scholarly journals A Viral Noncoding RNA Generated by cis-Element-Mediated Protection against 5′→3′ RNA Decay Represses both Cap-Independent and Cap-Dependent Translation

2008 ◽  
Vol 82 (20) ◽  
pp. 10162-10174 ◽  
Author(s):  
Hiro-oki Iwakawa ◽  
Hiroyuki Mizumoto ◽  
Hideaki Nagano ◽  
Yuka Imoto ◽  
Kazuma Takigawa ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Synthesis ◽  
Red Clover ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

scholarly journals Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome

2003 ◽  
Vol 77 (19) ◽  
pp. 10623-10629 ◽  
Author(s):  
Alexander A Khromykh ◽  
Natasha Kondratieva ◽  
Jean-Yves Sgro ◽  
Ann Palmenberg ◽  
Edwin G Westaway
Keyword(s):  
Rna Synthesis ◽  
Point Mutations ◽  
In Vitro Activity ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Double Stranded Rna ◽  
Positive Strand ◽  
Proposed Model

ABSTRACT Point mutations that resulted in a substitution of the conserved 3′-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3′-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3′-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of φ6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3′-penultimate cytidines and a 3′-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3′-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

scholarly journals Structural and functional characterization of the coxsackievirus B3 CRE(2C): role of CRE(2C) in negative- and positive-strand RNA synthesis

2006 ◽  
Vol 87 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Mark J. M. van Ooij ◽  
Dorothee A. Vogt ◽  
Aniko Paul ◽  
Christian Castro ◽  
Judith Kuijpers ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Coxsackievirus B3 ◽  
Viral Rna ◽  
Free System ◽  
Positive Strand ◽  
Negative Strand ◽  
Cell Extracts ◽  
Positive Strand Rna

A stem–loop element located within the 2C-coding region of the coxsackievirus B3 (CVB3) genome has been proposed to function as a cis-acting replication element (CRE). It is shown here that disruption of this structure indeed interfered with viral RNA replication in vivo and abolished uridylylation of VPg in vitro. Site-directed mutagenesis demonstrated that the previously proposed enteroviral CRE consensus loop sequence, R1NNNAAR2NNNNNNR3, is also applicable to CVB3 CRE(2C) and that a positive correlation exists between the ability of CRE(2C) mutants to serve as template in the uridylylation reaction and the capacity of these mutants to support viral RNA replication. To further investigate the effects of the mutations on negative-strand RNA synthesis, an in vitro translation/replication system containing HeLa S10 cell extracts was used. Similar to the results observed for poliovirus and rhinovirus, it was found that a complete disruption of the CRE(2C) structure interfered with positive-strand RNA synthesis, but not with negative-strand synthesis. All CRE(2C) point mutants affecting the enteroviral CRE consensus loop, however, showed a marked decrease in efficiency to induce negative-strand synthesis. Moreover, a transition (A5G) regarding the first templating adenosine residue in the loop was even unable to initiate complementary negative-strand synthesis above detectable levels. Taken together, these results indicate that the CVB3 CRE(2C) is not only required for the initiation of positive-strand RNA synthesis, but also plays an essential role in the efficient initiation of negative-strand RNA synthesis, a conclusion that has not been reached previously by using the cell-free system.

scholarly journals Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

2010 ◽  
Vol 84 (9) ◽  
pp. 4229-4242 ◽  
Author(s):  
Kenneth J. Ertel ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Synthesis ◽  
Cultured Cells ◽  
Wild Type Virus ◽  
Positive Strand ◽  
Negative Strand ◽  
Virus Rna ◽  
Hnrnp C ◽  
C Proteins ◽  
Positive Strand Rna

ABSTRACT The poliovirus 3′ noncoding region (3′ NCR) is necessary for efficient virus replication. A poliovirus mutant, PVΔ3′NCR, with a deletion of the entire 3′ NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5′ end of poliovirus negative-strand RNA (the complement of the genomic 3′ NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3′ end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5′ end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.

scholarly journals Role of the Alfalfa Mosaic Virus Methyltransferase-Like Domain in Negative-Strand RNA Synthesis

2002 ◽  
Vol 76 (22) ◽  
pp. 11321-11328 ◽  
Author(s):  
A. Corina Vlot ◽  
Aymeric Menard ◽  
John F. Bol
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Alfalfa Mosaic Virus ◽  
Positive Strand ◽  
Negative Strand ◽  
Putative Role ◽  
Associated Functions ◽  
Rna Accumulation ◽  
Positive Strand Rna

