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At least 469 records · Page 26Linked to original sources

PubMed1998

Conversion of dengue virus replicative form RNA (RF) to replicative intermediate (RI) by nonstructural proteins NS-5 and NS-3.

Dengue viruses infect more than 100 million people each year and cause serious clinical manifestations. It is important to understand the replication of these viruses so that therapeutic and/or prophylactic agents may be designed. Dengue virus type 2 nonstructural proteins NS-5 and NS-3 were produced by in vitro transcription and translation of cloned genes. Both proteins possessed RNA-dependent RNA polymerase activity as measured by their ability to convert purified replicative form (RF) RNA to replicative intermediate (RI). The recombinant proteins, however, required one or more cellular protein(s) for their activity. Examination of NS-3 protein sequence revealed heretofore unnoticed sequence similarities with other polymerases.

Amino Acid Sequence↗

PubMed1993

Protoplasts transiently expressing the 200K coding sequence of cowpea mosaic virus B-RNA support replication of M-RNA.

In order to identify the viral polymerase involved in cowpea mosaic virus (CPMV) RNA replication the 87K, 110K and 170K proteins as well as the complete 200K polyprotein of CPMV B-RNA have been produced in cowpea protoplasts, using expression vectors based on the 35S promoter of cauliflower mosaic virus. CPMV-specific proteins were obtained that were indistinguishable from proteins found in CPMV-infected protoplasts. Proteolytic processing of precursor proteins synthesized from the expression vectors proved that the 24K protease contained within these proteins is active. Moreover, it was established that protoplasts transfected with the expression vector containing the entire 200K coding sequence, but not those transfected with vectors containing the 170K, 110K or 87K coding sequences, were able to support replication of co-inoculated M-RNA. Despite the ability to support replication of M-RNA for protoplasts transiently expressing the 200K coding region, CPMV-specific RNA polymerase activity dependent on exogenous added template RNA could not be detected in extracts of these protoplasts in assays using poly(A).oligo(U) or other template/primer combinations. In contrast, extracts of protoplasts in which poliovirus polymerase was produced exhibited RNA polymerase activity in such assays. These results indicate that the CPMV polymerase, unlike the poliovirus polymerase, is not able to use oligo(U) as a primer or cannot function on exogenous template and primer RNA.

Base Sequence↗

PubMed2005

Translation and replication of FMDV RNA.

Foot-and-mouth disease virus (FMDV) RNA is infectious. After delivery of the RNA (about 8.3 kb) into the cytoplasm of a cell, the RNA must initially be translated to produce the viral proteins required for RNA replication and for the packaging of the RNA into new virions. Subsequently there has to be a switch in the function of the RNA; translation has to be stopped to permit RNA replication. The signals required for the control of the different roles of viral RNA must be included within the viral RNA sequence. Many cellular proteins interact with the viral RNA and probably also with the virus-encoded proteins. The functions of different RNA elements within the viral RNA and the various virus-encoded proteins in determining the efficiency of virus replication are discussed. Unique aspects of FMDV RNA translation and replication are emphasised.

3' Untranslated Regions↗

PubMed1993

Rescue of synthetic analogs of genomic RNA and replicative-intermediate RNA of human parainfluenza virus type 3.

