Efficient RNA virus targeting via CRISPR-CasRx in fish

2021 ◽  
Author(s):  
Qing Wang ◽  
Yun Liu ◽  
Chong Han ◽  
Min Yang ◽  
Fengqi Huang ◽  
...  
Keyword(s):  
Interference Effect ◽  
Rna Viruses ◽  
Rna Virus ◽  
Economic Losses ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus ◽  
Viral Pathogens ◽  
Positive Strand Rna ◽  
Rna Knockdown

The emergence of the CRISPR-Cas system as a technology has transformed our ability to modify nucleic acids, and the CRISPR-Cas13 system has been used to target RNA. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that may have an interference effect on RNA viruses. However, the RNA virus-targeting activity of CasRx still needs to be verified in vivo in vertebrates. In this study, we successfully engineered a highly effective CasRx system for fish virus interference. We designed synthetic mRNA coding for CasRx and used CRISPR RNAs to guide it to target the grouper nervous necrosis virus (RGNNV). This technique resulted in significant interference with virus infections both in vitro and in vivo . These results indicate that CRISPR/CasRx can be used to engineer interference against RNA viruses in fish, which provides a potential novel mechanism for RNA-guided immunity against other RNA viruses in vertebrates. Importance RNA viruses are most important viral pathogens infecting vertebrates and mammals. RNA virus populations are highly dynamic due to short generation times, large population sizes, and high mutation frequencies. Therefore, it is difficult to find a widely effective ways to inhibit RNA viruses. Therefore, we urgently need to develop effective antiviral methods. CasRx is a small sized type VI-D effector (Cas13d) with RNA knockdown efficiency that can have an interference effect on RNA viruses. Nervous necrosis virus (NNV), a non-enveloped positive-strand RNA virus, is one of the most serious viral pathogens infecting more than 40 cultured fish species resulting in huge economic losses worldwide. Here, we establish a novel efective CasRx system for RNA virus interference using NNV and grouper (Epinephelus coioices) as model. Our data show that CasRx have the most robust for RNA virus interference applications in fish and demonstrate its suitability for studying key questions relating to virus biology.

scholarly journals Interactions between the Structural Domains of the RNA Replication Proteins of Plant-Infecting RNA Viruses

1998 ◽  
Vol 72 (9) ◽  
pp. 7160-7169 ◽  
Author(s):  
Erin K. O’Reilly ◽  
Zhaohui Wang ◽  
Roy French ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Brome Mosaic Virus ◽  
Replication Proteins ◽  
Mutant Proteins ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Brome mosaic virus (BMV), a positive-strand RNA virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with N-terminal putative capping and C-terminal helicase-like sequences. These two proteins are part of a multisubunit complex which is necessary for viral RNA replication. We have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo RNA replication assays and biochemical assays of protein-protein interaction, thus permitting the identification of additional interacting domains. We now map an interaction found to take place between two 1a proteins. Using previously characterized 1a mutants, a perfect correlation was found between the in vivo phenotypes of these mutants and their abilities to interact with wild-type 1a (wt1a) and each other. Western blot analysis revealed that the stabilities of many of the noninteracting mutant proteins were similar to that of wt1a. Deletion analysis of 1a revealed that the N-terminal 515 residues of the 1a protein are required and sufficient for 1a-1a interaction. This intermolecular interaction between the putative capping domain and itself was detected in another tripartite RNA virus, cucumber mosaic virus (CMV), suggesting that the 1a-1a interaction is a feature necessary for the replication of tripartite RNA viruses. The boundaries for various activities are placed in the context of the predicted secondary structures of several 1a-like proteins of members of the alphavirus-like superfamily. Additionally, we found a novel interaction between the putative capping and helicase-like portions of the BMV and CMV 1a proteins. Our cumulative data suggest a working model for the assembly of the BMV RNA replicase.

scholarly journals Essential role of IPS-1 in innate immune responses against RNA viruses

2006 ◽  
Vol 203 (7) ◽  
pp. 1795-1803 ◽  
Author(s):  
Himanshu Kumar ◽  
Taro Kawai ◽  
Hiroki Kato ◽  
Shintaro Sato ◽  
Ken Takahashi ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Critical Role ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Innate Immune Responses ◽  
Antiviral Responses ◽  
Deficient Mice

IFN-β promoter stimulator (IPS)-1 was recently identified as an adapter for retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda5), which recognize distinct RNA viruses. Here we show the critical role of IPS-1 in antiviral responses in vivo. IPS-1–deficient mice showed severe defects in both RIG-I– and Mda5-mediated induction of type I interferon and inflammatory cytokines and were susceptible to RNA virus infection. RNA virus–induced interferon regulatory factor-3 and nuclear factor κB activation was also impaired in IPS-1–deficient cells. IPS-1, however, was not essential for the responses to either DNA virus or double-stranded B-DNA. Thus, IPS-1 is the sole adapter in both RIG-I and Mda5 signaling that mediates effective responses against a variety of RNA viruses.

