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At least 163 records · Page 9Linked to original sources
PubMed2003

Kunjin RNA replication and applications of Kunjin replicons.

The Kunjin virus (KUNV) has provided a useful laboratory model for Flavivirus RNA replication. The synthesis of progeny RNA(+) strands occurs via asymmetric and semiconservative replication on a template of recycling double-stranded RNA (dsRna) or replicative form (RF). Kinetics of viral RNA synthesis indicated a cycle period of about 15 min during which, on average, a single nascent RNA (+) strand displaces the pre-existing RNA(+) strand in the replicative intermediate. Data on the composition of the replication complex (RC) in KUNV-infected cells were obtained from several sources, including analyses of the partially-purified still active RC, immunogold labeling of cryosections using monospecific antibodies to the nonstructural proteins and to the dsRNA, radioimmunoprecipitations of cell lysates using antibodies to dsRNA and to an RC-associated cell marker, and pull-down assays of cell lysates using fusion proteins GST-NS2A and GST-NS4A. These results yeilded a consensus composition of NS1, NS2A, NS3, NS4A, and NS5 strongly associated with the dsRNA template. The RC was located in induced membranes described as vesicle packets. The RNA-dependent RNA polymerase activity late in infection did not require continuing protein synthesis. Replication of genomic RNA was completely dependent on the presence of conserved complementary or cyclization sequences near the 5' and 3' ends. Assembly of the RC during translation in cis and the relationships, particularly those of NS1 and NS5 among the components, were deduced from an extensive set of complementation experiments in trans involving mutations/deletions in all the nonstructural proteins and use of KUN or alphahavirus replicons as helpers. The KUN replicon has found useful applications also as a noncytopathic vector for the continuing expression of foreign genes, delivered either as packaged RNA or as plasmid DNA.

Amino Acid Sequence↗
PubMed1992

Mechanism of coronavirus transcription: duration of primary transcription initiation activity and effects of subgenomic RNA transcription on RNA replication.

Previously, we established a system whereby an intergenic region from mouse hepatitis virus (MHV) inserted into an MHV defective interfering (DI) RNA led to transcription of a subgenomic DI RNA in helper virus-infected cells. By using this system, the duration of a primary transcription initiation activity which transcribes subgenomic-size RNAs from the genomic-size RNA template in MHV-infected cells was examined. Efficient DI genomic and subgenomic RNA synthesis was observed when the DI RNA was transfected at 1, 3, 3.5, 5, and 6 h postinfection, indicating that all activities which are necessary for MHV RNA synthesis are present continuously during the first 6 h of infection. The effect of subgenomic DI RNA synthesis on DI genomic RNA replication was then examined. Replication efficiency of the DI genomic RNA which synthesized the subgenomic RNA was approximately 70% lower than that of DI genomic RNA which did not synthesize the subgenomic DI RNA in MHV-infected cells. Cotransfection of two different-size DI RNAs demonstrated that replication of the larger DI RNA was strongly inhibited by replication of the smaller genomic DI RNA. Cotransfection of two DI RNA species of the same length into MHV-infected cells demonstrated that reduced replication of the genomic DI RNA which synthesizes the subgenomic RNA did not affect the replication of cotransfected DI RNA, demonstrating that the reduction in DI genomic RNA replication works only in cis, not in trans. Therefore, the previously proposed hypothesis that coronavirus, subgenomic RNA synthesis may inhibit the replication of genomic RNA by competing for a limited amount of virus-derived factors seems unlikely. Possible mechanisms of coronavirus transcription are discussed.

Animals↗
PubMed1998

The effect of deletion of a conserved 11 nucleotide sequence on mouse hepatitis virus RNA replication.

A conserved 11 nt sequence present at near the 3' end of mouse hepatitis virus (MHV) genomic RNA binds to host proteins and is important for MHV RNA replication (Yu and Leibowitz, 1995). To better understand the role of this 11 nt sequence in positive-strand MHV RNA replication, we examined whether positive-strand MHV DI RNAs from negative-strand DI RNA transcripts lacking the 11 nt sequence were synthesized in MHV-infected cells. Positive-strand DI RNAs did not accumulate efficiently, indicating that the conserved 11 nt sequence was necessary for positive-strand MHV RNA synthesis.

Animals↗
PubMed1989

Analysis of sequences involved in cowpea mosaic virus RNA replication using site-specific mutants.

