Showing papers on "RNA-dependent RNA polymerase published in 2005"
TL;DR: It is shown that the sequestration of miR-122 in liver cells results in marked loss of autonomously replicating hepatitis C viral RNAs, suggesting that miR -122 may present a target for antiviral intervention.
Abstract: MicroRNAs are small RNA molecules that regulate messenger RNA (mRNA) expression. MicroRNA 122 (miR-122) is specifically expressed and highly abundant in the human liver. We show that the sequestration of miR-122 in liver cells results in marked loss of autonomously replicating hepatitis C viral RNAs. A genetic interaction between miR-122 and the 5' noncoding region of the viral genome was revealed by mutational analyses of the predicted microRNA binding site and ectopic expression of miR-122 molecules containing compensatory mutations. Studies with replication-defective RNAs suggested that miR-122 did not detectably affect mRNA translation or RNA stability. Therefore, miR-122 is likely to facilitate replication of the viral RNA, suggesting that miR-122 may present a target for antiviral intervention.
2,484 citations
TL;DR: By mutation of the two largest subunits (NRPD1a and NRPD2), it is shown that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3.
Abstract: Plants encode subunits for a fourth RNA polymerase (Pol IV) in addition to the well-known DNA-dependent RNA polymerases I, II, and III. By mutation of the two largest subunits (NRPD1a and NRPD2), we show that Pol IV silences certain transposons and repetitive DNA in a short interfering RNA pathway involving RNA-dependent RNA polymerase 2 and Dicer-like 3. The existence of this distinct silencing polymerase may explain the paradoxical involvement of an RNA silencing pathway in maintenance of transcriptional silencing.
749 citations
TL;DR: The structure of NS5A domain I at 2.5-Å resolution is reported, which contains a novel fold, a new zinc-coordination motif and a disulphide bond, and molecular surface analysis is used to suggest the location of protein-, RNA- and membrane-interaction sites.
Abstract: Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.
494 citations
TL;DR: The results suggest that c-Myc coordinates the activity of all three nuclear RNA polymerases, and thereby plays a key role in regulating ribosome biogenesis and cell growth.
Abstract: The c-Myc oncoprotein regulates transcription of genes that are associated with cell growth, proliferation and apoptosis(1). c-Myc levels are modulated by ubiquitin/proteasome-mediated degradation( ...
471 citations
TL;DR: A model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves is suggested.
Abstract: One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.
464 citations
TL;DR: It is demonstrated that a cellular peptidyl-prolyl cis-trans isomerase, cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus (HCV) genome.
463 citations
TL;DR: An RNA polymerase II complex that contains at least 12 novel subunits, termed the Integrator, in addition to core RNAPII subunits is described, which shows that Integrator is recruited to the U1 and U2 snRNA genes and mediates the snRNAs' 3' end processing.
445 citations
TL;DR: This study identified a novel RNA element within the NS5B coding sequence that is indispensable for replication and discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3′ NTR.
Abstract: The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3' NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3' NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3' end of the HCV genome that is essential for replication.
368 citations
TL;DR: Although many molecular details of the coronavirus life cycle remain to be investigated, the available information suggests that these viruses and their distant nidovirus relatives employ a unique collection of enzymatic activities and other protein functions to synthesize a set of 5′-leader-containing subgenomic mRNAs and to replicate the largest RNA virus genomes currently known.
Abstract: Coronavirus genome replication and transcription take place at cytoplasmic membranes and involve coordinated processes of both continuous and discontinuous RNA synthesis that are mediated by the viral replicase, a huge protein complex encoded by the 20-kb replicase gene. The replicase complex is believed to be comprised of up to 16 viral subunits and a number of cellular proteins. Besides RNA-dependent RNA polymerase, RNA helicase, and protease activities, which are common to RNA viruses, the coronavirus replicase was recently predicted to employ a variety of RNA processing enzymes that are not (or extremely rarely) found in other RNA viruses and include putative sequence-specific endoribonuclease, 3′-to-5′ exoribonuclease, 2′-O-ribose methyltransferase, ADP ribose 1′-phosphatase and, in a subset of group 2 coronaviruses, cyclic phosphodiesterase activities. This chapter reviews (1) the organization of the coronavirus replicase gene, (2) the proteolytic processing of the replicase by viral proteases, (3) the available functional and structural information on individual subunits of the replicase, such as proteases, RNA helicase, and the RNA-dependent RNA polymerase, and (4) the subcellular localization of coronavirus proteins involved in RNA synthesis. Although many molecular details of the coronavirus life cycle remain to be investigated, the available information suggests that these viruses and their distant nidovirus relatives employ a unique collection of enzymatic activities and other protein functions to synthesize a set of 5′-leader-containing subgenomic mRNAs and to replicate the largest RNA virus genomes currently known.