scholarly journals Capsid amino acids at positions 247 and 270 are involved in the virulence of betanodaviruses to European sea bass

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patricia Moreno ◽  
Sandra Souto ◽  
Rocio Leiva-Rebollo ◽  
Juan J. Borrego ◽  
Isabel Bandín ◽  
...  
Keyword(s):  
Amino Acids ◽  
Immune Response ◽  
Fish Species ◽  
Sea Bass ◽  
Fish Mortality ◽  
Wild Type Virus ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus ◽  
European Sea Bass

Abstract European sea bass (Dicentrarchus labrax) is severely affected by nervous necrosis disease, caused by nervous necrosis virus (NNV). Two out of the four genotypes of this virus (red-spotted grouper nervous necrosis virus, RGNNV; and striped jack nervous necrosis virus, SJNNV) have been detected in sea bass, although showing different levels of virulence to this fish species. Thus, sea bass is highly susceptible to RGNNV, whereas outbreaks caused by SJNNV have not been reported in this fish species. The role of the capsid protein (Cp) amino acids 247 and 270 in the virulence of a RGNNV isolate to sea bass has been evaluated by the generation of recombinant RGNNV viruses harbouring SJNNV-type amino acids in the above mentioned positions (Mut247Dl965, Mut270Dl965 and Mut247 + 270Dl965). Viral in vitro and in vivo replication, virus virulence and fish immune response triggered by these viruses have been analysed. Mutated viruses replicated on E-11 cells, although showing some differences compared to the wild type virus, suggesting that the mutations can affect the viral cell recognition and entry. In vivo, fish mortality caused by mutated viruses was 75% lower, and viral replication in sea bass brain was altered compared to non-mutated virus. Regarding sea bass immune response, mutated viruses triggered a lower induction of IFN I system and inflammatory response-related genes. Furthermore, mutations caused changes in viral serological properties (especially the mutation in amino acid 270), inducing higher seroconversion and changing antigen recognition.

scholarly journals Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists

2015 ◽  
Vol 89 (15) ◽  
pp. 8011-8025 ◽  
Author(s):  
Cindy Chiang ◽  
Vladimir Beljanski ◽  
Kevin Yin ◽  
David Olagnier ◽  
Fethia Ben Yebdri ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Inflammatory Responses ◽  
Rna Viruses ◽  
Rna Virus ◽  
A549 Cells ◽  
Antiviral Response ◽  
Interferon Response

ABSTRACTThe cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5′-triphosphate (5′ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5′ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5′pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5′pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replicationin vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatmentin vivoprolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5′pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses.IMPORTANCEThe development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral responsein vitroandin vivoand was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5′pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

scholarly journals Betanodavirus infection in the freshwater model fish medaka (Oryzias latipes)

2006 ◽  
Vol 87 (8) ◽  
pp. 2333-2339 ◽  
Author(s):  
Ryo Furusawa ◽  
Yasushi Okinaka ◽  
Toshihiro Nakai
Keyword(s):  
Marine Fish ◽  
Fish Species ◽  
Reverse Genetics ◽  
Developmental Stages ◽  
Rna Virus ◽  
Oryzias Latipes ◽  
Engineering System ◽  
Nervous Necrosis Virus ◽  
Necrosis Virus ◽  
Model Fish

Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite, positive-sense RNA genomes. As their genomes are the smallest and simplest among viruses, betanodaviruses have been studied in detail as model viruses by using a genetic-engineering system, as has occurred with the insect alphanodaviruses, the other members of the family Nodaviridae. However, studies of virus–host interactions have been limited, as betanodaviruses basically infect marine fish at early developmental stages (larval and juvenile). These fish are only available for a few months of the year and are not suitable for the construction of a reverse-genetics system. To overcome these problems, several freshwater fish species were tested for their susceptibility to betanodaviruses. It was found that adult medaka (Oryzias latipes), a well-known model fish, was susceptible to both Striped jack nervous necrosis virus (the type species of the genus Betanodavirus) and Redspotted grouper nervous necrosis virus (RGNNV), which have different host specificities in marine fish species. Infected medaka exhibited erratic swimming and the viruses were localized specifically in the brain, spinal cord and retina of the infected fish, similar to the pattern of infection in naturally infected marine fish. Moreover, medaka were susceptible to RGNNV at the larval stage. This is the first report of a model virus–model host infection system in fish. This system should facilitate elucidation of the mechanisms underlying RNA virus infections in fish.

