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Results for “RNA Replication”

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At least 1,081 records · Page 60Linked to original sources

PubMed1994

Identification of calreticulin as a rubella virus RNA binding protein.

Previously, we observed that sequences at the 3' end of rubella virus (RV) genomic RNA that form a stable stem-loop structure are necessary for initiation of RNA replication. A cytosolic protein found in Vero 76 cells (simian origin) specifically bound to the 3' (+)-stem-loop sequence. In the present study, we have purified the RNA binding protein and identified it as a simian homologue of human calreticulin. The purified calreticulin binds to the RV RNA with specificity similar to the protein present in cytosolic extracts. Human calreticulin antibodies recognize several forms of simian calreticulin, one of which is phosphorylated in vivo. A 2-fold increase in phosphorylation of this form of calreticulin is observed in RV-infected cells. Recombinant human calreticulin can bind RV 3' (+)-stem-loop RNA only after undergoing in vitro phosphorylation. This binding activity is abrogated by pretreatment of phosphorylated recombinant human calreticulin with alkaline phosphatase. The RV RNA was also immunoprecipitated from RV-infected UV-crosslinked Vero 76 cells by using calreticulin antibodies. Our results show that phosphorylated calreticulin is an RNA binding protein and phosphorylation is necessary for this activity. Specific binding of calreticulin to the cis-acting element of RV RNA in vivo suggests a possible role for this interaction in viral replication.

Amino Acid Sequence↗

PubMed1991

Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes.

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.

Capsid↗

PubMed1990

Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection.

RNAs 1 and 2 of the tripartite cowpea chlorotic mottle virus (CCMV) genome are sufficient for RNA replication in protoplasts, whereas systemic infection of cowpea plants additionally requires RNA3, which encodes the 3a noncapsid protein and coat protein. By using biologically active CCMV cDNA clones, we find that deletions in either RNA3 gene block systemic infection. Thus, though some plant RNA viruses are able to spread systemically without encapsidation, both the coat and 3a genes are required for systemic infection of cowpeas by CCMV. When plants were coinoculated with CCMV RNAs 1 and 2 and both the 3a and coat deletion mutants of RNA3, 30-60% rapidly developed systemic infection. Progeny RNA recovered from systemically infected leaves in such infections contained neither of the starting deletion mutants but rather a single full-length RNA3 component with both genes intact. Nucleotide substitutions introduced into the coat protein deletion mutant as an artificial marker were recovered in the full-length progeny RNA, confirming its recombinant nature. Intermolecular RNA recombination in planta can, therefore, rescue a complete infectious genome from coinoculated mutants independently disabled for systemic spread. These results have implications for the repair of defective genomes produced by frequent natural replication errors, the possible emergence of newly adapted RNA viruses upon coinfection of new hosts, and further studies of RNA virus recombination.

Capsid↗

PubMed1984

Divided genomes and intrinsic noise.

Segmental genomes (i.e., genomes in which the genetic information is dispersed between two or more discrete molecules) are abundant in RNA viruses, but virtually absent in DNA viruses. It has been suggested that the division of information in RNA viruses expands the pool of variation available to natural selection by providing for the reassortment of modular RNAs from different genetic sources. This explanation is based on the apparent inability of related RNA molecules to undergo the kinds of physical recombination that generate variation among related DNA molecules. In this paper we propose a radically different hypothesis. Self-replicating RNA genomes have an error rate of about 10(-3) - 10(-4) substitutions per base per generation, whereas for DNA genomes the corresponding figure is 10(-9) - 10(-11). Thus the level of noise in the RNA copier process is five to eight orders of magnitude higher than that in the DNA process. Since a small module of information has a higher chance of passing undamaged through a noisy channel than does a large one, the division of RNA viral information among separate small units increases its overall chances of survival. The selective advantage of genome segmentation is most easily modelled for modular RNAs wrapped up in separate viral coats. If modular RNAs are brought together in a common viral coat, segmentation is advantageous only when interactions among the modular RNAs are selective enough to provide some degree of discrimination against miscopied sequences. This requirement is most clearly met by the reoviruses.

