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

  2. In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination.

    PubMed

    Kwon, Hyung-Jun; Ryu, Young Bae; Kim, Young-Min; Song, Naaleum; Kim, Cha Young; Rho, Mun-Chual; Jeong, Jae-Ho; Cho, Kyoung-Oh; Lee, Woo Song; Park, Su-Jin

    2013-08-01

    Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2-5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1-5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection. PMID:23746631

  3. Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

    PubMed Central

    Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

    2015-01-01

    ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

  4. Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia.

    PubMed

    Anindita, Paulina Duhita; Sasaki, Michihito; Setiyono, Agus; Handharyani, Ekowati; Orba, Yasuko; Kobayashi, Shintaro; Rahmadani, Ibnu; Taha, Siswatiana; Adiani, Sri; Subangkit, Mawar; Nakamura, Ichiro; Sawa, Hirofumi; Kimura, Takashi

    2015-04-01

    Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus. PMID:25643817

  5. 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.

  6. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

    SciTech Connect

    Kim, Sunyoung; Baltimore, D. Massachusetts Institute of Technology, Cambridge ); Byrn, R.; Groopman, J. )

    1989-09-01

    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. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4{sup +} 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.

  7. Function of DNA polymerase I in RNA-primed synthesis of bacteriophage M-13 duplex DNA.

    PubMed Central

    Schneck, P K; Staudenbauer, W L; Hofschneider, P H

    1976-01-01

    Cell-free extracts from Escherichia coli contain a DNA polymerase activity resistant to SH-blocking agents, which is capable of synthesizing complementary strand DNA on a circular M-13 DNA template by extension of RNA primers. This activity is considered to be identical with DNA polymerase I (or some altered form of this enzyme) since it is missing in extracts from po1A- cells. DNA synthesis in the presence of SH-blocking agents occurs at a reduced rate as compared to untreated controls and leads to the formation of DNA chains of defined size (0.4-0.5 genome's length). It is concluded that efficient M-13 duplex DNA synthesis requires the cooperation of both DNA polymerase I and III. PMID:1272793

  8. 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. PMID:26899596

  9. CNP2 mRNA directs synthesis of both CNP1 and CNP2 polypeptides.

    PubMed

    O'Neill, R C; Minuk, J; Cox, M E; Braun, P E; Gravel, M

    1997-10-15

    The ribosome scanning model for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of two translational start sites on the mRNA encoding isoform 2 of the myelin marker enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in rat and mouse. We show that the CNP2 mRNA is able to direct synthesis of not only CNP2, but also CNP1 polypeptide. Immunoprecipitation experiments using a polyclonal antibody directed against CNP detect both CNP isoforms in tissues or cell lines expressing only the CNP2 transcript. Thus, the synthesis of CNP1 and CNP2 polypeptides must be encoded by the CNP2 transcript. In vitro translation of synthetic CNP2 mRNA demonstrates that both CNP isoforms are synthesized by initiation at different AUG codons. Furthermore, by introducing mutations to "switch off" translation from the second in-frame AUG codon in the CNP2 cDNA, and transfecting 293T cells with those constructs, we are able to correlate the production of CNP1 and CNP2 with different translational start sites. These results lead us to conclude that the CNP2 mRNA is able to produce both CNP1 and CNP2 polypeptides. This investigation has altered our understanding of the temporal expression of the CNP protein isoforms during development of the central nervous system (CNS). PMID:9373034

  10. Genetic diversity of coronaviruses in Miniopterus fuliginosus bats.

    PubMed

    Du, Jiang; Yang, Li; Ren, Xianwen; Zhang, Junpeng; Dong, Jie; Sun, Lilian; Zhu, Yafang; Yang, Fan; Zhang, Shuyi; Wu, Zhiqiang; Jin, Qi

