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At least 505 records · Page 28Linked to original sources
PubMed1992

Mutations affecting RNA-DNA hybrid formation of the ColE1 replication primer RNA. Restoration of RNA I sensitivity to a copy-number mutant by second-site mutations.

Certain high copy-number mutants of the ColE1 plasmid produce a primer RNA that, unlike the wild-type, is resistant to inhibition by the plasmid-encoded replication inhibitor RNA I. We show that this resistance is associated with the ability of mutant primer RNA to hybridize to the DNA template strand more efficiently than does the wild-type transcript in vitro. We have isolated two second-site intramolecular suppressor mutations that partially restore wild-type copy number behavior to the high copy-number mutant in vivo. Each of these mutations alters a second base in primer RNA near the original mutation. We show that the primer RNA made by the pseudo-revertants regained wild-type-like sensitivity to RNA I in vitro. Also, the efficiency of RNA-DNA hybrid formation by the pseudo-revertant primer RNAs is restored to a level similar to that of wild-type primer. Using non-denaturing gel electrophoresis as an indication of RNA conformation, we identified two primer RNA conformers, each of 550 nucleotides, whose equilibrium distribution differs between wild-type and the mutant plasmid. The pseudo-revertant plasmids have a conformer distribution similar to that of wild-type, indicating that these primer sequence changes have long-range effects on primer conformation. An oligonucleotide complementary to the primer domain containing the mutation reduced hybrid formation when present during primer elongation. These results indicate that the copy-number behavior of these plasmids is a consequence of conformational alterations in primer RNA that alter its hybridization efficiency with the DNA template strand and its sensitivity to inhibition by RNA I.

Bacteriocin Plasmids↗
PubMed2006

Expression of vector-based small interfering RNA against West Nile virus effectively inhibits virus replication.

RNA interference is one of the effective emerging anti-viral strategies to inhibit virus infection in cells. In this study, a small interfering RNA expressing vector (pSilencer-NS5) targeting the NS5 gene of West Nile virus (WNV) was employed to target and destroy WNV transcripts. Real-time PCR revealed drastic reduction in WNV RNA transcripts in pSilencer-NS5-transfected Vero cells. The virus infectious titre was also significantly reduced by 90% as determined by plaque assays. The resulting decrease in virus replication was shown to be specific since both scrambled and nucleotide(s) mismatch siRNA against WNV NS5 gene did not have any effect on WNV productive yields. Furthermore, Western immunoblot analysis on the expression of viral NS5 and envelope (E) proteins showed significant down-regulation on the expression of viral NS5 and envelope (E) proteins in virus-infected cells that were pre-transfected with pSilencer-NS5. These data clearly supported the notion that the expression of vector-based siRNA against WNV NS5 gene is able to exert its silencing effect on WNV-infected cells without inducing cytotoxicity, hence holding promise in therapeutic treatment of this important emerging infectious disease.

Animals↗
PubMed2004

Reconstitution in cultured cells of replicating HDV RNA from pairs of less than full-length RNAs.

The genome of hepatitis delta virus (HDV) is a small single-stranded circular RNA that is replicated via RNA-directed RNA synthesis. This makes use of a host RNA polymerase, probably pol II, that normally transcribes DNA templates. In vivo, the host polymerase can initiate replication from transfected linear RNAs using intramolecular template-switching. The present studies report that the polymerase could also achieve intermolecular switching leading to "reconstitution" of full-length HDV RNAs following transfection with two linear RNAs that were less than full length and yet lacking different regions of the genome. These two RNAs were synthesized in vitro, gel purified, pre-annealed, and then transfected into delta293, a cell line conditionally expressing the small delta antigen that is essential for HDV replication. Northern analyses of total RNA harvested from transfected cells detected the accumulation of full-length HDV genomic and antigenomic RNAs. Such reconstitution of full-length replicating HDV RNA was also achieved using nine other pairs of antigenomic RNAs and three pairs of genomic RNAs. Annealing of the RNAs prior to transfection was required for detectable HDV reconstitution. A second cell line, Huh7, also supported reconstitution when a pair of RNAs was cotransfected together with mRNA for the small delta protein. Taken together, these results support a model that observed genome reconstitution is a special form of recombination involving intermolecular template switches and they provide insights into the mechanism of RNA-directed RNA transcription catalyzed by a host RNA polymerase.

