Host functions used by hepatitis B virus to complete its life cycle: Implications for developing host-targeting agents to treat chronic hepatitis B

Similar to other mammalian viruses, the life cycle of hepatitis B virus (HBV) is heavily dependent upon and regulated by cellular (host) functions. These cellular functions can be generally placed in to two categories: (a) intrinsic host restriction factors and innate defenses, which must be evaded or repressed by the virus; and (b) gene products that provide functions necessary for the virus to complete its life cycle. Some of these functions may apply to all viruses, but some may be specific to HBV. In certain cases, the virus may depend upon the host function much more than does the host itself. Knowing which host functions regulate the different steps of a virus' life cycle, can lead to new antiviral targets and help in developing novel treatment strategies, in addition to improving a fundamental understanding of viral pathogenesis. Therefore, in this review we will discuss known host factors which influence key steps of HBV life cycle, and further elucidate therapeutic interventions targeting host-HBV interactions

Structure Guided Design of Potent and Selective Ponatinib-Based Hybrid Inhibitors for RIPK1

SummaryRIPK1 and RIPK3, two closely related RIPK family members, have emerged as important regulators of pathologic cell death and inflammation. In the current work, we report that the Bcr-Abl inhibitor and anti-leukemia agent ponatinib is also a first-in-class dual inhibitor of RIPK1 and RIPK3. Ponatinib potently inhibited multiple paradigms of RIPK1- and RIPK3-dependent cell death and inflammatory tumor necrosis factor alpha (TNF-α) gene transcription. We further describe design strategies that utilize the ponatinib scaffold to develop two classes of inhibitors (CS and PN series), each with greatly improved selectivity for RIPK1. In particular, we detail the development of PN10, a highly potent and selective “hybrid” RIPK1 inhibitor, capturing the best properties of two different allosteric RIPK1 inhibitors, ponatinib and necrostatin-1. Finally, we show that RIPK1 inhibitors from both classes are powerful blockers of TNF-induced injury in vivo. Altogether, these findings outline promising candidate molecules and design approaches for targeting RIPK1- and RIPK3-driven inflammatory pathologies

Exploring synergies and trade-offs among the sustainable development goals: collective action and adaptive capacity in marginal mountainous areas of India

Global environmental change (GEC) threatens to undermine the sustainable development goals (SDGs). Smallholders in marginal mountainous areas (MMA) are particularly vulnerable due to precarious livelihoods in challenging environments. Acting collectively can enable and constrain the ability of smallholders to adapt to GEC. The objectives of this paper are: (i) identify collective actions in four MMA of the central Indian Himalaya Region, each with differing institutional contexts; (ii) assess the adaptive capacity of each village by measuring livelihood capital assets, diversity, and sustainable land management practices. Engaging with adaptive capacity and collective action literatures, we identify three broad approaches to adaptive capacity relating to the SDGs: natural hazard mitigation (SDG 13), social vulnerability (SDG 1, 2 and 5), and social–ecological resilience (SDG 15). We then develop a conceptual framework to understand the institutional context and identify SDG synergies and trade-offs. Adopting a mixed method approach, we analyse the relationships between collective action and the adaptive capacity of each village, the sites where apparent trade-offs and synergies among SDGs occur. Results illustrate each village has unique socio-environmental characteristics, implying distinct development challenges, vulnerabilities and adaptive capacities exist. Subsequently, specific SDG synergies and trade-offs occur even within MMA, and it is therefore crucial that institutions facilitate locally appropriate collective actions in order to achieve the SDGs. We suggest that co-production in the identification, prioritisation and potential solutions to the distinct challenges facing MMA can increase understandings of the specific dynamics and feedbacks necessary to achieve the SDGs in the context of GEC

RIPK3 Is Largely Dispensable for RIG-I-Like Receptor- and Type I Interferon-Driven Transcriptional Responses to Influenza A Virus in Murine Fibroblasts

