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Results for “RNA Replication”

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At least 181 records · Page 10Linked to original sources

PubMed1993

Studies on dicistronic polioviruses implicate viral proteinase 2Apro in RNA replication.

A dicistronic poliovirus W1-P1/E/P2,3-1 with the genotype [PV]5'NTR-P1-[EMCV]IRES-[PV]P2,3-3'NTR (Molla, Jang, Paul, Reuer, and Wimmer, 1992, Nature 356, 255) was used to investigate whether the viral proteinase 2Apro, whose primary function in proteolytic processing was erased through the insertion of an internal ribosomal entry site (IRES) element into the ORF of the polyprotein, had other function(s) in viral replication. Deletion of 2Apro from W1-P1/E/P2,3-1 rendered the corresponding transcripts unable to replicate whereas partial deletion of 2Apro or an exchange of Cys109 (an amino acid of the catalytic triad of the proteinase) to Ala reduced RNA replication. No cytopathic effects were observed after transfection with any of the three dicistronic constructs containing mutant 2A, and no virus was recovered after attempts to expand a possibly low yield of mutant virus. In contrast, insertion of the IRES of encephalomyocarditis virus (EMCV) into the ORF of the poliovirus polyprotein at the cleavage site between 2Apro and 2B yielded the novel dicistronic virus W1-P1,2A/E/2BC,P3-1 with the genotype [PV]5'NTR-P1-2A-[EMCV]IRES-[PV]2BC-P3-3'NTR, expressing a small plaque phenotype. These results indicate that neither the intact P2 polypeptide nor the cleavage fragment 2AB of P2 is required for viral proliferation. On the other hand, 2Apro appears to be an essential component in RNA replication as no viral RNA synthesis can be observed by reverse transcription/PCR in cells transfected with dicistronic RNA lacking this viral polypeptide.

Base Sequence↗

PubMed1993

Ribozyme-catalyzed primer extension by trinucleotides: a model for the RNA-catalyzed replication of RNA.

The existence of RNA enzymes that catalyze phosphodiester transfer reactions suggests that RNA-catalyzed RNA replication might be possible. Indeed, it has been shown that the Tetrahymena and sunY self-splicing introns will catalyze the template-directed ligation of RNA oligonucleotides (Doudna et al., 1989, 1991). We have sought to develop a more general RNA replication system in which arbitrary template sequences could be copied by the assembly of a limited set of short oligonucleotides. Here we examine the use of tetranucleotides as substrates for a primer-extension reaction in which the 5'-nucleoside of the tetranucleotide is the leaving group, and the primer is extended by the remaining three nucleotides. When the 5'-nucleoside is guanosine, the reaction is quite efficient, but a number of competing side reactions occur, in which inappropriate guanosine residues in the primer or the template occupy the guanosine binding site of the ribozyme. We have blocked these side reactions by using the guanosine analogue 2-aminopurine riboside (2AP) as the leaving group, in a reaction catalyzed by a mutant sunY ribozyme that binds efficiently to 2AP and not to guanosine. We have begun to address the issue of the fidelity of the primer-extension reaction by measuring reaction rates with a number of different triplet/template combinations. Our results provide the basis for the further development of an RNA-catalyzed RNA replication system using short oligonucleotide substrates with novel leaving groups.

Animals↗

PubMed1999

Flaviviridae polymerase and RNA replication.

Sequence motifs within the non-structural protein NS5 or NS5B of the members of the family Flaviviridae suggest that this protein is the RNA-dependent RNA polymerase. This protein has now been expressed in various in vitro systems and used in polymerase assays. To understand the role of the RNA polymerase in RNA replication, this review will examine not only the polymerase protein but also the other proteins in the RNA replication complex. To date, several groups have investigated the interaction of these proteins both in vitro and in vivo and also the interaction of these proteins with the RNA signals at the 3' terminus of the RNA. Infectious clones and replicons containing the non-structural proteins have now been generated and these will be useful tools in understanding the processes of initiation and elongation of both positive and negative RNA synthesis.

Flaviviridae↗

PubMed1989

A point mutation in the poliovirus polymerase gene determines a complementable temperature-sensitive defect of RNA replication.

