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Results for “RNA Replication”

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At least 973 records · Page 54Linked to original sources

PubMed1989

Mitochondrial RNA polymerase: dual role in transcription and replication.

Mitochondrial RNA polymerases from humans, Xenopus laevis and Saccharomyces cerevisiae are very similar in protein composition and function. They consist of a nonspecific core RNA polymerase and a protein factor that confers promoter selectivity on the core component, and they participate in transcription as well as in DNA replication. Amino acid sequence comparisons indicate that the yeast mitochondrial core component is related to bacteriophage T3 and T7 RNA polymerases; mitochondrial and phage polymerases may therefore belong to a family of related polymerases.

Animals↗

PubMed2005

Quantitative analysis of the hepatitis C virus replication complex.

The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

Blotting, Northern↗

PubMed1989

The origin of polynucleotide-directed protein synthesis.

If protein synthesis evolved in an RNA world it was probably preceded by simpler processes by means of which interaction with amino acids conferred selective advantage on replicating RNA molecules. It is suggested that, at first, the simple attachment of amino acids to the 2'(3')-termini of RNA templates favored initiation of replication at the end of the template rather than at internal positions. The second stage in the evolution of protein synthesis would probably have been the association of pairs of charged RNA adaptors in such a way as to favor noncoded formation of peptides. Only after this process had become efficient could coded synthesis have begun.

Adenosine Monophosphate↗

PubMed1975

Studies of temperature sensitive mutants of bacteriophage Qbeta, defective in both replication and translation.

Temperature sensitive mutants of bacteriophage Qbeta have been isolated which fail in the synthesis of their virus RNA at the non-permissive temperature (42 degrees C). Nine mutants have been studied in some detail. Cells infected with these mutants at 37 degrees C and incubated long enough to produce substantial amounts of Qbeta RNA cease Qbeta RNA replication when shifted to 42 degrees C. The mutants can be classified into 3 groups according to the amount of Qbeta RNA replicase activity exhibited in extracts from infected cells isolated at various times after shift to 42 degrees C: in group 1 mutants, enzyme activity is the same, regardless of the time of isolation after shift; in group 2 mutants enzyme activity increases with time of isolation after shift; in group 3 mutants, enzyme activity decreases with time of isolation after shift. Synthesis of all virus proteins is suppressed at 42 degrees C in cells infected with group 2 of group 3 mutants. In cells infected with group 2 mutants, synthesis of Qbeta RNA replicase subunit beta is increased, but synthesis of other virus proteins is depressed at 42 degrees C. The inhibition of virus RNA and protein synthesis is reversible. A detailed analysis of these experiments suggests that a defective Qbeta RNA replicase is involved in the inhibition of both virus RNA and protein synthesis.

Coliphages↗

PubMed1994

Detection of minus-strand hepatitis C virus RNA in tumor tissues of hepatocellular carcinoma.

BACKGROUND: Recently, the detection of hepatitis C virus (HCV) antibody has been widely performed for clinical serum testing of HCV infection and can be identified in most hepatocellular carcinoma (HCC) cases in Japan. In the current study, the authors detected not only plus-strand but also minus-strand HCV RNA as a template for RNA replication in hepatocytes in the resected tumors of HCC, and they investigated those significant for HCC. METHODS: The plus-strand and minus-strand HCV RNA were detected using reverse transcription-polymerase chain reaction after the extraction of RNA from the tumor and nontumor tissues of hepatectomized liver, respectively. RESULTS: The detection of HCV RNA in liver tissues from hepatectomized specimens of 20 cases was as follows: in nontumor regions, both of the plus- and minus-strand HCV RNA were detected in 14 cases. Among those cases, plus- and minus-strand HCV RNA were detected in 11 cases, and in another 2 cases, only plus-strand HCV RNA was found in the tumor regions. CONCLUSIONS: These results suggest that HCV RNA is able to replicate in tumor tissues of HCC and may be involved in the development of HCC and also that HCV in remnant hepatocytes may cause the recurrence of HCC as secondary carcinogenesis after hepatectomy.

Adult↗

PubMed1996

Alphavirus-based expression vectors: strategies and applications.

