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The primary function of the immune system is to protect the host from invading pathogens. In response, microbial pathogens have developed various strategies to evade detection and destruction by the immune system. This tug-of-war between the host and the pathogen is a powerful force that shapes organismal evolution. Regulated cell death (RCD) is a host response that limits the reservoir for intracellular pathogens such as viruses. Since pathogen-specific T cell and B cell responses typically take several days and is therefore slow-developing, RCD of infected cells during the first few days of the infection is critical for organismal survival. This innate immune response not only restricts viral replication, but also serves to promote anti-viral inflammation through cell death-associated release of damage-associated molecular patterns (DAMPs). In recent years, necroptosis has been recognized as an important response against many viruses. The central adaptor for necroptosis, RIPK3, also exerts anti-viral effects through cell death-independent activities such as promoting cytokine gene expression. Here, we will discuss recent advances on how viruses counteract this host defense mechanism and the effect of necroptosis on the anti-viral inflammatory reaction.

The vaccine strain against smallpox, vaccinia virus (VACV), is highly immunogenic yet causes relatively benign disease. These attributes are believed to be caused by gene loss in VACV. Using a targeted small interfering RNA (siRNA) screen, we identified a viral inhibitor found in cowpox virus (CPXV) and other orthopoxviruses that bound to the host SKP1-Cullin1-F-box (SCF) machinery and the essential necroptosis kinase receptor interacting protein kinase 3 (RIPK3). This "viral inducer of RIPK3 degradation" (vIRD) triggered ubiquitination and proteasome-mediated degradation of RIPK3 and inhibited necroptosis. In contrast to orthopoxviruses, the distantly related leporipoxvirus myxoma virus (MYXV), which infects RIPK3-deficient hosts, lacks a functional vIRD. Introduction of vIRD into VACV, which encodes a truncated and defective vIRD, enhanced viral replication in mice. Deletion of vIRD reduced CPXV-induced inflammation, viral replication, and mortality, which were reversed in RIPK3- and MLKL-deficient mice. Hence, vIRD-RIPK3 drives pathogen-host evolution and regulates virus-induced inflammation and pathogenesis.

Necroptosis is an emerging mode of programmed cell death that is defined at the molecular level through involvement of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL). In contrast to apoptosis, phosphorylation is a key event in necroptosis. Necroptosis can be triggered in response to various cell-surface receptors, such as death receptors in the tumor necrosis factor receptor family, Toll-like receptors, and interferon receptors. Because apoptotic machinery is often impaired in cancer cells, necroptosis has garnered attention as a promising strategy to treat apoptosis-resistant cancer. Moreover, since necroptosis leads to release of intracellular immunogenic contents and is therefore highly immunogenic, it has the potential to promote robust antitumor adaptive immune response. In this chapter, we describe current understanding of the role of necroptosis in cancer progression and discuss strategies through which we can harness the power of necroptosis in cancer therapy.

Influenza virus remains one of the most important disease-causing viruses owing to its high adaptability and even higher contagious nature. Thus, it poses a constant threat of pandemic, engulfing a large population within the smallest possible time interval. A similar threat was anticipated with the identification of the novel H7N9 virus in China on 30 March 2013. Detection of transmission of the virus between humans has caused a stir with the identification of family clusters along with sporadic infections all across China. In this review we analyze the potential of the novel H7N9 virus as a probable cause of a pandemic and the possible consequences thereof.

Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified α‐actinin‐4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co‐immunoprecipitation studies, first in a coupled in vitro transcription‐translation assay and then in cells either transiently co‐expressing the two proteins or infected with whole IAV. Importantly, the NP–actinin‐4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin‐4 co‐localized largely around the nucleus and also in the cytoplasmic region of virus‐infected A549 cells. Silencing of actinin‐4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post‐infection with IAV, suggesting that actinin‐4 was critical for viral replication. Furthermore, actinin‐4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin‐4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin‐4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins. Structured digital abstract •http://www.uniprot.org/uniprot/P03466 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9512541, http://www.ebi.ac.uk/intact/interaction/EBI-9512553)•http://www.uniprot.org/uniprot/Q8JR21 and http://www.uniprot.org/uniprot/O43707 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0416 (http://www.ebi.ac.uk/intact/interaction/EBI-9514040)•http://www.uniprot.org/uniprot/Q91U50 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9514006)•http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007 (http://www.ebi.ac.uk/intact/interaction/EBI-9512166, http://www.ebi.ac.uk/intact/interaction/EBI-9512219)•http://www.uniprot.org/uniprot/C3W6D7 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513951)•http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007 (http://www.ebi.ac.uk/intact/interaction/EBI-9512237)•http://www.uniprot.org/uniprot/Q6DPG0 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513984) •http://www.uniprot.org/uniprot/B2BU63 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513930) •http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0018 (http://www.ebi.ac.uk/intact/interaction/EBI-9512145, http://www.ebi.ac.uk/intact/interaction/EBI-9512095) •http://www.uniprot.org/uniprot/C9S3S8 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513909), Human host proteins aiding the influenza A viral nucleoprotein in replication and transcription of virus and its intracellular shuttling are not well studied. The study presented here reports the dynamics of interaction between NP and actinin‐4 and the pivotal role of cytoskeletal protein in not only trafficking of NP, but also boosting its mRNA and vRNA levels, ultimately elevating viral titers.

Summary In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes.

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

The interplay between influenza virus and host factors to support the viral life cycle is well documented. Influenza A virus (IAV) proteins interact with an array of cellular proteins and hijack host pathways which are at the helm of cellular responses to facilitate virus invasion. The multifaceted nature of the ubiquitination pathway for protein regulation makes it a vulnerable target of many viruses including IAV. To this end we conducted a yeast two-hybrid screen to search for cellular ubiquitin ligases important for influenza virus replication. We identified host protein, RING finger protein 43 (RNF43), a RING-type E3 ubiquitin ligase, as a novel interactor of nucleoprotein (NP) of IAV and an essential partner to induce NP-driven p53-mediated apoptosis in IAV-infected cells. In this study, we demonstrate that IAV leads to attenuation of RNF43 transcripts and hence its respective protein levels in the cellular milieu whereas in RNF43 depleted cells, viral replication was escalated several folds. Moreover, RNF43 polyubiquitinates p53 which further leads to its destabilization resulting in a decrease in induction of the p53 apoptotic pathway, a hitherto unknown process targeted by NP for p53 stabilization and accumulation. Collectively, these results conclude that NP targets RNF43 to modulate p53 ubiquitination levels and hence causes p53 stabilization which is conducive to an enhanced apoptosis level in the host cells. In conclusion, our study unravels a novel strategy adopted by IAV for utilizing the much conserved ubiquitin proteasomal pathway.