<div><p>The kinase RIPK3 is a key regulator of cell death responses to a growing number of viral and microbial agents. We have found that influenza A virus (IAV)-mediated cell death is largely reliant on RIPK3 and that RIPK3-deficient mice are notably more susceptible to lethal infection by IAV than their wild-type counterparts. Recent studies demonstrate that RIPK3 also participates in regulating gene transcription programs during host pro-inflammatory and innate-immune responses, indicating that this kinase is not solely an inducer of cell death and that RIPK3-driven transcriptional responses may collaborate with cell death in promoting clearance of IAV. Here, we carried out DNA microarray analyses to determine the contribution of RIPK3 to the IAV-elicited host transcriptional response. We report that RIPK3 does not contribute significantly to the RLR-activated transcriptome or to the induction of type I IFN genes, although, interestingly, IFN-β production at a post-transcriptional step was modestly attenuated in IAV-infected <i>ripk3</i>^<i>-/-</i> fibroblasts. Overall, RIPK3 regulated the expression of <5% of the IAV-induced transcriptome, and no genes were found to be obligate RIPK3 targets. IFN-β signaling was also found to be largely normal in the absence of RIPK3. Together, these results indicate that RIPK3 is not essential for the host antiviral transcriptional response to IAV in murine fibroblasts.</p></div

Distinct Roles for the NF-κB RelA Subunit during Antiviral Innate Immune Responses▿ †

Production of type I interferons (IFNs; prominently, IFN-α/β) following virus infection is a pivotal antiviral innate immune response in higher vertebrates. The synthesis of IFN-β proceeds via the virus-induced assembly of the transcription factors IRF-3/7, ATF-2/c-Jun, and NF-κB on the ifnβ promoter. Surprisingly, recent data indicate that the NF-κB subunit RelA is not essential for virus-stimulated ifnβ expression. Here, we show that RelA instead sustains autocrine IFN-β signaling prior to infection. In the absence of RelA, virus infection results in significantly delayed ifnβ induction and consequently defective secondary antiviral gene expression. While RelA is not required for ifnβ expression after infection, it is nonetheless essential for fully one-fourth of double-stranded RNA (dsRNA)-activated genes, including several mediators of inflammation and immune cell recruitment. Further, RelA directly regulates a small subset of interferon-stimulated genes (ISGs). Finally, RelA also protects cells from dsRNA-triggered RIP1-dependent programmed necrosis. Taken together, our findings suggest distinct roles for RelA in antiviral innate immunity: RelA maintains autocrine IFN-β signaling in uninfected cells, facilitates inflammatory and adaptive immune responses following infection, and promotes infected-cell survival during this process

Normal IFN-β signaling in the absence of RIPK3.

<p><b>(A)</b> Heatmap showing expression profiles of genes upregulated by IFN-β in WT MEFs. Expression levels in untreated cells were normalized to one (2⁰, yellow), and genes demonstrating 4-fold or more induction at 6 h were considered induced and sorted based on fold-induction at 6 h. Heat bars shown to the left represent relative expression levels on a log₂ scale. <b>(B)</b> Heatmap displaying the behavior of IFN responsive genes in <i>ripk3</i>^<i>+/+</i> MEFs (column 1–4), <i>ripk3</i>^<i>+/+</i> MEFs treated with RIPK3 inhibitor (GSK’872 at 5μM, column 5–8), or <i>ripk3</i>^<i>-/-</i> MEFs (column 9–12) following PR8-ΔNS1 infection. Expression levels in mock infected in <i>ripk3</i>^<i>+/+</i> MEFs were normalized to one (2⁰, yellow) and genes displaying at least two-fold changes at 18 h were considered IAV regulated. Genes are sorted based on fold-induction at 18 h in <i>ripk3</i>^<i>+/+</i> MEFs. No genes were found to be dependent on RIPK3. <b>(C)</b> Levels of ISG-encoded proteins were compared in <i>ripk3</i>^<i>+/+</i> and <i>ripk3</i>^<i>-/-</i> MEFs by immunoblot analysis following treatment with IFN-β (1000U/mL) for the indicated times.</p

Role of RIPK3 in IAV-elicited RLR transcriptional responses.

