scholarly journals Endomembrane targeting of human OAS1 p46 augments antiviral activity

2021 ◽  
Author(s):  
Frank W. Soveg ◽  
Johannes Schwerk ◽  
Nandan S. Gokhale ◽  
Karen Cerosaletti ◽  
Julian R. Smith ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Rna Viruses ◽  
Important Determinant ◽  
Innate Immune ◽  
Viral Rna ◽  
Endomembrane System ◽  
Oligoadenylate Synthetase ◽  
Rna Sensors ◽  
Replication Sites ◽  
Positive Strand Rna

SUMMARYMany host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from host cell innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense viral RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flavivirus, picornavirus, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform strongly associates with COVID-19 severity. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

scholarly journals Endomembrane targeting of human OAS1 p46 augments antiviral activity

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Frank W Soveg ◽  
Johannes Schwerk ◽  
Nandan S Gokhale ◽  
Karen Cerosaletti ◽  
Julian R Smith ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Antiviral Activity ◽  
Rna Viruses ◽  
Important Determinant ◽  
Innate Immune ◽  
Endomembrane System ◽  
Oligoadenylate Synthetase ◽  
Rna Sensors ◽  
Replication Sites ◽  
Positive Strand Rna

Many host RNA sensors are positioned in the cytosol to detect viral RNA during infection. However, most positive-strand RNA viruses replicate within a modified organelle co-opted from intracellular membranes of the endomembrane system, which shields viral products from cellular innate immune sensors. Targeting innate RNA sensors to the endomembrane system may enhance their ability to sense RNA generated by viruses that use these compartments for replication. Here, we reveal that an isoform of oligoadenylate synthetase 1, OAS1 p46, is prenylated and targeted to the endomembrane system. Membrane localization of OAS1 p46 confers enhanced access to viral replication sites and results in increased antiviral activity against a subset of RNA viruses including flaviviruses, picornaviruses, and SARS-CoV-2. Finally, our human genetic analysis shows that the OAS1 splice-site SNP responsible for production of the OAS1 p46 isoform correlates with protection from severe COVID-19. This study highlights the importance of endomembrane targeting for the antiviral specificity of OAS1 and suggests that early control of SARS-CoV-2 replication through OAS1-p46 is an important determinant of COVID-19 severity.

scholarly journals Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes

2018 ◽  
Vol 92 (8) ◽  
Author(s):  
Maija K. Pietilä ◽  
Martijn J. van Hemert ◽  
Tero Ahola
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rna Viruses ◽  
Viral Rna ◽  
Cellular Membranes ◽  
Positive Strand ◽  
Membrane Markers ◽  
Highly Active ◽  
Replicase Proteins ◽  
Positive Strand Rna

ABSTRACTPositive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients. This revealed that SFV infection strongly modifies cellular membranes. We removed soluble proteins, the Golgi membranes, and most of the mitochondria, but plasma membrane, endoplasmic reticulum (ER), and late endosome markers were retained in the membrane fraction that contained viral RNA synthesizing activity, replicase proteins, and minus- and plus-strand RNA. Electron microscopy revealed that the purified membranes displayed spherule-like structures with a narrow neck. This membrane enrichment was specific to viral replication, as such a distribution of membrane markers was only observed after infection. Besides the plasma membrane, SFV infection remodeled the ER, and the cofractionation of the RC-carrying plasma membrane and ER suggests that SFV recruits ER proteins or membrane to the site of replication. The purified RCs were highly active in synthesizing both genomic and subgenomic RNA. Detergent solubilization destroyed the replication activity, demonstrating that the membrane association of the complex is essential. Most of the newly made RNA was in double-stranded replicative molecules, but the purified complexes also produced single-stranded RNA as well as released newly made RNA. This indicates that the purification established here maintained the functionality of RCs and thus enables further structural and functional studies of active RCs.IMPORTANCESimilar to all positive-strand RNA viruses, the arthropod-borne alphaviruses induce membranous genome factories, but little is known about the arrangement of viral replicase proteins and the presence of host proteins in these replication complexes. To improve our knowledge of alphavirus RNA-synthesizing complexes, we isolated and purified them from infected mammalian cells. Detection of viral RNA andin vitroreplication assays revealed that these complexes are abundant and highly active when located on the plasma membrane. After multiple purification steps, they remain functional in synthesizing and releasing viral RNA. Besides the plasma membrane, markers for the endoplasmic reticulum and late endosomes were enriched with the replication complexes, demonstrating that alphavirus infection modified cellular membranes beyond inducing replication spherules on the plasma membrane. We have developed here a gentle purification method to obtain large quantities of highly active replication complexes, and similar methods can be applied to other positive-strand RNA viruses.

scholarly journals Double-Stranded RNA Is Produced by Positive-Strand RNA Viruses and DNA Viruses but Not in Detectable Amounts by Negative-Strand RNA Viruses

2006 ◽  
Vol 80 (10) ◽  
pp. 5059-5064 ◽  
Author(s):  
Friedemann Weber ◽  
Valentina Wagner ◽  
Simon B. Rasmussen ◽  
Rune Hartmann ◽  
Søren R. Paludan
Keyword(s):  
Viral Infections ◽  
Rna Viruses ◽  
Innate Immune ◽  
Genome Replication ◽  
Dna Viruses ◽  
Double Stranded Rna ◽  
Positive Strand ◽  
Negative Strand ◽  
A Genome ◽  
Positive Strand Rna

ABSTRACT Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

scholarly journals Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

2016 ◽  
Vol 113 (8) ◽  
pp. E1064-E1073 ◽  
Author(s):  
Jiantao Zhang ◽  
Zhenlu Zhang ◽  
Vineela Chukkapalli ◽  
Jules A. Nchoutmboube ◽  
Jianhui Li ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Viruses ◽  
Critical Role ◽  
Brome Mosaic Virus ◽  
Alternate Host ◽  
Positive Strand ◽  
Cellular Processes ◽  
Replication Sites ◽  
Positive Strand Rna

