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Eric Jan - One of the best experts on this subject based on the ideXlab platform.
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transmission of Cricket Paralysis Virus via exosome like vesicles during infection of drosophila cells
Scientific Reports, 2018Co-Authors: Craig H Kerr, Udit Dalwadi, Nichollas E Scott, Calvin K Yip, Leonard J Foster, Eric JanAbstract:Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped Viruses (e.g. hepatitis A Virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket Paralysis Virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped Viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.
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disruption of stress granule formation by the multifunctional Cricket Paralysis Virus 1a protein
Journal of Virology, 2017Co-Authors: Anthony Khong, Craig H Kerr, Arabinda Nayak, Clarence H L Yeung, Kathleen Keatings, Douglas W Allan, Eric JanAbstract:Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several Viruses modulate SG formation, suggesting that SGs have an impact on Virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistroVirus Cricket Paralysis Virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) Virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA Viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistroVirus Cricket Paralysis Virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant Viruses containing mutations of CrPV-1A attenuate Virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in Virus-infected cells that permits productive Virus infection.
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molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis Virus infection
Scientific Reports, 2016Co-Authors: Craig H Kerr, Sunnie R Thompson, Zi Wang, Christopher J Jang, Eric JanAbstract:The dicistroVirus Cricket Paralysis Virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce Virus, demonstrating the physiological relevance of specific structural elements within the IRES for Virus infection. However, a subset of mutations still led to Virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during Virus infection.
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temporal regulation of distinct internal ribosome entry sites of the dicistroviridae Cricket Paralysis Virus
Viruses, 2016Co-Authors: Anthony Khong, Jennifer M Bonderoff, Ruth V Spriggs, Erik Tammpere, Craig H Kerr, Thomas J Jackson, Anne E Willis, Eric JanAbstract:Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA Viruses. Cricket Paralysis Virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5′untranslated region (5′UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5′UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.
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the 5 untranslated region of a novel infectious molecular clone of the dicistroVirus Cricket Paralysis Virus modulates infection
Journal of Virology, 2015Co-Authors: Craig H Kerr, Anthony Khong, Kathleen Keatings, Douglas W Allan, Calvin K Yip, Leonard J Foster, Qing S Wang, Eric JanAbstract:Dicistroviridae are a family of RNA Viruses that possesses a single-stranded positive-sense RNA genome containing two distinct open reading frames (ORFs), each preceded by an internal ribosome entry site that drives translation of the viral structural and nonstructural proteins, respectively. The type species, Cricket Paralysis Virus (CrPV), has served as a model for studying host-Virus interactions; however, investigations into the molecular mechanisms of CrPV and other dicistroViruses have been limited as an established infectious clone was elusive. Here, we report the construction of an infectious molecular clone of CrPV. Transfection of in vitro-transcribed RNA from the CrPV clone into Drosophila Schneider line 2 (S2) cells resulted in cytopathic effects, viral RNA accumulation, detection of negative-sense viral RNA, and expression of viral proteins. Transmission electron microscopy, viral titers, and immunofluorescence-coupled transwell assays demonstrated that infectious viral particles are released from transfected cells. In contrast, mutant clones containing stop codons in either ORF decreased Virus infectivity. Injection of adult Drosophila flies with Virus derived from CrPV clones but not UV-inactivated clones resulted in mortality. Molecular analysis of the CrPV clone revealed a 196-nucleotide duplication within its 5′ untranslated region (UTR) that stimulated translation of reporter constructs. In cells infected with the CrPV clone, the duplication inhibited viral infectivity yet did not affect viral translation or RNA accumulation, suggesting an effect on viral packaging or entry. The generation of the CrPV infectious clone provides a powerful tool for investigating the viral life cycle and pathogenesis of dicistroViruses and may further understanding of fundamental host-Virus interactions in insect cells. IMPORTANCE Dicistroviridae, which are RNA Viruses that infect arthropods, have served as a model to gain insights into fundamental host-Virus interactions in insect cells. Further insights into the viral molecular mechanisms are hampered due to a lack of an established infectious clone. We report the construction of the first infectious clone of the dicistroVirus, Cricket Paralysis Virus (CrPV). We show that transfection of the CrPV clone RNA into Drosophila cells led to production of infectious particles that resemble natural CrPV virions and result in cytopathic effects and expression of CrPV proteins and RNA in infected cells. The CrPV clone should provide insights into the dicistroVirus life cycle and host-Virus interactions in insect cells. Using this clone, we find that a 196-nucleotide duplication within the 5′ untranslated region of the CrPV clone increased viral translation in reporter constructs but decreased Virus infectivity, thus revealing a balance that interplays between viral translation and replication.
