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Showing papers on "RNA-dependent RNA polymerase published in 2016"


Journal ArticleDOI
07 Apr 2016-Cell
TL;DR: It is demonstrated that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells and establishes RCas9 as a means to track RNA inliving cells in a programmable manner without genetically encoded tags.

455 citations


Book ChapterDOI
TL;DR: Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.
Abstract: Coronaviruses are animal and human pathogens that can cause lethal zoonotic infections like SARS and MERS. They have polycistronic plus-stranded RNA genomes and belong to the order Nidovirales, a diverse group of viruses for which common ancestry was inferred from the common principles underlying their genome organization and expression, and from the conservation of an array of core replicase domains, including key RNA-synthesizing enzymes. Coronavirus genomes (~26-32 kilobases) are the largest RNA genomes known to date and their expansion was likely enabled by acquiring enzyme functions that counter the commonly high error frequency of viral RNA polymerases. The primary functions that direct coronavirus RNA synthesis and processing reside in nonstructural protein (nsp) 7 to nsp16, which are cleavage products of two large replicase polyproteins translated from the coronavirus genome. Significant progress has now been made regarding their structural and functional characterization, stimulated by technical advances like improved methods for bioinformatics and structural biology, in vitro enzyme characterization, and site-directed mutagenesis of coronavirus genomes. Coronavirus replicase functions include more or less universal activities of plus-stranded RNA viruses, like an RNA polymerase (nsp12) and helicase (nsp13), but also a number of rare or even unique domains involved in mRNA capping (nsp14, nsp16) and fidelity control (nsp14). Several smaller subunits (nsp7-nsp10) act as crucial cofactors of these enzymes and contribute to the emerging "nsp interactome." Understanding the structure, function, and interactions of the RNA-synthesizing machinery of coronaviruses will be key to rationalizing their evolutionary success and the development of improved control strategies.

454 citations


Journal ArticleDOI
TL;DR: Current knowledge of the structure of the influenza virus RNA polymerase is discussed, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors are discussed.
Abstract: The genomes of influenza viruses consist of multiple segments of single-stranded negative-sense RNA. Each of these segments is bound by the heterotrimeric viral RNA-dependent RNA polymerase and multiple copies of nucleoprotein, which form viral ribonucleoprotein (vRNP) complexes. It is in the context of these vRNPs that the viral RNA polymerase carries out transcription of viral genes and replication of the viral RNA genome. In this Review, we discuss our current knowledge of the structure of the influenza virus RNA polymerase, and insights that have been gained into the molecular mechanisms of viral transcription and replication, and their regulation by viral and host factors. Furthermore, we discuss how advances in our understanding of the structure and function of polymerases could help in identifying new antiviral targets.

329 citations


01 Jan 2016
TL;DR: In this paper, specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl transferase, EC 2.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before the RNA initialization site.
Abstract: Specific contacts between the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyl- transferase, EC 2.7.7.6) and the phosphates and purine bases of the A3 promoter of phage T7 cluster into three regions located approximately 10, 16, and 35 base pairs before the RNA initia- tion site. Two of these contain nucleotide sequences that are fairly conserved among many promoters, known as the "Prib- now box" and "-35 region" homologies; the third, just upstream from the Pribnow box, is not conserved. The polymerase binds preferentially to the coding strand and for the most part touches only one face of the DNA helix.

267 citations


Journal ArticleDOI
02 Jul 2016-eLife
TL;DR: It is shown that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV- 1 infection in cell lines and primary CD4+ T-cells, and suggests an important role of m 6A modification of HIV-3 RNA in viral infection and HIV-2 protein synthesis.
Abstract: The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4(+) T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis.

219 citations


Journal ArticleDOI
07 Jan 2016-Nature
TL;DR: It is shown that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells and is a candidate host target for novel antivirals.
Abstract: Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals.

201 citations


Journal ArticleDOI
TL;DR: This is the first application of ribosome profiling to an RNA virus and, contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site.
Abstract: Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus.