Cell Line↗
PubMed2006

The structure of a protein primer-polymerase complex in the initiation of genome replication.

Picornavirus RNA replication is initiated by the covalent attachment of a UMP molecule to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction is carried out by the viral RNA-dependent RNA polymerase (3D). Here, we report the X-ray structure of two complexes between foot-and-mouth disease virus 3D, VPg1, the substrate UTP and divalent cations, in the absence and in the presence of an oligoadenylate of 10 residues. In both complexes, VPg fits the RNA binding cleft of the polymerase and projects the key residue Tyr3 into the active site of 3D. This is achieved by multiple interactions with residues of motif F and helix alpha8 of the fingers domain and helix alpha13 of the thumb domain of the polymerase. The complex obtained in the presence of the oligoadenylate showed the product of the VPg uridylylation (VPg-UMP). Two metal ions and the catalytic aspartic acids of the polymerase active site, together with the basic residues of motif F, have been identified as participating in the priming reaction.

Crystallography, X-Ray↗
PubMed2000

Host protein interactions with the 3' end of bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication.

RNA viruses have 5' and 3' untranslated regions (UTRs) that contain specific signals for RNA synthesis. The coronavirus genome is capped at the 5' end and has a 3' UTR that consists of 300 to 500 nucleotides (nt) plus a poly(A) tail. To further our understanding of coronavirus replication, we have begun to examine the involvement of host factors in this process for two group II viruses, bovine coronavirus (BCV) and mouse hepatitis coronavirus (MHV). Specific host protein interactions with the BCV 3' UTR [287 nt plus poly(A) tail] were identified using gel mobility shift assays. Competition with the MHV 3' UTR [301 nt plus poly(A) tail] suggests that the interactions are conserved for the two viruses. Proteins with molecular masses of 99, 95, and 73 kDa were detected in UV cross-linking experiments. Less heavily labeled proteins were also detected in the ranges of 40 to 50 and 30 kDa. The poly(A) tail was required for binding of the 73-kDa protein. Immunoprecipitation of UV-cross-linked proteins identified the 73-kDa protein as the cytoplasmic poly(A)-binding protein (PABP). Replication of the defective genomes BCV Drep and MHV MIDI-C, along with several mutants, was used to determine the importance of the poly(A) tail. Defective genomes with shortened poly(A) tails consisting of 5 or 10 A residues were replicated after transfection into helper virus-infected cells. BCV Drep RNA that lacked a poly(A) tail did not replicate, whereas replication of MHV MIDI-C RNA with a deleted tail was detected after several virus passages. All mutants exhibited delayed kinetics of replication. Detectable extension or addition of the poly(A) tail to the mutants correlated with the appearance of these RNAs in the replication assay. RNAs with shortened poly(A) tails exhibited less in vitro PABP binding, suggesting that decreased interactions with the protein may affect RNA replication. The data strongly indicate that the poly(A) tail is an important cis-acting signal for coronavirus replication.

3' Untranslated Regions↗
PubMed1985

In vitro synthesis of infectious poliovirus RNA.

Replication of the infectious RNA genome of poliovirus is accomplished in cells by the viral RNA polymerase through negative-strand RNA intermediates. Full-length negative-strand poliovirus RNA was synthesized in vitro by transcription of infectious poliovirus cDNA with bacteriophage SP6 DNA-dependent RNA polymerase. When provided with this negative-strand RNA as template, the poliovirus RNA-dependent RNA polymerase synthesized full-length positive-strand molecules. The positive-strand RNAs synthesized in vitro were infectious when transfected into HeLa cells. In contrast, positive-strand copies of poliovirus RNA synthesized in vitro by SP6 polymerase, using a poliovirus cDNA template, were not infectious. Production of infectious positive-strand RNA by the poliovirus polymerase was not observed when magnesium or negative-strand RNA template was omitted from the reaction mixture. Infectivity of the product RNA was not destroyed by DNase treatment. The specific infectivity in HeLa cells of in vitro-synthesized positive-strand RNA was 4 X 10(4) plaque-forming units/micrograms of RNA.