Alphaviruses are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in insect and vertebrate cells. Through recombinant DNA technology, the alphavirus RNA replication machinery has been engineered for high-level expression of heterologous RNAs and proteins. Amplification of replication-competent alpha-virus RNAs (replicons) can be initiated by RNA or DNA transfection and a variety of packaging systems have been developed for producing high titers of infectious viral particles. Although normally cytocidal for vertebrate cells, variants with adaptive mutations allowing noncytopathic replication have been isolated from persistently infected cultures or selected using a dominant selectable marker. Such mutations have been mapped and used to create new alphavirus vectors for noncytopathic gene expression in mammalian cells. These vectors allow long-term expression at moderate levels and complement previous vectors designed for short-term high-level expression. Besides their use for a growing number of basic research applications, recombinant alphavirus RNA replicons may also facilitate genetic vaccination and transient gene therapy.

Alphavirus↗

PubMed2005

Two cis-acting elements in negative RNA strand of Hepatitis C virus involved in synthesis of positive RNA strand in vitro.

Sequences at the 3'-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3'-terminal region of negative RNA strand ((-)3'T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3'-cytidylate (3'C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3'-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3'-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3'C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3'-proximal stem-loop and the first 3'C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.

3' Untranslated Regions↗

PubMed2004

Two RNA polymerase complexes from vesicular stomatitis virus-infected cells that carry out transcription and replication of genome RNA.

By immunoaffinity column chromatography, we have purified two RNA polymerase complexes, the transcriptase and replicase, from vesicular stomatitis virus-infected baby hamster kidney cells. The transcriptase is a multiprotein complex, containing the virus-encoded RNA polymerase L and P proteins, and two cellular proteins, translation elongation factor-1alpha and heat-shock protein 60. In addition, the complex contains a submolar amount of cellular mRNA cap guanylyltransferase. The replicase, on the other hand, is a complex containing the viral proteins, L, P, and the nucleocapsid (N), but lacking elongation factor-1alpha, heat-shock protein 60, and guanylyltransferase. The transcriptase complex synthesizes capped mRNAs and initiates transcription at the first gene (N) start site, whereas the replicase complex initiates RNA synthesis at the precise 3' end of the genome RNA and synthesizes encapsidated replication products in the presence of the N-P complex. We propose that two RNA polymerase complexes that differ in their content of virally and host-encoded proteins are separately responsible for transcription and replication of vesicular stomatitis virus genome RNA.

Animals↗

PubMed1999

Fluorochrome-labeled RNA as a sensitive, strand-specific probe for direct fluorescence in situ hybridization.

Detection of target RNA by in situ hybridization (ISH) in the classic and confocal fluorescence microscope was performed using strand-specific single-stranded RNA probes labeled directly with the fluorochromes fluorescein isothiocyanate or Texas red. The probes, produced by in vitro transcription from PCR-generated templates with T7 RNA polymerase and fluorochromized UTP, gave ISH signals directly visible by fluorescence microscopy without the use of any immunological detection step. In avoiding antibodies, it was possible to strongly increase the sensitivity of the ISH since antibodies may contain RNase which can reduce hybridization signals considerably, even beyond the detection limit. Fluorescent RNA probes thus allowed for the detection of low numbers of target molecules per cell, such as minus strand intermediates in picornavirus RNA replication. Using appropriate denaturing conditions, the targets could be visualized in a double-stranded configuration as well as in the presence of a 100-fold excess of complementary RNA. Furthermore, double ISH for the simultaneous detection of two different RNA species, such as plus and minus strand RNA of poliovirus, or of different regions of the viral genomic RNA was possible with appropriate fluorescent strand-specific probes labeled with different fluorochromes. Combination of ISH and immunofluorescence was found feasible if RNA was present in relatively large amounts. In addition to the investigation of virus replication, possible applications of fluorochromized RNA probes might include antisense RNA detection as well as plant virus resistance and gene silencing.

Fluorescein-5-isothiocyanate↗

PubMed1991

Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus.

Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.

Base Sequence↗

PubMed1989

Budding efficiency of Sendai virus nucleocapsids: influence of size and ends of the RNA.

