Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.
TL;DR: These studies support a model whereby induction of the UPR through Cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and theUPR represent novel targets for future development of broad spectrum antiviral therapies.
Abstract: Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.
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TL;DR: Recent advances in norovirus animal models and in vitro cultivation systems have led to an increased understanding of norov virus molecular biology and replication, pathogenesis, cell tropism, and innate and adaptive immunity, and clinical trials of vaccines consisting of nonreplicating virus-like particles have shown promise.
184 citations
Cites background from "Antiviral activity of a small molec..."
...WP1130 is a promising small molecule inhibitor of a subset of deubiquitinases that reduces MuNoV-1 titers in the small intestine (Perry et al., 2012)....
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TL;DR: It is shown that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection, providing a proof-of-principle for the use of favipIRavir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.
Abstract: Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.
134 citations
Cites background from "Antiviral activity of a small molec..."
...Inhibitors with antiviral activity in vivo have been identified for multiple RNA viruses, including norovirus (Perry et al., 2012; Rocha-Pereira et al., 2013), encouraging further studies in this direction....
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TL;DR: The current state of the art of alphavirus host cell shut-off of viral transcription and translation is reviewed, and recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly are described.
Abstract: Alphaviruses cause debilitating disease in humans and animals and are transmitted by blood-feeding arthropods, typically mosquitoes. With a traditional focus on two models, Sindbis virus and Semliki Forest virus, alphavirus research has significantly intensified in the last decade partly due to the re-emergence and dramatic expansion of chikungunya virus in Asia, Europe, and the Americas. As a consequence, alphavirus–host interactions are now understood in much more molecular detail, and important novel mechanisms have been elucidated. It has become clear that alphaviruses not only cause a general host shut-off in infected vertebrate cells, but also specifically suppress different host antiviral pathways using their viral nonstructural proteins, nsP2 and nsP3. Here we review the current state of the art of alphavirus host cell shut-off of viral transcription and translation, and describe recent insights in viral subversion of interferon induction and signaling, the unfolded protein response, and stress granule assembly.
100 citations
Cites methods from "Antiviral activity of a small molec..."
...Similar to the use of IFNs, the replication of several RNA viruses, including SINV, was inhibited when the UPR was activated with a small molecule deubiquitinase inhibitor prior to infection [113,114]....
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TL;DR: A comprehensive summary of recent research in this field revealed that about 36 viruses trigger ER stress and differentially activate ER stress-related signaling pathways, which highlights the strategies evolved by viruses to modulate ER stress -related signaling networks including immune responses in order to ensure their survival and pathogenesis.
Abstract: The endoplasmic reticulum (ER) is a cellular membrane organelle that plays important roles in virus replication and maturation. Accumulating evidence indicates that virus infection often disturbs ER homeostasis and leads to ER stress, which is associated with a variety of prevalent diseases. To cope with the deleterious effects of virus-induced ER stress, cells activate critical signaling pathways including the unfolded protein response (UPR) and intrinsic mitochondrial apoptosis, which have complex effects on virus replication and pathogenesis. In this review, we present a comprehensive summary of recent research in this field, which revealed that about 36 viruses trigger ER stress and differentially activate ER stress-related signaling pathways. We also highlight the strategies evolved by viruses to modulate ER stress-related signaling networks including immune responses in order to ensure their survival and pathogenesis. Together, the knowledge gained from this field will shed light on unveiling the mechanisms of virus replication and pathogenesis and provide insight for future research as well as antiviral development.
94 citations
Cites background from "Antiviral activity of a small molec..."
...In addition, the IRE1XBP1 pathway inhibits the replication of Sindbis virus (SINV) and DENV through an unknown mechanism (Pena & Harris, 2011; Perry et al., 2012)....
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...The IRE1-XBP1 pathway agonist, WP1130, has broad antiviral effects against SINV, murine norovirus (MNV-1) and La Crosse virus (Perry et al., 2012)....
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TL;DR: This article is a thorough review/accounting of patented compounds targeting DUBs and stratifying/classifying the patented compounds based on: chemical-structures, nucleic-acid compositions, modes-of-action, and targeting sites.
Abstract: Introduction Deubiquitinating-enzymes (DUBs) are key components of the ubiquitin-proteasome system (UPS). The fundamental role of DUBs is specific removal of ubiquitin from substrates. DUBs contribute to activation/deactivation, recycling and localization of numerous regulatory proteins, and thus play major roles in diverse cellular processes. Altered DUB activity is associated with a multitudes of pathologies including cancer. Therefore, DUBs represent novel candidates for target-directed drug development. Areas covered The article is a thorough review/accounting of patented compounds targeting DUBs and stratifying/classifying the patented compounds based on: chemical-structures, nucleic-acid compositions, modes-of-action, and targeting sites. The review provides a brief background on the UPS and the involvement of DUBs. Furthermore, methods for assessing efficacy and potential pharmacological utility of DUB inhibitor (DUBi) are discussed. Expert opinion The FDA's approval of the 20S proteasome inhibitors (PIs): bortezomib and carfilzomib for treatment of hematological malignancies established the UPS as an anti-cancer target. Unfortunately, many patients are inherently resistant or develop resistance to PIs. One potential strategy to combat PI resistance is targeting upstream components of the UPS such as DUBs. DUBs represent a promising potential therapeutic target due to their critical roles in various cellular processes including protein turnover, localization and cellular homeostasis. While considerable efforts have been undertaken to develop DUB modulators, significant advancements are necessary to move DUBis into the clinic.
