Human Coronavirus 229E Infection Inactivates Pyroptosis Executioner Gasdermin D but Ultimately Leads to Lytic Cell Death Partly Mediated by Gasdermin E.

Human coronavirus 229E (HCoV-229E) is associated with upper respiratory tract infections and generally causes mild respiratory symptoms. HCoV-229E infection can cause cell death, but the molecular pathways that lead to virus-induced cell death as well as the interplay between viral proteins and cellular cell death effectors remain poorly characterized for HCoV-229E. Studying how HCoV-229E and other common cold coronaviruses interact with and affect cell death pathways may help to understand its pathogenesis and compare it to that of highly pathogenic coronaviruses. Here, we report that the main protease (Mpro) of HCoV-229E can cleave gasdermin D (GSDMD) at two different sites (Q29 and Q193) within its active N-terminal domain to generate fragments that are now unable to cause pyroptosis, a form of lytic cell death normally executed by this protein. Despite GSDMD cleavage by HCoV-229E Mpro, we show that HCoV-229E infection still leads to lytic cell death. We demonstrate that during virus infection caspase-3 cleaves and activates gasdermin E (GSDME), another key executioner of pyroptosis. Accordingly, GSDME knockout cells show a significant decrease in lytic cell death upon virus infection. Finally, we show that HCoV-229E infection leads to increased lytic cell death levels in cells expressing a GSDMD mutant uncleavable by Mpro (GSDMD Q29A+Q193A). We conclude that GSDMD is inactivated by Mpro during HCoV-229E infection, preventing GSDMD-mediated cell death, and point to the caspase-3/GSDME axis as an important player in the execution of virus-induced cell death. In the context of similar reported findings for highly pathogenic coronaviruses, our results suggest that these mechanisms do not contribute to differences in pathogenicity among coronaviruses. Nonetheless, understanding the interactions of common cold-associated coronaviruses and their proteins with the programmed cell death machineries may lead to new clues for coronavirus control strategies.

Viral sensing by epithelial cells involves PKR- and caspase-3-dependent generation of gasdermin E pores.

Viral sensing in myeloid cells involves inflammasome activation leading to gasdermin pore formation, cytokine release, and cell death. However, less is known about viral sensing in barrier epithelial cells, which are critical to the innate immune response to RNA viruses. Here, we show that poly(I:C), a mimic of viral dsRNA, is sensed by NLRP1 in human bronchial epithelial cells, leading to inflammasome-dependent gasdermin D (GSDMD) pore formation via caspase-1. DsRNA also stimulated a parallel sensing pathway via PKR which activated caspase-3 to cleave gasdermin E (GSDME) to form active pores. Influenza A virus (IAV) infection of cells caused GSDME activation, cytokine release, and cell death, in a PKR-dependent but NLRP1-independent manner, involving caspase-8 and caspase-3. Suppression of GSDMD and GSDME expression increased IAV replication. These data clarify mechanisms of gasdermin cleavage in response to viral sensing and reveal that gasdermin pore formation is intrinsically antiviral in human epithelial cells.

Non-canonical inflammasome activation mediates the adjuvanticity of nanoparticles.

The non-canonical inflammasome sensor caspase-11 and gasdermin D (GSDMD) drive inflammation and pyroptosis, a type of immunogenic cell death that favors cell-mediated immunity (CMI) in cancer, infection, and autoimmunity. Here we show that caspase-11 and GSDMD are required for CD8+ and Th1 responses induced by nanoparticulate vaccine adjuvants. We demonstrate that nanoparticle-induced reactive oxygen species (ROS) are size dependent and essential for CMI, and we identify 50- to 60-nm nanoparticles as optimal inducers of ROS, GSDMD activation, and Th1 and CD8+ responses. We reveal a division of labor for IL-1 and IL-18, where IL-1 supports Th1 and IL-18 promotes CD8+ responses. Exploiting size as a key attribute, we demonstrate that biodegradable poly-lactic co-glycolic acid nanoparticles are potent CMI-inducing adjuvants. Our work implicates ROS and the non-canonical inflammasome in the mode of action of polymeric nanoparticulate adjuvants and establishes adjuvant size as a key design principle for vaccines against cancer and intracellular pathogens.

Adaptation to host cell environment during experimental evolution of Zika virus.

