SEARCH · PubMed Health

Results for “RNA Replication”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 955 records · Page 53Linked to original sources

PubMed1995

Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations.

Poliovirus (PV) 2C protein is a nonstructural polypeptide involved in viral RNA replication, whose biochemical activity(ies) in this process has not been defined. By using site-directed mutagenesis, it was shown previously that disruption of nucleotide-binding motifs present in this protein abolished viral RNA synthesis (C. Mirzayan and E. Wimmer, Virology 189:547-555, 1992; N. L. Teterina, K. M. Kean, E. Gorbalenya, V. I. Agol, and M. Girard, J. Gen. Virol. 73:1977-1986, 1992). We have tested whether PV 2C or 2BC protein provided in trans could rescue the replication of these mutated genomes. Rescuing proteins were provided either by cotransfection with helper chimeric PV-coxsackievirus genomes or by expression in cells with a vaccinia virus-T7 RNA polymerase transient-expression system. We report here that replication of mutated RNAs genomes was poorly supported in trans both by helper genomes and by expressed 2C or 2BC proteins. Similarly, very inefficient complementation was observed for two mutated genomes with lethal lesions in 3D polymerase coding sequence. Our results indicate that poliovirus RNA replication shows marked preference for proteins contributed in cis.

Amino Acid Sequence↗

PubMed2004

cis-acting RNA signals in the NS5B C-terminal coding sequence of the hepatitis C virus genome.

The cis-replicating RNA elements in the 5' and 3' nontranslated regions (NTRs) of the hepatitis C virus (HCV) genome have been thoroughly studied before. However, no cis-replicating elements have been identified in the coding sequences of the HCV polyprotein until very recently. The existence of highly conserved and stable stem-loop structures in the RNA polymerase NS5B coding sequence, however, has been previously predicted (A. Tuplin, J. Wood, D. J. Evans, A. H. Patel, and P. Simmonds, RNA 8:824-841, 2002). We have selected for our studies a 249-nt-long RNA segment in the C-terminal NS5B coding region (NS5BCR), which is predicted to form four stable stem-loop structures (SL-IV to SL-VII). By deletion and mutational analyses of the RNA structures, we have determined that two of the stem-loops (SL-V and SL-VI) are essential for replication of the HCV subgenomic replicon in Huh-7 cells. Mutations in the loop and the top of the stem of these RNA elements abolished replicon RNA synthesis but had no effect on translation. In vitro gel shift and filter-binding assays revealed that purified NS5B specifically binds to SL-V. The NS5B-RNA complexes were specifically competed away by unlabeled homologous RNA, to a small extent by 3' NTR RNA, and only poorly by 5' NTR RNA. The other two stem-loops (SL-IV and SL-VII) of the NS5BCR domain were found to be important but not essential for colony formation by the subgenomic replicon. The precise function(s) of these cis-acting RNA elements is not known.

Base Sequence↗

PubMed2003

Identification of a ribavirin-resistant NS5B mutation of hepatitis C virus during ribavirin monotherapy.

Ribavirin (RBV), a guanosine analogue, has been suggested to exert an antiviral action against hepatitis C virus (HCV) by causing lethal mutations and suppressing RNA polymerase in vitro, but the mechanism of its clinical therapeutic effects is currently unknown. To test the hypothesis that RBV could act both as an RNA mutagen and inhibit viral RNA synthesis in vivo, we studied the evolution of the nucleotide sequences of HCV RNA at the nonstructural (NS) 5B region in patients receiving RBV, placebo, or interferon alfa (IFN-alpha) monotherapy. The RBV group showed a slightly more accelerated evolution rate of HCV RNA quasispecies than either the IFN-alpha or placebo group. RBV caused preferentially A-to-G and U-to-A mutations. Interestingly, an NS5B amino acid 415 Phe-to-Tyr (F415Y) mutation emerged in all (5 of 5) patients infected with HCV genotype 1a during the RBV treatment. Subsequently, the parental 415F strain reemerged in some patients after the treatment was discontinued. The effect of the amino acid substitution at NS5B415 on HCV RNA replication was then investigated using an HCV subgenomic replicon in Huh7 cells. We showed that treatment of replicon cells with RBV reduced the HCV RNA level of NS5B415F replicon, but not NS5B415Y, in a dose-dependent manner. Thus, NS5B F415Y mutation represents an RBV-resistant variant. The 3-dimensional modeling and structure analysis of NS5B protein revealed that the 415th amino acid is located at the P helix region of the thumb subdomain, which may interact with the minor groove of the template-primer duplex in the putative RNA-binding cleft. In conclusion, RBV could work as a weak mutagen for HCV RNA in HCV-infected patients. Furthermore, the selection of an RBV-resistant variant with a single amino acid substitution in NS5B suggested that RBV may directly interact with HCV RNA polymerase, thus interfering with its enzymatic activity.