We have previously described the isolation of a RNA- temperature-sensitive (ts) mutant of poliovirus type 1, ts035, after chemical mutagenesis by 5-fluorouracil. The ts defect of ts035 correlated with defective RNA replication, since the two characters corevert in the case of spontaneous revertants. The alteration of a trans-acting replication function of ts035 was suggested by significant rescue following mixed infection with another ts mutant, ts221, or with wild-type virus. Protein synthesis appeared normal at 39 degrees (nonpermissive temperature) in shift-up experiments and no defect of RNA elongation was evidenced when the activity of replication complexes or purified polymerase was measured at 39 degrees. These results provide circumstantial evidence that the initiation of ts035 RNA synthesis at 39 degrees is impaired. Molecular cloning of the ts035 genome allowed us to construct a recombinant virus with the same ts phenotype as ts035, by the transfer of a fragment of the mutant polymerase gene into the wild-type genome. Two mutations were present in this region of the ts035 genome but the determination of nucleotide sequences in the case of ts035 revertants indicated that only the substitution from A to G at nucleotide 7256 was necessary for the ts phenotype. This mutation replaces Asn 426 by an Asp in polypeptide 3D, the viral polymerase.

Base Sequence↗

PubMed2000

Efficient initiation of HCV RNA replication in cell culture.

Hepatitis C virus (HCV) infection is a global health problem affecting an estimated 170 million individuals worldwide. We report the identification of multiple independent adaptive mutations that cluster in the HCV nonstructural protein NS5A and confer increased replicative ability in vitro. Among these adaptive mutations were a single amino acid substitution that allowed HCV RNA replication in 10% of transfected hepatoma cells and a deletion of 47 amino acids encompassing the interferon (IFN) sensitivity determining region (ISDR). Independent of the ISDR, IFN-alpha rapidly inhibited HCV RNA replication in vitro. This work establishes a robust, cell-based system for genetic and functional analyses of HCV replication.

Amino Acid Substitution↗

PubMed2005

Synthesis of 9-(2-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives as inhibitors of HCV RNA replication.

A series of 9-(2'-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives were synthesized as potential inhibitors of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was used to further improve the potency of these compounds by increasing the intracellular levels of 5'-monophosphate metabolites. These nucleotide prodrugs showed much improved inhibitory activities of HCV RNA replication.

Antiviral Agents↗

PubMed2006

Evidence for functional protein interactions required for poliovirus RNA replication.

Poliovirus protein 2C contains a predicted N-terminal amphipathic helix that mediates association of the protein with the membranes of the viral RNA replication complex. A chimeric virus that contains sequences encoding the 18-residue core from the orthologous amphipathic helix from human rhinovirus type 14 (HRV14) was constructed. The chimeric virus exhibited defects in viral RNA replication and produced minute plaques on HeLa cell monolayers. Large plaque variants that contained mutations within the 2C-encoding region were generated upon subsequent passage. However, the majority of viruses that emerged with improved growth properties contained no changes in the region encoding 2C. Sequence analysis and reconstruction of genomes with individual mutations revealed changes in 3A or 2B sequences that compensated for the HRV14 amphipathic helix in the polio 2C-containing proteins, implying functional interactions among these proteins during the replication process. Direct binding between these viral proteins was confirmed by mammalian cell two-hybrid analysis.

Animals↗

PubMed1992

Turnip crinkle virus genes required for RNA replication and virus movement.

We have used infectious in vitro transcripts from mutagenized turnip crinkle virus (TCV) cDNA clones to identify the gene products required for viral RNA replication, virion assembly, and intercellular movement. Previous sequence analysis of the TCV genome revealed the presence of five open reading frames which had the potential to encode gene products of 88, 38, 28, 9, and 8 kDa. Inoculation of protoplasts with infectious RNA revealed that only the p28 and p88 gene products are required for viral RNA synthesis. Although the p8 and p9 gene products were dispensable for RNA replication and virion assembly in protoplasts, mutations in the p8 and p9 genes prevented the production of systemic infections in plants. No viral RNA or protein was observed in the inoculated or systemic leaves of plants inoculated with transcripts synthesized from p8 or p9 mutant cDNAs. In contrast to these results, viral RNA was recovered from the inoculated, but not the systemic leaves, of plants inoculated with an RNA lacking the coat protein (CP) gene. With the CP mutant, no symptoms were observed on normally systemic hosts, but small local lesions were induced on Chenopodium amaranticolor. These results indicate that p8, p9, and CP are required for viral movement.

Amino Acid Sequence↗

PubMed1998

Progress towards the construction of a transmissible gastroenteritis coronavirus self-replicating RNA using a two-layer expression system.