    2016-06-01

    Coronaviruses, such as severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus, pose significant public health threats. Bats have been suggested to act as natural reservoirs for both these viruses, and periodic monitoring of coronaviruses in bats may thus provide important clues about emergent infectious viruses. The Eastern bent-wing bat Miniopterus fuliginosus is distributed extensively throughout China. We therefore analyzed the genetic diversity of coronaviruses in samples of M. fuliginosus collected from nine Chinese provinces during 2011-2013. The only coronavirus genus found was Alphacoronavirus. We established six complete and five partial genomic sequences of alphacoronaviruses, which revealed that they could be divided into two distinct lineages, with close relationships to coronaviruses in Miniopterus magnater and Miniopterus pusillus. Recombination was confirmed by detecting putative breakpoints of Lineage 1 coronaviruses in M. fuliginosus and M. pusillus (Wu et al., 2015), which supported the results of topological and phylogenetic analyses. The established alphacoronavirus genome sequences showed high similarity to other alphacoronaviruses found in other Miniopterus species, suggesting that their transmission in different Miniopterus species may provide opportunities for recombination with different alphacoronaviruses. The genetic information for these novel alphacoronaviruses will improve our understanding of the evolution and genetic diversity of coronaviruses, with potentially important implications for the transmission of human diseases. PMID:27125516

  11. 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

  12. 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

  13. 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

  14. Identification of a human mitochondrial RNA that promotes tropomyosin synthesis and myocardial differentiation.

    PubMed

    Moses-Arms, Ashley; Kochegarov, Andrei; Arms, Jedidiah; Burlbaw, Shane; Lian, Will; Meyer, Jessica; Lemanski, Larry F

    2015-03-01

    Heart disease is the number one killer in the USA, making cardiogenesis and its related pathways a relevant area of study for improving health and life expectancy. The Mexican salamander (axolotl), Ambystoma mexicanum, provides an excellent vertebrate animal model for studying myofibrillogenesis due to its naturally occurring cardiac nonfunction mutation. Homozygous recessive embryos do not develop normal hearts due to a lack of myofibril formation. In previous studies, myofibril-inducing ribonucleic acid (MIR) from the normal wild-type axolotl genome was found to rescue mutant nonfunctioning hearts through restoration of tropomyosin levels followed by normal myofibril formation. Our purpose in this study is to identify and characterize functional homologs for the MIR from human fetal heart ribonucleic acid (RNA). After randomized cloning of human fetal heart RNA, 396 clones were analyzed for rescuing ability by using mutant heart rescue bioassays and confocal microscopy. By these analyses, we discovered a functional homolog of MIR from human fetal heart RNA, which is associated with the mitochondrial cytochrome c oxidase subunit II gene. This RNA came from our clone #30 and induces tropomyosin synthesis and myofibrillogenesis in mutant axolotl hearts which ordinarily do not synthesize tropomyosin or form organized myofibrils. Clone #30, a mitochondrial RNA molecule associated with human cytochrome c oxidase, serves as a functional homolog of MIR, leading to tropomyosin production, organized myofibrils, and beating cardiac tissue in mutant hearts. These findings hold great potential for the treatment and repair of damaged hearts in patients who have suffered from myocardial infarctions and other heart diseases. PMID:25408381

  15. Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor

    PubMed Central

    Burstein, David E.; Greene, Lloyd A.

    1978-01-01

    Studies on the mechanism of action of nerve growth factor (NGF) were carried out with PC12 rat pheochromocytoma cells. PC12 cells are uniquely useful for such studies because they respond to, but (unlike normal neurons) do not require, NGF and may undergo either generation or regeneration of neurites in response to NGF. Regeneration is defined here as NGF-dependent regrowth of neurites within 24 hr after subculture of NGF-treated PC12 cells. As in cultures of normal NGF-responsive neurons, neurite regeneration by PC12 cells occurs even in the presence of high concentrations of RNA synthesis inhibitors. Generation of neurites is defined as the de novo initiation of outgrowth when PC12 cells are exposed to NGF for the first time. In contrast to regeneration, neurite generation takes place with a lag of at least 24 hr and is blocked by low concentrations of RNA synthesis inhibitors. Such findings suggest that there are both RNA synthesis-dependent and -independent pathways in the mechanism whereby NGF stimulates neurite outgrowth. In addition, NGF-treated PC12 cells undergo a time-dependent loss of the capacity for neurite regeneration after pretreatment with RNA synthesis inhibitors or withdrawal of NGF. Such findings suggest that (i) initiation of neurite outgrowth requires NGF-stimulated, RNA synthesis-dependent accumulation of intracellular material(s), (ii) once such accumulation occurs, RNA synthesis-independent regeneration can occur (but only in the presence of NGF), and (iii) the turnover of such material(s) in the absence of their replacement leads to loss of the capacity for regeneration. A tentative sequence is presented for the events whereby NGF may stimulate neurite outgrowth. PMID:310552