175 citations


Journal ArticleDOI
TL;DR: In vitro evolution is reported of an improved RNA polymerase ribozyme that is able to synthesize structured functional RNAs, including aptamers and ribozymes, and replicate short RNA sequences in a protein-free form of the PCR, ensuring the two prerequisites of Darwinian life can now be accomplished with RNA in the complete absence of proteins.
Abstract: In all extant life, genetic information is stored in nucleic acids that are replicated by polymerase proteins. In the hypothesized RNA world, before the evolution of genetically encoded proteins, ancestral organisms contained RNA genes that were replicated by an RNA polymerase ribozyme. In an effort toward reconstructing RNA-based life in the laboratory, in vitro evolution was used to improve dramatically the activity and generality of an RNA polymerase ribozyme by selecting variants that can synthesize functional RNA molecules from an RNA template. The improved polymerase ribozyme is able to synthesize a variety of complex structured RNAs, including aptamers, ribozymes, and, in low yield, even tRNA. Furthermore, the polymerase can replicate nucleic acids, amplifying short RNA templates by more than 10,000-fold in an RNA-catalyzed form of the PCR. Thus, the two prerequisites of Darwinian life—the replication of genetic information and its conversion into functional molecules—can now be accomplished with RNA in the complete absence of proteins.

171 citations


Journal ArticleDOI
TL;DR: It is reported that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4).
Abstract: Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.

169 citations


Journal ArticleDOI
TL;DR: The different molecular mechanisms underlying the link betweenRNA G4s and human diseases are reviewed by proposing several overlapping models of deregulation emerging from recent research, including sequestration of RNA-binding proteins, aberrant expression or localization of RNA G4- binding proteins, and repeat associated non-AUG (RAN) translation.
Abstract: RNA G-quadruplexes (G4s) are formed by G-rich RNA sequences in protein-coding (mRNA) and non-coding (ncRNA) transcripts that fold into a four-stranded conformation. Experimental studies and bioinformatic predictions support the view that these structures are involved in different cellular functions associated to both DNA processes (telomere elongation, recombination and transcription) and RNA post-transcriptional mechanisms (including pre-mRNA processing, mRNA turnover, targeting and translation). An increasing number of different diseases have been associated with the inappropriate regulation of RNA G4s exemplifying the potential importance of these structures on human health. Here, we review the different molecular mechanisms underlying the link between RNA G4s and human diseases by proposing several overlapping models of deregulation emerging from recent research, including (i) sequestration of RNA-binding proteins, (ii) aberrant expression or localization of RNA G4-binding proteins, (iii) repeat associated non-AUG (RAN) translation, (iv) mRNA translational blockade and (v) disabling of protein-RNA G4 complexes. This review also provides a comprehensive survey of the functional RNA G4 and their mechanisms of action. Finally, we highlight future directions for research aimed at improving our understanding on RNA G4-mediated regulatory mechanisms linked to diseases.

164 citations


Journal ArticleDOI
21 Jul 2016-Nature
TL;DR: It is shown that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP).
Abstract: The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.

Journal ArticleDOI
TL;DR: Results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses.
Abstract: Positive-sense RNA viruses encode RNA-dependent RNA polymerases (RdRps) essential for genomic replication. With the exception of the large nidoviruses, such as coronaviruses (CoVs), RNA viruses lack proofreading and thus are dependent on RdRps to control nucleotide selectivity and fidelity. CoVs encode a proofreading exonuclease in nonstructural protein 14 (nsp14-ExoN), which confers a greater-than-10-fold increase in fidelity compared to other RNA viruses. It is unknown to what extent the CoV polymerase (nsp12-RdRp) participates in replication fidelity. We sought to determine whether homology modeling could identify putative determinants of nucleotide selectivity and fidelity in CoV RdRps. We modeled the CoV murine hepatitis virus (MHV) nsp12-RdRp structure and superimposed it on solved picornaviral RdRp structures. Fidelity-altering mutations previously identified in coxsackie virus B3 (CVB3) were mapped onto the nsp12-RdRp model structure and then engineered into the MHV genome with [nsp14-ExoN(+)] or without [nsp14-ExoN(−)] ExoN activity. Using this method, we identified two mutations conferring resistance to the mutagen 5-fluorouracil (5-FU): nsp12-M611F and nsp12-V553I. For nsp12-V553I, we also demonstrate resistance to the mutagen 5-azacytidine (5-AZC) and decreased accumulation of mutations. Resistance to 5-FU, and a decreased number of genomic mutations, was effectively masked by nsp14-ExoN proofreading activity. These results indicate that nsp12-RdRp likely functions in fidelity regulation and that, despite low sequence conservation, some determinants of RdRp nucleotide selectivity are conserved across RNA viruses. The results also indicate that, with regard to nucleotide selectivity, nsp14-ExoN is epistatic to nsp12-RdRp, consistent with its proposed role in a multiprotein replicase-proofreading complex. IMPORTANCE RNA viruses have evolutionarily fine-tuned replication fidelity to balance requirements for genetic stability and diversity. Responsibility for replication fidelity in RNA viruses has been attributed to the RNA-dependent RNA polymerases, with mutations in RdRps for multiple RNA viruses shown to alter fidelity and attenuate virus replication and virulence. Coronaviruses (CoVs) are the only known RNA viruses to encode a proofreading exonuclease (nsp14-ExoN), as well as other replicase proteins involved in regulation of fidelity. This report shows that the CoV RdRp (nsp12) likely functions in replication fidelity; that residue determinants of CoV RdRp nucleotide selectivity map to similar structural regions of other, unrelated RNA viral polymerases; and that for CoVs, the proofreading activity of the nsp14-ExoN is epistatic to the function of the RdRp in fidelity.