DNA↗
PubMed2005

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis.

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.

Animals↗
PubMed2001

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli.

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.

Base Sequence↗
PubMed2002

Initiation of hepatitis delta virus (HDV) replication: HDV RNA encoding the large delta antigen cannot replicate.

The hepatitis delta virus (HDV) nucleocapsid consists of a genomic-length RNA of 1.7 kb and approximately equimolar amounts of the small and large forms of the hepatitis delta antigen (S-HDAg and L-HDAg, respectively). Since HDV RNA particles contain not only a genomic RNA species encoding S-HDAg but also an RNA species encoding L-HDAg, which is produced by an RNA-editing process, the question arises as to whether RNAs encoding either L-HDAg or S-HDAg can initiate replication. To study this, two cDNA-free transfection methods were employed: HDV RNA cotransfected with either the S-HDAg-encoding mRNA species or the ribonucleocapsid protein complex, comprising HDV RNA and recombinant S-HDAg. Results showed that the genomic-sense RNA encoding S-HDAg could promote HDV replication, whereas the L-HDAg-encoding RNA species was unable to replicate under the same conditions. The antigenomic RNA species encoding either S-HDAg or L-HDAg could not replicate by either of these procedures. In addition, L-HDAg alone could not promote replication of the genomic RNA but, by supplementing an equal amount of S-HDAg, replication occurred. These data indicate that L-HDAg-encoding RNA species are probably not involved in the initiation of HDV RNA synthesis; instead, their main function may be to serve as template for producing L-HDAg, which regulates HDV RNA synthesis and virion assembly. These results suggest that the genomic RNA species encoding S-HDAg is the only functional genome for HDV infection and explain why the presence of the edited HDV RNA encoding L-HDAg does not interfere with HDV infection.

Animals↗
PubMed1998

Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) --> T, A(1762) --> T and G(1764) --> A mutations in the core promoter.

Hepatitis B virus (HBV) isolates with A-1762 to T and G-1764 to A mutations in the core promoter have been associated with active hepatitis, severe liver disease following liver transplantation, hepatocellular carcinoma and acute fulminant courses--in the latter case combined with a C-1653 to T mutation. In this study, a mutant core promoter region containing the T-1653, T-1762 and A-1764 mutations was placed into the context of a wild-type HBV genome and analysed by transfection. The mutations reduced the level of pre-C mRNA (by 55%) and e-antigen secretion. In contrast, no significant effects on the levels of pregenome/C and pre-S/S mRNAs, intracellular core, polymerase, and pre-S /S2 proteins and secreted S-antigen were observed. The amount of progeny virus DNA in the cells and in the culture medium was increased marginally, if at all.

Base Sequence↗
PubMed1985

Replication strategy of Kunjin virus: evidence for recycling role of replicative form RNA as template in semiconservative and asymmetric replication.

Only three forms of Kunjin virus-specified RNA were isolated from cytoplasm early after the latent period (about 15 hr) viz., 44 S genomic-sized single-stranded RNA, 20 S double-stranded "replicative form" (RF), and 20-28 S partially ribonuclease-resistant (about 70%) "replicative intermediate" (RI). The RF and RI were resolved by electrophoresis in aqueous-agarose gel only following LiCl fractionation. The RI did not enter urea-polyacrylamide gels. After denaturation of untreated or RNase-treated RI and RF, only 44 S RNA was present in electropherograms. RNA polymerase activity at 8 hr postinfection was detected by in vitro assays of cytoplasmic extracts and reached a maximum at 24 hr, the only major labeled product being RF; a trace amount of free 44 S RNA was also produced. These results, and the kinetics of incorporation of [3H]uridine into RI, RF, and 44 S RNA in pulse and pulse-chase experiments, formed the basis of a model in which flavivirus RF functions as a recycling template for semiconservative and (mainly) asymmetric replication, on which only one nascent strand is synthesized per cycle.