Zika virus (ZIKV) infection can cause important developmental and neurological defects in Humans. Type I/III interferon responses control ZIKV infection and pathological processes, yet the virus has evolved various mechanisms to defeat these host responses. Here, we established a pipeline to delineate at high-resolution the genetic evolution of ZIKV in a controlled host cell environment. We uncovered that serially passaged ZIKV acquired increased infectivity and simultaneously developed a resistance to TLR3-induced restriction. We built a mathematical model that suggests that the increased infectivity is due to a reduced time-lag between infection and viral replication. We found that this adaptation is cell-type specific, suggesting that different cell environments may drive viral evolution along different routes. Deep-sequencing of ZIKV populations pinpointed mutations whose increased frequencies temporally coincide with the acquisition of the adapted phenotype. We functionally validated S455L, a substitution in ZIKV envelope (E) protein, recapitulating the adapted phenotype. Its positioning on the E structure suggests a putative function in protein refolding/stability. Taken together, our results uncovered ZIKV adaptations to the cellular environment leading to accelerated replication onset coupled with resistance to TLR3-induced antiviral response. Our work provides insights into Zika virus adaptation to host cells and immune escape mechanisms.

Recent insights into innate immune nucleic acid sensing during viral infection.

Recent advances in our understanding of nucleic acid pattern-recognition receptor (PRR) sensing of viruses have revealed a previously unappreciated level of complexity of the host antiviral response. As well as direct recognition of viral nucleic acid by PRRs, viruses also induce the release of host nucleic acid from the nucleus and mitochondria into the cytosol, which boosts nucleic acid activation of antiviral PRRs. Crosstalk and cooperation between DNA- and RNA-recognition signaling pathways has also been revealed, as has direct restriction of viral genomes in an interferon-independent manner by PRRs, and new roles for inflammasomes in sensing viral nucleic acid. Further, newly identified viral-evasion strategies targeting PRR pathways emphasize the importance of nucleic acid detection during viral infection at the host-pathogen innate immune interface.

The Inflammasome Components NLRP3 and ASC Act in Concert with IRGM To Rearrange the Golgi Apparatus during Hepatitis C Virus Infection.

Hepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the roles of NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) and ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain [CARD]), two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and the Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. IRGM depletion in the ASC-silenced cells cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to nigericin treatment, but not in cells infected with another member of the Flaviviridae family, Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling during certain stimuli.IMPORTANCE Numerous pathogens can affect cellular homeostasis and organelle dynamics. Hepatitis C virus (HCV) triggers Golgi fragmentation through the immunity-related GTPase M (IRGM), a resident Golgi protein, to enhance its lipid supply for replication. Here, we reveal the role of the inflammasome components NLRP3 and ASC in this process, thus uncovering a new interplay between effectors of inflammation and viral infection or stress. We show that the inflammasome component ASC resides at the Golgi under homeostasis and associates with IRGM. Upon HCV infection, ASC is recruited to NLRP3 and dissociates from IRGM, causing Golgi fragmentation. Our results uncover that aside from their known function in the inflammation response, these host defense regulators also ensure the maintenance of intact intracellular structure in homeostasis, while their activation relieves factors leading to Golgi remodeling.

Detection of Viral Infections by Innate Immunity.

Pattern recognition receptors (PRRs) and inflammasomes are a key part of the anti-viral innate immune system as they detect conserved viral pathogen-associated molecular patterns (PAMPs). A successful host response to viral infections critically depend on the initial activation of PRRs by viruses, mainly by viral DNA and RNA. The signalling pathways activated by PRRs leads to the expression of pro-inflammatory cytokines, to recruit immune cells, and type I and type III interferons which leads to the induction of interferon stimulated genes (ISG), powerful virus restriction factors that establish the "antiviral state". Inflammasomes contribute to anti-viral responses through the maturation of interleukin (IL)-1 and IL-18 and through triggering pyroptotic cell death. The activity of the innate immune system along with the adaptive immune response normally leads to successful virus elimination, although disproportionate innate responses contribute to viral pathology. In this review we will discuss recent insights into the influence of PRR activation and inflammasomes on viral infections and what this means for the mammalian host. We will also comment on how specific PRRs and inflammasomes may be relevant to how SARS-CoV-2, the virus responsible for the current COVID-19 pandemic, interacts with host innate immunity.