Adult↗

PubMed2003

Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex.

Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.

Amino Acid Sequence↗

PubMed2001

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA.

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.

3' Untranslated Regions↗

PubMed1993

Characterization of defective interfering RNA components that increase symptom severity of broad bean mottle virus infections.

Several strains of the broad bean mottle virus (BBMV), an icosahedral tripartite plant RNA virus, which show distinct reactions on certain plant hosts have been described (K. M. Makkouk et al., Neth. J. Plant Pathol. 94, 195-212, 1988). Here we report defective interfering (DI) RNAs encapsidated in two BBMV strains from Morocco and Tunisia. While not effective in some plants, these DI RNAs exacerbated the severity of symptoms in others. The most dramatic, lethal effect of DI RNAs has been found on pea (Pisum sativum, cv. Rondo) seedlings. Sequence analysis has revealed that the DI RNAs were derived by single in-frame central deletions of 448 to 537 nt in the corresponding genomic RNA2 components. A comparison of the intensities of full-length RNA2 bands from DI molecule-containing and DI molecule-deficient virion RNA preparations revealed that the DI RNAs decreased the level of RNA2 components in total RNA preparations. The differences between corresponding virion RNAs were much smaller. This suggests an interference with RNA replication. In vitro assays and an analysis of the polyribosomal RNA fractions confirmed the translational activity of DI RNAs. This paper reports the first description of natural DI RNAs in tripartite isometric plant RNA viruses.

Base Sequence↗

PubMed1994

Replication of barley yellow dwarf virus satellite RNA transcripts in oat protoplasts.

A small RNA associated with an isolate of barley yellow dwarf virus (BYDV) has been described which has the physical properties of a satellite RNA (Miller et al., Virology 183, 711-720, 1991). Here we demonstrate that this RNA has the biological properties of a satellite RNA: it requires the presence of helper virus (BYDV genomic) RNA for replication and the helper RNA does not require the satellite. To obtain pure satellite RNA, a permuted dimeric clone was constructed from which infectious satellite RNA could be transcribed in vitro. The dimeric transcript self-cleaved to produce monomeric satellite RNA. When this RNA was coelectroporated with BYDV genomic RNA into oat protoplasts, replication of both RNAs was detected by Northern hybridization. The RPV, but not the PAV, serotype of BYDV supported satellite RNA replication. The presence of discrete oligomeric forms of (+) and (-) sense satellite RNA in infected protoplasts suggests that both strands replicate by a rolling circle mechanism.

Base Sequence↗

PubMed1987

Regulation of the replication of influenza virus RNA segments: partial suppression of protein synthesis restores the 'early' replication pattern.

The replication of influenza virus RNA was monitored by RNA-RNA hybridization with subsequent analysis of hybrid duplexes, as well as by immunosorption of viral nucleocapsids from extracts of [3H]uridine-labelled cells followed by the isolation and characterization of nucleocapsid-associated RNA. The nucleocapsid-associated RNA preparations contained mostly negative-strand genomic RNA. Electrophoresis of the hybrid RNA duplexes or single-stranded nucleocapsid-associated RNA in polyacrylamide gel revealed an 'early' replication pattern, with a predominance of the NP and NS gene segments, in cells labelled from 0 to 1 h post-infection. At later stages of infection the pattern changed to the 'late' one, with the M gene segment in excess of NS, and the NP gene no longer predominant. Cycloheximide added as late as 2 or 3 h post-infection suppressed RNA replication. Moderate concentrations of cycloheximide inhibited the replication of NS and NP gene segments to a lesser degree than the replication of the other RNA segments, thus restoring the 'early' replication pattern. Cycloheximide treatment resulted in a slight increase in the percentage of positive strands in nucleocapsid-associated RNA. The role of protein synthesis in the transition from the 'early' to the 'late' pattern of influenza virus RNA synthesis is discussed.

