Showing papers on "RNA-dependent RNA polymerase published in 2014"

One severe acute respiratory syndrome coronavirus protein complex integrates processive RNA polymerase and exonuclease activities

Abstract: In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

Structure of influenza A polymerase bound to the viral RNA promoter

Abstract: The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3′ and 5′ extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5′ extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design. The crystal structure of the bat-specific influenza A polymerase in complex with the viral RNA promoter is presented, revealing how binding of the 5′ end of the viral RNA is required to activate or enhance the polymerase allosterically. Stephen Cusack and colleagues have solved the crystal structure of the complete influenza polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. In the first of two papers they present the structure of the polymerase from a bat-specific influenza A virus, which is evolutionarily close to human/avian influenza A strains. The second paper presents the structure of the polymerase from a human isolate of influenza B. Together, the structures provide a wealth of information about how the influenza polymerase functions and how the different subunits interact with each other.

Structural insight into cap-snatching and RNA synthesis by influenza polymerase

Abstract: Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

The Pivotal Regulatory Landscape of RNA Modifications

Abstract: Posttranscriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information that is encoded in DNA. The modifications of RNA span all three phylogenetic domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and (less frequently) small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. However, many of these modifications, as well as their prevalence and impact, have only recently been discovered. Here, we examine both labile and permanent modifications, from simple methylation to complex transcript alteration (RNA editing and intron retention); detail the models for their processing; and highlight remaining questions in the field of the epitranscriptome.

Common and unique features of viral RNA-dependent polymerases.

Abstract: Eukaryotes and bacteria can be infected with a wide variety of RNA viruses. On average, these pathogens share little sequence similarity and use different replication and transcription strategies. Nevertheless, the members of nearly all RNA virus families depend on the activity of a virally encoded RNA-dependent polymerase for the condensation of nucleotide triphosphates. This review provides an overview of our current understanding of the viral RNA-dependent polymerase structure and the biochemistry and biophysics that is involved in replicating and transcribing the genetic material of RNA viruses.

Suppression of RNA silencing by a plant DNA virus satellite requires a host calmodulin-like protein to repress RDR6 expression.

Abstract: In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different effector molecules in the RNA silencing pathway. Evidence has shown that plants also encode endogenous suppressors of RNA silencing (ESRs) that function in proper regulation of RNA silencing. The possibility that these cellular proteins can be subverted by viruses to thwart host defense is intriguing but has not been fully explored. Here we report that the Nicotiana benthamiana calmodulin-like protein Nbrgs-CaM is required for the functions of the VSR βC1, the sole protein encoded by the DNA satellite associated with the geminivirus Tomato yellow leaf curl China virus (TYLCCNV). Nbrgs-CaM expression is up-regulated by the βC1. Transgenic plants over-expressing Nbrgs-CaM displayed developmental abnormities reminiscent of βC1-associated morphological alterations. Nbrgs-CaM suppressed RNA silencing in an Agrobacterium infiltration assay and, when over-expressed, blocked TYLCCNV-induced gene silencing. Genetic evidence showed that Nbrgs-CaM mediated the βC1 functions in silencing suppression and symptom modulation, and was required for efficient virus infection. Moreover, the tobacco and tomato orthologs of Nbrgs-CaM also possessed ESR activity, and were induced by betasatellite to promote virus infection in these Solanaceae hosts. We further demonstrated that βC1-induced Nbrgs-CaM suppressed the production of secondary siRNAs, likely through repressing RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) expression. RDR6-deficient N. benthamiana plants were defective in antiviral response and were hypersensitive to TYLCCNV infection. More significantly, TYLCCNV could overcome host range restrictions to infect Arabidopsis thaliana when the plants carried a RDR6 mutation. These findings demonstrate a distinct mechanism of VSR for suppressing PTGS through usurpation of a host ESR, and highlight an essential role for RDR6 in RNA silencing defense response against geminivirus infection.

Amino Acid Substitutions in Polymerase Basic Protein 2 Gene Contribute to the Pathogenicity of the Novel A/H7N9 Influenza Virus in Mammalian Hosts

Abstract: A novel avian-origin influenza A/H7N9 virus emerged in 2013 to cause more than 130 cases of zoonotic human disease, with an overall case fatality rate of around 30% in cases detected. It has been shown that an E-to-K amino acid change at residue 627 of polymerase basic protein 2 (PB2) occurred frequently in the H7N9 isolates obtained from humans but not in viruses isolated from poultry. Although this mutation has been reported to confer increased mammalian pathogenicity in other avian influenza subtypes, it has not been experimentally investigated in the H7N9 virus. In this study, we determined the contribution of PB2-E627K in H7N9 virus to its pathogenicity in mammalian hosts. In addition, the compensatory role of the PB2 mutations T271A, Q591K, and D701N in H7N9 virus was investigated. We characterized the activity of polymerase complexes with these PB2 mutations and found that they enhance the polymerase activity in human 293T cells. The rescued mutants enhanced growth in mammalian cells in vitro. Mice infected with the H7N9 mutant containing the avian signature protein PB2-627E showed a marked decrease in disease severity (weight loss) and pathology compared to mice infected with the wild-type strain (PB2-627K) or other PB2 mutants. Also, mutants with PB2-627E showed lower virus replication and proinflammatory cytokine responses in the lungs of the virus-infected mice, which may contribute to pathogenicity. Our results suggest that these amino acid substitutions contribute to mouse pathogenicity and mammalian adaptation. IMPORTANCE A novel avian H7N9 influenza A virus emerged in east China in 2013 to cause zoonotic human disease associated with significant mortality. It is important to understand the viral genetic markers of mammalian adaptation and disease severity in this H7N9 virus. Since many human (but not avian) H7N9 virus isolates have an amino acid substitution at position E627K in the polymerase basic protein 2 (PB2) gene, we investigated the role of this and other functionally related mutations for polymerase activity in vitro, virus replication competence, and pathogenicity in the mouse model. We found that E627K and functionally related mutations are associated with increased polymerase activity, increased viral replication competence, and increased disease severity in mice.

