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Results for “RNA Replication”

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PubMed1995

Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein.

A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

Base Sequence↗

PubMed1987

Self-cleavage of RNA in the replication of viroids and virusoids.

Viroids are infectious, circular RNA molecules of 246 to 375 nucleotides found in plants. Virusoids are of similar size and structure but they are dependent on, and encapsidated in, a helper virus. A rolling circle mechanism of replication is considered to account for the presence of greater-than-unit-length plus and minus RNAs of both viroids and virusoids found in infected plants. An essential feature of this mechanism is the specific processing or cleavage of high molecular weight intermediates to produce linear monomers which are then ligated to circular monomers. We have investigated the putative processing cleavage reactions using in vitro-synthesized RNA transcripts of dimeric cDNA clones of the 247-nucleotide avocado sunblotch viroid (ASBV) and of partial cDNA clones of the 324-nucleotide virusoid of lucerne transient streak virus (vLTSV). In both cases, there is a specific, non-enzymic, self-cleavage of plus as well as minus transcripts. The plus and minus sites of cleavage are in neighbouring parts of ASBV and of vLTSV and highly conserved two-dimensional structures can be drawn around the cleavage sites as well as around the putative or demonstrated cleavage sites of precursors of the virusoids of three other viruses and of the linear satellite RNA of tobacco ringspot virus. The results also indicate that the sole function of about one-third of the ASBV and vLTSV molecules is provision of sequences that allow the formation of the self-cleavage structures of both 'plus' and 'minus' RNA precursors during the replication cycle. Similar self-cleavage of 'plus' RNA transcripts of a dimeric cDNA clone of citrus exocortis virus (CEV) was not observed. However, the putative processing site for CEV precursors was located within three nucleotides by site-directed mutagenesis. No two-dimensional structures similar to those found for ASBV and vLTSV were found around the processing site. It is possible that a different type of self-cleavage or enzymic processing event occurs during the replication cycle of CEV and related viroids.

Base Sequence↗

PubMed1996

Mutations in the poliovirus 3CD proteinase S1-specificity pocket affect substrate recognition and RNA binding.

Sequence and structure comparisons with homologous trypsin-like serine proteases have predicted the S1-specificity pocket in picornavirus 3C proteinases. In this study, we examine the putative roles of such residues in poliovirus 3C substrate recognition. Single amino acid substitutions at 3C residues Thr-142, His-161, Gly-163, Gly-164, and Ala-172 were introduced into near full-length poliovirus cDNAs, and protein processing was examined in the context of authentic 3C cis cleavage activity. Our data are consistent with residues Thr-142, His-161, Gly-163, and Gly-164 acting as important determinants of 3C substrate specificity and support published models of 3C protein structure. An in vivo analysis of mutant viruses containing individual amino acid substitutions at 3C residues Thr-142 and Ala-172 suggests that such residues are important determinants for viral RNA replication. In addition, bacterially expressed, recombinant 3CD polypeptides containing amino acid substitutions at Thr-142 and Ala-172 show altered RNA binding properties in mobility shift assays that use a synthetic RNA corresponding to the poliovirus 5'-terminal sequences.

3C Viral Proteases↗

PubMed1998

Ribozyme gene therapy for hepatitis C virus infection.