  16. 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

  17. Analysis of putative recombination hot sites in the S gene of canine coronaviruses.

    PubMed

    Wang, Y Y; Lu, C P

    2009-01-01

    The S gene sequence of Canine coronavirus strain 1-71 (CCoV 1-71) was cloned, sequenced, and compared to those of other CCoVs, Transmissible gastroenteritis virus (TGEV), and Feline coronavirus (FCoV). The sequence analysis showed that CCoV 1-71 displayed a 98.8-99.8% identity with CCoVs strains V1, K378, and GP. Four putative recombination sites were found at the 5'-end of the S gene, namely at nt 53, 75, 425, 991. Both sequences flanking each site were significantly different. Three recombination hot regions were found on the S gene, namely at nt 337-437, 1545-3405, and 4203-4356, which shared a common recombination signal with Group 2 coronaviruses. The G/CTAAAAA/GT sequence downstream of the recombination site may represent a specific recombination signal in CCoVs. The CCoV 1-71 S protein sequence was found to be similar to those of other CCoVs except for several N-glycosylation sites at the N-terminus of the S protein, which could be related to the differences in virulence and cell tropism in individual CCoVs. This study indicated that the similarity of CCoVs in virulence and tropism was mostly acquired by the homologous RNA recombination and not only by simple mutation and selection. PMID:19537912

  18. Rapid Synthesis, RNA Binding, and Antibacterial Screening of a Peptidic-Aminosugar (PA) Library

    PubMed Central

    Jiang, Liuwei; Watkins, Derrick; Jin, Yi; Gong, Changjun; King, Ada; Washington, Arren Z.; Green, Keith D.; Garneau-Tsodikova, Sylvie; Oyelere, Adegboyega K.; Arya, Dev P.

    2016-01-01

    A 215-member mono- and diamino acid peptidic-aminosugar (PA) library, with neomycin as the model aminosugar, was systematically and rapidly synthesized via solid phase synthesis. Antibacterial activities of the PA library, on 13 bacterial strains (seven Gram-positive and six Gram-negative bacterial strains), and binding affinities of the PA library for a 27-base model of the bacterial 16S ribosomal A-site RNA were evaluated using high-throughput screening. The results of the two assays were correlated using Ribosomal Binding-Bacterial Inhibition Plot (RB-BIP) analysis to provide structure–activity relationship (SAR) information. From this work, we have identified PAs that can discriminate the E. coli A-site from the human A-site by up to a 28-fold difference in binding affinity. Aminoglycoside-modifying enzyme activity studies indicate that APH(2″)-Ia showed nearly complete removal of activity with a number of PAs. The synthesis of the compound library and screening can both be performed rapidly, allowing for an iterative process of aminoglycoside synthesis and screening of PA libraries for optimal binding and antibacterial activity for lead identification. PMID:25706406

  19. Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis.

    PubMed Central

    Tapprich, W E; Goss, D J; Dahlberg, A E

    1989-01-01

    A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535. The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E. coli and caused a retardation of cell growth. The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis. The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits. The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis. PMID:2662189

  20. 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. PMID:24571086

  1. The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

    PubMed Central

    Borucki, Monica K.; Allen, Jonathan E.; Chen-Harris, Haiyin; Zemla, Adam; Vanier, Gilda; Mabery, Shalini; Torres, Clinton; Hullinger, Pamela; Slezak, Tom