<p><b>(A)</b> qPCR determination of <i>ifnb1</i> and <i>ifna4</i> gene expression in <i>mavs</i>^<i>+/+</i> and <i>mavs</i>^<i>-/-</i> MEFs demonstrates that transfected poly(I:C) activates antiviral gene expression predominantly via the RLR pathway. <b>(B)</b> Heatmap showing expression profiles of genes upregulated by poly(I:C) in <i>ifnar</i>^<i>-/-</i> MEFs. Expression levels in untreated cells were normalized to one (2⁰, yellow), and genes demonstrating at least 6-fold induction at 6 h were designated the ‘core RLR transcriptomic signature’. Heat bars shown to the left represent relative expression levels on a log₂ scale. Genes were sorted based on fold-induction at 6 h. <b>(C)</b> Heatmap displaying the behavior of core RLR transcriptomic signature in <i>ripk3</i>^<i>+/+</i> MEFs (columns 1–4), <i>ripk3</i>^<i>+/+</i> MEFs treated with RIPK3 inhibitor (GSK’872 at 5μM, columns 5–8), or <i>ripk3</i>^<i>-/-</i> MEFs (column 9–12) following infection with PR8-ΔNS1 (m.o.i. = 1). Expression levels in mock infected in <i>ripk3</i>^<i>+/+</i> MEFs were normalized to one (2⁰, yellow) and genes displaying at least two-fold upregulation at 18 h were considered IAV-inducible; none of these were found to be RIPK3 dependent. Genes are sorted based on fold-induction at 18 h in <i>ripk3</i>^<i>+/+</i> MEFs. <b>(D)</b> Heatmap showing expression profiles of genes encoding type I IFNs, inflammatory cytokines and chemokines after infection with PR8-ΔNS1 in <i>ripk3</i>^<i>+/+</i> MEFs, <i>ripk3</i>^<i>+/+</i> treated with RIPK3 inhibitor (GSK’872, 5μM), or <i>ripk3</i>^<i>-/-</i> MEFs. Expression levels in mock infected in <i>ripk3</i>^<i>+/+</i> MEFs were normalized to one (2⁰, yellow) Heat bar, depicting relative expression levels on a log₂ scale, is shown to the left.</p

Anti-CD70 immunocytokines for exploitation of interferon-γ-induced RIP1-dependent necrosis in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel 'immunocytokine' chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC

Role of RIPK3 in IAV-activated transcriptional responses.

<p><b>(A)</b> Heatmap depicting behavior of IAV-regulated genes in <i>ripk3</i>^<i>+/+</i> MEFs (columns 1–4) or <i>ripk3</i>^<i>+/+</i> MEFs treated with the RIPK3 inhibitor (GSK’872 at 5μM, columns 5–8) or <i>ripk3</i>^<i>-/-</i> MEFs (columns 9–12) following PR8-ΔNS1 infection for the indicated times. All genes displaying two-fold or more change in expression following infection in <i>ripk3</i>^<i>+/+</i> MEFs at 18 h were considered IAV regulated. Expression levels in mock infected in <i>ripk3</i>^<i>+/+</i> MEFs were normalized to one (2⁰, yellow), and heat bars shown to the left represent relative expression levels on a log₂ scale. RIPK3-dependent genes, defined as those IAV targets which were (i) less than 1.5-fold differentially expressed in <i>ripk3</i>^<i>-/-</i> MEFs at 18 h and (ii) less than 1.two-fold differentially expressed in untreated <i>ripk3</i>^<i>-/-</i> MEFs at 18 h, are clustered together and indicated by asterisks. <b>(B)</b> Heatmap showing the top 20 genes whose upregulation by PR8-ΔNS1 is dependent on RIPK3. <b>(C)</b> Heatmap showing the top 20 genes whose downregulation by PR8-ΔNS1 is dependent on RIPK3. <b>(D)</b> Heatmap showing all the genes whose up- or downregulation by PR8-ΔNS1 requires the kinase activity of RIPK3.</p