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Results for “RNA Replication”

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At least 991 records · Page 55Linked to original sources

PubMed2000

Activation of the DNA replication checkpoint through RNA synthesis by primase.

When DNA replication is inhibited during the synthesis (S) phase of the cell cycle, a signaling pathway (checkpoint) is activated that serves to prevent mitosis from initiating before completion of replication. This replication checkpoint acts by down-regulating the activity of the mitotic inducer cdc2-cyclin B. Here, we report the relation between chromatin structure and induction of the replication checkpoint. Chromatin was competent to initiate a checkpoint response only after the DNA was unwound and DNA polymerase alpha had been loaded. Checkpoint induction did not require new DNA synthesis on the unwound template strand but did require RNA primer synthesis by primase. These findings identify the RNA portion of the primer as an important component of the signal that activates the replication checkpoint.

Animals↗

PubMed1995

Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle.

Hepatitis delta virus (HDV) is hepatotropic and frequently causes fulminant hepatitis in both human and nonhuman primate hosts. To understand the molecular basis of HDV tissue tropism and the mechanism of pathogenesis, transgenic mice in which replication-competent HDV dimeric RNA is expressed under the control of either liver-specific or universal transcriptional promoters were developed. The expressed RNA replicated efficiently in the liver and several tissues of nonhepatic origin. Surprisingly, maximal replication of HDV RNA occurred in skeletal muscle and was almost 100-fold greater than in the liver. These findings suggest that the hepatotropism of HDV is most likely a receptor-mediated restriction and that muscle-specific factors may facilitate HDV RNA replication. No evidence of cytopathology was apparent in most of the tissues examined, including the liver, supporting the contention that hepatocellular disease is not mediated by direct cytopathological effects associated with HDV RNA replication and gene expression. However, mild muscle atrophy in some of the transgenic mice was noted. Delta antigen was detected in the nuclei of myocytes. Only the small form, not the large form, of delta antigen was detected, suggesting that the RNA editing event which causes the conversion of delta antigen did not occur in transgenic mice. Furthermore, the 0.8-kb antigenomic RNA species, which is postulated to be the mRNA for delta antigen, was not detected in mice. The preferential replication of HDV RNA in skeletal muscle suggests that HDV RNA replication can be facilitated by certain muscle-specific factors.

Actins↗

PubMed2001

De novo initiation of viral RNA-dependent RNA synthesis.

RNA viruses use several initiation strategies to ensure that their RNAs are synthesized in appropriate amounts, have correct termini, and can be translated efficiently. Many viruses with genomes of single-stranded positive-, negative-, and double-stranded RNA initiate RNA synthesis by a de novo (primer-independent) mechanism. This review summarizes biochemical features and variations of de novo initiation in viral RNA replication.

Flaviviridae↗

PubMed2005

Hepatitis C virus and the related bovine viral diarrhea virus considerably differ in the functional organization of the 5' non-translated region: implications for the viral life cycle.

The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.

5' Untranslated Regions↗

PubMed2001

Deleterious effects of hepatitis delta virus replication on host cell proliferation.

Hepatitis delta virus (HDV) infection and spread in vivo are dependent upon coinfection by hepatitis B virus (HBV), and dual HDV/HBV infection is frequently more severe than HBV infection alone, raising the possibility that HDV infection may be deleterious to cells. Here we have examined the effects of HDV replication on the long-term growth of cultured cells. Our results show that most cells transfected with HDV cDNA do not give rise to stable cell lines expressing viral antigens or replicative intermediates; in addition, cotransfection of HDV replicons with a plasmid vector expressing a hygromycin resistance marker results in a dose-dependent impairment of hygromycin-resistant colony formation. When cells transfected with replication-competent HDV cDNA are followed prospectively, a progressive decline in viral RNA replication and a steady decrease in the proportion of cells expressing delta antigen are observed. However, in transient transfection assays, no evidence was found to link HDV replication to apoptosis or to cell cycle arrest, nor did HDV replication confer on host cells enhanced sensitivity to inducers of apoptosis. Thus, HDV replication does not appear to be acutely cytotoxic. However, in dividing cells HDV replication is associated with a subtler growth disadvantage, leading to selection in culture for cells displaying diminished HDV expression. This effect would not be expected to cause hepatitis in vivo but might contribute to impaired liver regeneration in the setting of ongoing hepatocellular injury.

Animals↗

PubMed2003

Coronavirus replication complex formation utilizes components of cellular autophagy.

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells. In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

Adenine↗

PubMed1975

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.

Adenoviridae↗

PubMed2003

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.

BACKGROUND & AIMS: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis. METHODS: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency. RESULTS: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/microg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/microg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay. CONCLUSIONS: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

Cell Line↗

PubMed1996

Functional significance of the nuclear-targeting and NTP-binding motifs of Semliki Forest virus nonstructural protein nsP2.