Peter Sarnow - One of the best experts on this subject based on the ideXlab platform.
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temperature protects insect cells from infection by Cricket Paralysis Virus
Journal of Virology, 2010Co-Authors: Randal C Cevallos, Peter SarnowAbstract:Heat shock is a well-known stress response characterized by a rapid synthesis of a set of proteins which are responsible for protection against stress. We examined the role of temperature on the growth of Cricket Paralysis Virus, a member of the family Dicistroviridae, in insect cells. Heat shock caused an induction of heat shock protein-encoding mRNAs in uninfected cells but not in infected cells. While viral RNA and protein were abundant during heat shock, virion formation was inhibited at higher temperatures. The different susceptibility to pathogens at different temperatures is likely a crucial feature of host-pathogen interaction in cold-blooded animals.
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The Imd pathway is involved in antiviral immune responses in Drosophila.
Public Library of Science (PLoS), 2009Co-Authors: Alexandre Costa, Peter Sarnow, Eric Jan, David SchneiderAbstract:Cricket Paralysis Virus (CrPV) is a member of the Dicistroviridae family of RNA Viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between Viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway
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initiation factor independent translation mediated by the hepatitis c Virus internal ribosome entry site
RNA, 2006Co-Authors: Alissa M Lancaster, Peter SarnowAbstract:The hepatitis C viral mRNA initiates translation using an internal ribosome entry site (IRES) located in the 5′ noncoding region of the viral genome. At physiological magnesium ion concentrations, the HCV IRES forms a binary complex with the 40S ribosomal subunit, recruits initiation factor eIF3 and the ternary eIF2/GTP/Met-tRNAi Met complex, and joins 60S subunits to assemble translation-competent 80S ribosomes. Here we show that in the presence of 5 mM MgCl2, the HCV IRES can initiate translation by an alternative mechanism that does not require known initiation factors. Specifically, the HCV IRES was shown to initiate translation in a reconstituted system consisting only of purified 40S and 60S subunits, elongation factors, and aminoacylated tRNAs at high magnesium concentration. Analyses of assembled complexes supported a mechanism by which preformed 80S ribosomes can assemble directly on the HCV IRES at high cation concentrations. This mechanism is reminiscent of that employed by the divergent IRES elements in the Dicistroviridae, exemplified by the Cricket Paralysis Virus, which mediates initiation of protein synthesis without initiator tRNA.
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factor independent assembly of elongation competent ribosomes by an internal ribosome entry site located in an rna Virus that infects penaeid shrimp
Journal of Virology, 2005Co-Authors: Randal C Cevallos, Peter SarnowAbstract:The Taura syndrome Virus (TSV), a member of the Dicistroviridae family of Viruses, is a single-stranded positive-sense RNA Virus which contains two nonoverlapping reading frames separated by a 230-nucleotide intergenic region. This intergenic region contains an internal ribosome entry site (IRES) which directs the synthesis of the TSV capsid proteins. Unlike other dicistroViruses, the TSV IRES contains an AUG codon that is in frame with the capsid region, suggesting that the IRES initiates translation at this AUG codon by using initiator tRNAmet. We show here that the TSV IRES does not use this or any other AUG codon to initiate translation. Like the IRES in Cricket Paralysis Virus (CrPV), the TSV IRES can assemble 80S ribosomes in the absence of initiation factors and can direct protein synthesis in a reconstituted system that contains only purified ribosomal subunits, eukaryotic elongation factors 1A and 2, and aminoacylated tRNAs. The functional conservation of the CrPV-like IRES elements in Viruses that can infect different invertebrate hosts suggests that initiation at non-AUG codons by an initiation factor-independent mechanism may be more prevalent.
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cryo em visualization of a viral internal ribosome entry site bound to human ribosomes the ires functions as an rna based translation factor
Cell, 2004Co-Authors: Peter Sarnow, Christian M T Spahn, Anke M Mulder, Robert A Grassucci, Joachim FrankAbstract:Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in Cricket Paralysis Virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.
Craig H Kerr - One of the best experts on this subject based on the ideXlab platform.