356 citations
TL;DR: Since sequence complementarity between the ends of the genome is required for dengue virus viability, it is proposed that cyclization of the RNA is a required conformation for viral replication.
Abstract: Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5′ and 3′ ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy reveled that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5′ and 3′ CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5′ and 3′ untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5′ and 3′ UAR (upstream AUG region). In order to investigate the functional role of 5′-3′ UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5′ or 3′ UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.
353 citations
01 Jan 2005
TL;DR: Methylation of Selenocysteine tRNA Governs Differential Expression of Mammalian Selenoproteins and Adenosine to Inosine RNA Editing in Animal Cells.
Abstract: Modification and Editing of RNA: an Overview- Biosynthesis and Function of tRNA Wobble Modifications- Editing and Modification in Trypanosomes : the Reshaping of Non-coding RNAs- Transfer RNA Modification and Modifying Enzymes in Yeast- Biosynthesis and Function of 1-methyladenosine in Transfer RNA- The Biosynthesis and Functional Roles of Methylated Nucleosides in Eukaryotic mRNA- Role of the 5'-cap in the Biosynthesis of Splicesomal snRNPs- Role of a Conserved Pseudouridine on the Structural and Electrostatic Features of the Splicesomal Pre-mRNA Branch Site- RNA-guided RNA Modification- Conserved Ribosomal RNA Modification and Their Putative Roles in Ribosome Biogenesis and Translation- Nucleotide Methylations in rRNA that Confer Resistance to Ribosome-targeting Antibiotics- Translational Recoding and RNA Modifications- Adenosine to Inosine RNA Editing in Animal Cells- Mammalian C-to-U Editing- Transfer RNA Modifications and DNA Editing in HIV-1 Reverse Transcription- Methylation of Selenocysteine tRNA Governs Differential Expression of Mammalian Selenoproteins
TL;DR: It is shown that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects ofRNA polymerase behavior and providing an alternative to the "transcriptomics" approach for studying global transcription patterns.
Abstract: Chromatin immunoprecipitation and high-density microarrays have been used to monitor the distribution of the global transcription regulator Escherichia coli cAMP-receptor protein (CRP) and RNA polymerase along the E. coli chromosome. Our results identify targets occupied by CRP and genes transcribed by RNA polymerase in vivo. Many of the loci of CRP binding are at known CRP regulated promoters. However, our results show that CRP also interacts with thousands of weaker sites across the whole chromosome and that this “background” binding can be used as a probe for organization within the E. coli folded chromosome. In rapidly growing cells, we show that the major sites of RNA polymerase binding are ≈90 transcription units that include genes needed for protein synthesis. Upon the addition of rifampicin, RNA polymerase is distributed among >500 functional promoters. We show that the chromatin immunoprecipitation and high-density-microarrays methodology can be used to study the redistribution of RNA polymerase induced by environmental stress, revealing previously uncharacterized aspects of RNA polymerase behavior and providing an alternative to the “transcriptomics” approach for studying global transcription patterns.
TL;DR: The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves the method developed to mine an Arabidopsis nonannotated, noncoding EST database to be a useful tool to uncover additional small regulatory RNAs.
Abstract: Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA–auxin response factor (tasiR-ARF), that shares a 21- and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3/ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs.
TL;DR: It is estimated that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-stranded RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.
Abstract: The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.
TL;DR: Results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHv small interfering RNAs into the RNA-induced silencing complex.
Abstract: As a counter-defense against antiviral RNA silencing during infection, the insect Flock House virus (FHV) expresses the silencing suppressor protein B2. Biochemical experiments show that B2 binds to double-stranded RNA (dsRNA) without regard to length and inhibits cleavage of dsRNA by Dicer in vitro. A cocrystal structure reveals that a B2 dimer forms a four-helix bundle that binds to one face of an A-form RNA duplex independently of sequence. These results suggest that B2 blocks both cleavage of the FHV genome by Dicer and incorporation of FHV small interfering RNAs into the RNA-induced silencing complex.
TL;DR: It is demonstrated that complementarity between sequences present at the 5' and 3' ends of the genome is essential for dengue virus RNA synthesis, while deletion of domains A2 or A3 within the 3'UTR resulted in replicons with decreased RNA amplification.
TL;DR: It is found that NS5A has the capacity to bind to the 3′-ends of HCV plus and minus strand RNAs, and this protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.
