Initiator cell death event induced by SARS-CoV-2 in the human airway epithelium.

Virus-induced cell death is a key contributor to COVID-19 pathology. Cell death induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is well studied in myeloid cells but less in its primary host cell type, angiotensin-converting enzyme 2 (ACE2)-expressing human airway epithelia (HAE). SARS-CoV-2 induces apoptosis, necroptosis, and pyroptosis in HAE organotypic cultures. Single-cell and limiting-dilution analysis revealed that necroptosis is the primary cell death event in infected cells, whereas uninfected bystanders undergo apoptosis, and pyroptosis occurs later during infection. Mechanistically, necroptosis is induced by viral Z-RNA binding to Z-DNA-binding protein 1 (ZBP1) in HAE and lung tissues from patients with COVID-19. The Delta (B.1.617.2) variant, which causes more severe disease than Omicron (B1.1.529) in humans, is associated with orders of magnitude-greater Z-RNA/ZBP1 interactions, necroptosis, and disease severity in animal models. Thus, Delta induces robust ZBP1-mediated necroptosis and more disease severity.

A Vitamin D-RelB/NF-κB Pathway Limits Chandipura Virus Multiplication by Rewiring the Homeostatic State of Autoregulatory Type 1 IFN-IRF7 Signaling.

Besides its functions in the skeletomuscular system, vitamin D is known to alleviate viral-inflicted pathologies. However, the mechanism underlying protective vitamin D function remains unclear. We examined the role of vitamin D in controlling cellular infections by Chandipura virus, an RNA virus implicated in human epidemics. How immune signaling pathways, including those regulating NF-κB and IFN regulatory factors (IRFs), are activated in virus-infected cells has been well studied. Our investigation involving human- and mouse-derived cells revealed that vitamin D instructs the homeostatic state of these antiviral pathways, leading to cellular resilience to subsequent viral infections. In particular, vitamin D provoked autoregulatory type 1 IFN-IRF7 signaling even in the absence of virus infection by downmodulating the expression of the IFN-inhibitory NF-κB subunit RelB. Indeed, RelB deficiency rendered vitamin D treatment redundant, whereas IRF7 depletion abrogated antiviral vitamin D action. In sum, immune signaling homeostasis appears to connect micronutrients to antiviral immunity at the cellular level. The proposed link may have a bearing on shaping public health policy during an outbreak.

Zika virus oncolytic activity requires CD8+ T cells and is boosted by immune checkpoint blockade.

Glioblastoma multiforme (GBM) is a fatal human cancer in part because GBM stem cells are resistant to therapy and recurrence is inevitable. Previously, we demonstrated Zika virus (ZIKV) targets GBM stem cells and prevents death of mice with gliomas. Here, we evaluated the immunological basis of ZIKV-mediated protection against GBM. Introduction of ZIKV into the brain tumor increased recruitment of CD8+ T and myeloid cells to the tumor microenvironment. CD8+ T cells were required for ZIKV-dependent tumor clearance because survival benefits were lost with CD8+ T cell depletion. Moreover, while anti-PD-1 antibody monotherapy moderately improved tumor survival, when coadministered with ZIKV, survival increased. ZIKV-mediated tumor clearance also resulted in durable protection against syngeneic tumor rechallenge, which also depended on CD8+ T cells. To address safety concerns, we generated an immune-sensitized ZIKV strain, which was effective alone or in combination with immunotherapy. Thus, oncolytic ZIKV treatment can be leveraged by immunotherapies, which may prompt combination treatment paradigms for adult patients with GBM.

Zinc Chelation Specifically Inhibits Early Stages of Dengue Virus Replication by Activation of NF-κB and Induction of Antiviral Response in Epithelial Cells.

Zinc is an essential micronutrient which regulates diverse physiological functions and has been shown to play a crucial role in viral infections. Zinc has a necessary role in the replication of many viruses, however, antiviral action of zinc has also been demonstrated in in vitro infection models most likely through induction of host antiviral responses. Therefore, depending on the host machinery that the virus employs at different stages of infection, zinc may either facilitate, or inhibit virus infection. In this study, we show that zinc plays divergent roles in rotavirus and dengue virus infections in epithelial cells. Dengue virus infection did not perturb the epithelial barrier functions despite the release of virus from the basolateral surface whereas rotavirus infection led to disruption of epithelial junctions. In rotavirus infection, zinc supplementation post-infection did not block barrier disruption suggesting that zinc does not affect rotavirus life-cycle or protects epithelial barriers post-infection suggesting the involvement of cellular pathways in the beneficial effect of zinc supplementation in enteric infections. Zinc depletion by N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) inhibited dengue virus and Japanese encephalitis virus (JEV) infection but had no effect on rotavirus. Time-of-addition experiments suggested that zinc chelation affected both early and late stages of dengue virus infectious cycle and zinc chelation abrogated dengue virus RNA replication. We show that transient zinc chelation induces ER stress and antiviral response by activating NF-kappaB leading to induction of interferon signaling. These results suggest that modulation of zinc homeostasis during virus infection could be a component of host antiviral response and altering zinc homeostasis may act as a potent antiviral strategy against flaviviruses.

Redox Sensitive Self-Assembling Dipeptide for Sustained Intracellular Drug Delivery.

The rational design and synthesis of molecules with functional supramolecular assemblies continues to be a challenging endeavor. Self-assembled nano- and microstructures from natural building blocks are considered more appropriate for medical applications due to their biocompatible nature. We report for the first time a simple redox-responsive dipeptide that self-assembles to form vesicles in aqueous medium. The experimental results based on the control compound and all-atom molecular dynamics (MD) simulations support the mechanism of association through intermolecular π-π interactions between the indole rings of tryptophan residues. These peptide vesicles showed a DOX loading capacity of ∼16% (w/w) and redox-triggered controlled release of the packaged drug. The drug-loaded vesicles were able to penetrate into MDA-MB-231 and HeLa cells, and release payload, suggesting their putative use as chemotherapeutic delivery vehicles. These natural peptide-based carriers disassemble inside cells due to the high cytosolic GSH concentration, and the resultant Cys-Trp dipeptide is degradable. The minimalistic peptide design presented here, coupled with the propensity to form vesicles that can encapsulate the chemotherapeutic drug, opens up unlimited potential for engineering targeted sustained-release drug delivery vehicles.

Chandipura Virus Utilizes the Prosurvival Function of RelA NF-κB for Its Propagation.

Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-ß) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-ß in response to virus infections. Unexpectedly, infection of Rela-/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.IMPORTANCE RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-ß, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.

A TNF-p100 pathway subverts noncanonical NF-κB signaling in inflamed secondary lymphoid organs.

Lymphotoxin-beta receptor (LTßR) present on stromal cells engages the noncanonical NF-κB pathway to mediate RelB-dependent expressions of homeostatic chemokines, which direct steady-state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF-κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection-inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non-infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTßR-stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF-mediated inhibitions in inflamed SLOs of immunized Nfkb2-/- mice. In sum, we reveal that an inhibitory TNF-p100 pathway modulates the adaptive compartment during immune responses.