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transmission of Cricket Paralysis Virus via exosome like vesicles during infection of drosophila cells
Scientific Reports, 2018Co-Authors: Craig H Kerr, Udit Dalwadi, Nichollas E Scott, Calvin K Yip, Leonard J Foster, Eric JanAbstract:Viruses are classically characterized as being either enveloped or nonenveloped depending on the presence or absence of a lipid bi-layer surrounding their proteinaceous capsid. In recent years, many studies have challenged this view by demonstrating that some nonenveloped Viruses (e.g. hepatitis A Virus) can acquire an envelope during infection by hijacking host cellular pathways. In this study, we examined the role of exosome-like vesicles (ELVs) during infection of Drosophilia melanogaster S2 cells by Cricket Paralysis Virus (CrPV). Utilizing quantitative proteomics, we demonstrated that ELVs can be isolated from both mock- and CrPV-infected S2 cells that contain distinct set of proteins compared to the cellular proteome. Moreover, 40 proteins increased in abundance in ELVs derived from CrPV-infected cells compared to mock, suggesting specific factors associate with ELVs during infection. Interestingly, peptides from CrPV capsid proteins (ORF2) and viral RNA were detected in ELVs from infected cells. Finally, ELVs from CrPV-infected cells are infectious suggesting that CrPV may hijack ELVs to acquire an envelope during infection of S2 cells. This study further demonstrates the diverse strategies of nonenveloped Viruses from invertebrates to vertebrates to acquire an envelope in order to evade the host response or facilitate transmission.
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a viral protein restricts drosophila rnai immunity by regulating argonaute activity and stability
Cell Host & Microbe, 2018Co-Authors: Craig H Kerr, Arabinda Nayak, Dong Young Kim, Michael J Trnka, Peter V Lidsky, David J Stanley, Brianna M Rivera, Alma L BurlingameAbstract:Summary The dicistroVirus, Cricket Paralysis Virus (CrPV) encodes an RNA interference (RNAi) suppressor, 1A, which modulates viral virulence. Using the Drosophila model, we combined structural, biochemical, and virological approaches to elucidate the strategies by which CrPV-1A restricts RNAi immunity. The atomic resolution structure of CrPV-1A uncovered a flexible loop that interacts with Argonaute 2 (Ago-2), thereby inhibiting Ago-2 endonuclease-dependent immunity. Mutations disrupting Ago-2 binding attenuates viral pathogenesis in wild-type but not Ago-2-deficient flies. CrPV-1A also contains a BC-box motif that enables the Virus to hijack a host Cul2-Rbx1-EloBC ubiquitin ligase complex, which promotes Ago-2 degradation and Virus replication. Our study uncovers a viral-based dual regulatory program that restricts antiviral immunity by direct interaction with and modulation of host proteins. While the direct inhibition of Ago-2 activity provides an efficient mechanism to establish infection, the recruitment of a ubiquitin ligase complex enables CrPV-1A to amplify Ago-2 inactivation to restrict further antiviral RNAi immunity.
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disruption of stress granule formation by the multifunctional Cricket Paralysis Virus 1a protein
Journal of Virology, 2017Co-Authors: Anthony Khong, Craig H Kerr, Arabinda Nayak, Clarence H L Yeung, Kathleen Keatings, Douglas W Allan, Eric JanAbstract:Stress granules (SGs) are cytosolic ribonucleoprotein aggregates that are induced during cellular stress. Several Viruses modulate SG formation, suggesting that SGs have an impact on Virus infection. However, the mechanisms and impact of modulating SG assembly in infected cells are not completely understood. In this study, we identify the dicistroVirus Cricket Paralysis Virus 1A (CrPV-1A) protein that functions to inhibit SG assembly during infection. Moreover, besides inhibiting RNA interference, CrPV-1A also inhibits host transcription, which indirectly modulates SG assembly. Thus, CrPV-1A is a multifunctional protein. We identify a key R146A residue that is responsible for these effects, and mutant CrPV(R146A) Virus infection is attenuated in Drosophila melanogaster S2 cells and adult fruit flies and results in increased SG formation. Treatment of CrPV(R146A)-infected cells with actinomycin D, which represses transcription, restores SG assembly suppression and viral yield. In summary, CrPV-1A modulates several cellular processes to generate a cellular environment that promotes viral translation and replication.IMPORTANCE RNA Viruses encode a limited set of viral proteins to modulate an array of cellular processes in order to facilitate viral replication and inhibit antiviral defenses. In this study, we identified a viral protein, called CrPV-1A, within the dicistroVirus Cricket Paralysis Virus that can inhibit host transcription, modulate viral translation, and block a cellular process called stress granule assembly. We also identified a specific amino acid within CrPV-1A that is important for these cellular processes and that mutant Viruses containing mutations of CrPV-1A attenuate Virus infection. We also demonstrate that the CrPV-1A protein can also modulate cellular processes in human cells, suggesting that the mode of action of CrPV-1A is conserved. We propose that CrPV-1A is a multifunctional, versatile protein that creates a cellular environment in Virus-infected cells that permits productive Virus infection.
