scholarly journals Strand-Specific RNA Synthesis Defects in a Poliovirus with a Mutation in Protein 3A

2003 ◽  
Vol 77 (23) ◽  
pp. 12679-12691 ◽  
Author(s):  
Natalya L. Teterina ◽  
Mario S. Rinaudo ◽  
Ellie Ehrenfeld
Keyword(s):  
Rna Synthesis ◽  
Binding Activity ◽  
Viral Rna ◽  
Wild Type ◽  
Positive Strand ◽  
Polyprotein Processing ◽  
Delayed Onset ◽  
Methionine Residue ◽  
Cell Extracts ◽  
Positive Strand Rna

ABSTRACT Substitution of a methionine residue at position 79 in poliovirus protein 3A with valine or threonine caused defective viral RNA synthesis, manifested as delayed onset and reduced yield of viral RNA, in HeLa cells transfected with a luciferase-containing replicon. Viruses containing these same mutations produced small or minute plaques that generated revertants upon further passage, with either wild-type 3A sequences or additional nearby compensating mutations. Translation and polyprotein processing were not affected by the mutations, and 3AB proteins containing the altered amino acids at position 79 showed no detectable loss of membrane-binding activity. Analysis of individual steps of viral RNA synthesis in HeLa cell extracts that support translation and replication of viral RNA showed that VPg uridylylation and negative-strand RNA synthesis occurred normally from mutant viral RNA; however, positive-strand RNA synthesis was specifically reduced. The data suggest that a function of viral protein 3A is required for positive-strand RNA synthesis but not for production of negative strands.

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 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.

scholarly journals Mutation of Host dnaJ Homolog Inhibits Brome Mosaic Virus Negative-Strand RNA Synthesis

2003 ◽  
Vol 77 (5) ◽  
pp. 2990-2997 ◽  
Author(s):  
Yuriko Tomita ◽  
Tomomitsu Mizuno ◽  
Juana Díez ◽  
Satoshi Naito ◽  
Paul Ahlquist ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Brome Mosaic Virus ◽  
Wild Type ◽  
Positive Strand ◽  
Replication Complex ◽  
Negative Strand ◽  
Rna Accumulation ◽  
Positive Strand Rna ◽  
Mutant Cells

ABSTRACT The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex.

scholarly journals Phosphorylation of Rubella Virus Capsid Regulates Its RNA Binding Activity and Virus Replication

2003 ◽  
Vol 77 (3) ◽  
pp. 1764-1771 ◽  
Author(s):  
Lok Man J. Law ◽  
Jason C. Everitt ◽  
Martin D. Beatch ◽  
Charles F. B. Holmes ◽  
Tom C. Hobman
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Binding ◽  
Virus Assembly ◽  
Binding Activity ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Wild Type ◽  
Positive Strand Rna

ABSTRACT Rubella virus is an enveloped positive-strand RNA virus of the family Togaviridae. Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.

scholarly journals Poliovirus cis-Acting Replication Element-Dependent VPg Uridylylation Lowers the Km of the Initiating Nucleoside Triphosphate for Viral RNA Replication

2008 ◽  
Vol 82 (19) ◽  
pp. 9400-9408 ◽  
Author(s):  
Benjamin P. Steil ◽  
David J. Barton
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Viral Rna ◽  
Nucleoside Triphosphate ◽  
Independent Manner ◽  
Positive Strand ◽  
Negative Strand ◽  
Phosphodiester Bond ◽  
Low Concentrations ◽  
Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5′ terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5′ end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpUOH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpUOH-independent manner, CRE-dependent VPgpUpUOH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the Km of UTP required for viral RNA replication and that CRE-dependent VPgpUpUOH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.

scholarly journals Poliovirus Requires a Precise 5′ End for Efficient Positive-Strand RNA Synthesis

2000 ◽  
Vol 74 (14) ◽  
pp. 6394-6400 ◽  
Author(s):  
Jens Herold ◽  
Raul Andino
Keyword(s):  
Cell Culture ◽  
Rna Synthesis ◽  
Hammerhead Ribozyme ◽  
In Vitro Translation ◽  
Positive Strand ◽  
Viral Genomes ◽  
Cell Extracts ◽  
Phage Dna ◽  
Positive Strand Rna