Animals↗

PubMed1997

RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase: evidence of discrimination between DNA and RNA substrates.

The RNA dependent DNA replication fidelity of HIV-1 reverse transcriptase has been investigated using pre-steady-state kinetics under single turnover conditions. In contrast to previous estimates of low replication fidelity of HIV-1 reverse transcriptase, the present study finds the enzyme to be more highly discriminating when an RNA/DNA template-primer is employed as compared with the corresponding DNA/DNA template-primer. The basis of this selectivity is due to extremely slow polymerization kinetics for incorporation of an incorrect deoxynucleotide. The maximum rates for misincorporation (kpol) of dGTP, dCTP, and dTTP opposite a template uridine were 0.2, 0.03, and 0.003 s-1, respectively. The equilibrium dissociation constants (Kd) for the incorrect nucleotide opposite a template uridine were 1.0, 1.1, and 0.7 mM for dGTP, dCTP, and dTTP, respectively. These kinetic values provide fidelity estimates of 26 000 for discrimination against dGTP, 176 000 for dCTP, and 1 x 10(6) for dTTP misincorporation at this position. Similar observations were obtained when incorrect nucleotide misincorporation was examined opposite a template adenine. Thus in a direct comparison of RNA/DNA and DNA/DNA template-primer substrates, HIV-1 RT exhibits approximately a 10-60-fold increase in fidelity. This study augments our current understanding of the similarities and differences of catalytic activity of HIV-1 reverse transcriptase using RNA and DNA substrates. Moreover, these studies lend further support for a model for nucleotide incorporation by HIV-1 reverse transcriptase involving a two-step binding mechanism governed by a rate-limiting conformational change for correct incorporation.

DNA Primers↗

PubMed1998

Cell proteins bind to a 67 nucleotide sequence within the 3' noncoding region (NCR) of simian hemorrhagic fever virus (SHFV) negative-strand RNA.

The 3'NCR of the SHFV negative-strand RNA [SHFV 3'(-)NCR RNA] is thought to be the initiation site of full-length and possibly also subgenomic positive-strand RNA and so is likely to contain cis-acting signals for viral RNA replication. Cellular and viral proteins may specifically interact with this region to form replication complexes. When in vitro transcribed SHFV 3'(-)NCR RNA was used as a probe in gel mobility shift assays, two RNA-protein complexes were detected with MA104 S100 cytoplasmic extracts. The specificity of thes RNA-protein interactions was demonstrated by competition gel mobility shift assays. Four MA104 protein (103, 86, 55, and 36 kDa) were detected by UV-induced cross-linking assays and three proteins (103, 55, and 36 kDa) were detected by northwestern blotting assays. The binding sites for these proteins were mapped to the region between nucleotides 117 to 184 on the SHFV 3'(-)NCR RNA. Four cellular proteins with identical molecular masses to those of the proteins that bind to the SHFV 3'(-)NCR RNA were detected by the 3'(-)NCR of another arterivirus, LDV-C, suggesting that divergent arteriviruses utilize the same set of conserved cell protein domains.

Animals↗

PubMed1990

Sequence conservation and divergence of hepatitis delta virus RNA.