A cross-chiral RNA polymerase ribozyme.

Abstract: Here, a cross-chiral RNA polymerase is developed—an RNA enzyme that can catalyse the templated polymerization of activated mononucleotides that are of the opposite handedness—shedding light on how RNA-based life could have emerged. It is widely assumed that homochirality is a requirement for life and that biological macromolecules must be of the same stereochemical 'handedness' to interact efficiently. Working with Leslie Orgel and others, Gerald Joyce extended this idea in 1984 to suggest that homochirality may also be essential for the origins of life, as templated polymerization of RNA occurs readily in a homochiral system but is impaired in racemic mixtures. Now Joyce and co-author Jonathan Sczepanski show that RNAs of opposing chirality can work together. They devised a D-RNA enzyme that catalyses the polymerization of L-RNA on a L-RNA template — and vice versa. The catalytic potency of this ribozyme is sufficient for it to synthesize its own enantiomer by joining 11 component oligonucleotides. The ribozyme is thought to interact with its substrates via tertiary contacts rather than Watson–Crick base pairing. This unexpected finding will add a new dimension to thoughts on how life could have emerged in an 'RNA world'. Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer1. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes before the origin of RNA-based life3,4. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a d-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme’s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.

Viroids: Survivors from the RNA World?

Abstract: Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario—the so-called RNA world—existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G + C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.

Tomato yellow leaf curl virus resistance by Ty-1 involves increased cytosine methylation of viral genomes and is compromised by cucumber mosaic virus infection

Abstract: Tomato yellow leaf curl virus (TYLCV) and related begomoviruses are a major threat to tomato production worldwide and, to protect against these viruses, resistance genes from different wild tomato species are introgressed. Recently, the Ty-1 resistance gene was identified, shown to code for an RNA-dependent RNA polymerase and to be allelic with Ty-3. Here we show that upon TYLCV challenging of resistant lines carrying Ty-1 or Ty-3, low virus titers were detected concomitant with the production of relatively high levels of siRNAs whereas, in contrast, susceptible tomato Moneymaker (MM) revealed higher virus titers but lower amounts of siRNAs. Comparative analysis of the spatial genomic siRNA distribution showed a consistent and subtle enrichment for siRNAs derived from the V1 and C3 genes in Ty-1 and Ty-3. In plants containing Ty-2 resistance the virus was hardly detectable, but the siRNA profile resembled the one observed in TYLCV-challenged susceptible tomato (MM). Furthermore, a relative hypermethylation of the TYLCV V1 promoter region was observed in genomic DNA collected from Ty-1 compared with that from (MM). The resistance conferred by Ty-1 was also effective against the bipartite tomato severe rugose begomovirus, where a similar genome hypermethylation of the V1 promoter region was discerned. However, a mixed infection of TYLCV with cucumber mosaic virus compromised the resistance. The results indicate that Ty-1 confers resistance to geminiviruses by increasing cytosine methylation of viral genomes, suggestive of enhanced transcriptional gene silencing. The mechanism of resistance and its durability toward geminiviruses under natural field conditions is discussed.

Transactivation of programmed ribosomal frameshifting by a viral protein.

Abstract: Programmed −1 ribosomal frameshifting (−1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes −1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual −2 frameshifting (−2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of −1 PRF, yielding a third, truncated nsp2 variant named “nsp2N.” Remarkably, we now show that both −2 and −1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β’s papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection.

MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells

Abstract: MicroRNAs (miRNAs) are small regulatory RNAs that play significant roles in most cellular processes. In the seemingly endless arms race between hosts and pathogens, viruses also encode miRNAs that facilitate successful infection. In search of functional miRNAs or viral small RNAs (vsRNAs) encoded by Dengue virus (DENV), deep sequencing data of virus-infected Aedes aegypti mosquitoes were used. From six vsRNAs, with candidate stem-loop structures in the 5′ and 3′ untranslated regions of the viral genomic RNA, inhibition of DENV–vsRNA-5 led to significant increases in viral replication. Silencing of RNA interference (RNAi)/miRNA pathways’ associated proteins showed that Argonaute 2 is mainly involved in DENV–vsRNA-5 biogenesis. Cloning of the precursor stem loop, immunoprecipitations, ectopic expression and detection in RNAi-deficient C6/36, and the mammalian Vero cell lines further confirmed DENV–vsRNA-5 production. Furthermore, significant impact of synthetic mimic and inhibitor of DENV–vsRNA-5 on DENV RNA levels revealed DENV–vsRNA-5’s role in virus autoregulation by targeting the virus nonstructural protein 1 gene. Notably, DENV–vsRNA-5 homologous mimics from DENV serotypes 1 and 4, but not 3, inhibited DENV-2 replication. The results revealed that DENV is able to encode functional vsRNAs, and one of those, which resembles miRNAs, specifically targets a viral gene, opening an avenue for possible utilization of the small RNA to limit DENV replication.

