Showing papers on "RNA-dependent RNA polymerase published in 2001"
TL;DR: In this article, the authors demonstrate that 21 and 22-nt RNA fragments are the sequence-specific mediators of RNA interference in a Drosophila in vitro system, and provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.
Abstract: Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3 ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.
3,980 citations
TL;DR: Analysis of small interfering RNAs produced during RNAi in C. elegans revealed a substantial fraction that cannot derive directly from input dsRNA, and appeared to derive from the action of a cellular RNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted by the RNAi mechanism.
1,372 citations
TL;DR: The crystal structure of Thermus aquaticus core RNAP complexed with Rif explains the effects of Rif on RNAP function and indicates that the inhibitor acts by directly blocking the path of the elongating RNA when the transcript becomes 2 to 3 nt in length.
1,286 citations
TL;DR: It is suggested that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP- dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target.
1,274 citations
Patent•
29 Nov 2001
TL;DR: In this article, the authors demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNA interference in a Drosophila in vitro system, and provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNA complex.
Abstract: Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3′ ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.
1,119 citations
TL;DR: A null mutation indicer-1 (dcr-1), a gene proposed to encode the enzyme that generates short RNAs, is described and it is found that Mutant animals have defects in RNAi under some, but not all, conditions.
Abstract: An early event in RNA interference (RNAi) is the cleavage of the initiating double-stranded RNA (dsRNA) to short pieces, 21 to 23 nucleotides in length. Here we describe a null mutation in dicer-1 (dcr-1), a gene proposed to encode the enzyme that generates these short RNAs. We find that dcr-1(-/-) animals have defects in RNAi under some, but not all, conditions. Mutant animals have germ line defects that lead to sterility, suggesting that cleavage of dsRNA to short pieces is a requisite event in normal development.
935 citations
TL;DR: The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 Å resolution and protein–nucleic acid contacts help explain DNA and RNA strand contacts, the specificity of RNA synthesis, “abortive cycling” during transcription initiation, and RNA and DNA translocation during transcription elongation.
Abstract: The crystal structure of RNA polymerase II in the act of transcription was determined at 3.3 A resolution. Duplex DNA is seen entering the main cleft of the enzyme and unwinding before the active site. Nine base pairs of DNA-RNA hybrid extend from the active center at nearly right angles to the entering DNA, with the 3′ end of the RNA in the nucleotide addition site. The 3′ end is positioned above a pore, through which nucleotides may enter and through which RNA may be extruded during back-tracking. The 5′-most residue of the RNA is close to the point of entry to an exit groove. Changes in protein structure between the transcribing complex and free enzyme include closure of a clamp over the DNA and RNA and ordering of a series of “switches” at the base of the clamp to create a binding site complementary to the DNA-RNA hybrid. Protein–nucleic acid contacts help explain DNA and RNA strand separation, the specificity of RNA synthesis, “abortive cycling” during transcription initiation, and RNA and DNA translocation during transcription elongation.
886 citations
TL;DR: Genetic studies have expanded the biology of RNAi to cosuppression, transposon silencing, and the first hints of relationships to regulation of translation and development, as well as expanding the possible roles of RNA-dependent RNA polymerase (RdRp) in RNAi.
Abstract: In the few years since the discovery of RNA interference (RNAi; Fire et al. 1998), it has become clear that this process is ancient. RNAi, the oldest and most ubiquitous antiviral system, appeared before the divergence of plants and animals. Because aspects of RNAi, known as cosuppression, also control the expression of transposable elements and repetitive sequences (Ketting et al. 1999; Tabara et al. 1999), the interplay of RNAi and transposon activities have almost certainly shaped the structure of the genome of most organisms. Surprisingly, we are only now beginning to explore the molecular processes responsible for RNAi and to appreciate the breadth of its function in biology. Practical applications of this knowledge have allowed rapid surveys of gene functions (see Fraser et al. 2000 and Gönczey et al. 2000 for RNAi analysis of genes on chromosome I and III of Caenorhabditis elegans) and will possibly result in new therapeutic interventions. Genetic studies have expanded the biology of RNAi to cosuppression, transposon silencing, and the first hints of relationships to regulation of translation and development. The possible roles of RNA-dependent RNA polymerase (RdRp) in RNAi have been expanded. Many experiments indicate that dsRNA directs gene-specific methylation of DNA and, thus, regulation at the stage of transcription in plants. Cosuppression may involve regulation by polycomb complexes at the level of transcription in C. elegans and Drosophila. This article will review these topics and primarily summarize advances in the study of RNAi over the past year.
774 citations
TL;DR: Cell culture-adaptive mutations enhance HCV RNA replication and are corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase.
Abstract: Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.
651 citations
TL;DR: An RNA molecule is described that catalyzes the type of polymerization needed for RNA replication, which uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides.