TL;DR: A model of the role of NS5A phosphorylation in the viral life cycle is proposed and a deletion encompassing all highly conserved serine residues in the C-terminal region ofNS5A that are involved in basal phosphorylated did not significantly affect RNA replication but reduced formation of p56.
Abstract: Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.
TL;DR: This review will highlight the current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA‐membrane interactions observed between the virus families Flaviviridae and Picornaviridae.
Abstract: All single-stranded 'positive-sense' RNA viruses that infect mammalian, insect or plant cells rearrange internal cellular membranes to provide an environment facilitating virus replication. A striking feature of these unique membrane structures is the induction of 70-100 nm vesicles (either free within the cytoplasm, associated with other induced vesicles or bound within a surrounding membrane) harbouring the viral replication complex (RC). Although similar in appearance, the cellular composition of these vesicles appears to vary for different viruses, implying different organelle origins for the intracellular sites of viral RNA replication. Genetic analysis has revealed that induction of these membrane structures can be attributed to a particular viral gene product, usually a non-structural protein. This review will highlight our current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA-membrane interactions observed between the virus families Flaviviridae and Picornaviridae.
TL;DR: Increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.
Abstract: The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.
TL;DR: In this paper, the authors identify >100 new 6S RNA homologs in diverse eubacterial lineages, including Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs.
Abstract: 6S RNA is an abundant noncoding RNA in Escherichia coli that binds to σ70 RNA polymerase holoenzyme to globally regulate gene expression in response to the shift from exponential growth to stationary phase. We have computationally identified >100 new 6S RNA homologs in diverse eubacterial lineages. Two abundant Bacillus subtilis RNAs of unknown function (BsrA and BsrB) and cyanobacterial 6Sa RNAs are now recognized as 6S homologs. Structural probing of E. coli 6S RNA and a B. subtilis homolog supports a common secondary structure derived from comparative sequence analysis. The conserved features of 6S RNA suggest that it binds RNA polymerase by mimicking the structure of DNA template in an open promoter complex. Interestingly, the two B. subtilis 6S RNAs are discoordinately expressed during growth, and many proteobacterial 6S RNAs could be cotranscribed with downstream homologs of the E. coli ygfA gene encoding a putative methenyltetrahydrofolate synthetase. The prevalence and robust expression of 6S RNAs emphasize their critical role in bacterial adaptation.
TL;DR: This work shows that ADAR2- but not ADAR1-mediated RNA editing occurs in the nucleolus, and identifies a brain-specific small RNA, termed MBII-52, which was predicted to function as a nucleolar C/D RNA, thereby targeting an A-to-I editing site (C-site) within the 5-HT2C serotonin receptor pre-mRNA for 2′-O-methylation.
Abstract: Posttranscriptional, site-specific adenosine to inosine (A-to-I) base conversions, designated as RNA editing, play significant roles in generating diversity of gene expression. However, little is known about how and in which cellular compartments RNA editing is controlled. Interestingly, the two enzymes that catalyze RNA editing, adenosine deaminases that act on RNA (ADAR) 1 and 2, have recently been demonstrated to dynamically associate with the nucleolus. Moreover, we have identified a brain-specific small RNA, termed MBII-52, which was predicted to function as a nucleolar C/D RNA, thereby targeting an A-to-I editing site (C-site) within the 5-HT2C serotonin receptor pre-mRNA for 2′-O-methylation. Through the subcellular targeting of minigenes that contain natural editing sites, we show that ADAR2- but not ADAR1-mediated RNA editing occurs in the nucleolus. We also demonstrate that MBII-52 forms a bona fide small nucleolar ribonucleoprotein particle that specifically decreases the efficiency of RNA editing by ADAR2 at the targeted C-site. Our data are consistent with a model in which C/D small nucleolar RNA might play a role in the regulation of RNA editing.
TL;DR: Various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification are designed, which demonstrate low variability and can be applied in various RT- PCR assays.
Abstract: Diagnostic systems based on reverse transcription (RT)-PCR are widely used for the detection of viral genomes in different human specimens. The application of internal controls (IC) to monitor each step of nucleic acid amplification is necessary to prevent false-negative results due to inhibition or human error. In this study, we designed various real-time RT-PCRs utilizing the coliphage MS2 replicase gene, which differ in detection format, amplicon size, and efficiency of amplification. These noncompetitive IC assays, using TaqMan, hybridization probe, or duplex scorpion probe techniques, were tested on the LightCycler and Rotorgene systems. In our approach, clinical specimens were spiked with the control virus to monitor the efficiency of extraction, reverse transcription, and amplification steps. The MS2 RT-PCR assays were applied for internal control when using a second target hepatitis C virus RNA in duplex PCR in blood donor screening. The 95% detection limit was calculated by probit analysis to 44.9 copies per PCR (range, 38.4 to 73.4). As demonstrated routinely, application of MS2 IC assays exhibits low variability and can be applied in various RT-PCR assays. MS2 phage lysates were obtained under standard laboratory conditions. The quantification of phage and template RNA was performed by plating assays to determine PFU or via real-time RT-PCR. High stability of the MS2 phage preparations stored at −20°C, 4°C, and room temperature was demonstrated.