91 citations
Cites methods from "Antiviral activity of a small molec..."
...5 WPI 130 (WO2012040527A2) The inventors [77] disclose compounds useful as DUB inhibitors and methods for use of the same....
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References
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TL;DR: It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease.
Abstract: Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, conside...
3,990 citations
"Antiviral activity of a small molec..." refers background in this paper
...including proteasomal degradation [18] and the unfolded protein response (UPR) (e....
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TL;DR: The results suggest that thapsigargin increases the concentration of cytosolic free Ca2+ in sensitive cells by an acute and highly specific arrest of the endoplasmic reticulum Ca 2+ pump, followed by a rapid Ca2+.
Abstract: Thapsigargin, a tumor-promoting sesquiterpene lactone, discharges intracellular Ca2+ in rat hepatocytes, as it does in many vertebrate cell types. It appears to act intracellularly, as incubation of isolated rat liver microsomes with thapsigargin induces a rapid, dose-dependent release of stored Ca2+. The thapsigargin-releasable pool of microsomal Ca2+ includes the pools sensitive to inositol 1,4,5-trisphosphate and GTP. Thapsigargin pretreatment of microsomes blocks subsequent loading with 45Ca2+, suggesting that its target is the ATP-dependent Ca2+ pump of endoplasmic reticulum. This hypothesis is strongly supported by the demonstration that thapsigargin causes a rapid inhibition of the Ca2(+)-activated ATPase activity of rat liver microsomes, with an identical dose dependence to that seen in whole cell or isolated microsome Ca2+ discharge. The inhibition of the endoplasmic reticulum isoform of the Ca2(+)-ATPase is highly selective, as thapsigargin has little or no effect on the Ca2(+)-ATPases of hepatocyte or erythrocyte plasma membrane or of cardiac or skeletal muscle sarcoplasmic reticulum. These results suggest that thapsigargin increases the concentration of cytosolic free Ca2+ in sensitive cells by an acute and highly specific arrest of the endoplasmic reticulum Ca2+ pump, followed by a rapid Ca2+ leak from at least two pharmacologically distinct Ca2+ stores. The implications of this mechanism of action for the application of thapsigargin in the analysis of Ca2+ homeostasis and possible forms of Ca2+ control are discussed.
3,153 citations
"Antiviral activity of a small molec..." refers methods in this paper
...Pharmacologic activation of the UPR with thapsigargin, an inhibitor of the sarco/endoplasmic reticulum calcium ATPase [39], also significantly inhibited MNV1 infection....
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TL;DR: Malfolded proteins in the endoplasmic reticulum induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs), and Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and I RE1alpha-/- fibroblasts were impaired in JNK activation by ER stress.
Abstract: Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1alpha-/- fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.
2,774 citations
TL;DR: It is demonstrated that mutations in either ire-1 or the transcription-factor-encoding xbp-1 gene abolished the UPR in Caenorhabditis elegans, suggesting that physiological ER load regulates a developmental decision in higher eukaryotes.
Abstract: The unfolded protein response (UPR), caused by stress, matches the folding capacity of endoplasmic reticulum (ER) to the load of client proteins in the organelle. In yeast, processing of HAC1 mRNA by activated Ire1 leads to synthesis of the transcription factor Hac1 and activation of the UPR. The responses to activated IRE1 in metazoans are less well understood. Here we demonstrate that mutations in either ire-1 or the transcription-factor-encoding xbp-1 gene abolished the UPR in Caenorhabditis elegans. Mammalian XBP-1 is essential for immunoglobulin secretion and development of plasma cells, and high levels of XBP-1 messenger RNA are found in specialized secretory cells. Activation of the UPR causes IRE1-dependent splicing of a small intron from the XBP-1 mRNA both in C. elegans and mice. The protein encoded by the processed murine XBP-1 mRNA accumulated during the UPR, whereas the protein encoded by unprocessed mRNA did not. Purified mouse IRE1 accurately cleaved XBP-1 mRNA in vitro, indicating that XBP-1 mRNA is a direct target of IRE1 endonucleolytic activity. Our findings suggest that physiological ER load regulates a developmental decision in higher eukaryotes.
2,643 citations
"Antiviral activity of a small molec..." refers background in this paper
...The active endonuclease domain of IRE1 splices the mRNA encoding XBP-1, which leads to expression of the active XBP-1 transcription factor [46]....
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TL;DR: Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles.
2,503 citations
"Antiviral activity of a small molec..." refers methods in this paper
...Ribavirin, MG132, Bortezomib, and thapsigargin were obtained from Sigma-Aldrich....
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...As anticipated the proteasome inhibitors MG132 and Bortezomib significantly reduced proteasome activity, while WP1130 treatment did not (Fig....
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...To confirm that WP1120 did not affect proteasome activity in murine macrophages, we tested for chymotrypsin-like activity in RAW cells treated with WP1130, using two known proteasome inhibitors MG132 and Bortezomib [44,45], or DMSO control as described [34] (Fig....
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