The genome of human parainfluenza virus type 3 (PIV3) is a single negative-sense RNA strand (vRNA) that is 15,463 nucleotides in length. A cDNA was constructed to encode an 898-nucleotide, internally deleted version of PIV3 vRNA, PIV3-CAT vRNA, in which the viral genes were replaced with the bacterial chloramphenicol acetyltransferase (CAT) reporter gene. The CAT gene was flanked in turn by sequences representing (i) nontranslated sequences of the first and last genes in the PIV3 genome, (ii) PIV3 gene-start and gene-end sequences, which are presumed to be transcription signals, and (iii) 3' extracistronic (leader) and 5' extracistronic (trailer) terminal regions of PIV3 vRNA. A second cDNA was constructed to encode the exact complement of PIV3-CAT vRNA; this positive-sense RNA, PIV3-CAT vcRNA, would correspond to the predicted replicative intermediate of PIV3-CAT vRNA. When synthesized in vitro by runoff transcription with T7 RNA polymerase and transfected separately into PIV3-infected cells, both PIV3-CAT vRNA and vcRNA were rescued with similar efficiencies; that is, they were expressed to yield CAT and were packaged into particles that could be used to infect fresh cells. Rescue of PIV3-CAT vRNA was strictly dependent on complementation by PIV3; PIV3 could not be replaced by respiratory syncytial virus or, unexpectedly, by a bovine strain of PIV3. Passage was blocked by prior incubation with neutralizing monoclonal antibodies specific to the PIV3 attachment protein. Also, during nine serial passages, the expression of CAT by PIV3-CAT vRNA increased more than 3,000-fold. These results indicated that the 3'-terminal 111 nucleotides and the 5'-terminal 115 nucleotides of PIV3 vRNA, which are present in PIV3-CAT vRNA, contained all of the cis-acting RNA sequences required for replication, gene expression, and transmission.

Base Sequence↗

PubMed2006

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA.

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

3C Viral Proteases↗

PubMed2001

Apoptosis by influenza viruses correlates with efficiency of viral mRNA synthesis.

A mutant influenza virus, A/NWS-Mvi, grows well in the presence of exogenous sialidase activity sufficient to remove all cell surface sialic acids. Related wild-type viruses grow very poorly under these conditions, although mutant and wild-type viruses bind to desialylated cells with similar efficiency and show similar reduction of binding to sialidase-treated cells compared to native cells. Here we examine entry, transcription, translation, and RNA replication and find that, although the viruses appear to utilize the same entry pathway, the mutant NWS-Mvi transcribes and replicates RNA to higher levels than the wild-type strains. The kinetics of replication in multi-cycle infection show that this enhancement of RNA synthesis facilitates growth where entry is restricted. The hemagglutinin (HA) protein of NWS-Mvi lyses red blood cells 0.1 pH unit higher than wild-type viruses. This higher fusion pH may allow more efficient release of nucleocapsids from endosomes and contribute to the enhanced RNA synthesis. The efficient RNA synthesis assists virus survival at low inocula or under stringent growth conditions, such as the presence of antiviral agents. NWS-Mvi induces apoptosis in infected cells more readily than wild-type viruses, apparently as a consequence of enhanced production of viral mRNA. Since growth of NWS-Mvi is more efficient, apoptosis may play a positive role in viral replication by removing cells that have already been infected from those capable of making more virus.

Animals↗

PubMed1980

Interaction of host-coded and virus-coded polypeptides in RNA phage replication.

The enzymes responsible for replication of the RNA of the single-stranded RNA bacteriophages contain, in addition to one phage-coded polypeptide, three host-coded polypeptides taken from the protein biosynthetic machinery: ribosomal protein S1 and the elongation factors Tu and Ts. While S1 performs a function in RNA replication derived from its protein synthetic function, mRNA binding, the reactions catalysed by the elongation factors in protein synthesis are apparently dispensible for RNA replication. In the replicase, these polypeptides, acting as the EF-Tu . Ts complex, play a fundamental structural role. Replacement of the endogenous EF-Tu with mutant EF-Tu, itself stable, causes the RNA replicase to become unstable. The possibility that EF-Tu . Ts is solely a structural protein in the RNA replicase is suggested by experiments showing that a variety of modifications of the elongation factors can be tolerated without loss of RNA synthetic capacity. In fact, EF-Tu . Ts from distantly related bacterial species can substitute for E. coli EF-Tu . Ts in RNA replicase. Evidence is presented that the high in vitro template specificity of Q beta replicase may be accomplished through modulation of the level of GTP required for initiation of transcription. Different natural and synthetic RNAs require quite different GTP concentrations. Mn2+ ions, which extend the range of templates transcribed by Q beta replicase, lower the requirement for GTP. High ionic strength, which alters the conformation of Q beta replicase such that template specificity is increased, raises the GTP requirement. An additional host coded protein required for in vitro Q beta RNA replication, host factor (HF), interacts specifically with Q beta RNA. This polypeptide acts by allowing Q beta replicase to initiate RNA synthesis with Q beta RNA at reduced GTP concentration.