BACKGROUND: The development of antiviral drugs for hepatitis C virus (HCV) infection represents a substantial challenge. Similar to human immunodeficiency virus (HIV), HCV is highly prone to mutation. It is, therefore, expected that potential HCV therapeutics currently under development, such as protease inhibitors, will suffer from the same shortcomings of HIV therapeutic drugs; the emergence of drug resistant viral mutants. Ribozymes (Rz) are enzymatic RNA molecules that can be engineered to specifically target any given RNA molecule. A therapeutic Rz can be manufactured and administered as a drug, or a Rz gene can be delivered and expressed intracellularly by gene therapy. For HCV therapeutics, we favour the gene therapy approach as delivery and in vivo expression of Rz genes will result in a constant and continuous supply of multiple intracellular Rz, offering less opportunity for the development of drug-resistant viral variants. OBJECTIVES: To utilise direct intravenous injection of hepatotropic viral vectors to transfer Rz genes directly into the hepatocytes of HCV-infected patients, resulting in degradation of the HCV positive strand RNA genome, the viral mRNAs, and even the negative strand RNA replication intermediate. We plan to circumvent the emergence of drug-resistant viral mutants by targeting multiple, highly conserved HCV RNA sequences simultaneously with multiple Rz genes expressed from a single vector. STUDY DESIGN: Rzs targeting conserved regions of the HCV positive and negative RNAs were transcribed in vitro and used to cleave HCV target RNAs. The most effective Rzs identified were then incorporated into adeno associated viral (AAV) vectors and adenoviral (AV) vectors and tested for their ability to inhibit HCV core expression in a tissue culture model. RESULTS: Several Rzs targeting highly conserved HCV sequences effectively degraded positive and negative strands of HCV RNA in vitro. Furthermore, substantial inhibition of HCV gene expression was observed in tissue culture using viral vectors to deliver and express Rz genes. CONCLUSIONS: Rz gene therapy has potential for the production of anti-viral drugs directed against HCV. Initial studies employing Rz gene therapy to produced anti-viral drugs against HCV have proved successful. Rz gene therapy may be a useful approach to overcome problems associated with anti-HCV drug design, such as the emergence of drug-resistant mutants.

Adenoviridae↗

PubMed2002

Subgenomic replicon derived from a cell line infected with the hepatitis C virus.

Recently, cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. To see whether a HCV sequence derived from HCV-infected cultured cell sequence can be used for the construction of a functional replicon, a HCV subgenomic RNA carrying a neomycin-resistant gene was constructed using the HCV genome RNA obtained from cultured cells infected with HCV. After transfection, G418-resistant Huh-7 cells were selected and subcloned. Finally, the production of HCV proteins and de novo synthesis of subgenomic RNA were confirmed in the selected cell clone, indicating that this subgenomic RNA replicated in cultured cells and functioned as a replicon. These results suggest that the HCV genome obtained from an in vitro HCV infection system with cultured cells can be used to develop a subgenomic replicon system with diverse HCV sequences.

Cell Line↗

PubMed1997

Nonhomologous RNA recombination in a cell-free system: evidence for a transesterification mechanism guided by secondary structure.

Extensive nonhomologous recombinations occur between the 5' and 3' fragments of a replicable RNA in a cell-free system composed of pure Qbeta phage replicase and ribonucleoside triphosphates, providing direct evidence for the ability of RNAs to recombine without DNA intermediates and in the absence of host cell proteins. The recombination events are revealed by the molecular colony technique that allows single RNA molecules to be cloned in vitro. The observed nonhomologous recombinations are entirely dependent on the 3' hydroxyl group of the 5' fragment, and are due to a splicing-like reaction in which RNA secondary structure guides the attack of this 3' hydroxyl on phosphoester bonds within the 3' fragment.

Base Sequence↗

PubMed2000

The bulk of the phosphorylation of human respiratory syncytial virus phosphoprotein is not essential but modulates viral RNA transcription and replication.

The ability of variants of the human respiratory syncytial virus (HRSV) phosphoprotein (P protein) to support RNA transcription and replication has been studied by using HRSV-based subgenomic replicons. The serine residues normally phosphorylated in P during HRSV infection have been replaced by other residues. The results indicate that the bulk of phosphorylation of P (98%) is not essential for viral RNA transcription or replication but that phosphorylation can modulate these processes.

HN Protein↗

PubMed2004

Analysis of the 3' cis-acting elements of rubella virus by using replicons expressing a puromycin resistance gene.

A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3' cis-acting elements (3' CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3' CSE varied. The 3' region necessary for optimal relative survival consisted of the 3' 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3' CSE. Within the 3' CSE, deletions in the approximately 245 nt that overlap the 3' end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the approximately 60-nt 3' untranslated region (UTR) were lethal. None of the 3' CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5' proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3' CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3' CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3' CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.