Three transmissible gastroenteritis coronavirus (TGEV) defective interfering RNAs of 21, 10.6 and 9.7 kb (DI-A, DI-B and DI-C, respectively) were isolated. Dilution experiments showed that the largest DI RNA, DI-A, is a self-replicating RNA (replicon), and thus codes for a functional RNA polymerase and all the necessary replication signals. In order to engineer a cDNA encoding the RNA replicon a strategy based on the cloning of DI-C cDNA, followed by the insertion of the sequences required to complete the DI-A sequence has been developed. A cDNA complementary to DI-C RNA was cloned under the control of the CMV promoter (pDI-C-CMV) and rescued with a helper virus. In the ORF 1a of polymerase gene pDI-C-CMV contained a 10 kb deletion and in ORF 1b a 1.1 kb deletion. The consensus sequence corresponding to the deleted regions was cloned, and the deletions in pDI-C-CMV were replaced to yield a complete cDNA clone of DI-A, pDI-A-21-CMV, containing a full-length TGEV polymerase, driven by a CMV promoter. Expression of a functional TGEV polymerase is being investigated.

Animals↗

PubMed2026

O'nyong-nyong virus adaptive mutations in non-structural protein 1 and 3 enhance RNA replication and overcome FHL1 requirement.

Arthritogenic alphaviruses, like o'nyong-nyong virus (ONNV), cause debilitating musculoskeletal diseases and are geographically expanding. To predict their emergence, we seek to better understand evolutionary mechanisms that enable changes in virus tropism. Here, we identify adaptive mutations in the ONNV non-structural proteins (nsPs) that arose during cellular serial passaging and enabled ONNV to infect non-permissive Lunet cells. Using shotgun proteomics, we show that this human hepatoma cell line lacks the four-and-a-half-LIM domain protein 1 (FHL1), an essential host factor in ONNV RNA replication. Individual single nucleotide mutations in the nsP1 ring-aperture membrane-binding and oligomerization domain, the nsP3 macrodomain, and the nsP3 opal stop codon overcome FHL1 deficiency in Lunet cells by enhanced RNA replication. These findings demonstrate how subtle genomic changes in nsPs can profoundly influence alphavirus replication and tropism.

LIM Domain Proteins↗

PubMed2006

Inducible yeast system for Viral RNA recombination reveals requirement for an RNA replication signal on both parental RNAs.

To facilitate RNA recombination studies, we tested whether Saccharomyces cerevisiae, which supports brome mosaic virus (BMV) replication, also supports BMV RNA recombination. Yeast strains expressing BMV RNA replication proteins 1a and 2a(pol) were engineered to transiently coexpress two independently inducible, overlapping, nonreplicating derivatives of BMV genomic RNA3. B3Delta3' lacked the coat protein gene and negative-strand RNA promoter. B3Delta5' lacked the positive-strand RNA promoter and had the coat gene replaced by the selectable URA3 gene. After 12 to 72 h of induction, B3Delta3' and B3Delta5' transcription was repressed and Ura(+) yeast cells were selected. All Ura(+) cells contained recombinant RNA3 replicons expressing URA3. Most replicons arose by intermolecular homologous recombination between B3Delta3' and B3Delta5'. Such recombinants were isolated only when 1a and 2a(pol) were expressed and after transient transcription of both B3Delta3' and B3Delta5', showing that recombination occurred at the RNA, not DNA, level. A minority of URA3-expressing replicons were derived from B3Delta5', independently of B3Delta3', by 5' truncation and modification, generating novel positive-strand promoters and demonstrating that BMV can give rise to subgenomic RNA replicons. Intermolecular B3Delta3'-B3Delta5' recombination occurred only when both parental RNAs bore a functional, cis-acting template recognition and recruitment element targeting viral RNAs to replication complexes. The results imply that recombination occurred in RNA replication complexes to which parental RNAs were independently recruited. Moreover, the ability to obtain intermolecular recombinants at precisely measurable, reproducible frequencies, to control genetic background and induction conditions, and other features of this system will facilitate further studies of virus and host functions in RNA recombination.

Base Sequence↗

PubMed2002

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles.

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

Animals↗

PubMed2003

The RNA replication enhancer element of tombusviruses contains two interchangeable hairpins that are functional during plus-strand synthesis.

Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the approximately 80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.

Base Sequence↗

PubMed1988

A deletion mutant of L-A double-stranded RNA replicates like M1 double-stranded RNA.

X double-stranded RNA (dsRNA) is a 0.52-kilobase dsRNA molecule that arose spontaneously in a nonkiller strain of Saccharomyces cerevisiae originally containing L-A and L-BC dsRNAs (L-BC is the same size as L-A but shares no homology with it). X hybridized with L-A, and direct RNA sequencing of X showed that the first 5' 25 base pairs (of the X positive strand) and at least the last 110 base pairs of the 3' end were identical to the ends of L-A dsRNA. X showed cytoplasmic inheritance and, like M1, was dependent on L-A for its maintenance. X was encapsidated in viruslike particles whose major coat protein was provided by L-A (as is true for M1), and X was found in viruslike particles with one to eight X molecules per particle. This finding confirms our "head-full replication" model originally proposed for M1 and M2. Like M1 or M2, X lowers the copy number of L-A, especially in a ski host. Surprisingly, X requires many chromosomal MAK genes that are necessary for M1 but not for L-A.

