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At least 19 recordsLinked to original sources
PubMed2003

Brome mosaic virus RNA replication: revealing the role of the host in RNA virus replication.

The replication of positive-strand RNA viruses is a complex multi-step process involving interactions between the viral genome, virus-encoded replication factors, and host factors. The plant virus brome mosaic virus (BMV) has served as a model for positive-strand RNA virus replication, recombination, and virion assembly. This review addresses recent findings on the identification and characterization of host factors in BMV RNA replication. To date, all characterized host factors facilitate steps that lead to assembly of a functional BMV RNA replication complex. Some of these host factors are required for regulation of viral gene expression. Others are needed to co-regulate BMV RNA translation and recruitment of BMV RNAs from translation to viral RNA replication complexes on the endoplasmic reticulum. Other host factors provide essential lipid modifications in the endoplasmic reticulum membrane or function as molecular chaperones to activate the replication complex. Characterizing the functions of these host factors is revealing basic aspects of virus RNA replication and helping to define the normal functions of these factors in the host.

Base Sequence↗
PubMed2003

Membrane synthesis, specific lipid requirements, and localized lipid composition changes associated with a positive-strand RNA virus RNA replication protein.

Multifunctional RNA replication protein 1a of brome mosaic virus (BMV), a positive-strand RNA virus, localizes to the cytoplasmic face of endoplasmic reticulum (ER) membranes and induces ER lumenal spherules in which viral RNA synthesis occurs. We previously showed that BMV RNA replication in yeast is severely inhibited prior to negative-strand RNA synthesis by a single-amino-acid substitution in the ole1w allele of yeast Delta9 fatty acid (FA) desaturase, which converts saturated FAs (SFAs) to unsaturated FAs (UFAs). Here we further define the relationships between 1a, membrane lipid composition, and RNA synthesis. We show that 1a expression increases total membrane lipids in wild-type (wt) yeast by 25 to 33%, consistent with recent results indicating that the numerous 1a-induced spherules are enveloped by invaginations of the outer ER membrane. 1a did not alter total membrane lipid composition in wt or ole1w yeast, but the ole1w mutation selectively depleted 18-carbon, monounsaturated (18:1) FA chains and increased 16:0 SFA chains, reducing the UFA-to-SFA ratio from approximately 2.5 to approximately 1.5. Thus, ole1w inhibition of RNA replication was correlated with decreased levels of UFA, membrane fluidity, and plasticity. The ole1w mutation did not alter 1a-induced membrane synthesis, 1a localization to the perinuclear ER, or colocalization of BMV 2a polymerase, nor did it block spherule formation. Moreover, BMV RNA replication templates were still recovered from cell lysates in a 1a-induced, 1a- and membrane-associated, and nuclease-resistant but detergent-susceptible state consistent with spherules. However, unlike nearby ER membranes, the membranes surrounding spherules in ole1w cells were not distinctively stained with osmium tetroxide, which interacts specifically with UFA double bonds. Thus, in ole1w cells, spherule-associated membranes were locally depleted in UFAs. This localized UFA depletion helps to explain why BMV RNA replication is more sensitive than cell growth to reduced UFA levels. The results imply that 1a preferentially interacts with one or more types of membrane lipids.

Bromovirus↗
PubMed1998

Population dynamics in the evolution of RNA viruses.

RNA virus quasispecies are subjected to processes of positive Darwinian selection, to a very active and continuous negative selection and to random genetic drift. The course of RNA virus evolution is often unpredictable, and recent results suggest that even highly conserved motifs, once regarded as essential for infectivity, may be rendered dispensable by singular evolutionary events. An immediate consequence of the quasispecies genetic organization of RNA viruses is a surprising ability to gain fitness once a minimal replication ability is established in a biological environment. The unique features of RNA genetics should not be underestimated since they are at the basis of the emergence of new viral diseases and of the current difficulties to control many diseases associated variable viruses.

Animals↗
PubMed2004

Evolutionary potential of an RNA virus.

RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi 6, containing a beta-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.

Adaptation, Physiological↗
PubMed1997

Is group selection a factor modulating the virulence of RNA viruses?