Acetyltransferases↗

PubMed2005

Nitric oxide radical suppresses replication of wild-type dengue 2 viruses in vitro.

Nitric oxide is well accepted as one of the defenses for inhibiting viral dissemination. Macrophages and cells in the macrophage lineage are professional nitric oxide producers which sub-serve as target for dengue virus. The interaction between nitric oxide and dengue virus in such target cell is unknown. In this report, the impact of nitric oxide on infectious dengue virus serotype 2 production and RNA replication was investigated in vitro. Primary isolates of dengue virus serotype 2 from dengue patients were replicated in mouse neuroblastoma cells in the presence of an exogenous nitric oxide donor, s-nitroso-N-acethylpennicillamine, SNAP, at the concentration of 50 or 75 or 100 microM. Nitric oxide inhibited viral replication in a dose and a multiplicity of infection dependent manner. Nitric oxide from 50 and 75 microM SNAP delayed and suppressed replication of dengue virus isolates while higher concentration of nitric oxide, 100 microM SNAP, completely inhibited production of infectious particles up to 36 hr study. Twenty-four out of forty tested isolates, 60%, were susceptible to 50 microM SNAP inhibitory effect. The mechanism of inhibition was investigated at the level of RNA synthesis and was found that RNA production was suppressed which correlated to production of the infectious particles. Down-regulation of the RNA synthesis resulted in reduction of protein synthesis which was detected by lower level of NS1 protein synthesis using immunoblotting. In conclusion, nitric oxide from exogenous nitric oxide donor down regulated replication of dengue virus serotype 2 isolates from dengue patients. The suppression was clearly shown at the level of viral RNA and protein synthesis resulting in reduction of viral progenies production. This phenomenon implies that nitric oxide may serve as a defense which diminishes viral load in patients.

Animals↗

PubMed2006

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

3' Untranslated Regions↗

PubMed1991

Conserved genes encode guide RNAs in mitochondria of Crithidia fasciculata.

RNA editing is the post-transcriptional alteration of the nucleotide sequence of RNA, which in trypanosome mitochondria is characterized by the insertion and deletion of uridine residues. It has recently been proposed that the information for the sequence alteration in Leishmania tarentolae is provided by small guide (g) RNAs encoded in the mitochondrial DNA [Blum et al. (1990) Cell, 60, 189-198]. We are studying the mechanism of RNA editing in the insect trypanosome Crithidia fasciculata and report that: (i) a full length, conventional DNA gene or an independently replicating RNA gene that could encode the edited MURF3 transcript is absent when probed for in sensitive, calibrated assay systems; (ii) in all cases (seven) investigated in C. fasciculata so far, putative gRNA genes are found in a position in the mitochondrial DNA virtually identical to that in L. tarentolae and (iii) also in C. fasciculata, the putative gRNA genes are transcribed into small RNAs with discrete 5' ends. These results provide strong evolutionary evidence in support of the participation of gRNAs in RNA editing. Remarkably, in C. fasciculata the basepaired region of some putative gRNA:mRNA hybrids contains a C:A non-Watson-Crick basepair.

Animals↗

PubMed1980

Dependence of the activity of the poliovirus replicase on the host cell protein.

Two poliovirus-specific RNA polymerase activities have been identified: a poly(U) polymerase that copies poly(A).oligo(U) and a replicase that copies natural heteropolymers with some preference for poliovirus RNA. Both activities purified together until a step of a salt gradient elution from poly(U)-agarose, when poly(U) polymerase but no replicase was recovered. Addition of a salt wash fraction from the ribisomes of uninfected cells to this poly(U) polymerase fraction reconstituted replicase activity, and a host factor was purified 50 fold from the ribosomal salt wash. None of the available initiation factors or elongation factors for protein synthesis were able to reconstitute replicase activity. Host factor activity could be supplied by adding oligo(U), suggesting that the factor acts at the initiation step of RNA replication. With the purified replicase-host factor combination, only poly(A)-containing RNAs were copied, and a preference for poliovirus RNA was shown.