Base Sequence↗

PubMed1997

Complete nucleotide sequence of tobacco necrosis virus strain DH and genes required for RNA replication and virus movement.

The complete genome sequence of tobacco necrosis virus strain D (Hungarian isolate, TNV-DH) was determined. The genome (3762 nt) has an organization identical to that reported for TNV-D. Highly infectious synthetic transcripts from a full-length TNV-DH cDNA clone were prepared, the first infectious necrovirus transcript reported. This clone was used for reverse genetic studies to map the viral genes required for replication and movement. Protoplast inoculation with delta 22 and delta 82 mutants revealed that both the 22 kDa and 82 kDa gene products are required for RNA replication. Although the products of three small central genes (p7(1), p7a and p7b) were not essential for RNA replication in protoplasts, mutations in these ORFs prevented infection of plants. In contrast, viral RNA accumulation and cell-to-cell movement were observed in the inoculated, but not the systemically infected, leaves of Nicotiana benthamiana challenged with RNA lacking the intact coat protein (CP) gene. These results strongly suggest that p7(1), p7a, p7b and CP are involved in TNV-DH cell-to-cell and long-distance movement, respectively.

Base Sequence↗

PubMed2002

Domains I and II in the 5' nontranslated region of the HCV genome are required for RNA replication.

Hepatitis C virus (HCV), a hepacivirus member of the Flaviviridae family, has a positive-stranded RNA genome, which consists of a single open reading frame (ORF) and nontranslated regions (NTRs) at the 5' and 3' ends. The 5'NTR was found to contain an internal ribosomal entry site (IRES), which is required for the translation of HCV mRNA. Moreover, the 5'NTR is likely to play a key role in the replication of viral RNA. To identify the cis-acting element required for viral RNA replication, chimeric subgenomic replicons of HCV were generated. Dissection of the replication element from the translation element was accomplished by inserting the polioviral IRES between the serially deleted 5'NTR of HCV and ORF encoding neomycin phosphotransferase. The deletions of the 5'NTR of HCV were performed according to the secondary structure of HCV. Replicons containing domains I and II supported RNA replication and further deletion toward the 5' end abolished replication. The addition of domain III and the pseudoknot structure of the 5'NTR to domains I and II augmented the colony-forming efficiency of replicons by 100-fold. This indicates that domains I and II are necessary and sufficient for replication of RNA and that almost all of the 5'NTR is required for efficient RNA replication.

5' Untranslated Regions↗

PubMed1990

Studies of coronavirus DI RNA replication using in vitro constructed DI cDNA clones.

Sequence analysis of an intracellular defective-interfering (DI) RNA, DIssE, of mouse hepatitis virus (MHV) revealed that it is composed of three noncontiguous genomic regions, representing the first 864 nucleotides of the 5'-end, an internal 748 nucleotides of the polymerase gene, and 601 nucleotides from the 3'-end of the parental MHV genome. DIssE had three base substitutions within the leader sequence and also a deletion of nine nucleotides located at the junction of the leader and the remaining genomic sequence. A system was developed for generating DI RNAs to study the mechanism of MHV RNA replication. A cDNA copy of DIssE RNA was placed downstream of T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA is utilized for the replication of MHV RNA.

Animals↗

PubMed2002

Kissing interaction between 3' noncoding and coding sequences is essential for porcine arterivirus RNA replication.

We used an infectious cDNA clone of porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the presence of essential replication elements in the region of the genome encoding the structural proteins. Deletion analysis showed that a stretch of 34 nucleotides (14653 to 14686) within ORF7, which encodes the nucleocapsid protein, is essential for RNA replication. Strand-specific reverse transcription-PCR analysis of viral RNA isolated from transfected BHK-21 cells revealed that this region is required for negative-strand genomic RNA synthesis. The 34-nucleotide stretch is highly conserved among PRRSV isolates and folds into a putative hairpin. A 7-base sequence within the loop of this structure was suggested to base-pair with a sequence present in the loop of a hairpin located in the 3' noncoding region, resulting in a kissing interaction. Mutational analyses confirmed that this kissing interaction is required for RNA replication.

3' Untranslated Regions↗