RNA viruses consist of populations of extremely high genetic heterogeneity called quasispecies. Based on theoretical considerations, it has been suggested that the unit of selection in such complex genetic populations is not the single viral particle but a set of genetically related particles which form the quasispecies. In the present study we carried out a set of experiments with the vesicular stomatitis virus (VSV) dealing with the evolution of life-history characters under selection acting at two factors either in the same or in opposite directions. The two factors at which selective pressure is applied are the individual and the group. We show evidence that group selection modulates the virulence of VSV populations, in opposition to an unlimited increase in virulence by competitive optimization promoted by individual selection. The results are of relevance for understanding the evolution of parasite virulence.

Genetics, Population↗
PubMed1974

Induction of type-C RNA virus by cycloheximide: increased expression of virus-specific RNA.

Mouse cells contain the genetic information for multiple endogenous type-C RNA viruses. The mechanisms by which the cell controls expression of these naturally integrated viruses are not yet known. Recently, chemicals that inhibit protein synthesis have been shown to induce a specific type-C virus at high frequency from BALB/c mouse embryo cells. In the present studies, virus activation in response to a representative translational inhibitor, cycloheximide, is demonstrated to be transient, with virus release primarily occurring within the first 12-24 hr following drug exposure. Analysis of virus-specific RNA in cells by molecular hybridization revealed an absolute increase in viral RNA concentration in cycloheximide-treated cells. This was blocked by simultaneous exposure of the cells to actinomycin D. Further, inhibition of RNA synthesis during but not subsequent to cycloheximide exposure prevented virus activation. These findings show that virus induction by cycloheximide requires de novo RNA synthesis during but not after drug exposure and suggest that the required RNA species may be that of the virus itself. The present results are consistent with the hypothesis that translational inhibitors prevent synthesis of a labile protein whose normal action is to inhibit viral RNA transcription or to cause degradation of viral RNA.

Animals↗
PubMed1989

A mutation in the short 5'-proximal open reading frame on Rous sarcoma virus RNA alters virus production.

The 5'-proximal open reading frame on Rous sarcoma virus RNA encodes a seven-amino-acid peptide and is conserved in all avian sarcoma-leukosis retroviruses. Ribosome-binding site analysis in intact chick cells showed that the 5'-proximal AUG codon is a strong site for initiation of translation in vivo. Removal of the 5'-proximal AUG codon by site-specific mutagenesis resulted in a virus with a reduced ability either to replicate or to transform a population of chicken embryo fibroblasts. These results establish a procedure for determining sites of ribosome binding and initiation of translation on mRNAs in intact eucaryotic cells and strongly suggest that the 5'-proximal open reading frame (or its AUG codon) on Rous sarcoma virus RNA has an important role in regulating viral activity.

Animals↗
PubMed2005

Identification of an RNA silencing suppressor from a plant double-stranded RNA virus.

RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus (RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of beta-glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.

Plant Diseases↗
PubMed1999

RNA elements required for RNA recombination function as replication enhancers in vitro and in vivo in a plus-strand RNA virus.

RNA replication requires cis-acting elements to recruit the viral RNA-dependent RNA polymerase (RdRp) and facilitate de novo initiation of complementary strand synthesis. Hairpins that are hot spots for recombination in the genomic RNA of turnip crinkle virus (TCV) and satellite (sat)-RNA C, a parasitic RNA associated with TCV infections, stimulate RNA synthesis 10-fold from a downstream promoter sequence in an in vitro assay using partially purified TCV RdRp. Artificial hairpins had an inhibitory effect on transcription. RNA accumulation in single cells was enhanced 5- to 10-fold when the natural stem-loop structures were inserted into a poorly accumulating sat-RNA. The effect of the stem-loop structures on RNA replication was additive, with insertion of three stem-loop RNA elements increasing sat-RNA accumulation to the greatest extent (25-fold). These stem-loop structures do not influence the stability of the RNAs in vivo, but may serve to recruit the RdRp to the template.

Arabidopsis↗
PubMed2001

Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells.