Coliphages↗

PubMed1993

Replication of hepatitis delta virus RNA: effect of mutations of the autocatalytic cleavage sites.

Hepatitis delta virus (HDV) contains a circular RNA genome of 1.7 kb. HDV RNA replication is thought to proceed via a rolling-circle mechanism that is dependent on autocatalytic cleavage and ligation reactions. However, it has never been established that these ribozyme activities are indeed involved in HDV RNA replication. To investigate the possible biological significance of HDV RNA self-cleavage, we constructed several HDV dimer cDNAs containing single-base substitutions of the 3' nucleotide of the genomic and the antigenomic self-cleavage sites. These mutations were known to affect self-cleavage in vitro to various extents. The effects of these mutations on HDV RNA replication were examined in hepatic and nonhepatic cell lines. The results showed that all of the mutants which had lost the in vitro self-cleavage activity could not replicate. The only mutant which retained full cleavage activity replicated as efficiently as the wild-type RNA. Thus, this study established that self-cleavage activity is required for HDV RNA replication in cells. Interestingly, the level of HDV RNA detected in cells transfected with this replication-competent mutant and that detected in cells transfected with the wild-type construct were similar in COS-7 cells but vastly different in HepG2 and Huh-7 cells, suggesting that HDV RNA self-cleavage activity may be modulated by cell-specific factors. We also compared the effects of mutations when the primary transcripts of these constructs were of either genomic or antigenomic sense. In constructs which synthesize primary transcripts of genomic sense, all of the antigenomic self-cleavage mutants produced as much hepatitis delta antigen (HDAg) as did the wild-type construct, even in the absence of detectable HDV RNA replication, whereas the genomic self-cleavage mutants produced very little HDAg. These and other data suggest that (i) the primary HDV RNA transcripts of both genomic and antigenomic polarities must first be processed to serve as a template for HDV RNA transcription, (ii) efficient cleavage at the antigenomic self-cleavage site is not required for HDAg expression, and (iii) HDV RNA replication most likely occurs by a double-rolling-circle mechanism.

Animals↗

PubMed2005

An authentic 3' noncoding region is necessary for efficient poliovirus replication.

Picornavirus RNA replication involves the specific synthesis of negative-strand intermediates followed by an accumulation of positive-strand viral RNA in the presence of a multitude of cellular mRNAs. Previously, in an effort to identify cis-acting elements required for initiation of negative-strand RNA synthesis, we deleted the entire 3' noncoding regions from human rhinovirus and poliovirus genomic RNAs. These deletion mutation transcripts displayed a severe delay in RNA accumulation following transfection of HeLa cells. Interestingly, in subsequent infection of HeLa cells, the deletion-mutant poliovirus displayed only a moderate deficiency in RNA synthesis. These data suggested that the delay in the production of cytopathic effects after transfection may have been due to an RNA replication defect overcome by the accumulation of a compensatory mutation(s) generated during initial rounds of RNA synthesis. In this study, we have sequenced the entire genome of the deletion-mutant virus and found only two nucleotide changes from the parental clone. Transfection analysis of these sequence variants revealed that the sequence changes did not provide compensatory functions for the 3' noncoding region deletion mutation replication defect. Further examination of the deletion mutant phenotype revealed that the severe replication defect following RNA transfection is due, in part, to nonviral terminal sequences present in the in vitro-derived deletion mutation transcripts. Our data suggest that poliovirus RNA harboring a complete 3' noncoding region deletion mutation is infectious (not merely quasi-infectious).

3' Untranslated Regions↗

PubMed2004

The emergence of competition between model protocells.

The transition from independent molecular entities to cellular structures with integrated behaviors was a crucial aspect of the origin of life. We show that simple physical principles can mediate a coordinated interaction between genome and compartment boundary, independent of any genomic functions beyond self-replication. RNA, encapsulated in fatty acid vesicles, exerts an osmotic pressure on the vesicle membrane that drives the uptake of additional membrane components, leading to membrane growth at the expense of relaxed vesicles, which shrink. Thus, more efficient RNA replication could cause faster cell growth, leading to the emergence of Darwinian evolution at the cellular level.