Animals↗
PubMed1995

Mutations in the helicase-like domain of protein 1a alter the sites of RNA-RNA recombination in brome mosaic virus.

A system that uses engineered heteroduplexes to efficiently direct in vivo crossovers between brome mosaic virus (BMV) RNA1 and RNA3 (P. Nagy and J. Bujarski, Proc. Natl. Acad. Sci. USA 90:6390-6394, 1993) has been used to explore the possible involvement of BMV 1a protein, an essential RNA replication factor, in RNA recombination. Relative to wild-type 1a, several viable amino acid insertion mutations in the helicase-like domain of BMV 1a protein affected the nature and distribution of crossover sites in RNA3-RNA1 recombinants. At 24 degrees C, mutants PK19 and PK21 each increased the percentage of asymmetric crossovers, in which the RNA1 and RNA3 sites joined by recombination were not directly opposite each other on the engineered RNA3-RNA1 heteroduplex used to target recombination but rather were separated by 4 to 85 nucleotides. PK21 and another 1a mutant, PK14, also showed increases in the fraction of recombinants containing nontemplated U residues at the recombination junction. At 33 degrees C, the highest temperature that permitted infections with PK19, which is temperature sensitive for RNA replication, the mean location of RNA1-RNA3 crossovers in recombinants recovered from PK19 infections was shifted by nearly 25 bp into the energetically less stable side of the RNA1-RNA3 heteroduplex. Thus, mutations in the putative helicase domain of the 1a protein can influence BMV RNA recombination. The results are discussed in relation to models for recombination by template switching during pausing of RNA replication at a heteroduplexed region in the template.

Base Sequence↗
PubMed1997

Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.

The mechanisms that direct positive-stranded RNA virus replication complexes to plant and animal cellular membranes are poorly understood. We describe a specific interaction between a replication protein of an RNA plant virus and membranes in vitro and in live cells. The tobacco etch virus (TEV) 6 kDa protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. In the presence or absence of other viral proteins, fluorescent fusion proteins containing the 6 kDa protein associated with large vesicular compartments derived from the endoplasmic reticulum (ER). Infection by TEV was associated with a collapse of the ER network into a series of discrete aggregated structures. Viral RNA replication complexes from infected cells were also associated with ER-like membranes. Targeting of TEV RNA replication complexes to membranous sites of replication is proposed to involve post-translational interactions between the 6 kDa protein and the ER.

Amino Acid Sequence↗
PubMed1996

Replication of murine coronavirus defective interfering RNA from negative-strand transcripts.

The positive-strand defective interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV), when introduced into MHV-infected cells, results in DI RNA replication and accumulation. We studied whether the introduction of negative-strand transcripts of MHV DI RNA would also result in replication. At a location downstream of the T7 promoter and upstream of the human hepatitis delta virus ribozyme domain, we inserted a complete cDNA clone of MHV DI RNA in reverse orientation; in vitro-synthesized RNA from this plasmid yielded a negative-strand RNA copy of the MHV DI RNA. When the negative-strand transcripts of the DI RNA were expressed in MHV-infected cells by a vaccinia virus T7 expression system, positive-strand DI RNAs accumulated in the plasmid-transfected cells. DI RNA replication depended on the expression of T7 polymerase and on the presence of the T7 promoter. Transfection of in vitro-synthesized negative-strand transcripts into MHV-infected cells and serial passage of virus samples from RNA-transfected cells also resulted in accumulation of the DI RNA. Positive-strand DI RNA transcripts were undetectable in sample preparations of the in vitro-synthesized negative-strand DI RNA transcripts, and DI RNA did not accumulate after cotransfection of a small amount of positive-strand DI RNA and truncated-replication-disabled negative-strand transcripts; clearly, the DI RNA replicated from the transfected negative-strand transcripts and not from minute amounts of positive-strand DI RNAs that might be envisioned as artifacts of T7 transcription. Sequence analysis of positive-strand DI RNAs in the cells transfected with negative-strand transcripts showed that DI RNAs maintained the DI-specific unique sequences introduced within the leader sequence. These data indicated that positive-strand DI RNA synthesis occurred from introduced negative-strand transcripts in the MHV-infected cells; this demonstration, using MHV, of DI RNA replication from transfected negative-strand DI RNA transcripts is the first such demonstration among all positive-stranded RNA viruses.