Semliki Forest virus-specific polypeptide nsP2 is a nonstructural protein involved in multiple steps during viral RNA replication. It was recently shown to possess single-stranded RNA-stimulated ATPase and GTPase activities. Replacement of the highly conserved lysine (Lys-192) within the classical nucleotide-binding motif A/GXXGXGKS/T with asparagine abolished its NTP-hydrolyzing activity. Also, about half of nsP2 is transported into the nucleus during viral infection. Substitution of the second arginine in its nuclear localization signal (P648RRRV) with aspartic acid rendered nsP2 totally cytoplasmic. To assess the functional importance of these sequence motifs, the same mutations were introduced into a cDNA clone of Semliki Forest virus, from which infectious RNA can be produced in vitro. Transfection of an RNA encoding Lys-192 --> Asn mutation into BHK cells did not promote viral infection. However, revertants encoding the wild-type amino acid were obtained. Cells transfected with RNA coding for Arg-649 --> Asp mutation gave rise to infectious virus termed SFV-RDR. Indirect immunofluorescence and subcellular fractionation of SFV-RDR-infected cells confirmed the cytoplasmic localization of nsP2. Measurement of host DNA synthesis late in infection revealed that infection with the parental virus inhibited DNA synthesis to 10% of control cells. In contrast, infection with SFV-RDR led only to a partial shutoff of cellular DNA synthesis. Mice experiments indicated that the pathogenicity of SFV-RDR was attenuated.

Adenosine Triphosphate↗

PubMed2004

Effect of mutation in the hepatitis C virus nonstructural 5B region on HCV replication.

BACKGROUND: We have reported that the presence of a mutation at the hepatitis C virus (HCV) nonstructural protein 5B (NS5B), defined as a change in amino acids at sites specific for a different reported genotype, was related to complete response (CR) to interferon (IFN) therapy in patients with chronic hepatitis C (CHC) with genotype 1b. The present study assessed the impact of the NS5B mutation on the replication of HCV in these patients. METHODS: Genotype-specific mutations of HCV NS5B were determined by direct sequencing. We measured HCV-RNA titers in serum by real-time detected polymerase chain reaction (PCR), and serum HCV core protein levels (as a marker of HCV-RNA replication) were measured using an enzyme immunoassay in patients with CHC genotype 1b. RNA-dependent RNA polymerase (RdRp) activity was measured by Behrens' method in liver cirrhosis patients infected with HCV (n = 13) and in those infected with hepatitis B virus (HBV; n = 2). RESULTS: The titers of HCV-RNA (n = 44) and the levels of HCV core protein (n = 41) were significantly lower in patients with the HCV genotype 1b mutant compared with wild-type HCV (P < 0.05). RdRp activity in liver tissue did not show any correlation with the HCV NS5B mutation. CONCLUSIONS: HCV NS5B genotype-specific mutations in HCV genotype 1b may influence HCV replication.

DNA Mutational Analysis↗

PubMed1975

RNA of replication-defective strains of Rous sarcoma virus.

The RNA of a replication-defective (rd) mutant, isolated from stocks of nondefective (nd) Schmidt-Ruppin Rous sarcoma virus of subgroup A (SR-A) and termed SR-N8, was compared to the RNAs of SR-A, of a transformation-defective derivative of SR-A (td SR-A) and of rd Bryan Rous sarcoma virus, RSV (minus). The molecular mass of the 30-40S species of SR-N8 RNA was estimated to be 21% (congruent to 7.5 to 8 times 10-5 daltons) smaller than that of SR-A by (i) electrophoresis in polyacrylamide gels and (ii) analyses of RNA complexity based on RNase T1-resistant oligonucleotides. ST-N8 shares probably all (=14) of its large RNase T1-resistant oligonucleotides with the RNA of SR-A as judged from the chromatographic distribution and the RNase A-resistant fragments obtained from RNase T1-resistant oligonucleotides. However, SR-N8 RNA lacked six large oligonucleotides which were present in the RNAs of SR-A and td SR-A. Conversely, the RNAs of SR-A, and of SR-N8 contained two oligonucleotides not found in td SR-A. The RNA of SR-N8 was found to differ from that of RSV (minus) in its electrophoretic mobility and its fingerprint pattern. It is concluded that the RNA of SR-N8 was generated by a deletion of SR-A. The extent of this deletion is compatible with the notion that the genetic information for the large viral envelope glycoprotein (molecular mass = 70,000-85,000 daltons) has been lost from the RNA of SR-A to yield SR-N8 RNA. From a comparison of td and rd deletion mutants, it appears that loss of different functions corresponds to the absence of different oligonucleotides in their RNA.