scholarly journals Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

2015 ◽  
Vol 89 (12) ◽  
pp. 6184-6195 ◽  
Author(s):  
Xiuling Cao ◽  
Xuejiao Jin ◽  
Xiaofeng Zhang ◽  
Ying Li ◽  
Chunyan Wang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rna Viruses ◽  
Rna Virus ◽  
Three Dimensional ◽  
Replication Protein ◽  
Positive Strand ◽  
Content Type ◽  
Positive Strand Rna ◽  
Tomographic Analysis ◽  
Beet Black Scorch Virus

ABSTRACTAll well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of theBeet black scorch virus(BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infectedNicotiana benthamianacells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes.IMPORTANCEAssembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes.Beet black scorch virus, a necrovirus in the familyTombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed inN. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

scholarly journals Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

2014 ◽  
Vol 89 (1) ◽  
pp. 275-286 ◽  
Author(s):  
Grace Campagnola ◽  
Seth McDonald ◽  
Stéphanie Beaucourt ◽  
Marco Vignuzzi ◽  
Olve B. Peersen
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Mutation Rates ◽  
Rapid Adaptation ◽  
Replication Fidelity ◽  
Virus Growth ◽  
Polymerase Structure ◽  
Positive Strand Rna

ABSTRACTViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates andin vivofidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects onin vitronucleotide discrimination, and these effects are strongly correlated with elongation rates andin vivomutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.IMPORTANCEPositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects onin vitronucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growthin vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

scholarly journals Upper-limit mutation rate estimation for a plant RNA virus

2009 ◽  
Vol 5 (3) ◽  
pp. 394-396 ◽  
Author(s):  
Rafael Sanjuán ◽  
Patricia Agudelo-Romero ◽  
Santiago F. Elena
Keyword(s):  
Mutation Rate ◽  
Rna Viruses ◽  
Rna Virus ◽  
Plant Viruses ◽  
Mutation Rates ◽  
Methodological Error ◽  
Direct Estimate ◽  
Rate Estimation ◽  
Mutation Rate Estimation

It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus , we calculate an upper-bound mutation rate estimation of 3×10 −5 per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.

scholarly journals Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11664-11676 ◽  
Author(s):  
David J. Miller ◽  
Michael D. Schwartz ◽  
Paul Ahlquist
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Protein A ◽  
Rna Replication ◽  
Yeast Cells ◽  
Flock House Virus ◽  
Mitochondrial Membranes ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection ofDrosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

scholarly journals A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses

2008 ◽  
Vol 83 (4) ◽  
pp. 1981-1991 ◽  
Author(s):  
Huiquan Liu ◽  
Yanping Fu ◽  
Daohong Jiang ◽  
Guoqing Li ◽  
Jun Xie ◽  
...  
Keyword(s):  
Sclerotinia Sclerotiorum ◽  
Hepatitis E Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Hepatitis E ◽  
Genomic Rna ◽  
Human Virus ◽  
Positive Strand ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna

ABSTRACT Previously, we reported that three double-stranded RNA (dsRNA) segments, designated L-, M-, and S-dsRNAs, were detected in Sclerotinia sclerotiorum strain Ep-1PN. Of these, the M-dsRNA segment was derived from the genomic RNA of a potexvirus-like positive-strand RNA virus, Sclerotinia sclerotiorum debilitation-associated RNA virus. Here, we present the complete nucleotide sequence of the L-dsRNA, which is 6,043 nucleotides in length, excluding the poly(A) tail. Sequence analysis revealed the presence of a single open reading frame (nucleotide positions 42 to 5936) that encodes a protein with significant similarity to the replicases of the “alphavirus-like” supergroup of positive-strand RNA viruses. A sequence comparison of the L-dsRNA-encoded putative replicase protein containing conserved methyltransferase, helicase, and RNA-dependent RNA polymerase motifs showed that it has significant sequence similarity to the replicase of Hepatitis E virus, a virus infecting humans. Furthermore, we present convincing evidence that the virus-like L-dsRNA could replicate independently with only a slight impact on growth and virulence of its host. Our results suggest that the L-dsRNA from strain Ep-1PN is derived from the genomic RNA of a positive-strand RNA virus, which we named Sclerotinia sclerotiorum RNA virus L (SsRV-L). As far as we know, this is the first report of a positive-strand RNA mycovirus that is related to a human virus. Phylogenetic and sequence analyses of the conserved motifs of the RNA replicase of SsRV-L showed that it clustered with the rubi-like viruses and that it is related to the plant clostero-, beny- and tobamoviruses and to the insect omegatetraviruses. Considering the fact that these related alphavirus-like positive-strand RNA viruses infect a wide variety of organisms, these findings suggest that the ancestral positive-strand RNA viruses might be of ancient origin and/or they might have radiated horizontally among vertebrates, insects, plants, and fungi.

Sign in / Sign up

Export Citation Format

Share Document