Abstract: The RNA world hypothesis regarding the early evolution of life relies on the premise that some RNA sequences can catalyze RNA replication. In support of this conjecture, we describe here an RNA molecule that catalyzes the type of polymerization needed for RNA replication. The ribozyme uses nucleoside triphosphates and the coding information of an RNA template to extend an RNA primer by the successive addition of up to 14 nucleotides-more than a complete turn of an RNA helix. Its polymerization activity is general in terms of the sequence and the length of the primer and template RNAs, provided that the 3' terminus of the primer pairs with the template. Its polymerization is also quite accurate: when primers extended by 11 nucleotides were cloned and sequenced, 1088 of 1100 sequenced nucleotides matched the template.
628 citations
TL;DR: The results provide a working model for the initiation of replication and transcription of double-stranded RNA viruses and flaviviruses and suggest a mechanism by which the incoming double-Stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route.
Abstract: In most RNA viruses, genome replication and transcription are catalysed by a viral RNA-dependent RNA polymerase. Double-stranded RNA viruses perform these operations in a capsid (the polymerase complex), using an enzyme that can read both single- and double-stranded RNA. Structures have been solved for such viral capsids, but they do not resolve the polymerase subunits in any detail. Here we show that the 2 A resolution X-ray structure of the active polymerase subunit from the double-stranded RNA bacteriophage straight phi6 is highly similar to that of the polymerase of hepatitis C virus, providing an evolutionary link between double-stranded RNA viruses and flaviviruses. By crystal soaking and co-crystallization, we determined a number of other structures, including complexes with oligonucleotide and/or nucleoside triphosphates (NTPs), that suggest a mechanism by which the incoming double-stranded RNA is opened up to feed the template through to the active site, while the substrates enter by another route. The template strand initially overshoots, locking into a specificity pocket, and then, in the presence of cognate NTPs, reverses to form the initiation complex; this process engages two NTPs, one of which acts with the carboxy-terminal domain of the protein to prime the reaction. Our results provide a working model for the initiation of replication and transcription.
TL;DR: It is shown that GFP and Pp-Luc siRNAs, isolated from a protein complex in Drosophila embryo extract, target mRNA degradation in vitro and serve as primers to transform the target mRNA into dsRNA.
TL;DR: It is shown that a long-range interaction between ribonucleoprotein (RNP) complexes formed at the ends of the viral genome is necessary for RNA replication, and RNA circularization may be a general replication mechanism for positive stranded RNA viruses.
TL;DR: The first identification of signals in the 5′ NTR of HCV RNA essential for replication of this virus is provided, and sequences upstream of the HCV IRES are essential for RNA replication.
Abstract: 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.
TL;DR: Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases, and will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle.
Abstract: Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes. Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.
TL;DR: A reverse-genetic system is described for the generation of recombinant coronaviruses based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus.
Abstract: The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.
TL;DR: Analysis of murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA suggests that RNA is a structural element in retrovirus particles.
Abstract: A single retroviral protein, Gag, is sufficient for virus particle assembly While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as “scaffolding” in the particle To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA We found that these particles contain cellular mRNA in place of genomic RNA This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles Furthermore, we found that the retroviral cores were disrupted by treatment with RNase Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles
TL;DR: The results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.
Abstract: Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.
TL;DR: Data suggest that ribavirin can exert a direct effect on HCV replication, which is mediated by the HCV RNA polymerase, and the implications of this work on the development of nucleoside analogs for treatment of HCV infection.
TL;DR: It is shown that the purified NS5 alone is sufficient for the synthesis of the two products and that the template-length RNA is the product of de novo initiation and the incubation temperature during initiation, but not elongation phase of RNA synthesis modulates the relative amounts of the hairpin and de noVO RNA products.
TL;DR: Results indicate that the 5′ cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases, and proposes a replication model in which the5′ and 3′ ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliov virus RNA.
Abstract: A cloverleaf structure at the 5' terminus of poliovirus RNA binds viral and cellular proteins. To examine the role of the cloverleaf in poliovirus replication, we determined how cloverleaf mutations affected the stability, translation and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Mutations within the cloverleaf destabilized viral RNA in these reactions. Adding a 5' 7-methyl guanosine cap fully restored the stability of the mutant RNAs and had no effect on their translation. These results indicate that the 5' cloverleaf normally protects uncapped poliovirus RNA from rapid degradation by cellular nucleases. Preinitiation RNA replication complexes formed with the capped mutant RNAs were used to measure negative-strand synthesis. Although the mutant RNAs were stable and functional mRNAs, they were not active templates for negative-strand RNA synthesis. Therefore, the 5' cloverleaf is a multifunctional cis-acting replication element required for the initiation of negative-strand RNA synthesis. We propose a replication model in which the 5' and 3' ends of viral RNA interact to form a circular ribonucleoprotein complex that regulates the stability, translation and replication of poliovirus RNA.