HeLa Cells↗

PubMed2004

Susceptibility of peritoneal macrophages to infection by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) strains fall into two groups: high-neurovirulence GDVII virus results in rapidly fatal encephalitis, while low-neurovirulence BeAn and DA viruses produce persistent central nervous system (CNS) infection and inflammatory demyelinating disease. Because macrophages (Mphis) are key components in BeAn virus-induced demyelinating disease, we examined the susceptibility of primary peritoneal macrophages (pMphis) to BeAn infection in vitro. Freshly isolated, thioglycollate-elicited pMphis were resistant to BeAn virus infection even at high multiplicity of infection. In contrast, after incubation of thioglycollate-elicited pMphis at 37 degrees C for 4 days before infection, approximately half of the cells expressed virus antigen(s) and contained nicked DNA indicative of apoptosis. However, BeAn virus RNA replication and virus yields were highly restricted. Interestingly, about one-third of the cells were apoptotic but negative for virus RNA and antigen(s). Tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha) were elevated in BeAn-infected pMphi cultures suggesting that bystander killing may be responsible for the apoptosis seen in BeAn virus antigen-negative cells. These data show for the first time that pMphis are susceptible to BeAn virus infection, although the infection is highly restricted and most of these cells undergo BeAn-induced apoptosis.

Animals↗

PubMed1996

Evidence for the existence of a pseudoknot structure at the 3' terminus of the flavivirus genomic RNA.

The 3'-terminal nucleotides of the flavivirus genomic RNA form conserved secondary structures that may function as cis-acting signals for RNA replication. Here we provide evidence for the existence of a conserved pseudoknot structure at the 3' terminus of the flavivirus genomic RNA. A truncated version of the West Nile virus (WNV) 3'-terminal RNA sequence was used as the model for these studies. Circular dichroism spectra indicated the presence of a highly structured RNA conformation with a significant amount of A-form helix. Ribonuclease probing not only confirmed the presence of the predicted secondary structure, which consists of a long stem-loop (SL1) and a shorter stem-loop (SL2), but also suggested that base pairing occurs between nucleotides in the loop of SL2 and those in an internal loop strand located on the 5' side of SL1. Analysis of three mutant RNAs further supported the existence of pseudoknot interactions. UV-melting analysis of the WNV 3' model RNA showed three transitions with significant hyperchromicity at approximately 46, 62, and 79 degrees C. UV-melting analysis with either SL1 or SL2 RNA alone suggested that the 62 and 79 degree C transitions represent the unfolding of SL2 and SL1, respectively. The 46 degree C transition is most likely due to the opening of the proposed tertiary structure. A similar melting curve was obtained for another flavivirus (dengue-3 virus) 3'-terminal RNA, providing further support for the conservation of the structure among flaviviruses. Molecular modeling of the RNA indicated that a pseudoknot structure is a stereochemically and energetically reasonable model for the 3' terminus of flavivirus genomic RNA.

Base Sequence↗

PubMed1996

RNA-catalysed RNA polymerization using nucleoside triphosphates.

The hypothesis that certain RNA molecules may be able to catalyse RNA replication is central to current theories of the early evolution of life. In support of this idea, we describe here an RNA that synthesizes RNA using the same reaction as that employed by protein enzymes that catalyse RNA polymerization. In the presence of the appropriate template RNA and nucleoside triphosphates, the ribozyme extends an RNA primer by successive addition of up to six mononucleotides. The added nucleotides are joined to the growing RNA chain by 3',5'-phosphodiester linkages. The ribozyme shows marked template fidelity: extension by nucleotides complementary to the template is up to 1,000 times more efficient than is extension by mismatched nucleotides.