Biological Evolution↗

PubMed2002

Persistent and transient replication of full-length hepatitis C virus genomes in cell culture.

The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

Culture Media, Conditioned↗

PubMed1991

The course of giardiavirus infection in the Giardia lamblia trophozoites.

The subcellular distribution of Giardia lamblia virus RNA in infected G. lamblia trophozoites was examined by in situ hybridization using biotinylated DNA probe and riboprobe. In G. lamblia Portland I strain, which is chronically infected by G. lamblia viruses, the viral RNA was detected in the cytoplasm as well as in the twin nuclei. When riboprobe was used to examine the course of virus infection in WB strain, accumulation of viral RNA was detected only in the cytoplasm prior to the first 72 hr of infection. Using DNA probe, further accumulation of viral RNA in increasing number of cells occurred after the 72nd hr of infection, with the RNA found in both the cytoplasm and nuclei. Eventually, the cell nuclei showed damaged morphology that deteriorated rapidly toward the final stage of infection. These observations indicate that early phase of viral RNA replication may take place in the cytoplasm of infected G. lamblia, but the nuclei are also involved during the late phase of viral replication.

Animals↗

PubMed2005

Antiviral mode of action of a synthetic brassinosteroid against Junin virus replication.

The antiviral mode of action of the synthetic brassinosteroid (22S,23S)-3beta-bromo-5alpha,22,23-trihydroxystigmastan-6-one (6b) against Junin virus replication in Vero cells was investigated. Time-related experiments showed that 6b mainly affects an early event of virus growth cycle. Neither adsorption nor internalization of viral particles was the target of the inhibitory action. The analysis of the effect of 6b on viral RNA synthesis demonstrated that the presence of the compound adversely affects virus RNA replication by preventing the synthesis of full length antigenomic RNA. Although 6b was most effective the earlier it was added to the cells after infection with JV, a high level of inhibition of JV yield and fusion activity of newly synthesized viral glycoproteins was still detected when the compound was present during the last hours of infection. Therefore, we cannot rule out an inhibitory action of 6b on later events of JV replicative cycle.

Animals↗

PubMed1998

A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene.

A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pO delta CP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pO delta CP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.

Blotting, Northern↗

PubMed2006

A GCUA tetranucleotide loop found in the poliovirus oriL by in vivo SELEX (un)expectedly forms a YNMG-like structure: Extending the YNMG family with GYYA.

The cloverleaf structure in the 5'-untranslated region of enterovirus RNA that regulates viral RNA replication contains an evolutionarily conserved YNMG tetraloop closed by a Y-G base pair. This loop is believed to interact specifically with the viral protease 3C. To further characterize the specificity of this interaction, the tetraloop and two flanking base pairs of the poliovirus RNA were randomized, and viable viral clones were obtained using in vivo SELEX. Among many different mutants with the canonical YNMG sequences to be described elsewhere, a large-plaque-forming clone contained a deviating uGCUAg sequence. The NMR structure of a small hairpin capped with uGCUAg that we present here shows that the GCUA tetraloop adopts a novel fold, which is highly similar to that of the YNMG tetraloop with common stacking properties and hydrogen-bond interactions including an unusual syn conformation of the adenosine. Thermodynamic studies show moderate stabilities of hairpins with canonical YNMG and the novel GCUA loops, which, together with the similarity of spatial structures, illustrates that the tetraloop structure itself is crucial for the RNA-protein interaction required for the viral replication. A re-evaluation of the ribosomal secondary structure database reveals a hairpin containing a GCUA loop, which covaries with YNMG and is involved in a tertiary interaction, and in the 50S ribosomal subunit from Haloarcula marismortui the structurally comparable apex of stem-loop 35a is a recognition site for protein L2. These observations show a more general occurrence and importance of the so-far unrecognized GYYA hairpin loops.

Base Pairing↗

PubMed1998

Complex formation of NS5B with NS3 and NS4A proteins of hepatitis C virus.

At present, the mechanism of replication of the HCV genome remains unclear. Recently, NS5B and NS3 of HCV have been shown to exhibit RNA-dependent RNA polymerase and helicase activities, respectively, both of which are indispensable for virus RNA replication. In this study, we examined the complex formation of NS5B with NS3 and NS4A, a cofactor for NS3. We show here that NS5B forms a complex with NS3 through an amino-terminal portion of NS3. The NS3-NS5B complex formation took place both in the presence and absence of NS4A. We also demonstrate that NS5B form a complex with NS4A in the absence of NS3. These results suggest that NS3, NS4A and NS5B interact with each other to form a complex that functions as part of the replication machinery of HCV.

Amino Acid Sequence↗

PubMed1985

Replication of the vesicular stomatitis virus genome in permissive and nonpermissive host cells.

Permissive infections of BHK cells and nonpermissive infections of Raji cells were probed for the accumulation of vesicular stomatitis virus intracellular RNAs. In Raji cells, the onset of vesicular stomatitis virus transcription and replication was delayed when compared to BHK cells, and the accumulation of plus and minus sense leader RNAs was significantly reduced. In contrast, full length plus and minus strand replicative RNAs accumulated in Raji cells to levels approximately equivalent to those in BHK cells. In both cell types, approximately four times as many minus strands as plus strands were detected late in the infections. At 16 h postinfection, 12% of the total genomic RNA synthesized in BHK cells was packaged and released whereas only 0.8% was released from Raji cells. In addition, of those particles released by Raji cells, only 1% were infectious whereas 77% of those released by BHK cells were infectious. The virions released from both cell types contained similar amounts of the five viral proteins, however. Analysis of virions from Raji cells revealed a faster electrophoretic mobility of the glycoprotein than the glycoprotein in virions released from BHK cells. These results suggest that Raji cells may be restricted in their ability to support a complete infection at the level of virus assembly rather than at the level of RNA replication.

Animals↗

PubMed1999

RNA sequence and secondary structural determinants in a minimal viral promoter that directs replicase recognition and initiation of genomic plus-strand RNA synthesis.

Viral RNA replication provides a useful system to study the structure and function of RNAs and the mechanism of RNA synthesis from RNA templates. Previously we demonstrated that a 27 nt RNA from brome mosaic virus (BMV) can direct correct initiation of genomic plus-strand RNA synthesis by the BMV replicase. In this study, using biochemical, nuclear magnetic resonance, and thermodynamic analyses, we determined that the secondary structure of this 27 nt RNA can be significantly altered and retain the ability to direct RNA synthesis. In contrast, we find that position-specific changes in the RNA sequence will affect replicase recognition, modulate the polymerization process, and contribute to the differential accumulation of viral RNAs. These functional results are in agreement with the phylogenetic analysis of BMV and related viral sequences and suggest that a similar mechanism of RNA synthesis takes place for members of the alphavirus superfamily.

Base Sequence↗

PubMed1989

Inhibition of murine coronavirus RNA synthesis by hydroxyguanidine derivatives.

A series of hydroxyguanidine derivatives, which are substituted salicylaldehyde Schiff-bases of 1-amino-3- hydroxyguanidine tosylate, were tested for the inhibition of RNA synthesis of mouse hepatitis virus (MHV). It was shown that these compounds could selectively inhibit virus-specific RNA synthesis. Every aspect of viral RNA synthesis, including synthesis of negative-stranded RNA, subgenomic mRNA transcription and genomic RNA replication, was inhibited to roughly the same extent. These compounds are the first known inhibitors of coronaviral RNA synthesis and should prove useful for understanding the mechanism of viral RNA synthesis.

Antiviral Agents↗

PubMed2003

The 5'-end sequence of the genome of Aichi virus, a picornavirus, contains an element critical for viral RNA encapsidation.