The crystal structure of TDP-43 RRM1-DNA complex reveals the specific recognition for UG- and TG-rich nucleic acids

Abstract: TDP-43 is an important pathological protein that aggregates in the diseased neuronal cells and is linked to various neurodegenerative disorders. In normal cells, TDP-43 is primarily an RNA-binding protein; however, how the dimeric TDP-43 binds RNA via its two RNA recognition motifs, RRM1 and RRM2, is not clear. Here we report the crystal structure of human TDP-43 RRM1 in complex with a single-stranded DNA showing that RRM1 binds the nucleic acid extensively not only by the conserved β-sheet residues but also by the loop residues. Mutational and biochemical assays further reveal that both RRMs in TDP-43 dimers participate in binding of UG-rich RNA or TG-rich DNA with RRM1 playing a dominant role and RRM2 playing a supporting role. Moreover, RRM1 of the amyotrophic lateral sclerosis-linked mutant D169G binds DNA as efficiently as the wild type; nevertheless, it is more resistant to thermal denaturation, suggesting that the resistance to degradation is likely linked to TDP-43 proteinopathies. Taken together all the data, we suggest a model showing that the two RRMs in each protomer of TDP-43 homodimer work together in RNA binding and thus the dimeric TDP-43 recognizes long clusters of UG-rich RNA to achieve high affinity and specificity.

Regulation of the hepatitis C virus RNA replicase by endogenous lipid peroxidation

Abstract: Oxidative tissue injury often accompanies viral infection, yet there is little understanding of how it influences virus replication. We show that multiple hepatitis C virus (HCV) genotypes are exquisitely sensitive to oxidative membrane damage, a property distinguishing them from other pathogenic RNA viruses. Lipid peroxidation, regulated in part through sphingosine kinase-2, severely restricts HCV replication in Huh-7 cells and primary human hepatoblasts. Endogenous oxidative membrane damage lowers the 50% effective concentration of direct-acting antivirals in vitro, suggesting critical regulation of the conformation of the NS3-4A protease and the NS5B polymerase, membrane-bound HCV replicase components. Resistance to lipid peroxidation maps genetically to transmembrane and membrane-proximal residues within these proteins and is essential for robust replication in cell culture, as exemplified by the atypical JFH1 strain of HCV. Thus, the typical, wild-type HCV replicase is uniquely regulated by lipid peroxidation, providing a mechanism for attenuating replication in stressed tissue and possibly facilitating long-term viral persistence.

The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

Abstract: Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K.

Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

Abstract: The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001.

Endogenous polyamine function—the RNA perspective

Abstract: Recent progress with techniques for monitoring RNA structure in cells such as ‘DMS-Seq’ and ‘Structure-Seq’ suggests that a new era of RNA structure-function exploration is on the horizon. This will also include systematic investigation of the factors required for the structural integrity of RNA. In this context, much evidence accumulated over 50 years suggests that polyamines play important roles as modulators of RNA structure. Here, we summarize and discuss recent literature relating to the roles of these small endogenous molecules in RNA function. We have included studies directed at understanding the binding interactions of polyamines with polynucleotides, tRNA, rRNA, mRNA and ribozymes using chemical, biochemical and spectroscopic tools. In brief, polyamines bind RNA in a sequence-selective fashion and induce changes in RNA structure in context-dependent manners. In some cases the functional consequences of these interactions have been observed in cells. Most notably, polyamine-mediated effects on RNA are frequently distinct from those of divalent cations (i.e. Mg2+) confirming their roles as independent molecular entities which help drive RNA-mediated processes.

The RNA exosome promotes transcription termination of backtracked RNA polymerase II

Abstract: The exosome is an RNA-decay complex that constantly monitors transcription and contributes to post-transcriptional turnover of faulty mRNAs. Yet how nuclear RNA surveillance by the exosome is coordinated with transcription is still unknown. Here we show that the RNA exosome of Schizosaccharomyces pombe can target the transcription machinery by terminating transcription events associated with paused and backtracked RNA polymerase II (RNAPII); this is contrary to the notion that the exosome acts exclusively on RNAs that have been released by RNAPII. Our data support a mechanism by which RNAPII backtracking provides a free RNA 3' end for the core exosome, which results in transcription termination with concomitant degradation of the associated transcript. These findings uncover a mechanism of cotranscriptional RNA surveillance whereby termination of transcription by the exosome prevents formation of aberrant readthrough RNAs and transcriptional interference at neighboring genes.