Sendai Virus Infection Induces Expression of Novel RNAs in Human Cells.

Innate antiviral immune responses are driven by virus-induced changes in host gene expression. While much research on antiviral effectors has focused on virus-inducible mRNAs, recent genome-wide analyses have identified hundreds of novel target sites for virus-inducible transcription factors and RNA polymerase. These sites are beyond the known antiviral gene repertoire and their contribution to innate immune responses is largely unknown. In this study, RNA-sequencing of mock-infected and Sendai virus-infected cells was performed to characterize the virus-inducible transcriptome and identify novel virus-inducible RNAs (nviRNAs). Virus-inducible transcription was observed throughout the genome resulting in expression of 1755 previously RefSeq-annotated RNAs and 1545 nviRNAs. The previously-annotated RNAs primarily consist of protein-coding mRNAs, including several well-known antiviral mRNAs that had low sequence conservation but were highly virus-inducible. The previously-unannotated nviRNAs were mostly noncoding RNAs with poor sequence conservation. Independent analyses of nviRNAs based on infection with Sendai virus, influenza virus, and herpes simplex virus 1, or direct stimulation with IFNα revealed a range of expression patterns in various human cell lines. These phylogenetic and expression analyses suggest that many of the nviRNAs share the high inducibility and low sequence conservation characteristic of well-known primary antiviral effectors and may represent dynamically evolving antiviral factors.

Constitutively Active MDA5 Proteins Are Inhibited by Paramyxovirus V Proteins.

Excessive interferon (IFN) production and signaling can lead to immunological and developmental defects giving rise to autoimmune diseases referred to collectively as "type I interferonopathies." A subset of these diseases is caused by monogenic mutations affecting proteins involved in nucleic acid sensing, homeostasis, and metabolism. Interferonopathic mutations in the cytosolic antiviral sensor MDA5 render it constitutively hyperactive, resulting in chronic IFN production and IFN-stimulated gene expression. Few therapeutic options are available for patients with interferonopathic diseases, but a large number of IFN evasion and antagonism strategies have evolved in viral pathogens that can counteract IFN production and signaling to enhance virus replication. To test the hypothesis that these natural IFN suppressors could be used to subdue the activity of interferonopathic signaling proteins, hyperactive MDA5 variants were assessed for susceptibility to a family of viral MDA5 inhibitors. In this study, Paramyxovirus V proteins were tested for their ability to counteract constitutively active MDA5 proteins. Results indicate that the V proteins are able to bind to and disrupt the signaling activity of these MDA5 proteins, irrespective of their specific mutations, reducing IFN production and IFN-stimulated gene expression to effectively suppress the hyperactive antiviral response.

RNA sensor LGP2 inhibits TRAF ubiquitin ligase to negatively regulate innate immune signaling.

The production of type I interferon (IFN) is essential for cellular barrier functions and innate and adaptive antiviral immunity. In response to virus infections, RNA receptors RIG-I and MDA5 stimulate a mitochondria-localized signaling apparatus that uses TRAF family ubiquitin ligase proteins to activate master transcription regulators IRF3 and NFκB, driving IFN and antiviral target gene expression. Data indicate that a third RNA receptor, LGP2, acts as a negative regulator of antiviral signaling by interfering with TRAF family proteins. Disruption of LGP2 expression in cells results in earlier and overactive transcriptional responses to virus or dsRNA LGP2 associates with the C-terminus of TRAF2, TRAF3, TRAF5, and TRAF6 and interferes with TRAF ubiquitin ligase activity. TRAF interference is independent of LGP2 ATP hydrolysis, RNA binding, or its C-terminal domain, and LGP2 can regulate TRAF-mediated signaling pathways in trans, including IL-1ß, TNFα, and cGAMP These findings provide a unique mechanism for LGP2 negative regulation through TRAF suppression and extend the potential impact of LGP2 negative regulation beyond the IFN antiviral response.

Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway.

STAT1 and STAT2 proteins are key mediators of type I and type III interferon (IFN) signaling, and are essential components of the cellular antiviral response and adaptive immunity. They associate with IFN regulatory factor 9 (IRF9) to form a heterotrimeric transcription factor complex known as ISGF3. The regulation of IFN-stimulated gene (ISG) expression has served as a model of JAK-STAT signaling and mammalian transcriptional regulation, but to date has primarily been analyzed at the single gene level. While many aspects of ISGF3-mediated gene regulation are thought to be common features applicable to several ISGs, there are also many reports of distinct cases of non-canonical STAT1 or STAT2 signaling events and distinct patterns of co-regulators that contribute to gene-specific transcription. Recent genome-wide studies have begun to uncover a more complete profile of ISG regulation, moving toward a genome-wide understanding of general mechanisms that underlie gene-specific behaviors.

Extensive cooperation of immune master regulators IRF3 and NFκB in RNA Pol II recruitment and pause release in human innate antiviral transcription.

Transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NFκB) are activated by external stimuli, including virus infection, to translocate to the nucleus and bind genomic targets important for immunity and inflammation. To investigate RNA polymerase II (Pol II) recruitment and elongation in the human antiviral gene regulatory network, a comprehensive genome-wide analysis was conducted during the initial phase of virus infection. Results reveal extensive integration of IRF3 and NFκB with Pol II and associated machinery and implicate partners for antiviral transcription. Analysis indicates that both de novo polymerase recruitment and stimulated release of paused polymerase work together to control virus-induced gene activation. In addition to known messenger-RNA-encoding loci, IRF3 and NFκB stimulate transcription at regions not previously associated with antiviral transcription, including abundant unannotated loci that encode novel virus-inducible RNAs (nviRNAs). These nviRNAs are widely induced by virus infections in diverse cell types and represent a previously overlooked cellular response to virus infection.

RAS mutations affect tumor necrosis factor-induced apoptosis in colon carcinoma cells via ERK-modulatory negative and positive feedback circuits along with non-ERK pathway effects.

More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. When the dynamics of phosphorylated ERK response to TNFalpha were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-alpha. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-alpha autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFalpha treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence.