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At least 253 records · Page 14Linked to original sources

PubMed2001

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging.

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.

3' Untranslated Regions↗

PubMed1995

Significance of strand configuration in self-replicating RNA molecules.

The kinetic theory of replication has been extended to include dual mechanisms for conversion of self-annealed single-strand RNA to double-strand molecules, which do not replicate. An analysis of experimental results established that the replicate-template annealing reaction during transcription significantly retarded replication in vitro among three RNA variants copied by Q beta replicase. Annealing between complementary RNA strands free in solution had far less significance. The finding that an RNA variant can be replicated in a multiple hairpin configuration, but not as its single, long hairpin conformer, the correlation between stability of strand secondary structure and replicative fitness, and a lack of homology in the internal sequence of RNA variants copied by Q beta replicase support the conclusion that template competence depends on strand configuration, independent of most of the underlying base sequence. Occurrence of self-annealed strands in the Q beta replicase system was attributed to its reliance on RNA-driven strand separation, in the absence of enzyme catalysed strand unwinding. A 'configuration before sequence' path to self-replication exhibited a substantially lower combinatorial barrier than standard sequence-dependent evolution. RNA-dependent RNA synthesis in the Q beta system thus displays features of an RNA World and, interestingly, they reveal a rapid path for evolution of the first self-replicating molecule on Earth.

Base Sequence↗

PubMed1988

The onset of macroscopically detectable amplification of template concentration for self-replicating RNA.

We calculate the strength of fluctuations in concentrations and rates for a self-replicating RNA system catalyzed by the Q beta-replicase at very low initial template concentration (1-10(3) strands/ml). The work is centered upon the derivation of the induction periods which must elapse in order for the rate-correlation size to become comparable to a kinetic barrier determined by the width of the probability distribution about the invariant portion of the concentration space. This surface is identified by a center manifold and corresponds to the subordination of relaxing kinetic modes to the overall growth of the total (free and complexed) template concentration. The results are compared with the experimental data for the onset of a macroscopically detectable amplification of template concentration and a satisfactory agreement is observed.

Kinetics↗

PubMed2001

Self-replicative RNA vaccines elicit protection against influenza A virus, respiratory syncytial virus, and a tickborne encephalitis virus.

In genetic vaccination, recipients are immunized with antigen-encoding nucleic acid, usually DNA. This study addressed the possibility of using the recombinant alpha virus RNA molecule, which replicates in the cytoplasm of transfected cells, as a novel approach for genetic vaccination. Mice were immunized with recombinant Semliki Forest virus RNA-encoding envelope proteins from one of 3 viruses: influenza A virus, a tickborne flavivirus (louping ill virus), or respiratory syncytial virus (RSV). Serologic analyses showed that antigen-specific antibody responses were elicited. IgG isotyping indicated that predominantly Th1 type immune responses were induced after immunization with RSV F protein-encoding RNA, which is relevant for protection against RSV infection. Challenge infection showed that RNA immunization had elicited significant levels of protection against the 3 model virus diseases.

Animals↗

PubMed1995

[Effect of Astragalus membranaceus on Ca2+ influx and coxsackie virus B3 RNA replication in cultured neonatal rat heart cells].

The effect of Astragalus membranaceus (AM) on Ca2+ influx across the myocardial plasma membrane and coxsackie virus B3(CVB3)-RNA replication in cultured neonatal rat heart cells infected with CVB3 was investigated. It was found that the Ca2+ influx could be inhibited significantly (P < 0.01) by AM after infection of heart cells for 48 h. In addition, when the cultured heart cells infected with CVB3 and treated with AM for 48 h, the Ca2+ influx of infected heart cells also could be inhibited by AM (P < 0.05) and the amounts of CVB3-RNA in myocytes were significantly decreased than that in infected control group (P < 0.001). These phenomena suggested that AM could exert the effects of decreasing the secondary Ca2+ damages, and improving the abnormal myocardial electric activity, and inhibiting replication of CVB3-RNA in myocardium. Thus, it is a rational choice to treat patients with AM in viral myocarditis.

Animals↗

PubMed1993

RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication.

Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J. H. Lin, M. F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai, J. Virol. 64:4051-4058, 1990). By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins. The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids. Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg. Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity. Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro. Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity. The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants. The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells. It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication. We conclude that the trans-activating function of HDAg requires its binding to HDV RNA.

Amino Acid Sequence↗

PubMed2005

Analysis of the 5' end structure of HCV subgenomic RNA replicated in a Huh7 cell line.

OBJECTIVE: Recently, HCV subgenomic RNA that replicates in vitro in a certain cell line have been elucidated. Since the 5' end of the genome of positive strand RNA viruses is often modified with a cap structure or a covalently linked protein, we have assessed structural feature of the HCV genome obtained from Huh7 cells in which HCV subgenomic RNA has been shown to efficiently self-replicate. METHODS: HCV subgenomic RNA was obtained from the Huh7 and was analyzed for its 5' end. RESULTS: Phosphorylation of the genomic RNA by polynucleotide kinase was observed only after treatment with phosphatase. The labeling efficiency of the genome with polynucleotide kinase was not enhanced by treatment with pyrophosphatase. CONCLUSION: It is suggested that the 5' end of HCV genomic RNA obtained from HCV replicon cells is not modified except phosphorylation. Furthermore, analysis of the 5' end of the HCV RNA obtained from the HCV subgenome self-replicating cells revealed the presence of two types of subgenomic RNA that contained either guanylate or adenylate at the 5' end. This result indicates that the 5' end of the subgenome in Huh7 cells is redundant and there is no significant evolutionary advantage between the two genomes.

Adenosine Monophosphate↗

PubMed2003

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules.

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.

3' Untranslated Regions↗

PubMed1997

Replication signals in the genome of vesicular stomatitis virus and its defective interfering particles: identification of a sequence element that enhances DI RNA replication.

We have analyzed the role of terminal sequences of a defective interfering (DI) particle RNA of vesicular stomatitis virus (VSV) in replication. A series of internal deletion mutants of DI cDNA was generated to obtain DI genomic RNAs that differed from one another by the presence of different lengths of 3'-terminal and/or 5'-terminal sequences. Analyses of the mutant. RNAs for their ability to replicate in cells transfected with the corresponding plasmids suggested that distinct regions at the termini of DI RNA are important for RNA replication. Region I, encompassing nucleotides 1-24, is absolutely required for replication since DI RNA genomes lacking any part of this region failed to replicate. Region II, spanning nucleotides 25-45, is not essential for replication but it functions as an enhancer of replication in that the presence of these specific sequences confers high efficiency of replication to the template. Deleting these specific sequences from both termini of DI RNA but maintaining the length of terminal complementarity as seen in wild-type DI RNA resulted in a template that replicated poorly (about 20-fold less efficiently). Furthermore, insertion or substitution of these sequences into the 3'-terminus of a VSV minigenome resulted in a template that replicated more efficiently (at least 4-fold to as high as 15-fold) than the parental minigenome. These results strongly support the conclusion that the presence of specific sequences rather than the extent of complementarity at the termini of DI RNA is a major determinant of the efficiency of replication. The presence of the specific sequences at the 3'-terminus of both genomic and antigenomic DI RNAs may explain in part the replicative dominance of DI RNA over the full-length VSV genome which contains these sequences only at the 3'-terminus of the antigenome.

Animals↗

PubMed2001

Rubella virus RNA replication is cis-preferential and synthesis of negative- and positive-strand RNAs is regulated by the processing of nonstructural protein.