The complete RNA sequence of the hepatitis delta virus (HDV) obtained from the Nauru Island in the Pacific was determined by cDNA cloning and amplification by polymerase chain reaction (PCR). The sequence showed 14-17% divergence from the two known HDV RNA sequences. There are three highly conserved domains: the region around the autocatalytic cleavage site of the genomic RNA (nucleotides 659 to 772), the region around the autocatalytic cleavage site of the antigenomic-sense RNA (nucleotides 847 to 966), and the region around the middle one-third domain of the open reading frame (ORF) encoding the hepatitis delta antigen on the antigenomic RNA (nucleotides 1267 to 1347). The two autocatalytic activities are required for the cleavage and ligation of HDV RNA during RNA replication. The third conserved domain codes for the RNA-binding domain of HDAg, which specifically interacts with HDV RNA. Three nucleotide changes within the genomic catalytic sequence are present but did not alter the catalytic cleavage activity of the HDV RNA. Microheterogeneity of the RNA sequences was also detected. One of these occurred within the coding region of the delta antigen, creating an amber termination codon in some of the RNA species. Thus, this HDV strain contains two different RNA species, one of which encodes a delta antigen of 214 amino acids and the other 195 amino acids. These two protein species were detected by immunoblotting of the patient's plasma. In contrast to other HDV strains, only three ORFs capable of encoding more than 100 amino acids each are present in this HDV RNA. We recommend that oligonucleotides complementary to the highly conserved sequences should be used as primers for PCR in clinical detection assays of hepatitis delta virus infection.

Adult↗

PubMed1996

Interaction of retroviral nucleocapsid proteins with transfer RNAPhe: a lead ribozyme and 1H NMR study.

In the initiation of reverse transcription in retroviruses, nucleocapsid (NC) protein accelerates the rate of annealing of transfer RNA replication primer to a complementary sequence on the genomic RNA. In this report, we have probed the conformational changes induced by HIV-1 NC protein and domain deletion mutants in a structurally well-characterized transfer RNA, yeast tRNAPhe, as a model for the natural primer. One molar equivalent of recombinant 71 amino acid HIV-1 nucleocapsid protein (NC 1-71) is sufficient to completely inhibit the Pb2(+)-ribozyme activity of tRNAPhe at 25 degrees C, pH 7.0 and 15 mM MgCl2, Zn2 HIV-1 NC proteins which lack one or both flexible terminal domains also inhibit the ribozyme activity. 1H NMR spectra acquired for Mg(2+)-tRNAPhe suggest that NC 1-71 and NC 12-55 (lacking residues 1-11 and 56-71) inhibit the lead-ribozyme activity by only modestly altering the active site region rather than inducing large-scale unfolding of the molecule. In the absence of Mg2+, the extent of destabilization of tRNAPhe is greater but appears to be confined to internal regions of the acceptor and T psi C helices, as evidenced by the selectively enhanced exchange rates for imino protons associated with these base pairs. These findings show that NC destabilizes the folded form of tRNAPhe and by extension, other complex RNAs, in tertiary and secondary structural regions most susceptible to thermally-induced denaturation.

Capsid↗

PubMed1981

Role of RNA transcripts in replication incompatibility and copy number control in antibiotic resistance plasmid derivatives.

The genes required for autonomous replication and incompatibility in the antibiotic resistance plasmids R100 and R1 have been located within a 2.5-kilobase region of the 90-kilobase genome, within which the incompatibility gene occupies a 1.3-kilobase region excluding the replication origin. We now report that three RNA species are synthesized in vitro from the 2.5-kilobase region, which R100 and R1 have in common. One, a long RNA molecule which is transcribed in the direction of DNA replication, probably acts as a messenger or a protein required for plasmid replication. The second RNA species, only 91 nucleotides long, is transcribed in the opposite direction, from a region of the DNA entirely contained within the first and known to specify incompatibility and copy control functions. The third RNA species, 150 bases long, is transcribed from a region including the replication origin; it may be a primer of DNA synthesis or, in conjunction with the second of the three RNA species, an influence in the control of replication.

Chromosome Mapping↗

PubMed2001

Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus.

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.

Animals↗

PubMed1982

Partial characterization of temperature-sensitive mutants of foot-and-mouth disease virus, O1 caseros strain.