    2013-01-01

    The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation. PMID:23308119

  2. Molecular pathology of emerging coronavirus infections

    PubMed Central

    Gralinski, Lisa E; Baric, Ralph S

    2015-01-01

    Respiratory viruses can cause a wide spectrum of pulmonary diseases, ranging from mild, upper respiratory tract infections to severe and life-threatening lower respiratory tract infections, including the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Viral clearance and subsequent recovery from infection require activation of an effective host immune response; however, many immune effector cells may also cause injury to host tissues. Severe acute respiratory syndrome (SARS) coronavirus and Middle East respiratory syndrome (MERS) coronavirus cause severe infection of the lower respiratory tract, with 10% and 35% overall mortality rates, respectively; however, >50% mortality rates are seen in the aged and immunosuppressed populations. While these viruses are susceptible to interferon treatment in vitro, they both encode numerous genes that allow for successful evasion of the host immune system until after high virus titres have been achieved. In this review, we discuss the importance of the innate immune response and the development of lung pathology following human coronavirus infection. PMID:25270030

  3. Synthesis of reinitiated transcripts by mammalian RNA polymerase II is controlled by elongation factor SII.

    PubMed Central

    Szentirmay, M N; Sawadogo, M

    1993-01-01

    Previous studies have revealed that the in vitro synthesis of reinitiated transcripts by RNA polymerase II requires an additional activity, designated reinitiation transcription factor (RTF), which is distinct from all of the general class II initiation factors. While further characterizing this activity, it was found that RTF displays properties indistinguishable from those of the RNA polymerase II elongation factor SII. In addition, Western blot analysis using SII-specific antibodies revealed that human SII is a major component in purified RTF preparations. The functional equivalence of the two proteins was established using recombinant SII, which proved fully capable of substituting for RTF in the reinitiation assay. In these reconstituted reactions, transcription complexes resulting from reinitiation events required SII to proceed through a 400 bp G-free cassette, while complexes resulting from the first round of initiations were SII-independent. Reinitiations can take place in the absence of SII; however, addition of the elongation factor is essential for full extension of the reinitiated transcripts. These results suggest that events taking place at the promoter (e.g. first-round initiations versus reinitiations) can create marked differences in the properties of RNA polymerase II elongation complexes. Images PMID:8223477

  4. Intersection of RNA Processing and the Type II Fatty Acid Synthesis Pathway in Yeast Mitochondria▿

    PubMed Central

    Schonauer, Melissa S.; Kastaniotis, Alexander J.; Hiltunen, J. Kalervo; Dieckmann, Carol L.

    2008-01-01

    Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5′ processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNAPro accumulated dramatically. Subsequent Pet127-driven 5′ processing of RPM1 was blocked. The FAS II pathway defects resulted in the loss of lipoic acid attachment to subunits of three key mitochondrial enzymes, which suggests that the octanoic acid produced by the pathway is the sole precursor for lipoic acid synthesis and attachment. The protein component of yeast mitochondrial RNase P, Rpm2, is not modified by lipoic acid in the wild-type strain, and it is imported in FAS II mutant strains. Thus, a product of the FAS II pathway is required for RNase P RNA maturation, which positively affects RNase P activity. In addition, a product is required for lipoic acid production, which is needed for the activity of pyruvate dehydrogenase, which feeds acetyl-coenzyme A into the FAS II pathway. These two positive feedback cycles may provide switch-like control of mitochondrial gene expression in response to the metabolic state of the cell. PMID:18779316

  5. 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. PMID:25209234

  6. 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

  7. Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

    PubMed Central

    Ruzsics, Zsolt; Friedel, Caroline C.; Koszinowski, Ulrich H.; Dölken, Lars

    2013-01-01

    The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated. We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms. PMID:23963265

  8. Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA(Leu(UUR)) and tRNA(Lys).

    PubMed

    Leszczynska, Grazyna; Leonczak, Piotr; Wozniak, Karolina; Malkiewicz, Andrzej

    2014-06-01

    5-Taurinomethyluridine (τm(5)U) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) are located at the wobble position of human mitochondrial (hmt) tRNA(Leu(UUR)) and tRNA(Lys), respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys) are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm(5)U and τm(5)s(2)U into 17-mers related to the sequence of the anticodon arms hmt-tRNA(Leu(UUR)) and hmt-tRNA(Lys), respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF3) are compatible with the blockage of the canonical monomeric units. The synthesis of τm(5)s(2)U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data. PMID:24757169

  9. 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. PMID:24447852

  10. Protease inhibitors targeting coronavirus and filovirus entry.

    PubMed

    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

    2015-04-01

    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