Rubella virus (RV) genome encodes nonstructural protein (NSP) in a large open reading frame at its 5' end. It is translated into p200 and further processed into p150 and p90. The NSPs are responsible for viral RNA replication, during which a full-length negative-strand RNA serves as the intermediate for the replication of positive-strand genomic RNA and the transcription of subgenomic RNA. Using complementation experiments, we demonstrated that RV negative-strand RNA is synthesized preferentially in cis while positive-strand RNAs can be synthesized both in cis and in trans but with higher efficiency in cis. During virus infection, negative-strand RNA accumulates until 10 hours postinfection (hpi) and remains nearly constant thereafter. In contrast, positive-strand RNAs (both genomic and subgenomic RNA) do not increase much before 10 hpi and accumulate rapidly thereafter. Previously we demonstrated that p200 synthesizes negative- but not positive-strand RNA, whereas cleavage products p150/p90 are required for efficient production of positive-strand RNAs. In this study, we present evidence demonstrating that a higher concentration of p150/p90 is associated with lower production of negative-strand RNA. Our data support the hypothesis that p200 is the principal replicase for negative-strand RNA, as is p150/p90 for positive-strand RNA. The switch from the synthesis of negative- to positive-strand RNA is thus regulated by NSP processing, which not only activates the efficient production of positive-strand RNA, but also disables negative-strand RNA synthesis. A mechanism for NSP translation, processing, and regulation of RV RNA synthesis is proposed.

Animals↗

PubMed2002

Coalescence of the sites of cowpea mosaic virus RNA replication into a cytopathic structure.

Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.

Comovirus↗

PubMed2003

Fine mapping of a cis-acting sequence element in yellow fever virus RNA that is required for RNA replication and cyclization.

We present fine mapping of a cis-acting nucleotide sequence found in the 5' region of yellow fever virus genomic RNA that is required for RNA replication. There is evidence that this sequence interacts with a complementary sequence in the 3' region of the genome to cyclize the RNA. Replicons were constructed that had various deletions in the 5' region encoding the capsid protein and were tested for their ability to replicate. We found that a sequence of 18 nucleotides (residues 146 to 163 of the yellow fever virus genome, which encode amino acids 9 to 14 of the capsid protein) is essential for replication of the yellow fever virus replicon and that a slightly longer sequence of 21 nucleotides (residues 146 to 166, encoding amino acids 9 to 15) is required for full replication. This region is larger than the core sequence of 8 nucleotides conserved among all mosquito-borne flaviviruses and contains instead the entire sequence previously proposed to be involved in cyclization of yellow fever virus RNA.

Base Sequence↗

PubMed1978

Recovery of tobacco mosaic virus RNA Replication after incubation at 40 degrees.

Incubation of tobacco mosaic virus (TMV)-infected tissue at 40 degrees for 12 h destroyed the capacity for TMV RNA replication. Upon shift of the tissue from 40 to 25 degrees, synthesis of TMV RNA did not resume. Incubation at 40 degrees for 1 h did not destory the TMV RNA replicase, because viral RNA synthesis resumed upon return to 25 degrees. However, upon further incubation at 25 degrees, the synthesis rate of TMV RNA gradually declined. Upon still further incubation at 25 degrees (16-20 h), the synthesis of TMV RNA in tissue incubated at 40 degrees for either 1 or 12 h recovered. This recovery was inhibited by cycloheximide but not by 2-thiouracil.

Cycloheximide↗

PubMed1977

An Escherichia coli mutant with a temperature-sensitive function affecting bacteriophage Qbeta RNA replication.

We report the isolation of E. coli mutant capable of supporting replication of bacteriophage Qbeta at 33 degrees, but not at 40 degrees. Coliphages f2, R23, fd, and yamma formed plaques on mutant cells at both temperatures. Temperature-shift experiments showed that bacteriophage Q beta replication was blocked in the mutant within the first 20-30 min of infection. The defect did not prevent translation of the Qbeta polymerase gene or assembly of catalytically active Qbeta replicase molecules. In fact, mutant cells infected at 40 degrees hyperinduced replicase active both in vivo and in vitro. However, zone sedimentation of the in vivo RNA product showed it to consist of partially double-stranded material sedimenting at 9 S, with little or no viral 32S RNA. The 9S RNA was also found, along with a predominant peak of 32S RNA in parental cells infected at 40 degrees, but not in cells infected at 33 degrees. It thus appears that the temperature-sensitive component is required for viral RNA replication, but not for other RNA synthesis catalyzed by the replicase. Uninfected mutant cells grew normally at 40 degrees in nutrient broth, but not in glucose- or glycerol-minimal media. Revertants selected for their abillity to grow in minimal medium at 40 degrees also supported bacteriophage Qbeta replication at 40 degrees.

Coliphages↗

PubMed2004

High-resolution structure of a picornaviral internal cis-acting RNA replication element (cre).

Picornaviruses constitute a medically important family of RNA viruses in which genome replication critically depends on a small RNA element, the cis-acting replication element (cre), that templates 3D(pol) polymerase-catalyzed uridylylation of the protein primer for RNA synthesis, VPg. We report the solution structure of the 33-nt cre of human rhinovirus 14 under solution conditions optimal for uridylylation in vitro. The cre adopts a stem-loop conformation with an extended duplex stem supporting a novel 14-nt loop that derives stability from base-stacking interactions. Base-pair interactions are absent within the loop, and base substitutions within the loop that favor such interactions are detrimental to viral RNA replication. Conserved adenosines in the 5' loop sequence that participate in a slide-back mechanism of VPg-pUpU synthesis are oriented to the inside of the loop but are available for base templating during uridylation. The structure explains why substitutions of the 3' loop nucleotides have little impact on conformation of the critical 5' loop bases and accounts for wide variation in the sequences of cres from different enteroviruses and rhinoviruses.

Base Pairing↗

PubMed1988

Influenza virus RNA replication in vitro: synthesis of viral template RNAs and virion RNAs in the absence of an added primer.

The two steps in influenza virus RNA replication are (i) the synthesis of template RNAs, i.e., full-length copies of the virion RNAs, and (ii) the copying of these template RNAs into new virion RNAs. We prepared nuclear extracts from infected HeLa cells that catalyzed both template RNA and virion RNA synthesis in vitro in the absence of an added primer. Antibody depletion experiments implicated nucleocapsid protein molecules not associated with nucleocapsids in template RNA synthesis for antitermination at the polyadenylation site used during viral mRNA synthesis. Experiments with the WSN influenza virus temperature-sensitive mutant ts56 containing a defect in the nucleocapsid protein proved that the nucleocapsid protein was indeed required for template RNA synthesis both in vivo and in vitro. Nuclear extracts prepared from mutant virus-infected cells synthesized template RNA at the permissive temperature but not at the nonpermissive temperature, whereas the synthesis of mRNA-size transcripts was not decreased at the nonpermissive temperature. Antibody depletion experiments showed that nucleocapsid protein molecules not associated with nucleocapsids were also required for the copying of template RNA into virion RNA. In contrast to the situation with the synthesis of transcripts complementary to virion RNA, no discrete termination product(s) were made during virion RNA synthesis in vitro in the absence of nucleocapsid protein molecules.

Capsid↗

PubMed1977

Host factor for coliphage Qbeta RNA replication as an aid in elucidating phylogenetic relationships: the genus Pseudomonas.

Host Factor (HF) is a heat-stable RNA-binding protein required along with Qbeta replicase for in vitro transcription of coliphage Qbeta RNA. We have found that HF activity and antigenicity are conserved among certain Gram-negative bacterial species. We examined selected species within the genus Pseudomonas for the presence of the HF polypeptide's antigenicity and Qbeta RNA replication function. While we were unable to detect either of these in Pseudomonas diminuta or Pseudomonas vesicularis, the other eleven species tested contained cross-reacting material to Escherichia coli HF. Furthermore, in six of these eleven species we were able to detect HF activity. The detection of HF structure and function allowed the examined species to be grouped into three categories which we have called 'sets'. The results correlate well with those of previous studies on ribosomal RNA homology (Palleroni et al., 1973).

Antigens, Bacterial↗

PubMed2005

Wrapping things up about virus RNA replication.

All single-stranded 'positive-sense' RNA viruses that infect mammalian, insect or plant cells rearrange internal cellular membranes to provide an environment facilitating virus replication. A striking feature of these unique membrane structures is the induction of 70-100 nm vesicles (either free within the cytoplasm, associated with other induced vesicles or bound within a surrounding membrane) harbouring the viral replication complex (RC). Although similar in appearance, the cellular composition of these vesicles appears to vary for different viruses, implying different organelle origins for the intracellular sites of viral RNA replication. Genetic analysis has revealed that induction of these membrane structures can be attributed to a particular viral gene product, usually a non-structural protein. This review will highlight our current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA-membrane interactions observed between the virus families Flaviviridae and Picornaviridae.

Animals↗