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molecular analysis of the factorless internal ribosome entry site in Cricket Paralysis Virus infection
Scientific Reports, 2016Co-Authors: Craig H Kerr, Sunnie R Thompson, Zi Wang, Christopher J Jang, Eric JanAbstract:The dicistroVirus Cricket Paralysis Virus contains a unique dicistronic RNA genome arrangement, encoding two main open reading frames that are driven by distinct internal ribosome entry sites (IRES). The intergenic region (IGR) IRES adopts an unusual structure that directly recruits the ribosome and drives translation of viral structural proteins in a factor-independent manner. While structural, biochemical, and biophysical approaches have provided mechanistic details into IGR IRES translation, these studies have been limited to in vitro systems and little is known about the behavior of these IRESs during infection. Here, we examined the role of previously characterized IGR IRES mutations on viral yield and translation in CrPV-infected Drosophila S2 cells. Using a recently generated infectious CrPV clone, introduction of a subset of mutations that are known to disrupt IRES activity failed to produce Virus, demonstrating the physiological relevance of specific structural elements within the IRES for Virus infection. However, a subset of mutations still led to Virus production, thus revealing the key IRES-ribosome interactions for IGR IRES translation in infected cells, which highlights the importance of examining IRES activity in its physiological context. This is the first study to examine IGR IRES translation in its native context during Virus infection.
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temporal regulation of distinct internal ribosome entry sites of the dicistroviridae Cricket Paralysis Virus
Viruses, 2016Co-Authors: Anthony Khong, Jennifer M Bonderoff, Ruth V Spriggs, Erik Tammpere, Craig H Kerr, Thomas J Jackson, Anne E Willis, Eric JanAbstract:Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA Viruses. Cricket Paralysis Virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5′untranslated region (5′UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5′UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.
Christian M T Spahn - One of the best experts on this subject based on the ideXlab platform.
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cryo em of ribosomal 80s complexes with termination factors reveals the translocated Cricket Paralysis Virus ires
Molecular Cell, 2015Co-Authors: Margarita Muhs, Tatyana V Pestova, T Hilal, Thorsten Mielke, Maxim A Skabkin, Karissa Y Sanbonmatsu, Christian M T SpahnAbstract:Summary The Cricket Paralysis Virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jumpstarts translation in the elongation phase from the ribosomal A site. Here, we present cryoelectron microscopy (cryo-EM) maps of 80S⋅CrPV-STOP⋅eRF1⋅eRF3⋅GMPPNP and 80S⋅CrPV-STOP⋅eRF1 complexes, revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event, the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time, our structural analysis provides information about the binding modes of eRF1⋅eRF3⋅GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.
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Structure of the ribosome-bound Cricket Paralysis Virus IRES RNA.
Nature Structural and Molecular Biology, 2006Co-Authors: Martin Schüler, Eric Westhof, Thorsten Mielke, Sean R Connell, Aurelie Lescoute, Jan Giesebrecht, Marylena Dabrowski, Birgit Schroeer, Pawel A Penczek, Christian M T SpahnAbstract:Internal ribosome entry sites (IRESs) facilitate an alternative, end-independent pathway of translation initiation. A particular family of dicistroviral IRESs can assemble elongation-competent 80S ribosomal complexes in the absence of canonical initiation factors and initiator transfer RNA. We present here a cryo-EM reconstruction of a dicistroviral IRES bound to the 80S ribosome. The resolution of the cryo-EM reconstruction, in the subnanometer range, allowed the molecular structure of the complete IRES in its active, ribosome-bound state to be solved. The structure, harboring three pseudoknot-containing domains, each with a specific functional role, shows how defined elements of the IRES emerge from a compactly folded core and interact with the key ribosomal components that form the A, P and E sites, where tRNAs normally bind. Our results exemplify the molecular strategy for recruitment of an IRES and reveal the dynamic features necessary for internal initiation.
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cryo em visualization of a viral internal ribosome entry site bound to human ribosomes the ires functions as an rna based translation factor
Cell, 2004Co-Authors: Peter Sarnow, Christian M T Spahn, Anke M Mulder, Robert A Grassucci, Joachim FrankAbstract:Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in Cricket Paralysis Virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.