TL;DR: New biochemical and genetic evidence now shows that an RNA-directed RNA polymerase chain reaction, primed by siRNA, amplifies the interference caused by a small amount of "trigger" dsRNA.
TL;DR: The results suggest that a reduced ability of the polymerase complex of avian viruses to ensure replication of the viral genome at 33°C could contribute to their inability to grow efficiently in humans.
Abstract: Human influenza A viruses replicate in the upper respiratory tract at a temperature of about 33°C, whereas avian viruses replicate in the intestinal tract at a temperature close to 41°C. In the present study, we analyzed the influence of low temperature (33°C) on RNA replication of avian and human viruses in cultured cells. The kinetics of replication of the NP segment were similar at 33 and 37°C for the human A/Puerto-Rico/8/34 and A/Sydney/5/97 viruses, whereas replication was delayed at 33°C compared to 37°C for the avian A/FPV/Rostock/34 and A/Mallard/NY/6750/78 viruses. Making use of a genetic system for the in vivo reconstitution of functional ribonucleoproteins, we observed that the polymerase complexes derived from avian viruses but not human viruses exhibited cold sensitivity in mammalian cells, which was determined mostly by residue 627 of PB2. Our results suggest that a reduced ability of the polymerase complex of avian viruses to ensure replication of the viral genome at 33°C could contribute to their inability to grow efficiently in humans.
TL;DR: The assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV) is described and a paradigm for generating genetically defined vaccines for IBV is established.
Abstract: Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.
TL;DR: It is shown that a 48-nt stem-loop structure within the 1.3-kb TLC1 RNA is necessary and sufficient for disrupting telomeric silencing and shortening telomeres and this short RNA sequence appears to function through an interaction with the conserved DNA end-binding protein Ku.
Abstract: The telomerase enzyme lengthens telomeres, an activity essential for chromosome stability in most eukaryotes. The enzyme is composed of a specialized reverse transcriptase and a template RNA (ref. 1). In Saccharomyces cerevisiae, overexpression of TLC1, the telomerase RNA gene, disrupts telomeric structure2. The result is both shortened telomere length and loss of a special chromatin structure that normally silences telomere-proximal genes. Because telomerase function is not required for telomeric silencing, we postulated that the dominant-negative effect caused by overexpression of TLC1 RNA originates in a normal interaction between the RNA and an unknown telomeric factor important for silencing; the overexpressed RNA presumably continues to bind the factor and compromises its function3. Here we show that a 48-nt stem-loop structure within the 1.3-kb TLC1 RNA is necessary and sufficient for disrupting telomeric silencing and shortening telomeres. Moreover, this short RNA sequence appears to function through an interaction with the conserved DNA end-binding protein Ku (ref. 4). We propose that, in addition to its roles in telomeric silencing, homologous recombination and non-homologous end-joining (NHEJ), S. cerevisiae Ku also helps to recruit or activate telomerase at the telomere through an interaction with this stem-loop of TLC1 RNA.
TL;DR: These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence.
TL;DR: It is concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.
Abstract: The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.
TL;DR: The results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.
Abstract: Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not singlestranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation. Gene sequences derived from different plant viruses have been introduced into a wide variety of plant species to produce transgenic plants protected against virus infection. In a number of cases, it is known that the mechanism of resistance is a posttranscriptional, RNA-mediated process that targets both the viral RNA and the transgene mRNA for degradation in a sequence-specific manner (11, 19). RNA-mediated virus resistance is a manifestation of posttranscriptional gene silencing (PTGS), a more general phenomenon which was first described as a coordinated and reciprocal inactivation of host gene and transgenes encoding the same sense RNA (reviewed in references 33 and 41). More recently, three components in the dynamics of PTGS have been proposed: initiation, propa
TL;DR: A molecular map of the five known essential active sites of the influenza viral polymerase is provided, showing that at least one of the tryptophans functions in cap binding, indicating that this active site is probably similar to that of other known cap‐binding proteins.
Abstract: The cap-dependent endonuclease of the influenza viral RNA polymerase, which produces the capped RNA primers that initiate viral mRNA synthesis, is comprised of two active sites, one for cap binding and one for endonuclease cleavage. We identify the amino acid sequences that constitute these two active sites and demonstrate that they are located on different polymerase subunits. Binding of the 5' terminal sequence of virion RNA (vRNA) to the polymerase activates a tryptophan-rich, cap-binding sequence on the PB2 subunit. At least one of the tryptophans functions in cap binding, indicating that this active site is probably similar to that of other known cap-binding proteins. Endonuclease cleavage, which is activated by the subsequent binding of the 3' terminal sequence of vRNA, resides in a PB1 sequence that contains three essential acidic amino acids, similar to the active sites of other enzymes that cut polynucleotides to produce 3'-OH ends. These results, coupled with those of our previous study, provide a molecular map of the five known essential active sites of the influenza viral polymerase.