ABSTRACT Poliovirus infectious RNA can be synthesized in vitro using phage DNA-dependent RNA-polymerases. These synthetic transcripts contain several extra nucleotides at the 5′ end, which are deleted during replication to generate authentic viral genomes. We removed those 5′-end extra nucleotides utilizing a hammerhead ribozyme to produce transcripts with accurate 5′ ends. These transcripts replicate substantially more rapidly in cell culture, demonstrating no lag before replication; they also replicate more efficiently in Xenopus laevis oocytes and in in vitro translation-replication cell extracts. In both systems, an exact 5′ end is necessary for synthesis of positive-strand RNA but not negative-strand RNA.

scholarly journals Template Requirements for De Novo RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase on the Viral X RNA

2002 ◽  
Vol 76 (14) ◽  
pp. 6944-6956 ◽  
Author(s):  
Meehyein Kim ◽  
Hajeong Kim ◽  
Seong-Pil Cho ◽  
Mi-Kyung Min
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
De Novo ◽  
Nonstructural Protein ◽  
Initiation Site ◽  
Viral Rna ◽  
Positive Strand ◽  
Esherichia Coli ◽  
Positive Strand Rna

ABSTRACT The hepatitis C virus (HCV)-encoded NS5B protein is an RNA-dependent RNA polymerase which plays a substantial role in viral replication. We expressed and purified the recombinant NS5B of an HCV genotype 3a from Esherichia coli, and we investigated its ability to bind to the viral RNA and its enzymatic activity. The results presented here demonstrate that NS5B interacts strongly with the coding region of positive-strand RNA, although not in a sequence-specific manner. It was also determined that more than two molecules of polymerase bound sequentially to this region with the direction 3′ to 5′. Also, we attempted to determine the initiation site(s) of de novo synthesis by NS5B on X RNA, which contains the last 98 nucleotides of HCV positive-strand RNA. The initiation site(s) on X RNA was localized in the pyrimidine-rich region of stem I. However, when more than five of the nucleotides of stem I in X RNA were deleted from the 3′ end, RNA synthesis initiated at another site of the specific ribonucleotide. Our study also showed that the efficiency of RNA synthesis, which was directed by X RNA, was maximized by the GC base pair at the penultimate position from the 3′ end of the stem. These results will provide some clues to understanding the mechanism of HCV genomic RNA replication in terms of viral RNA-NS5B interaction and the initiation of de novo RNA synthesis.

scholarly journals The 5′-Terminal Region of the Aichi Virus Genome Encodes cis-Acting Replication Elements Required for Positive- and Negative-Strand RNA Synthesis

2005 ◽  
Vol 79 (11) ◽  
pp. 6918-6931 ◽  
Author(s):  
Shigeo Nagashima ◽  
Jun Sasaki ◽  
Koki Taniguchi
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Virus Genome ◽  
Encephalomyocarditis Virus ◽  
Viral Rna ◽  
Aichi Virus ◽  
Pseudoknot Structure ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5′ end) formed at the 5′ end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5′-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5′-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5′-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5′-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5′ end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3′ end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5′ end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.

scholarly journals Cell-Dependent Role for the Poliovirus 3′ Noncoding Region in Positive-Strand RNA Synthesis

2004 ◽  
Vol 78 (3) ◽  
pp. 1344-1351 ◽  
Author(s):  
David M. Brown ◽  
Steven E. Kauder ◽  
Christopher T. Cornell ◽  
Gwendolyn M. Jang ◽  
Vincent R. Racaniello ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Rna Replication ◽  
Mutant Virus ◽  
Noncoding Region ◽  
Viral Rna ◽  
Wild Type Virus ◽  
Genomic Rna ◽  
Virus Growth ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACT We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3′ noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3′ noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3′ noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3′ noncoding region is involved in positive-strand synthesis during genome replication.

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