Picornavirus positive-strand RNAs are selectively encapsidated despite the coexistence of viral negative-strand RNAs and cellular RNAs in infected cells. However, the precise mechanism of the RNA encapsidation process in picornaviruses remains unclear. Here we report the first identification of an RNA element critical for encapsidation in picornaviruses. The 5' end of the genome of Aichi virus, a member of the family Picornaviridae, folds into three stem-loop structures (SL-A, SL-B, and SL-C, from the most 5' end). In the previous study, we constructed a mutant, termed mut6, by exchanging the seven-nucleotide stretches of the middle part of the stem in SL-A with each other to maintain the base pairings of the stem. mut6 exhibited efficient RNA replication and translation but formed no plaques. The present study showed that in cells transfected with mut6 RNA, empty capsids were accumulated, but few virions containing RNA were formed. This means that mut6 has a severe defect in RNA encapsidation. Site-directed mutational analysis indicated that as the mutated region was narrowed, the encapsidation was improved. As a result, the mutation of the 7 bp of the middle part of the stem in SL-A was required for abolishing the plaque-forming ability. Thus, the 5'-end sequence of the Aichi virus genome was shown to play an important role in encapsidation.

5' Untranslated Regions↗

PubMed1999

Intracellular determinants of picornavirus replication.

Viruses replicate in a restricted number of hosts and tissues. In addition to viral receptors, several intracellular factors can be involved in determining tissue tropism. Many proteins have recently been implicated in picornavirus translation and RNA replication. Although the functional role of these proteins has not been established in vivo, it is possible that they determine cell-type tropism and the pathogenic outcome of the infection.

Animals↗

PubMed2002

Effect of actinomycin D on simian rotavirus (SA11) replication in cell culture.

Rotaviruses are the major cause of viral diarrhea in humans and animals. Actinomycin D (Act D) is an antibiotic that intercalates DNA and therefore inhibits DNA-dependent transcription. The current study was carried out to assess the influence of Act D on the replication of simian rotavirus (SA11) in cell culture. Virus-infected MA-104 cell cultures were studied in the presence of Act D at concentrations of 1.25 and 2.5 microg/ml. Treatment of rotavirus-infected cells with 2.5 microg/ml Act D 48 h post-infection reduced the cytoplasmic metachromasia after staining with acridine orange by 25%. Viral RNA labeled with 3H-uridine in the presence of the drug was separated by polyacrylamide gel electrophoresis. Viral RNA replication was not affected by Act D, but increased 3H-uridine uptake was demonstrable by infected cells in the presence of the drug. This possibly was due to the inhibition of cellular RNA synthesis by Act D, which thus enhances incorporation of the radionuclide into the viral RNA. Act D reduced the number of infected cells presenting virus-specific fluorescence 48 h post-infection by more than 50%. These data suggest that Act D may have complexed with viral RNA and prevented newly synthesized mRNA from being translated, but may not have prevented early replication.

Animals↗

PubMed1991

Replication of TMV-L and Lta1 RNAs and their recombinants in TMV-resistant Tm-1 tomato protoplasts.

Tm-1 is a gene that provides resistance to tomato plants against tobacco mosaic virus (TMV) infection. In tomato cells carrying the Tm-1 gene, multiplication of TMV is inhibited. From previous analysis of resistance-breaking mutants, the involvement of the 130- and 180-kDa proteins, putative viral replicases, in the resistance conferred by the Tm-1 gene was suggested. When wild-type TMV RNA was co-inoculated with a resistance-breaking mutant RNA, replication of the wild-type TMV genomic RNA could not be rescued by the 130- and 180-kDa proteins of a resistance-breaking strain, Lta1. To investigate how the putative resistance factor interacts with the 130- and 180-kDa proteins, we expressed the wild-type TMV protein sequence that is associated with the resistance-breaking phenomenon as part of a recombinant virus derived from Lta1 in Tm-1/Tm-1 protoplasts. No specific degradation of wild-type TMV protein sequences was observed, suggesting that the mechanism of the resistance does not involve the instability of a viral protein.

Amino Acid Sequence↗

PubMed1994

Priming of HIV replication by tRNA(Lys3): role of reverse transcriptase.

The fundamental role played by reverse transcriptase in the replication of retroviruses has stimulated the study of the mechanism of action of this enzyme. The reverse transcriptase of the type 1 human immunodeficiency virus forms a stable complex with its cognate transfer RNA replication primer (tRNA(Lys3)). Here, we outline the role of this enzyme in the selection of its primer tRNA, the annealing of primer tRNA to the complementary region of the retroviral genome, and the first attempts to use the reverse-transcriptase-tRNA complex as a new target for antiviral agents.

Base Sequence↗