01 Jan 2016
Abstract: Human immunodeficiency virus type 1 (HIV-1) is genetically highly variable. This is attributed to the error-prone nature of HIV-1 replication and its proclivity for recombination. During replication and recombination, reverse transcriptase (RT) must polymerize DNA to the 5' ends of multiple RNA and DNA template termini while converting HIV-1 RNA to double-stranded DNA. We have determined the fidelity of HIV-1 RT in vitro during polymerization to the 5' ends of HIV-1 long terminal repeat DNA template sequences and to the end of a partial HIV-1 genomic RNA template that mimics a recombination intermediate. HIV-1 RT readily extended recessed DNA primers to form full-length blunt-end DNA-DNA and DNA-RNA duplexes. In addition, HIV-1 RT catalyzed high yields of products with one to four extra nucleotides at the 3' ends of the nascent DNAs. These products were formed processively via a nontemplated mechanism that is highly specific for the addition of purine nucleotides (A > G >> T > or = C). Thus, HIV-1 RT is extremely unfaithful at both DNA and RNA template ends, introducing errors (extra nucleotides) in one out of every two or three nascent strands processively polymerized. This error rate is 1000 times higher than for HIV-1 RT-catalyzed errors at internal template positions. Blunt-end additions were also catalyzed by other retroviral RTs at relative rates of HIV-1 approximately Moloney murine leukemia virus > avian myeloblastosis virus. These data suggest a potentially important mechanism for retroviral mutation mediated by nontemplated blunt-end addition of purines prior to forced copy-choice recombination.

Journal ArticleDOI
TL;DR: The major progress that has been made in recent years in unravelling the structure and functions of this protein complex is reviewed, enabling structure‐aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap‐binding PB2 subunit.
Abstract: Influenza viruses cause seasonal epidemics and pandemic outbreaks associated with significant morbidity and mortality, and a huge cost. Since resistance to the existing anti-influenza drugs is rising, innovative inhibitors with a different mode of action are urgently needed. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. We here review the major progress that has been made in recent years in unravelling the structure and functions of this protein complex, enabling structure-aided drug design toward the core regions of the PA endonuclease, PB1 polymerase, or cap-binding PB2 subunit. Alternatively, inhibitors may target a protein-protein interaction site, a cellular factor involved in viral RNA synthesis, the viral RNA itself, or the nucleoprotein component of the viral ribonucleoprotein. The latest advances made for these diverse pharmacological targets have yielded agents in advanced (i.e., favipiravir and VX-787) or early clinical testing, besides several experimental inhibitors in various stages of development, which are all covered here.

Journal ArticleDOI
TL;DR: A FluB polymerase structure with a bound complementary cRNA 5′ end that exhibits a major rearrangement of the subdomains within the C-terminal two-thirds of PB2 (PB2-C) is presented.

Journal ArticleDOI
TL;DR: Seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template–product duplex.
Abstract: Viral RNA-dependent RNA polymerases (RdRPs) play essential roles in viral genome replication and transcription. We previously reported several structural states of the poliovirus RdRP nucleotide addition cycle (NAC) that revealed a unique palm domain-based active site closure mechanism and proposed a six-state NAC model including a hypothetical state representing translocation intermediates. Using the RdRP from another human enterovirus, enterovirus 71, here we report seven RdRP elongation complex structures derived from a crystal lattice that allows three NAC events. These structures suggested a key order of events in initial NTP binding and NTP-induced active site closure and revealed a bona fide translocation intermediate featuring asymmetric movement of the template–product duplex. Our work provides essential missing links in understanding NTP recognition and translocation mechanisms in viral RdRPs and emphasizes the uniqueness of the viral RdRPs compared with other processive polymerases.

Journal ArticleDOI
TL;DR: In this paper, a pan-serotype and cell-active DENV RdRp inhibitors are proposed to hinder the conformational changes during its transition from initiation to elongation.
Abstract: Flaviviruses comprise major emerging pathogens such as dengue virus (DENV) or Zika virus (ZIKV). The flavivirus RNA genome is replicated by the RNA-dependent-RNA polymerase (RdRp) domain of non-structural protein 5 (NS5). This essential enzymatic activity renders the RdRp attractive for antiviral therapy. NS5 synthesizes viral RNA via a "de novo" initiation mechanism. Crystal structures of the flavivirus RdRp revealed a "closed" conformation reminiscent of a pre-initiation state, with a well ordered priming loop that extrudes from the thumb subdomain into the dsRNA exit tunnel, close to the "GDD" active site. To-date, no allosteric pockets have been identified for the RdRp, and compound screening campaigns did not yield suitable drug candidates. Using fragment-based screening via X-ray crystallography, we found a fragment that bound to a pocket of the apo-DENV RdRp close to its active site (termed "N pocket"). Structure-guided improvements yielded DENV pan-serotype inhibitors of the RdRp de novo initiation activity with nano-molar potency that also impeded elongation activity at micro-molar concentrations. Inhibitors exhibited mixed inhibition kinetics with respect to competition with the RNA or GTP substrate. The best compounds have EC50 values of 1-2 μM against all four DENV serotypes in cell culture assays. Genome-sequencing of compound-resistant DENV replicons, identified amino acid changes that mapped to the N pocket. Since inhibitors bind at the thumb/palm interface of the RdRp, this class of compounds is proposed to hinder RdRp conformational changes during its transition from initiation to elongation. This is the first report of a class of pan-serotype and cell-active DENV RdRp inhibitors. Given the evolutionary conservation of residues lining the N pocket, these molecules offer insights to treat other serious conditions caused by flaviviruses.

Journal ArticleDOI
TL;DR: The results significantly advance the understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus.
Abstract: Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus.

Journal ArticleDOI
TL;DR: Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization.
Abstract: Chromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a “non-canonical” form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM. We performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs. Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.

Journal ArticleDOI
TL;DR: These findings provide the first evidence that G-quadruplex RNA is present in a negative-sense RNA virus and may represent a new therapeutic strategy against Ebola virus disease.

Journal ArticleDOI
TL;DR: A potential role for CoV nsp14 ExoN activity in counteracting the antiviral response is proposed, which could serve as a novel target for the design of antiviral strategies.
Abstract: UNLABELLED Coronavirus (CoV) nonstructural protein 14 (nsp14) is a 60-kDa protein encoded by the replicase gene that is part of the replication-transcription complex. It is a bifunctional enzyme bearing 3'-to-5' exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) activities. ExoN hydrolyzes single-stranded RNAs and double-stranded RNAs (dsRNAs) and is part of a proofreading system responsible for the high fidelity of CoV replication. nsp14 N7-MTase activity is required for viral mRNA cap synthesis and prevents the recognition of viral mRNAs as "non-self" by the host cell. In this work, a set of point mutants affecting different motifs within the ExoN domain of nsp14 was generated, using transmissible gastroenteritis virus as a model of Alphacoronavirus Mutants lacking ExoN activity were nonviable despite being competent in both viral RNA and protein synthesis. A specific mutation within zinc finger 1 (ZF-C) led to production of a viable virus with growth and viral RNA synthesis kinetics similar to that of the parental virus. Mutant recombinant transmissible gastroenteritis virus (TGEV) ZF-C (rTGEV-ZF-C) caused decreased cytopathic effect and apoptosis compared with the wild-type virus and reduced levels of dsRNA accumulation at late times postinfection. Consequently, the mutant triggered a reduced antiviral response, which was confirmed by evaluating different stages of the dsRNA-induced antiviral pathway. The expression of beta interferon (IFN-β), tumor necrosis factor (TNF), and interferon-stimulated genes in cells infected with mutant rTGEV-ZF-C was reduced compared to the levels seen with the parental virus. Overall, our data revealed a potential role for CoV nsp14 in modulation of the innate immune response. IMPORTANCE The innate immune response is the first line of antiviral defense that culminates in the synthesis of interferon and proinflammatory cytokines to control viral replication. CoVs have evolved several mechanisms to counteract the innate immune response at different levels, but the role of CoV-encoded ribonucleases in preventing activation of the dsRNA-induced antiviral response has not been described to date. The introduction of a mutation in zinc finger 1 of the ExoN domain of nsp14 led to production of a virus that induced a weak antiviral response, most likely due to the accumulation of lower levels of dsRNA in the late phases of infection. These observations allowed us to propose a novel role for CoV nsp14 ExoN activity in counteracting the antiviral response, which could serve as a novel target for the design of antiviral strategies.