ABSTRACT RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus (AMV) encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, which has a putative role in capping the viral RNAs. Six residues in this domain that are highly conserved in the methyltransferase domains of alphavirus-like viruses were mutated individually in AMV P1. None of the mutants was infectious to plants. Mutant RNA 1 was coexpressed with wild-type (wt) RNAs 2 and 3 from transferred DNA vectors in Nicotiana benthamiana by agroinfiltration. Mutation of His-100 or Cys-189 in P1 reduced accumulation of negative- and positive-strand RNA in the infiltrated leaves to virtually undetectable levels. Mutation of Asp-154, Arg-157, Cys-182, or Tyr-266 in P1 reduced negative-strand RNA accumulation to levels ranging from 2 to 38% of those for the wt control, whereas positive-strand RNA accumulation by these mutants was 2% or less. The (transiently) expressed replicases of the six mutants were purified from the agroinfiltrated leaves. Polymerase activities of these preparations in vitro ranged from undetectable to wt levels. The data indicate that, in addition to its putative role in RNA capping, the methyltransferase-like domain of P1 has distinct roles in replication-associated functions required for negative-strand RNA synthesis. The defect in negative-strand RNA synthesis of the His-100 and Cys-189 mutants could be complemented in trans by coexpression of wt P1.

scholarly journals Intracellular location and translocation of silent and active poliovirus replication complexes

2005 ◽  
Vol 86 (3) ◽  
pp. 707-718 ◽  
Author(s):  
Denise Egger ◽  
Kurt Bienz
Keyword(s):  
Viral Protein ◽  
Rna Synthesis ◽  
Nuclear Periphery ◽  
Lag Phase ◽  
Intracellular Location ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna ◽  
Intracellular Translocation

Replication of poliovirus (PV) genomic RNA in HeLa cells has previously been found to start at distinct sites at the nuclear periphery. In the present study, the earliest steps in the virus replication cycle, i.e. the appearance and intracellular translocation of viral protein and negative-strand RNA prior to positive-strand RNA synthesis, were followed. During translation, positive-strand RNA and newly synthesized viral protein presented as a dispersed endoplasmic reticulum (ER)-like pattern. Concomitant with translation, individual PV vesicle clusters emerged at the ER and formed nascent replication complexes, which contained newly synthesized negative-strand RNA. The complexes rapidly moved centripetally, in a microtubule-dependent way, to the perinuclear area to engage in positive-strand viral RNA synthesis. Replication complexes made transcriptionally silent with guanidine/HCl followed the anterograde membrane pathway to the Golgi complex within the microtubule-organizing centre (MTOC), whereas replication complexes active in positive-strand RNA synthesis were retained at the nuclear periphery. If the silent replication complexes that had accumulated at the MTOC were released from the guanidine block, transcription was not readily resumed. Rather, positive-strand RNA was redistributed back to the ER to start, after a lag phase, translation, followed by negative- and positive-strand RNA synthesis in replication complexes migrating to the nuclear periphery. As some of the findings appear to be in contrast to events reported in cell-free guanidine-synchronized translation/transcription systems, implications for the comparison of in vitro systems with the living cell are discussed.

scholarly journals Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes

2005 ◽  
Vol 79 (6) ◽  
pp. 3254-3266 ◽  
Author(s):  
Jo Ellen Brunner ◽  
Joseph H. C. Nguyen ◽  
Holger H. Roehl ◽  
Tri V. Ho ◽  
Kristine M. Swiderek ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Cellular Protein ◽  
Rna Recognition Motif ◽  
Positive Strand ◽  
Negative Strand ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
In Vitro Replication ◽  
Positive Strand Rna ◽  
Nuclear Ribonucleoprotein

ABSTRACT We had previously demonstrated that a cellular protein specifically interacts with the 3′ end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3Dpol, as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1ΔC) diminished these protein-protein interactions. When GST/hnRNP C1ΔC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.

scholarly journals Poliovirus CRE-Dependent VPg Uridylylation Is Required for Positive-Strand RNA Synthesis but Not for Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (8) ◽  
pp. 4739-4750 ◽  
Author(s):  
Kenneth E. Murray ◽  
David J. Barton
Keyword(s):  
Rna Structure ◽  
Rna Synthesis ◽  
Critical Role ◽  
Rna Replication ◽  
Stem Loop ◽  
Positive Strand ◽  
Negative Strand ◽  
Step Model ◽  
Positive Strand Rna