Biological Evolution↗

PubMed2002

The amplicon-plus system for high-level expression of transgenes in plants.

Many biotechnological applications require high-level expression of transgenes in plants. One strategy to achieve this goal was the production of potato virus X (PVX) "amplicon" lines: transgenic lines that encode a replicating RNA virus vector carrying a gene of interest. The idea was that transcription of the amplicon transgene would initiate viral RNA replication and gene expression, resulting in very high levels of the gene product of interest. This approach failed, however, because every amplicon transgene, in both tobacco and Arabidopsis thaliana, was subject to post-transcriptional gene silencing (PTGS). In PTGS, the transgene is transcribed but the transcripts fail to accumulate as a result of sequence-specific targeting and destruction. Even though the amplicon locus is silenced, the level of beta-glucuronidase (GUS) activity in a PVX/GUS line is similar to that in some transgenic lines expressing GUS from a conventional (not silenced) GUS locus. This result suggested that the very high levels of expression originally envisioned for amplicons could be achieved if PTGS could be overcome and if the resulting plants did not suffer from severe viral disease. Here we report that high-level transgene expression can be achieved by pairing the amplicon approach with the use of a viral suppressor of PTGS, tobacco etch virus (TEV) helper component proteinase (HC-Pro). Leaves of mature tobacco plants co-expressing HC-Pro and a PVX/GUS amplicon accumulate GUS to approximately 3% of total protein. Moreover, high-level expression occurs without viral symptoms and, when HC-Pro is expressed from a mutant transgene, without detrimental developmental phenotypes.

Cysteine Endopeptidases↗

PubMed1983

Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.

The association of newly synthesized vesicular stomatitis virus proteins into nucleocapsid structures was examined in a cell-free system that supports concurrent viral protein synthesis, transcription, and RNA replication. The vesicular stomatitis virus proteins synthesized by this system associated with the newly replicated RNA to form structures that banded in CsCl gradients with marker nucleocapsids. In reactions lacking nucleocapsid templates to program RNA synthesis, the newly synthesized proteins did not associate into nucleocapsid structures. The newly synthesized proteins associated with nucleocapsids were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate after separation from non-associated proteins by chromatography on Bio-Gel A15M agarose columns. The results of this analysis showed that newly synthesized L, NS, and N proteins associated into nucleocapsids in the in vitro system. In addition, a small amount of newly synthesized M protein was stably bound to the nucleocapsids. The molar ratio of the associated, newly synthesized proteins was 2:350:1,000:10 (L:NS:N:M). More than 90% of the newly synthesized NS protein that associated with nucleocapsids in vitro was of the NS2 subspecies, as assayed by DEAE-cellulose column chromatography. The stability of the association of the newly synthesized proteins with nucleocapsids in the system mimicked that of the association of viral proteins with nucleocapsids from infected cells as measured by salt sensitivity. These data indicate that nucleocapsids were assembled from newly synthesized proteins within our in vitro system and that the molar ratio of assembled proteins was similar to that observed for virion nucleocapsids.

Capsid↗

PubMed2005

Aptamers directed to HIV-1 reverse transcriptase display greater efficacy over small hairpin RNAs targeted to viral RNA in blocking HIV-1 replication.

RNA molecules can be powerful inhibitors of HIV-1 replication. To determine the relative efficacy of siRNAs and RNA aptamers, a direct comparison of three anti-HIV reverse transcriptase aptamers and three shRNAs targeted to HIV-1(R3b) was made. U6 promoter-driven anti-HIV genes were delivered into CEMx174 cells via a retroviral vector, and transduced cells were sorted out via green fluorescent protein function and challenged with HIV. The results show that, at low virus input, shRNAs can block HIV as efficiently as aptamers. When expressed in target cells, both classes of inhibitors blocked early events of reverse transcription, suggesting they are both able to access intracellular reverse transcription complexes. However, at higher multiplicities of infection (m.o.i. of 50), while the aptamers could efficiently inhibit HIV replication, shRNAs did not. RNase protection assays indicated similar steady-state levels or nucleocytoplasmic distribution showing that the differential efficacy was not a reflection of intracellular concentration. The higher potency of anti-RT aptamers could be due to their ability to inhibit two successive rounds of reverse transcription owing to their unique ability to be encapsidated into virion particles. Furthermore, anti-RT aptamers expressed in T cells afforded protection against high-dose infection by chimeric RT-SHIV viruses.