Journal ArticleDOI
TL;DR: This work has shown that chemical labeling coupled with LC-MS enables the sensitive and simultaneous detection of the oxidative products of 5-methylcytosine in RNA of mammals.
Abstract: Similar to the reversible epigenetic modifications on DNA, dynamic RNA modifications were recently considered to constitute another realm for biological regulation in the form of "RNA epigenetics" 5-Methylcytosine (5-mC) has long been known to be present in RNA from all three kingdoms of life However, the functions of 5-mC in RNA have not been fully understood, especially for the RNA demethylation mechanism The discovery of 5-hydroxymethylcytosine (5-hmC) in RNA together with our recently reported 5-formylcytosine (5-foC) in RNA indicated that 5-mC in RNA may undergo the same cytosine oxidation demethylation pathway with generating intermediates 5-hmC, 5-foC, and 5-carboxylcytosine (5-caC) by ten-eleven translocation (Tet) proteins as that in DNA However, endogenous 5-caC in RNA has not been observed so far In the current study, we established a method using chemical labeling coupled with liquid chromatography-mass spectrometry analysis for the sensitive and simultaneous determination of the oxidative products of 5-mC Our results demonstrated that the detection sensitivities of 5-mC, 5-hmC, 5-foC and 5-caC in RNA increased by 70-313 folds upon 2-bromo-1-(4-diethylaminophenyl)-ethanone (BDEPE) labeling Using this method, we discovered the existence of 5-caC in the RNA of mammals In addition, we found the 5-mC occurs in all RNA species including mRNA, 28S rRNA, 18S rRNA and small RNA (<200 nt) However, 5-hmC, 5-foC and 5-caC mainly occur in mRNA, and barely detected in other types of RNA Furthermore, we found that the content of 5-hmC in the RNA of human colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) tissues significantly decreased compared to tumor adjacent normal tissues, suggesting that 5-hmC in RNA may play certain functional roles in the regulation of cancer development and formation

Journal ArticleDOI
TL;DR: A patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype is presented and viral mutations associated with treatment failure are identified and investigated to investigate the underlying causes of treatment failure.

Journal ArticleDOI
TL;DR: The approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.
Abstract: To decrypt the regulatory code of the genome, sequence elements must be defined that determine the kinetics of RNA metabolism and thus gene expression. Here, we attempt such decryption in an eukaryotic model organism, the fission yeast S. pombe. We first derive an improved genome annotation that redefines borders of 36% of expressed mRNAs and adds 487 non-coding RNAs (ncRNAs). We then combine RNA labeling in vivo with mathematical modeling to obtain rates of RNA synthesis and degradation for 5,484 expressed RNAs and splicing rates for 4,958 introns. We identify functional sequence elements in DNA and RNA that control RNA metabolic rates and quantify the contributions of individual nucleotides to RNA synthesis, splicing, and degradation. Our approach reveals distinct kinetics of mRNA and ncRNA metabolism, separates antisense regulation by transcription interference from RNA interference, and provides a general tool for studying the regulatory code of genomes.

Journal ArticleDOI
Hao-Ying Zhang1, Jun Xiong1, Bao-Ling Qi1, Yu-Qi Feng1, Bi-Feng Yuan1 
TL;DR: A novel strategy by oxidation-derivatization combined mass spectrometry analysis for the determination of 5-hydroxymethylcytosine and 5-formylcyTosine in both DNA and RNA is developed.

Journal ArticleDOI
TL;DR: The X-ray structure of NS5 presented here suggests thatMTase and RdRp activities could be coordinated as a dimer during viral genome replication, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RoadRp domains.
Abstract: Flavivirus nonstructural protein 5 (NS5) consists of methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) domains, which catalyze 5’-RNA capping/methylation and RNA synthesis, respectively, during viral genome replication. Although the crystal structure of flavivirus NS5 is known, no data about the quaternary organization of the functional enzyme are available. We report the crystal structure of dengue virus full-length NS5, where eight molecules of NS5 are arranged as four independent dimers in the crystallographic asymmetric unit. The relative orientation of each monomer within the dimer, as well as the orientations of the MTase and RdRp domains within each monomer, is conserved, suggesting that these structural arrangements represent the biologically relevant conformation and assembly of this multi-functional enzyme. Essential interactions between MTase and RdRp domains are maintained in the NS5 dimer via inter-molecular interactions, providing evidence that flavivirus NS5 can adopt multiple conformations while preserving necessary interactions between the MTase and RdRp domains. Furthermore, many NS5 residues that reduce viral replication are located at either the inter-domain interface within a monomer or at the inter-molecular interface within the dimer. Hence the X-ray structure of NS5 presented here suggests that MTase and RdRp activities could be coordinated as a dimer during viral genome replication.