Using a full-length cDNA clone of cowpea mosaic virus (CPMV) B-RNA from which infectious transcripts can be generated, we examined the influence of a sequence of 11 nucleotides, UUUUAUUAAAA, comprising the nucleotides 5883 to 5893 in the 3' noncoding region of B-RNA, on viral RNA replication. This sequence is not only present in B-RNA but also in M-RNA and represents the 7 nucleotides preceding the poly(A) tail and the first four A residues of the poly(A) tail. Replication of B-RNA transcripts derived from a series of mutants in this region was tested in cowpea plants and protoplasts. Only mutant transcripts with minor modifications appeared able to replicate, which indicates that the region has a function in viral RNA replication. In addition, the results suggest the existence of a hairpin loop in this region. Those transcripts with deletions which disturb the putative hairpin structure have decreased specific infectivities. Mutant transcripts reversed stepwise to the wild-type sequence during replication in plants. This observation strengthens the idea that the sequence of 11 nucleotides has a function in viral RNA replication.

Base Sequence↗
PubMed2006

Selective repression of translation by the brome mosaic virus 1a RNA replication protein.

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.

5' Untranslated Regions↗
PubMed1994

A cis-acting function for the coronavirus leader in defective interfering RNA replication.

To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that, in contrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements originating from discontinuous downstream regions within the polymerase gene for replication or for packaging.

Amino Acid Sequence↗
PubMed2005

Human astrovirus C-terminal nsP1a protein is involved in RNA replication.

Human astrovirus nonstructural C-terminal nsP1a protein, which contains a hypervariable region (HVR) and colocalizes with the endoplasmic reticulum and viral RNA, has been suggested to be involved in the RNA replication process. Four viruses differing only in their C-terminal nsP1a protein, corresponding to HVR-derived genotypes IV, V, VI, and XII, were all able to replicate in CaCo-2 cells but displayed differences in their RNA replication and growth properties. Two overall patterns of replication were observed: types IV and V on one side, and types VI and XII on the other. The main detected differences were on the levels of antigenomic and subgenomic RNAs, being the latter significantly higher in types IV and V. Accordingly, quantification of viral RNA load in feces from children with gastroenteritis showed that HVR-derived genotypes IV and V occur in significantly higher numbers. In consequence, it may be concluded that the variability of the C-terminal nsP1a gene affects the virus replication phenotype.

Amino Acid Sequence↗
PubMed1990

Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement.

Barley stripe mosaic hordeivirus (BSMV) has a tripartite positive-sense RNA genome which encodes seven major polypeptides. Infectious in vitro transcripts derived from full-length wild-type and mutant cDNA clones have been used to investigate the contribution made by various BSMV gene products to viral RNA replication and systemic movement. We show that whereas all three of the BSMV RNA components are required for plant infection, RNAs alpha and gamma can replicate together in barley protoplasts, and therefore RNA beta must encode functions required for systemic invasion of plants. The alpha a and gamma a proteins, which contain helicase and RNA polymerase sequence motifs, together comprise the essential virus-encoded components of BSMV RNA replicase. A second BSMV protein (beta b) which contains a helicase motif is not required for RNA replication. A small cysteine-rich protein (gamma b) is dispensable for infection of plants, but in its absence the accumulation of viral coat (beta a) and beta b proteins is significantly reduced. In addition, mutations in both the gamma b and gamma a (replicase) proteins can affect the systemic movement phenotype.

Drug Combinations↗
PubMed2001

Efficient hepatitis delta virus RNA replication in avian cells requires a permissive factor(s) from mammalian cells.

Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses.

Animals↗
PubMed2003

Aichi virus leader protein is involved in viral RNA replication and encapsidation.

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.

Animals↗
PubMed1990

Use of bromovirus RNA2 hybrids to map cis- and trans-acting functions in a conserved RNA replication gene.

Brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) are related positive-strand RNA viruses with tripartite genomes. RNA replication by either virus requires genomic RNAs 1 and 2, which encode protein 1a and the polymeraselike, 94-kilodalton 2a protein, respectively. Proteins 1a and 2a share extensive sequence similarity with proteins encoded by a wide range of other positive-strand RNA viruses of animals and plants. Heterologous combinations of BMV and CCMV RNAs 1 and 2 do not support viral RNA replication, and although BMV RNA2 is amplified in CCMV-infected cells, CCMV RNA2 is not amplified by BMV. Construction of hybrids by precise exchange of segments between BMV and CCMV RNA2 has now allowed preliminary mapping of such virus-specific replication functions in RNA2 and the 2a protein. The ability to support replication in trans with BMV RNA1 segregated with a 5' BMV RNA2 fragment encoding the first 358 2a gene amino acids, while a 5' fragment extending over 281 BMV 2a codons transferred only cis-acting competence for RNA2 amplification in cells coinfected with wild-type BMV. Successful trans-acting function with CCMV RNA1 segregated with a CCMV RNA2 3' fragment that included the last 206 2a gene codons. Thus, the less conserved N- and C-terminal 2a segments appear to be involved in required interaction(s) of this polymeraselike protein with the 1a protein or RNA1 or both. Moreover, when individual hybrid RNA2 molecules that function with either BMV or CCMV RNA1 were tested, BMV- and CCMV-specific differences in recognition and amplification of RNA3 templates appeared to segregate with RNA1.