Ronald P. Van Rij - One of the best experts on this subject based on the ideXlab platform.
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Peroxisome-associated Sgroppino links fat metabolism with survival after RNA Virus infection in Drosophila.
Scientific reports, 2019Co-Authors: Sarah H. Merkling, Gijs J. Overheul, Annette Schenck, Human Riahi, Ronald P. Van RijAbstract:The fruit fly Drosophila melanogaster is a valuable model organism for the discovery and characterization of innate immune pathways, but host responses to Virus infection remain incompletely understood. Here, we describe a novel player in host defense, Sgroppino (Sgp). Genetic depletion of Sgroppino causes hypersensitivity of adult flies to infections with the RNA Viruses Drosophila C Virus, Cricket Paralysis Virus, and Flock House Virus. Canonical antiviral immune pathways are functional in Sgroppino mutants, suggesting that Sgroppino exerts its activity via an as yet uncharacterized process. We demonstrate that Sgroppino localizes to peroxisomes, organelles involved in lipid metabolism. In accordance, Sgroppino-deficient flies show a defect in lipid metabolism, reflected by higher triglyceride levels, higher body mass, and thicker abdominal fat tissue. In addition, knock-down of Pex3, an essential peroxisome biogenesis factor, increases sensitivity to Virus infection. Together, our results establish a genetic link between the peroxisomal protein Sgroppino, fat metabolism, and resistance to Virus infection.
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convergent evolution of argonaute 2 slicer antagonism in two distinct insect rna Viruses
PLOS Pathogens, 2012Co-Authors: Joël T. Van Mierlo, Gijs J. Overheul, Christophe Antoniewski, Alfred W Bronkhorst, Sajna Anand Sadanandan, Jensola Ekstrom, Marco Heestermans, Dan Hultmark, Ronald P. Van RijAbstract:RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA Viruses. We show here that Nora Virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora Virus infected Drosophila. Furthermore, we demonstrate that the Nora Virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis Virus in an RNAi dependent manner. Nora Virus VP1 and the viral suppressor of RNAi of Cricket Paralysis Virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA Viruses highlights the importance of AGO2 in antiviral defense.
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Identification of viral suppressors of RNAi by a reporter assay in Drosophila S2 cell culture.
Antiviral RNAi, 2011Co-Authors: Koen W. R. Van Cleef, Joël T. Van Mierlo, Marius Van Den Beek, Ronald P. Van RijAbstract:The RNA interference (RNAi) pathway plays an important role in antiviral immunity in insects. To -counteract the RNAi-mediated immune response of their hosts, several insect Viruses, such as Flock house Virus, Drosophila C Virus, and Cricket Paralysis Virus, encode potent viral suppressors of RNAi (VSRs). Because of the importance of RNAi in antiviral defense in insects, other insect Viruses are likely to encode VSRs as well. In this chapter, we describe a detailed protocol for an RNAi reporter assay in Drosophila S2 cells for the identification of VSR activity.
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the rna silencing endonuclease argonaute 2 mediates specific antiviral immunity in drosophila melanogaster
Genes & Development, 2006Co-Authors: Ronald P. Van Rij, Christophe Antoniewski, Mariacarla Saleh, Bassam Berry, Catherine K Foo, Andrew R Houk, Raul AndinoAbstract:Most organisms have evolved defense mechanisms to protect themselves from Viruses and other pathogens. Arthropods lack the protein-based adaptive immune response found in vertebrates. Here we show that the central catalytic component of the RNA-induced silencing complex (RISC), the nuclease Argonaute 2 (Ago-2), is essential for antiviral defense in adult Drosophila melanogaster. Ago-2-defective flies are hypersensitive to infection with a major fruit fly pathogen, Drosophila C Virus (DCV), and with Cricket Paralysis Virus (CrPV). Increased mortality in ago-2 mutant flies was associated with a dramatic increase in viral RNA accumulation and Virus titers. The physiological significance of this antiviral mechanism is underscored by our finding that DCV encodes a potent suppressor of RNA interference (RNAi). This suppressor binds long double-stranded RNA (dsRNA) and inhibits Dicer-2-mediated processing of dsRNA into short interfering RNA (siRNA), but does not bind short siRNAs or disrupt the microRNA (miRNA) pathway. Based on these results we propose that RNAi is a major antiviral immune defense mechanism in Drosophila.