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting theCHO2gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.

scholarly journals Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Kirsi Hellström ◽  
Katri Kallio ◽  
Age Utt ◽  
Tania Quirin ◽  
Eija Jokitalo ◽  
...  
Keyword(s):  
Rna Viruses ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Complex Assembly ◽  
Positive Strand ◽  
Replication Complex ◽  
Active Replication ◽  
Replicase Proteins ◽  
Polyprotein Precursor ◽  
Positive Strand Rna

ABSTRACT Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures. However, we now find that specific partially processed forms of the replicase proteins alone can give rise to membrane invaginations in the absence of RNA or replication. The minimal requirement for spherule formation was the expression of properly cleaved nsP4, together with either uncleaved P123 or with the combination of nsP1 and uncleaved P23. These inactive spherules were morphologically less regular than replication-induced spherules. In the presence of template, nsP1 plus uncleaved P23 plus nsP4 could efficiently assemble active replication spherules producing both negative-sense and positive-sense RNA strands. P23 alone did not have membrane affinity, but could be recruited to membrane sites in the presence of nsP1 and nsP4. These results define the set of viral components required for alphavirus replication complex assembly and suggest the possibility that it could be reconstituted from separately expressed nonstructural proteins. IMPORTANCE All positive-strand RNA viruses extensively modify host cell membranes to serve as efficient platforms for viral RNA replication. Alphaviruses and several other groups induce protective membrane invaginations (spherules) as their genome factories. Most positive-strand viruses produce their replicase as a polyprotein precursor, which is further processed through precise and regulated cleavages. We show here that specific cleavage intermediates of the alphavirus replicase can give rise to spherule structures in the absence of viral RNA. In the presence of template RNA, the same intermediates yield active replication complexes. Thus, partially cleaved replicase proteins play key roles that connect replication complex assembly, membrane deformation, and the different stages of RNA synthesis.

scholarly journals The Small-Compound Inhibitor K22 Displays Broad Antiviral Activity against Different Members of the Family Flaviviridae and Offers Potential as a Panviral Inhibitor

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Obdulio García-Nicolás ◽  
Philip V'kovski ◽  
Nathalie J. Vielle ◽  
Nadine Ebert ◽  
Roland Züst ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Host Cell ◽  
Rna Viruses ◽  
Close Association ◽  
Viral Inhibition ◽  
Virus Family ◽  
Positive Strand ◽  
Content Type ◽  
Small Compound ◽  
Positive Strand Rna

ABSTRACT The virus family Flaviviridae encompasses several viruses, including (re)emerging viruses which cause widespread morbidity and mortality throughout the world. Members of this virus family are positive-strand RNA viruses and replicate their genome in close association with reorganized intracellular host cell membrane compartments. This evolutionarily conserved strategy facilitates efficient viral genome replication and contributes to evasion from host cell cytosolic defense mechanisms. We have previously described the identification of a small-compound inhibitor, K22, which exerts a potent antiviral activity against a broad range of coronaviruses by targeting membrane-bound viral RNA replication. To analyze the antiviral spectrum of this inhibitor, we assessed the inhibitory potential of K22 against several members of the Flaviviridae family, including the reemerging Zika virus (ZIKV). We show that ZIKV is strongly affected by K22. Time-of-addition experiments revealed that K22 acts during a postentry phase of the ZIKV life cycle, and combination regimens of K22 together with ribavirin (RBV) or interferon alpha (IFN-α) further increased the extent of viral inhibition. Ultrastructural electron microscopy studies revealed severe alterations of ZIKV-induced intracellular replication compartments upon infection of K22-treated cells. Importantly, the antiviral activity of K22 was demonstrated against several other members of the Flaviviridae family. It is tempting to speculate that K22 exerts its broad antiviral activity against several positive-strand RNA viruses via a similar mechanism and thereby represents an attractive candidate for development as a panviral inhibitor.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

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.

Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication

2021 ◽  
Author(s):  
Sari Mattila ◽  
Pirjo Merilahti ◽  
Sarah Wazir ◽  
Tania Quirin ◽  
Mirko M. Maksimainen ◽  
...  
Keyword(s):  
Semliki Forest Virus ◽  
Rna Viruses ◽  
Virus Production ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Cells ◽  
Febrile Disease ◽  
Replication Stage ◽  
Replication Systems ◽  
Positive Strand Rna

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human MacroD1 protein inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression. The inhibitor was similarly active in alphavirus trans -replication systems, indicating that it targets the viral RNA replication stage.

scholarly journals Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity

2017 ◽  
Vol 92 (1) ◽  
Author(s):  
James Brett Case ◽  
Yize Li ◽  
Ruth Elliott ◽  
Xiaotao Lu ◽  
Kevin W. Graepel ◽  
...  
Keyword(s):  
Immune Response ◽  
Virus Replication ◽  
Innate Immune Response ◽  
Rna Viruses ◽  
Hepatitis Virus ◽  
Innate Immune ◽  
Viral Rna ◽  
Wild Type ◽  
Murine Hepatitis Virus ◽  
Antiviral State

ABSTRACTCoronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3′-to-5′ exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the orderNidovirales, arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(−)] are sensitive to cellular pretreatment with interferon beta (IFN-β) in a dose-dependent manner. In addition, ExoN(−) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR−/−) BMMs. ExoN(−) virus replication did not result in IFN-β gene expression, and in the presence of an IFN-β-mediated antiviral state, ExoN(−) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(−) virus generated from IFN-β-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(−) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity.IMPORTANCECoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-β and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

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