Advances in Norovirus Biology

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.

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Favipiravir elicits antiviral mutagenesis during virus replication in vivo

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.

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Alphavirus Infection: Host Cell Shut-Off and Inhibition of Antiviral Responses.

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.

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The expanding roles of endoplasmic reticulum stress in virus replication and pathogenesis

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.

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Deubiquitinases (DUBs) and DUB inhibitors: a patent review

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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Mohammad Ahmed

Papers

Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.