The preliminary characterization of four temperature-sensitive (ts) mutants of the O1 Caseros strain of foot-and-mouth disease virus is described. Two mutants, ts 6 and ts 40, showed a very low RNA synthesis rate at nonpermissive temperature and were classified phenotypically as RNA(-). Shift-up experiments demonstrated an incapacity to organize the RNA replicative process at nonpermissive temperature. Another mutant (ts 139) behaved phenotypically as RNA(+) and its virions were more thermolabile than the wild type virus, so the defect in this mutant is likely to be in one of its structural proteins. Finally, mutant ts 5 was phenotypically RNA(+); its leak production at nonpermissive temperature was high, and the shift-up experiment showed a defect that was expressed either late in the replicative cycle or continuously.

Animals↗

PubMed2004

Quantitative parameters determining whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus and an engineered defective RNA.

In this study, quantitative parameters affecting in vitro acquisition and whitefly (Bemisia tabaci) transmission of Lettuce infectious yellows virus (LIYV) were examined and B. tabaci transmission of an engineered defective RNA (D-RNA) was demonstrated. Virions purified from virus- and virion RNA-inoculated Chenopodium murale plants and protoplasts of Nicotiana tabacum, respectively, were consistently transmitted to plants by B. tabaci when virion concentrations were 0.1 ng microl(-1) or greater. Transmission efficiency increased with increasing virion concentration and number of whiteflies used for inoculation. When in vitro-derived transcripts of the M5gfp D-RNA (engineered to express the green fluorescent protein, GFP) were co-inoculated to protoplasts with wild-type LIYV virion RNAs, the resulting virions were transmissible to plants. LIYV and the M5gfp D-RNA systemically invaded inoculated plants; however, GFP expression was not detected in these plants. Unlike LIYV, the M5gfp D-RNA was not subsequently transmitted by B. tabaci from the initially infected plants, but, when high concentrations of virions from plants infected by LIYV and the M5gfp D-RNA were used for in vitro acquisition by whiteflies, both were transmitted to plants. Quantitative and qualitative analyses showed that, although the M5gfp D-RNA replicated within and systemically invaded plants along with LIYV, compared with LIYV RNA 2 it was not as abundant in plants or in the resulting virions, and concentration of encapsidated RNAs is an important factor affecting transmission efficiency.

Animals↗

PubMed1985

Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution.

The sequence of the entire RNA genome of the type flavivirus, yellow fever virus, has been obtained. Inspection of this sequence reveals a single long open reading frame of 10,233 nucleotides, which could encode a polypeptide of 3411 amino acids. The structural proteins are found within the amino-terminal 780 residues of this polyprotein; the remainder of the open reading frame consists of nonstructural viral polypeptides. This genome organization implies that mature viral proteins are produced by posttranslational cleavage of a polyprotein precursor and has implications for flavivirus RNA replication and for the evolutionary relation of this virus family to other RNA viruses.

Base Sequence↗

PubMed1989

Circular RNAs: relics of precellular evolution?

The demonstration of enzymatic capabilities of certain RNAs, in addition to their well-known template properties, has led to the recognition that RNAs are the only biological macromolecules that can function both as genotype and phenotype, hence raising the possibility of Darwinian selection and precellular evolution at the RNA level in the absence of DNA or protein. Recent models of such precellular RNA systems are patterned after the properties of intron-derived ribozymes. On the basis of a phylogenetic analysis and known properties of certain small plant pathogenic RNAs (viroids and viroid-like satellite RNAs), I suggest that these plant RNAs are more plausible candidates than introns as "living fossils" of a precellular RNA world. Their small size and circularity would have enhanced probability of their survival in error-prone, primitive self-replicating RNA systems and assured complete replication without the need for initiation or termination signals. All of these RNAs possess efficient mechanisms for the precise cleavage of monomers from oligomeric replication intermediates. Some (most viroids) require a host factor, but others (viroid-like satellite RNAs and one viroid) function as self-cleaving RNA enzymes far smaller and simpler than those derived from introns. The question is raised whether introns could have evolved from viroids or viroid-like satellite RNAs rather than vice versa, as has been widely speculated.

Base Sequence↗