Journal ArticleDOI
TL;DR: The recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide–resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states, is provided.
Abstract: The transcription cycle of RNA polymerase II (Pol II) correlates with changes to the phosphorylation state of its large subunit C-terminal domain (CTD). We recently developed Native Elongation Transcript sequencing using mammalian cells (mNET-seq), which generates single-nucleotide-resolution genome-wide profiles of nascent RNA and co-transcriptional RNA processing that are associated with different CTD phosphorylation states. Here we provide a detailed protocol for mNET-seq. First, Pol II elongation complexes are isolated with specific phospho-CTD antibodies from chromatin solubilized by micrococcal nuclease digestion. Next, RNA derived from within the Pol II complex is size fractionated and Illumina sequenced. Using mNET-seq, we have previously shown that Pol II pauses at both ends of protein-coding genes but with different CTD phosphorylation patterns, and we have also detected phosphorylation at serine 5 (Ser5-P) CTD-specific splicing intermediates and Pol II accumulation over co-transcriptionally spliced exons. With moderate biochemical and bioinformatic skills, mNET-seq can be completed in ∼6 d, not including sequencing and data analysis.

Book ChapterDOI
TL;DR: This work reviews the current understanding of coronavirus cis-acting RNA elements, focusing on elements required for genome replication and packaging and discusses the structural and functional features of these cis- acting RNA elements and their specific functions in coronav virus RNA synthesis.
Abstract: Coronaviruses have exceptionally large RNA genomes of approximately 30 kilobases. Genome replication and transcription is mediated by a multisubunit protein complex comprised of more than a dozen virus-encoded proteins. The protein complex is thought to bind specific cis-acting RNA elements primarily located in the 5'- and 3'-terminal genome regions and upstream of the open reading frames located in the 3'-proximal one-third of the genome. Here, we review our current understanding of coronavirus cis-acting RNA elements, focusing on elements required for genome replication and packaging. Recent bioinformatic, biochemical, and genetic studies suggest a previously unknown level of conservation of cis-acting RNA structures among different coronavirus genera and, in some cases, even beyond genus boundaries. Also, there is increasing evidence to suggest that individual cis-acting elements may be part of higher-order RNA structures involving long-range and dynamic RNA-RNA interactions between RNA structural elements separated by thousands of nucleotides in the viral genome. We discuss the structural and functional features of these cis-acting RNA elements and their specific functions in coronavirus RNA synthesis.

Journal ArticleDOI
TL;DR: Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture, which advances the understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.
Abstract: RNA-dependent RNA polymerases (RdRps) are used by RNA viruses to replicate and transcribe their RNA genomes(1). They adopt a closed, right-handed fold with conserved subdomains called palm, fingers and thumb(1,2). Conserved RdRp motifs A-F coordinate the viral RNA template, NTPs and magnesium ions to facilitate nucleotide condensation(1). For the initiation of RNA synthesis, most RdRps use either a primer-dependent or de novo mechanism(3). The influenza A virus RdRp, in contrast, uses a capped RNA oligonucleotide to initiate transcription, and a combination of terminal and internal de novo initiation for replication(4). To understand how the influenza A virus RdRp coordinates these processes, we analysed the function of a thumb subdomain β-hairpin using initiation, elongation and single-molecule Forster resonance energy transfer (sm-FRET) assays. Our data indicate that this β-hairpin is essential for terminal initiation during replication, but not necessary for internal initiation and transcription. Analysis of individual residues in the tip of the β-hairpin shows that PB1 proline 651 is critical for efficient RNA synthesis in vitro and in cell culture. Overall, this work advances our understanding of influenza A virus RNA synthesis and identifies the initiation platform of viral replication.

Journal ArticleDOI
TL;DR: Interplay between two RNA viruses is reported, in which the capsidless (+)ssRNA virus (YkV1), hijacks the Capsid protein of the ds RNA virus ( YnV1, and replicates as if it were a dsRNA virus.
Abstract: Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal totiviruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+)ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.