Base Composition↗

PubMed1994

Requirement of the 5'-end genomic sequence as an upstream cis-acting element for coronavirus subgenomic mRNA transcription.

We have developed a defective interfering (DI) RNA containing a chloramphenicol acetyltransferase reporter gene, placed behind an intergenic sequence, for studying subgenomic mRNA transcription of mouse hepatitis virus (MHV), a prototype coronavirus. Using this system, we have identified the sequence requirement for MHV subgenomic mRNA transcription. We show that this sequence requirement differs from that for RNA replication. In addition to the previously identified requirement for an intergenic (promoter) sequence, additional sequences from the 5' end of genomic RNA are required for subgenomic mRNA transcription. These upstream sequences include the leader RNA and a spacer sequence between the leader and intergenic sequence, which is derived from the 5' untranslated region and part of gene 1. The spacer sequence requirement is specific, since only the sequence derived from the 5' end of RNA genome, but not from other MHV genomic regions or heterologous sequences, could initiate subgenomic transcription from the intergenic sequence. These results strongly suggest that the wild-type viral subgenomic mRNAs (mRNA2 to mRNA7) and probably their counterpart subgenomic negative-sense RNAs cannot be utilized for mRNA amplification. Furthermore, we have demonstrated that a partial leader sequence present at the 5' end of genome, which lacks the leader-mRNA fusion sequence, could still support subgenomic mRNA transcription. In this case, the leader sequences of the subgenomic transcripts were derived exclusively from the wild-type helper virus, indicating that the MHV leader RNA initiates in trans subgenomic mRNA transcription. Thus, the leader sequence can enhance subgenomic transcription even when it cannot serve as a primer for mRNA synthesis. These results taken together suggest that the 5'-end leader sequence of MHV not only provides a trans-acting primer for mRNA initiation but also serves as a cis-acting element required for the transcription of subgenomic mRNAs. The identification of an upstream cis-acting element for MHV subgenomic mRNA synthesis defines a novel sequence requirement for regulating mRNA synthesis in RNA viruses.

Animals↗

PubMed1985

Antiviral activity of natural and recombinant human leukocyte interferons in tobacco protoplasts.

Several purified species of human leukocyte interferon, including recombinant interferons, inhibit the multiplication of tobacco mosaic virus (TMV) in tobacco protoplasts derived from various cultivars. Viral RNA accumulation was determined by dot-blot hybridization to specific cDNA probes, and virus antigen was determined serologically. Interferon apparently inhibited both TMV-RNA replication and its expression into coat protein. However, these effects were of limited duration. Maximum effect was obtained when interferon was applied to the cells either prior to inoculation or within the first hour after inoculation. Antibodies to interferon abolished its antiviral activity in protoplasts. Tobacco protoplasts were about 1000 times more responsive to interferon than the reference animal viral-cell system and showed an "antiviral state" at a ratio of 1 molecule of interferon per cell.

DNA Replication↗

PubMed1984

Vaccinia virus RNA polymerase associated with nuclei of infected HeLa cells.

Though vaccinia virus DNA and RNA replication take place predominantly in the cytoplasm of an infected cell, virus formation requires the presence of a functional nucleus in a yet undefined manner. When the nuclei from cells infected for 3 h are isolated and purified, they are found to synthesize five times more RNA in vitro than do corresponding nuclei from noninfected cells. Fifty percent of the RNA synthesized in vitro by nuclei from infected cells is vaccinia specific, and this vaccinia RNA synthesis is resistant to alpha-amanitin concentrations up to 100 micrograms/ml. Furthermore, when the RNA polymerase activities of these nuclei are separated on DEAE-Sephadex columns, 56% of the total nuclear enzyme activity is found to be the vaccinia-specific RNA polymerase known to be alpha-amanitin resistant. The nucleus associated vaccinia RNA polymerase represents 18% of the total cellular vaccinia RNA polymerase. This synthesis of vaccinia RNA in the nucleus may explain the nuclear requirement for vaccinia virus maturation.

Cell Compartmentation↗