Amino Acid Sequence↗
PubMed2005

Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A.

Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.

Cell Line, Tumor↗
PubMed1993

Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34.

A vaccinia virus transient expression system was used to determine which of the Sindbis virus (SIN) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsP4, the putative polymerase, versus P34 in RNA replication. We generated vaccinia virus recombinants in which the cDNA for the entire SIN nonstructural coding region as well as cDNA copies of the individual nonstructural proteins (nsPs) and several intermediate polyproteins were placed downstream of the promoter for T7 RNA polymerase and the encephalomyocarditis virus 5' untranslated region. The proteins expressed by the vaccinia virus recombinants comigrate with authentic proteins synthesized in SIN-infected cells, and the polyproteins appear to be processed to the individual proteins of the correct size. To examine the replication efficiencies of different protein combinations, a vaccinia virus recombinant was designed to express an engineered substrate RNA which could serve as a template for replication and subgenomic mRNA transcription by the SIN nsPs. Expression of the entire SIN nonstructural coding region resulted in the synthesis of high levels of both genomic and subgenomic RNAs derived from the engineered template. No RNA replication could be detected during coexpression of the four individual nsPs, although the proteins were indistinguishable, in terms of electrophoretic mobility, from those synthesized in SIN-infected cells. Coexpression of polyproteins P12, P23, and/or P34 with the individual nsPs also did not result in detectable levels of RNA replication. However, when P123 and P34 were coexpressed, efficient RNA replication and subgenomic mRNA transcription of the substrate RNA was observed. Coexpression of nsP4 with P123 resulted in the synthesis of only minus-strand RNAs. These studies show that expression of both P123 and P34 is necessary for establishment of functional RNA replication and transcription complexes and raise the possibility that the polyproteins themselves may be functional components of these complexes. In addition, these data indicate that an nsP4 moiety expressed independently with an additional N-terminal methionine is capable of functioning in minus-strand but not plus-strand RNA synthesis.

Animals↗
PubMed1993

Structure of the maize mitochondrial replicon RNA b and its relationship with other autonomously replicating RNA species.

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and > 4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5' terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b-oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3' termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Q beta and "variant" RNAs that serve as in vitro templates for the Q beta replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Base Sequence↗
PubMed1979

Sindbis virus RNA replication. I. Properties of the 38s RNA species.

Four species of single-stranded virus RNA (49S, 38S, 33S and 26S) were detected in chick embryo fibroblasts infected with Sindbis virus. The relative amounts of these RNAs were unaffected by the m.o.i. There was also no significant difference in the molar proportions of the four RNA species when purified virion RNA was used as the inoculum. These findings suggest that the 38S and 33S species represent products of the transcription of non-defective virion RNAs. Kinetic analyses of RNA synthesis indicated that during a 1 min pulse more radioactivity was associated with the 38S than with the 49S RNA and as the length of the pulse increased, the ratio of 38S/49S decreased, with the 49S appearing as the predominant species. Furthermore, addition of cycloheximide within the first 3 h p.i. resulted in detection of only the 49S species. Synthese of all four species was unaffected when the drug was added after this time period. These data suggest that the 38S species may represent newly synthesized 49S molecules and some protein(s) synthesized within the first 3 h p.i. is necessary for maintaining the 38S conformational form.

Animals↗
PubMed2001

Sequences in the 5' nontranslated region of hepatitis C virus required for RNA replication.

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.

5' Untranslated Regions↗
PubMed1993

Hepatitis delta virus cDNA monomer can be used in transfection experiments to initiate viral RNA replication.

The complementary DNA (cDNA) of hepatitis delta virus (HDV), constructed as tandem repeats under appropriate exogenous promoters, has been used to initiate viral replication in transfection experiments. Whether the structure of tandem repeats is essential has not yet been systematically examined. In this study, expression vectors containing only an HDV cDNA monomer, permutated at unique position of the genome, were shown still able to initiate viral replication. Furthermore, HDV cDNA monomer, separated from plasmid sequences by restriction enzyme digestion, also could be used to initiate viral RNA replication. The competence of HDV cDNA alone to direct viral RNA production suggested the presence of cryptic internal promoter-like elements. Such elements actually demonstrated by the chloramphenicol acetyltransferase assay. Therefore, HDV cDNA, in contrast to that of viroids, could be used to initiate viral RNA replication in monomeric form. This observation simplified the use of HDV cDNA for studying viral biology in transfection systems.

Cell Line↗