TL;DR: It is demonstrated for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain, indicating that it targets actively transcribing Pol II.
Abstract: Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.
TL;DR: RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.
Abstract: RNA interference represents an exciting new technology that could have therapeutic applications for the treatment of viral infections. Hepatitis C virus (HCV) is a major cause of chronic liver disease and affects over 270 million individuals worldwide. The HCV genome is a single-stranded RNA that functions as both an mRNA and a replication template, making it an attractive target for therapeutic approaches using short interfering RNA (siRNA). We have shown previously that double-stranded siRNA molecules designed to target the HCV genome block gene expression and RNA synthesis from hepatitis C replicons propagated in human liver cells. However, we now show that this block is not complete. After several treatments with a highly effective siRNA, we have shown growth of replicon RNAs that are resistant to subsequent treatment with the same siRNA. However, these replicon RNAs were not resistant to siRNA targeting another part of the genome. Sequence analysis of the siRNA-resistant replicons showed the generation of point mutations within the siRNA target sequence. In addition, the use of a combination of two siRNAs together severely limited escape mutant evolution. This suggests that RNA interference activity could be used as a treatment to reduce the devastating effects of HCV replication on the liver and the use of multiple siRNAs could prevent the emergence of resistant viruses.
TL;DR: The structures of the NS5B polymerase/non-nucleoside inhibitor complexes bind at a common binding site, which is nearly 35 Å away from the polymerase active site and is located in the thumb domain, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions.
TL;DR: The data suggest that the Arabidopsis RDR6-associated epigenetic pathway and the AS1-AS2 pathway synergistically repress BP and MIR165/166 for proper plant development.
Abstract: The Arabidopsis thaliana ASYMMETRIC LEAVES1 (AS1) and AS2 genes are important for repressing class I KNOTTED1-like homeobox (KNOX) genes and specifying leaf adaxial identity in leaf development. RNA-dependent RNA polymerases (RdRPs) are critical for posttranscriptional and transcriptional gene silencing in eukaryotes; however, very little is known about their functions in plant development. Here, we show that the Arabidopsis RDR6 gene (also called SDE1 and SGS2) that encodes a putative RdRP, together with AS1 and AS2, regulates leaf development. rdr6 single mutant plants displayed only minor phenotypes, whereas rdr6 as1 and rdr6 as2 double mutants showed dramatically enhanced as1 and as2 phenotypes, with severe defects in the leaf adaxial-abaxial polarity and vascular development. In addition, the double mutant plants produced more lobed leaves than the as1 and as2 single mutants and showed leaf-like structures associated on a proportion of leaf blades. The abnormal leaf morphology of the double mutants was accompanied by an extended ectopic expression of a class I KNOX gene BREVIPEDICELLUS (BP) and high levels of microRNA165/166 that may lead to mRNA degradation of genes in the class III HD-ZIP family. Taken together, our data suggest that the Arabidopsis RDR6-associated epigenetic pathway and the AS1-AS2 pathway synergistically repress BP and MIR165/166 for proper plant development.
TL;DR: HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR, which has a highly folded stem-bulge-loop structure, which also binds cellular proteins to form ribonucleoprotein complexes.
Abstract: HIV-1 TAR RNA is the binding site of the viral protein Tat, the trans-activator of the HIV-1 LTR. It is present at the 5' end of all HIV-1 spliced and unspliced mRNAs in the nucleus as well as in the cytoplasm. It has a highly folded stem-bulge-loop structure, which also binds cellular proteins to form ribonucleoprotein complexes. The Tat-Cyclin T1-CDK9 complex is the main component in the trans-activation of HIV-1 and its affinity for TAR is regulated through Tat acetylation by histone acetyl transferases. Recent studies show that this complex is able to recruit other cellular partners to mediate efficient transcriptional elongation. TRBP, PKR and La bind directly to the TAR RNA structure and influence translation of HIV-1 in either positive or negative manners. Some mutations in TAR RNA severely impair HIV-1 trans-activation, translation and viral production, showing its functional importance. The overexpression or suppression of several TAR RNA- binding proteins has a strong impact on viral replication pointing out their major role in the viral life cycle. TAR RNA has been the target of drug development to inhibit viral replication. Recent data using small molecules or RNA-based technologies show that acting on the TAR RNA or on its viral and cellular binding factors effectively decreases virion production.