The budding efficiency of Sendai virus antigenomes, as well as of defective interfering (DI) nucleocapsids of the deletion and copy-back types, was compared to that of the viral genome during infections of baby hamster kidney (BHK) cells. The antigenomes were shown to bud into virus particles as efficiently as the genomes, arguing for the irrelevance of the nucleocapsid-RNA ends in regulating the efficiency of budding. The DI nucleocapsids, however, were restricted in their budding by factors inversely proportional to their size, arguing for an effect of nucleocapsid size in this process. This restriction in budding, however, appeared to be only expressed under conditions of very efficient DI-RNA replication.

Animals↗

PubMed1988

Minus-strand RNA synthesis in the spinal cords of mice persistently infected with Theiler's virus.

Theiler's virus, a murine picornavirus, causes a chronic neurological disease characterized by primary demyelination in SJL/J mice. The lesions are very reminiscent of those of multiple sclerosis. Theiler's virus persists in oligodendrocytes and to a lesser extent in astrocytes and macrophages throughout the disease. Viral RNA and capsid protein syntheses are minimal in these cells. This restriction could play a central role in the mechanism of virus persistence. By quantitating plus- and minus-strand RNAs in infected central nervous system cells, we showed that RNA replication was blocked at the level of minus-strand RNA synthesis.

Animals↗

PubMed1996

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.

Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (D. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3' end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNA-protein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3' end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3' end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.

Cell Line↗

PubMed2002

Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles.

Little is known about the function of flavivirus nonstructural protein NS2A. Two forms of NS2A are found in yellow fever virus-infected cells. Full-length NS2A (224 amino acids) is the product of cleavage at the NS1/2A and NS2A/2B sites. NS2Aalpha, a C-terminally truncated form of 190 amino acids, results from partial cleavage by the viral NS2B-3 serine protease at the sequence QK /T within NS2A. Exchange of serine for lysine at this site (QKT-->QST) blocks the production of both NS2Aalpha and infectious virus. The present study reveals that this defect is not at the level of RNA replication. Despite normal structural region processing, infectious particles containing genome RNA and capsid protein were not released from cells transfected with the mutant RNA. Nevertheless, production of subviral prM/M- and E-containing particles was unimpaired. The NS2A defect could be complemented in trans by providing NS1-2A or NS1-2Aalpha. However, trans complementation was not observed when the C-terminal lysine of NS1-2Aalpha was replaced with serine. In addition to true reversions, NS2Aalpha cleavage site mutations could be suppressed by two classes of second-site changes. The first class consisted of insertions at the NS2Aalpha cleavage site that restored its basic character and cleavability. A second class of suppressors occurred in the NS3 helicase domain, in which NS3 aspartate 343 was replaced with an uncharged residue (either valine, alanine, or glycine). These mutations in NS3 restored infectious-virus production in the absence of cleavage at the mutant NS2Aalpha site. Taken together, our results reveal an unexpected role for NS2A and NS3 in the assembly and/or release of infectious flavivirus particles.

Animals↗

PubMed2002

Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis.

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C(pro). The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C(pro) to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C(pro)-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C(pro) and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C(pro)-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.

Animals↗

PubMed1994

Identification and characterization of host factor interactions with cis-acting elements of rubella virus RNA.

We have analyzed the function of cis-acting elements of rubella virus RNA and the components which interact with these elements in viral RNA replication. We demonstrated that the 5'- and 3'-terminal sequences from RV RNA promote translation and negative-strand RNA synthesis of chimeric chloroamphenicol acetyltransferase (CAT) RNAs. These sequences have a potential to form stem-loop (SL) structures and bind cellular proteins specifically in RNA gel-shift and UV cross-linking assays. The 5' end binding proteins were identified to be Ro/SSA-associated antigens by virtue of being recognized in an RNA complex by an autoimmune patient serum with Ro antigen type specificity. Purification and sequence analysis of the 3' end binding protein revealed that it is a homologue of human calreticulin. The role of host protein in RV replication is discussed.

Autoantigens↗

PubMed1981

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.

Animals↗

PubMed2006

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

Cell Line, Tumor↗