Avian Sarcoma Viruses↗

PubMed1997

Translational activation in coliphage Qbeta: on a polycistronic messenger RNA, repression of one gene can activate translation of another.

We present evidence for translational activation of the Qbeta coliphage maturation cistron, mediated by the presence of Qbeta replicase. This activation does not require RNA replication, translation of a second gene, or any direct protein-RNA binding at the maturation gene initiation site. Our data support a model in which the Qbeta maturation gene remains translationally "off" by two means: (1) the thermodynamic stability of an RNA structure that greatly discourages, but does not eliminate, ribosome access at the maturation start site; and (2) the presence of the stronger, proximal coat gene ribosome binding site. Moreover, maturation gene expression is switched "on" when ribosome entry at the coat initiation site, present on the same polycistronic RNA molecule, is repressed by Qbeta replicase, thereby allowing ribosomes to compete for the weaker, upstream maturation start site.

Allolevivirus↗

PubMed2000

Recognition of nucleoside triphosphates during RNA-catalyzed primer extension.

In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K(m) and a higher k(cat), suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the gamma-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k(cat) of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5'-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.

Animals↗

PubMed2002

A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA.

Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.

3' Untranslated Regions↗

PubMed2006

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.

Anticholesteremic Agents↗

PubMed2006

Interaction of rotavirus polymerase VP1 with nonstructural protein NSP5 is stronger than that with NSP2.

Rotavirus morphogenesis starts in intracellular inclusion bodies called viroplasms. RNA replication and packaging are mediated by several viral proteins, of which VP1, the RNA-dependent RNA polymerase, and VP2, the core scaffolding protein, were shown to be sufficient to provide replicase activity in vitro. In vivo, however, viral replication complexes also contain the nonstructural proteins NSP2 and NSP5, which were shown to be essential for replication, to interact with each other, and to form viroplasm-like structures (VLS) when coexpressed in uninfected cells. In order to gain a better understanding of the intermediates formed during viral replication, this work focused on the interactions of NSP5 with VP1, VP2, and NSP2. We demonstrated a strong interaction of VP1 with NSP5 but only a weak one with NSP2 in cotransfected cells in the absence of other viral proteins or viral RNA. By contrast, we failed to coimmunoprecipitate VP2 with anti-NSP5 antibodies or NSP5 with anti-VP2 antibodies. We constructed a tagged form of VP1, which was found to colocalize in viroplasms and in VLS formed by NSP5 and NSP2. The tagged VP1 was able to replace VP1 structurally by being incorporated into progeny viral particles. When applying anti-tag-VP1 or anti-NSP5 antibodies, coimmunoprecipitation of tagged VP1 with NSP5 was found. Using deletion mutants of NSP5 or different fragments of NSP5 fused to enhanced green fluorescent protein, we identified the 48 C-terminal amino acids as the region essential for interaction with VP1.

Animals↗

PubMed1984

A revised replication cycle for viroids: the role of longer than unit length RNA in viroid replication.

Longer than unit length plus and minus strand RNAs were detected in hop stunt viroid (HSV) infected cucumber leaf tissues by Northern blot hybridization analysis using strand-specific probes. To elucidate the role of these longer than unit length RNAs in the viroid replication cycle, we synthesized tandemly repeated plus and minus strand HSV RNAs in vitro from cloned HSV cDNA and assayed their infectivities. Two and four unit tandemly repeated plus strand RNAs were infectious, but one unit plus, and one, two and four unit minus strands were noninfectious. Taking these data into consideration, we propose a revised rolling circle model for viroid replication.

Cloning, Molecular↗

PubMedDate not supplied

Cis-acting signals and trans-acting factors involved in influenza virus RNA synthesis.

Influenza A virus RNA replication and expression is directed from cis-acting sequences present on the viral RNAs with the help of trans-acting factors encoded by the virus. Ribonucleoprotein (RNP) complexes reconstituted from synthetic cDNA-derived RNA and purified viral proteins have facilitated the dissection of these cis-acting signals and trans-acting factors. Prior to these studies influenza viruses and other negative-strand RNA viruses were refractory to molecular genetic manipulations. These reverse genetic studies have helped in defining the promoter and polyadenylation signals required for viral RNA synthesis. Studies involving the use of reconstituted RNP complexes have revealed that the viral proteins PB1, PB2, PA, and the nucleoprotein (NP) are necessary for replication and expression of influenza virus RNA. Inroads have also been made in determining the cellular proteins that participate in influenza virus gene expression and replication. The yeast interactive trap system has been used to identify and clone a gene (NPI-1), which encodes a protein that interacts with the influenza virus NP suggesting that this cellular protein is a trans-acting factor functioning in viral RNA synthesis.

Base Sequence↗