Animals↗
PubMed1991

Control of adenovirus alternative RNA splicing: effect of viral DNA replication on RNA splice site choice.

The primary transcripts of most adenovirus transcription units are processed into multiple, alternatively spliced mRNAs. The relative concentrations of such differentially processed mRNAs changes during the infectious cycle. The factors that control this temporal shift in mRNA abundance have not yet been characterized. In the experiments presented here we have examined mRNA synthesis from three viral transcription units: two early regions E1a and E1b, and late region L1. We show that viral DNA replication plays a key role in the control of cytoplasmic mRNA expression from these regions. In the absence of efficient late protein synthesis, viral DNA replication was sufficient to induce a substantial fraction of the E1a, E1b and L1 transcripts to shift from the early to the late pattern of mRNA structure. The shift was not complete under the conditions used, suggesting that viral proteins, although not essential for the process, play an important regulatory role. The requirement for late viral protein synthesis differed between the three transcription units examined. This dependence was most pronounced for correct L1 mRNA production. Viral DNA replication was sufficient to trigger a significant shift in L1 alternative 3' splice site selection. However, in the absence of late translation the L1 pre-mRNA was aberrantly spliced.

Adenovirus Early Proteins↗
PubMed1995

The molecular biology of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA genome, the only animal viral RNA of its kind. It possesses a ribozyme activity, which can autocatalytically cleave and ligate itself. The ribozyme has a unique structural requirement different from other known ribozymes. HDV RNA undergoes RNA-dependent RNA replication via a double rolling circle mechanism, which is probably mediated by cellular RNA polymerase II, utilizing modified cellular transcription machineries. HDV RNA encodes a single protein, hepatitis delta antigen, which is a nuclear, RNA-binding phosphoprotein and required for viral RNA replication. During replication, HDV RNA undergoes a specific RNA editing event to extend its open reading frame and produce a longer, isoprenylated delta antigen, which suppresses RNA replication and initiates viral particle assembly. Ribozyme, cell-mediated RNA-dependent RNA replication, and RNA editing are some of the unique properties and unresolved issues of the molecular biology of HDV.

Animals↗
PubMed2004

Alternate, virus-induced membrane rearrangements support positive-strand RNA virus genome replication.

All positive-strand RNA [(+)RNA] viruses replicate their RNA on intracellular membranes, often in association with spherular invaginations of the target membrane. For brome mosaic virus, we previously showed that such spherules serve as compartments or mini-organelles for RNA replication and that their assembly, structure, and function have similarities to the replicative cores of retrovirus and double-stranded RNA virus virions. Some other (+)RNA viruses conduct RNA replication in association with individual or clustered double-membrane vesicles, appressed double membranes, or other structures whose possible relationships to the spherular invaginations are unclear. Here we show that modulating the relative levels and interactions of brome mosaic virus replication factors 1a and 2a polymerase (2apol) shifted the membrane rearrangements associated with RNA replication from small invaginated spherules to large, karmellae-like, multilayer stacks of appressed double membranes that supported RNA replication as efficiently as spherules. Spherules were induced by expressing 1a, which has functional similarities to retrovirus virion protein Gag, or 1a plus low levels of 2apol. Double-membrane layers were induced by 1a plus higher levels of 2apol and were suppressed by deleting the major 1a-interacting domain from 2apol. The stacked, double-membrane layers alternated with spaces that, like spherule interiors, were 50-60 nm wide, connected to the cytoplasm, and contained 1a and 2apol. These and other results suggest that seemingly diverse membrane rearrangements associated with RNA replication by varied (+)RNA viruses may represent topologically and functionally related structures formed by similar protein-protein and protein-membrane interactions and interconverted by altering the balances among those interactions.

Bromovirus↗