TL;DR: The data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA, and a target sequence located within the 5′-untranslated region of the HIV- 1 RNA was identified to be necessary and sufficient for efficient APo3G packaging.
Abstract: APOBEC3G (APO3G) is a host cytidine deaminase that is incorporated into human immunodeficiency virus type 1 (HIV-1) particles. We report here that viral RNA promotes stable association of APO3G with HIV-1 nucleoprotein complexes (NPC). A target sequence located within the 5′-untranslated region of the HIV-1 RNA was identified to be necessary and sufficient for efficient APO3G packaging. Fine mapping revealed a sequence normally involved in viral genomic RNA dimerization and Gag binding to be important for APO3G packaging and association with viral NPC. Our data suggest that packaging of APO3G into HIV-1 NPC is enhanced by viral RNA.
TL;DR: It is proposed that the p33:IRE interaction provides a mechanism to selectively recruit viral RNAs into cognate viral replicase complexes and may be relevant to other members of this large virus family.
Abstract: Plus-strand RNA viruses replicate their genomes in infected cells by using a replicase complex comprised of viral and host proteins that assembles in association with cellular membranes (1, 4, 15). In infected cells, viral replicases are able to specifically recognize and selectively replicate their cognate viral RNAs from a heterogeneous pool of cellular RNA molecules. In contrast, in vitro studies have shown that many purified viral replicase complexes are able to utilize heterologous promoter or initiation elements quite efficiently (11, 28, 39). These conflicting findings between in vivo and in vitro data have led to models that attribute selective recognition of viral templates to host proteins (4, 13). Phage Qbeta utilizes this type of mechanism whereby the S1 ribosomal protein and elongation factor Tu in the four-subunit replicase complex mediate viral template recognition (3, 13).
There is also evidence that virally encoded proteins can facilitate selective template recruitment to the viral replicase complex. Examples in this category include the 1a protein of Brome mosaic virus and the 126-kDa protein of Tomato mosaic virus (5, 17, 33). However, in both of these cases it is not known whether the respective viral RNAs are recognized directly by these replicase proteins or require assistance from host proteins (6). In Poliovirus, specific binding of 3CD protein to the 5′-terminal cloverleaf-like structure has been reported, and this interaction likely contributes to template selection into replication (9, 38). However, the host poly(C) binding protein 2 also interacts specifically with the same 5′-terminal RNA structure, and it, too, is proposed to be involved in mediating template selection (9, 38). In general, the contribution of viral and cellular proteins to RNA template recognition by cognate replicases is largely unknown in most viral systems.
Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the large family Tombusviridae. Its genome encodes two proteins involved in viral RNA replication, the prereadthrough product p33 and the readthrough product p92 (Fig. 1A and B). Both of these proteins are essential for RNA replication (18, 22), they are part of the viral replicase complex (23), and they accumulate in vivo at a ratio of 20:1, respectively (23, 31). The less plentiful p92 functions as the viral RNA-dependent RNA polymerase (RdRp), while the role of the more abundant prereadthrough p33 is undefined (37). In other tombusviruses, the orthologues of TBSV p33 have been shown to be targeted to mitochondrial or peroxisomal membranes, the presumed sites of tombusvirus RNA replication (29). In TBSV, both p33 and p92 are membrane associated (23, 31). RNA-binding domains have also been identified in these proteins (22, 26), and the ability of p33 to interact with itself and p92 has been demonstrated (27). The cumulative data support an essential role for both TBSV p33 and p92 in viral RNA replication, with p92 comprising the catalytic subunit responsible for RNA synthesis and p33 playing a critical but unknown auxiliary role.
FIG. 1.
Specific binding of the recombinant p33 replicase protein to RII(+)-SL in vitro. (A) Schematic representation of the p33 and p92 replicase proteins of TBSV. The sequence of p33 is identical to the N-terminal overlapping (prereadthrough) domain ...
In this paper we tested the binding of a recombinant TBSV p33 to four conserved regions of the viral genome known to affect replication (37). We demonstrate that p33 binds selectively in vitro to a conserved RNA motif within the p92 coding region of the viral genome. The specific recognition of this RNA element is dependent on a C · C mismatch positioned within a helix, and this key determinant of p33 binding is also essential for TBSV replication in host cells. We propose that this interaction directs viral template recruitment into replication and suggest that this mechanism may also apply to other members of the large family Tombusviridae.