Base Pairing↗

PubMed1982

Replication of mouse hepatitis virus: negative-stranded RNA and replicative form RNA are of genome length.

There are seven virus-specific mRNA species in mouse hepatitis virus-infected cells (Lai et al., J. Virol. 39:823-834, 1981). In this study, we examined virus-specific negative-stranded RNA to determine whether there are corresponding multiple negative-stranded RNAs. Intracellular RNA from mouse hepatitis virus-infected cells was separated by agarose gel electrophoresis, transferred to nitrocellulose membranes, and hybridized to positive-stranded genomic 60S [32P]RNA. Only a single RNA species of genomic size was detected under these conditions. This RNA was negative stranded. No negative-stranded subgenomic RNA was detected. We also studied double-stranded replicative-form RNA in the infected cells. Only one replicative-form of genomic size was detected. When the double-stranded RNA isolated without RNase treatment was analyzed, again only one RNA species of genomic size was detectable. Furthermore, most of the virus-specific mRNAs could be released from this RNA species upon heating. These results suggest that all of the mouse hepatitis virus-specific RNAs are transcribed from a single species of negative-stranded RNA template of genomic size.

Animals↗

PubMed1990

Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants.

The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs. One RNA is the primer precursor and the other is a small counter-transcript called RNA I. The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation. We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I. We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription. We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription. During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type. Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed2003

Systematic, genome-wide identification of host genes affecting replication of a positive-strand RNA virus.

Positive-strand RNA viruses are the largest virus class and include many pathogens such as hepatitis C virus and the severe acute respiratory syndrome coronavirus (SARS). Brome mosaic virus (BMV) is a representative positive-strand RNA virus whose RNA replication, gene expression, and encapsidation have been reproduced in the yeast Saccharomyces cerevisiae. By using traditional yeast genetics, host genes have been identified that function in controlling BMV translation, selecting BMV RNAs as replication templates, activating the replication complex, maintaining a lipid composition required for membrane-associated RNA replication, and other steps. To more globally and systematically identify such host factors, we used engineered BMV derivatives to assay viral RNA replication in each strain of an ordered, genome-wide set of yeast single-gene deletion mutants. Each deletion strain was transformed to express BMV replicase proteins and a BMV RNA replication template with the capsid gene replaced by a luciferase reporter. Luciferase expression, which is dependent on viral RNA replication and RNA-dependent mRNA synthesis, was measured in intact yeast cells. Approximately 4500 yeast deletion strains ( approximately 80% of yeast genes) were screened in duplicate and selected strains analyzed further. This functional genomics approach revealed nearly 100 genes whose absence inhibited or stimulated BMV RNA replication and/or gene expression by 3- to >25-fold. Several of these genes were shown previously to function in BMV replication, validating the approach. Newly identified genes include some in RNA, protein, or membrane modification pathways and genes of unknown function. The results further illuminate virus and cell pathways. Further refinement of virus screening likely will reveal contributions from additional host genes.

Bromovirus↗

PubMed1998

Cellular factors in the transcription and replication of viral RNA genomes: a parallel to DNA-dependent RNA transcription.

Viral RNA replication and transcription involves not only viral RNA-dependent RNA polymerases, but also cellular proteins, the majority of which are subverted from the RNA-processing or translation machineries of host cells. These factors interact with viral RNA or polymerases to form transcription or replication ribonucleoprotein complexes and may provide template specificity for RNA-dependent RNA synthesis, suggesting a close parallel to the mechanism of DNA-dependent RNA synthesis. The types of cellular proteins involved and their modes of action are reviewed.

Animals↗

PubMed2000

RNA-Dependent replication and transcription of hepatitis delta virus RNA involve distinct cellular RNA polymerases.

Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of alpha-amanitin (<3 microgram/ml) and could be partially restored by an alpha-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by alpha-amanitin to a concentration higher than 25 microgram/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

Animals↗