ABSTRACT The cis-acting replication element (CRE) is a 61-nucleotide stem-loop RNA structure found within the coding sequence of poliovirus protein 2C. Although the CRE is required for viral RNA replication, its precise role(s) in negative- and positive-strand RNA synthesis has not been defined. Adenosine in the loop of the CRE RNA structure functions as the template for the uridylylation of the viral protein VPg. VPgpUpUOH, the predominant product of CRE-dependent VPg uridylylation, is a putative primer for the poliovirus RNA-dependent RNA polymerase. By examining the sequential synthesis of negative- and positive-strand RNAs within preinitiation RNA replication complexes, we found that mutations that disrupt the structure of the CRE prevent VPg uridylylation and positive-strand RNA synthesis. The CRE mutations that inhibited the synthesis of VPgpUpUOH, however, did not inhibit negative-strand RNA synthesis. A Y3F mutation in VPg inhibited both VPgpUpUOH synthesis and negative-strand RNA synthesis, confirming the critical role of the tyrosine hydroxyl of VPg in VPg uridylylation and negative-strand RNA synthesis. trans-replication experiments demonstrated that the CRE and VPgpUpUOH were not required in cis or in trans for poliovirus negative-strand RNA synthesis. Because these results are inconsistent with existing models of poliovirus RNA replication, we propose a new four-step model that explains the roles of VPg, the CRE, and VPgpUpUOH in the asymmetric replication of poliovirus RNA.

scholarly journals Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras

2007 ◽  
Vol 81 (10) ◽  
pp. 5270-5283 ◽  
Author(s):  
Marco Binder ◽  
Doris Quinkert ◽  
Olga Bochkarova ◽  
Rahel Klein ◽  
Nikolina Kezmic ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Negative Strand ◽  
Virus Rna ◽  
Nontranslated Region ◽  
Genotype 2 ◽  
Positive Strand Rna

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

scholarly journals Differential Rescue of Poliovirus RNA Replication Functions by Genetically Modified RNA Polymerase Precursors

2004 ◽  
Vol 78 (23) ◽  
pp. 13007-13018 ◽  
Author(s):  
Christopher T. Cornell ◽  
Jo Ellen Brunner ◽  
Bert L. Semler
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Genetically Modified ◽  
Rna Replication ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Wild Type ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We have previously described the RNA replication properties of poliovirus transcripts harboring chimeric RNA polymerase sequences representing suballelic exchanges between poliovirus type 1 (PV1) and coxsackievirus B3 (CVB3) utilizing an in vitro translation and RNA replication assay (C. Cornell, R. Perera, J. E. Brunner, and B. L. Semler, J. Virol. 78:4397-4407, 2004). We showed that three of the seven chimeras were capable of RNA replication in vitro, although replication levels were greatly reduced compared to that of wild-type transcripts. Interestingly, one of the replication-competent transcripts displayed a strand-specific RNA synthesis defect suggesting (i) a differential replication complex assembly mechanism involving 3D and/or precursor molecules (i.e., 3CD) required for negative- versus positive-strand RNA synthesis or (ii) effect(s) on the ability of the 3D polymerase to form higher-ordered structures required for positive-strand RNA synthesis. In this study, we have attempted to rescue defective RNA replication in vitro by cotranslating nonstructural proteins from a transcript encoding a large precursor polyprotein (P3) to complement 3D polymerase and/or precursor polypeptide functions altered in each of the chimeric constructs. Utilization of a wild-type P3 construct revealed that all transcripts containing chimeric PV1/CVB3 polymerase sequences can be complemented in trans for both negative- and positive-strand RNA synthesis. Furthermore, data from experiments utilizing genetically modified forms of the P3 polyprotein, containing mutations within 3C or 3D sequences, strongly suggest the existence of different protein-protein and protein-RNA interactions required for positive- versus negative-strand RNA synthesis. These results, combined with data from in vitro RNA elongation assays, indicate that the delivery of active 3D RNA polymerase to replication complexes requires a series of macromolecular interactions that rely on the presence of specific 3D amino acid sequences.

scholarly journals Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis

2000 ◽  
Vol 74 (11) ◽  
pp. 5133-5141 ◽  
Author(s):  
Yuying Liang ◽  
Shirley Gillam
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Synthesis ◽  
Rnase Protection Assay ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

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