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

Animals↗
PubMed2002

Detection and stability of Japanese encephalitis virus RNA and virus viability in dead infected mosquitoes under different storage conditions.

A semi-nested polymerase chain reaction (PCR) was evaluated for detection of Japanese encephalitis (JE) virus in infected mosquitoes stored under simulated northern Australian summer conditions. The effect of silica gel, thymol, and a combination of the two on RNA stability and virus viability in dead mosquitoes were also examined. While JE virus RNA was relatively stable in mosquitoes held for up to 14 days after death, viable virus was not detected after day 1. Thymol vapor inhibited fungal contamination. Detection of single mosquitoes infected with JE virus in large pools of mosquitoes was also investigated. Single laboratory-infected mosquitoes were detected in pools of < or = 200 mosquitoes and in pools diluted to 0.2/100 and 0.1/100 mosquitoes, using the semi-nested PCR. However, the ability to detect live virus decreased as pool size increased. The semi-nested PCR proved more expensive than virus isolation for pools of 100 mosquitoes. However, the semi-nested PCR was faster and more economical using larger pools. Results indicate that surveillance of JE virus in mosquitoes using the semi-nested PCR is an alternative to monitoring seroconversions in sentinel pigs.

Animals↗
PubMed2005

A plant RNA virus suppresses RNA silencing through viral RNA replication.

RNA interference (RNAi) is a post-transcriptional gene-regulatory mechanism that operates in many eukaryotes. RNAi is induced by double-stranded RNA (dsRNA) and is mainly involved in defence against transposons and viruses. To counteract RNAi, viruses have RNAi suppressors. Here we show a novel mechanism of RNAi suppression by a plant virus Red clover necrotic mosaic virus (RCNMV). To suppress RNAi, RCNMV needs multiple viral components, which include viral RNAs and putative RNA replicase proteins. A close relationship between the RNA elements required for negative-strand RNA synthesis and RNAi suppression suggests a strong link between the viral RNA replication machinery and the RNAi machinery. In a transient assay, RCNMV interferes with the accumulation of small-interfering RNA (siRNAs) in RNAi induced by a hairpin dsRNA and it also interferes with microRNA (miRNA) biogenesis. An Arabidopsis dcl1 mutant showed reduced susceptibility to RCNMV infection. Based on these results, we propose a model in which, to replicate, RCNMV deprives the RNAi machinery of Dicer-like enzymes that are involved in both siRNA and miRNA biogenesis.

3' Untranslated Regions↗
PubMed2006

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication.

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

Adenosine Monophosphate↗
PubMed2006

Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication.

Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5' triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

Base Pairing↗
PubMed2006

Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses.

Viruses are divided into seven classes on the basis of differing strategies for storing and replicating their genomes through RNA and/or DNA intermediates. Despite major differences among these classes, recent results reveal that the non-virion, intracellular RNA-replication complexes of some positive-strand RNA viruses share parallels with the structure, assembly and function of the replicative cores of extracellular virions of reverse-transcribing viruses and double-stranded RNA viruses. Therefore, at least four of seven principal virus classes share several underlying features in genome replication and might have emerged from common ancestors. This has implications for virus function, evolution and control.

Biological Evolution↗
PubMed2000

A fast one-step reverse transcription and polymerase chain reaction (RT-PCR) amplification procedure providing highly specific complementary DNA from plant virus RNA.

Reverse transcription and polymerase chain reaction (RT-PCR) are being used increasingly for detection and typing RNA viruses. For this purpose, metal block thermal cyclers (MBTC) are considered to provide higher DNA yield, whereas air thermal cyclers (ATC) allow PCR amplification in a much shorter time. A fast ATC protocol (0 s denaturation, 0 s annealing, and 4-8 s elongation) was developed to amplify genomic segments from two RNA viruses, which allowed increasing the number of cycles without a parallel increase of non-specific DNA fragments. Under these conditions, 80-90 cycles with the ATC provided a DNA yield close to that of a standard 40-cycles MBTC protocol in about half the time. The DNA synthesised by the new procedure was highly specific and could be cloned readily.

Citrus↗