scholarly journals IWS1 phosphorylation by AKT3 controls nuclear export of type I IFN mRNAs and sensitivity to oncolytic viral infection, by regulating the alternative RNA splicing of U2AF2

2020 ◽  
Author(s):  
Georgios I. Laliotis ◽  
Adam D. Kenney ◽  
Evangelia Chavdoula ◽  
Arturo Orlacchio ◽  
Abdul K. Kaba ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Viral Infection ◽  
Nuclear Export ◽  
Rna Splicing ◽  
Proper Function ◽  
Type I ◽  
Exon 2 ◽  
Type I Ifns ◽  
Intronless Genes ◽  
In Cells

AbstractType I IFNs orchestrate the antiviral response. Interestingly, IFNA1 and IFNB1 genes are naturally intronless. Based on previous work, the splicing factor U2 Associated Factor 65 (U2AF65), encoded by U2AF2, and pre-mRNA Processing factor 19 (Prp19) function on the Cytoplasmic Accumulation Region Elements (CAR-E), affecting the nuclear export of intronless genes. We have previously shown that the loss of IWS1 phosphorylation by AKT3, promotes the alternative RNA splicing of U2AF2, resulting in novel transcripts lacking exon 2. This exon encodes part of the Serine-Rich (RS) domain of U2AF65, which is responsible for its binding with Prp19. Here, we show that IWS1 phosphorylation and the U2AF2 RNA splicing pattern affect the nuclear export of introless mRNAs. We also demonstrate that the same axis is required for the proper function of the CAR-Es. Mechanistically, whereas both U2AF65 isoforms bind CAR-E, the recruitment of Prp19 occurs only in cells expressing phosphorylated IWS, promoting intronless genes export. Moreover, analysis of Lung adenocarcinoma patients showed that high p-IWS1 activity correlates with the assembly of the U2AF65/Prp19 complex and export of intronless genes, in vivo. Accordingly, the expression of type I IFNs was decreased in cells deficient in IWS1 phosphorylation and the viral infection was increased. Furthermore, following infection with oncolytic virus, we observed reduced activation of p-STAT1 and expression of Interferon Stimulated Genes (ISG), in cells stimulated by shIWS1-derived supernatant, or cells treated with the pan-AKT inhibitor, MK2206. Consistently, killing curves and apoptosis assays after infection with oncolytic viruses, revealed increased susceptibility upon the loss of IWS1, with subsequent activation of Caspase-mediated death. The treatment of the lung adenocarcinoma cells with MK2206, phenocopied the loss of IWS1 phosphorylation. These data identify a novel mechanism by which the AKT/p-IWS1 axis, by hijacking the epigenetic regulation of RNA splicing and processing, contributes to the resistance to oncolytic viral infection, suggesting that combined inhibition of the splicing machinery and AKT/p-IWS1 signals would sensitize tumors to oncolytic viral treatment.

scholarly journals Phosphor-IWS1-dependent U2AF2 splicing regulates trafficking of CAR-E-positive intronless gene mRNAs and sensitivity to viral infection

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Georgios I. Laliotis ◽  
Adam D. Kenney ◽  
Evangelia Chavdoula ◽  
Arturo Orlacchio ◽  
Abdul Kaba ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Rna Splicing ◽  
Target Genes ◽  
Apoptotic Cell ◽  
Oncolytic Viruses ◽  
Poly I:C ◽  
Type I ◽  
Exon 2 ◽  
Intronless Gene ◽  
Enhanced Sensitivity

AbstractAKT-phosphorylated IWS1 promotes Histone H3K36 trimethylation and alternative RNA splicing of target genes, including the U2AF65 splicing factor-encoding U2AF2. The predominant U2AF2 transcript, upon IWS1 phosphorylation block, lacks the RS-domain-encoding exon 2, and encodes a protein which fails to bind Prp19. Here we show that although both U2AF65 isoforms bind intronless mRNAs containing cytoplasmic accumulation region elements (CAR-E), only the RS domain-containing U2AF65 recruits Prp19 and promotes their nuclear export. The loading of U2AF65 to CAR-Elements was RS domain-independent, but RNA PolII-dependent. Virus- or poly(I:C)-induced type I IFNs are encoded by genes targeted by the pathway. IWS1 phosphorylation-deficient cells therefore, express reduced levels of IFNα1/IFNβ1 proteins, and exhibit enhanced sensitivity to infection by multiple cytolytic viruses. Enhanced sensitivity of IWS1-deficient cells to Vesicular Stomatitis Virus and Reovirus resulted in enhanced apoptotic cell death via caspase activation. Inhibition of this pathway may therefore sensitize cancer cells to oncolytic viruses.

scholarly journals Role of Alternative Splicing in Regulating Host Response to Viral Infection

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1720
Author(s):  
Kuo-Chieh Liao ◽  
Mariano A. Garcia-Blanco
Keyword(s):  
Innate Immunity ◽  
Alternative Splicing ◽  
Immune Responses ◽  
Viral Infection ◽  
Rna Splicing ◽  
Type I ◽  
Host Immune Responses ◽  
Transcriptional Regulatory Mechanisms ◽  
Host Virus Interactions

The importance of transcriptional regulation of host genes in innate immunity against viral infection has been widely recognized. More recently, post-transcriptional regulatory mechanisms have gained appreciation as an additional and important layer of regulation to fine-tune host immune responses. Here, we review the functional significance of alternative splicing in innate immune responses to viral infection. We describe how several central components of the Type I and III interferon pathways encode spliced isoforms to regulate IFN activation and function. Additionally, the functional roles of splicing factors and modulators in antiviral immunity are discussed. Lastly, we discuss how cell death pathways are regulated by alternative splicing as well as the potential role of this regulation on host immunity and viral infection. Altogether, these studies highlight the importance of RNA splicing in regulating host–virus interactions and suggest a role in downregulating antiviral innate immunity; this may be critical to prevent pathological inflammation.

scholarly journals Type I interferons act directly on nociceptors to produce pain sensitization: Implications for viral infection-induced pain

2019 ◽  
Author(s):  
Paulino Barragan-Iglesias ◽  
Úrzula Franco-Enzástiga ◽  
Vivekanand Jeevakumar ◽  
Andi Wangzhou ◽  
Vinicio Granados-Soto ◽  
...  
Keyword(s):  
Viral Infection ◽  
Viral Infections ◽  
Mrna Translation ◽  
Type I Interferons ◽  
Poly I:C ◽  
Type I ◽  
Drg Neurons ◽  
Pain Hypersensitivity ◽  
Type I Ifns ◽  
Pain Sensitization

ABSTRACTOne of the first signs of viral infection is body-wide aches and pain. While this type of pain usually subsides, at the extreme, viral infections can induce painful neuropathies that can last for decades. Neither of these types of pain sensitization are well understood. A key part of the response to viral infection is production of interferons (IFNs), which then activate their specific receptors (IFNRs) resulting in downstream activation of cellular signaling and a variety of physiological responses. We sought to understand how type I IFNs (IFN-α and IFN-β) might act directly on nociceptors in the dorsal root ganglion (DRG) to cause pain sensitization. We demonstrate that type I IFNRs are expressed in small/medium DRG neurons and that their activation produces neuronal hyper-excitability and mechanical pain in mice. Type I IFNs stimulate JAK/STAT signaling in DRG neurons but this does not apparently result in PKR-eIF2α activation that normally induces an anti-viral response by limiting mRNA translation. Rather, type I interferons stimulate MNK-mediated eIF4E phosphorylation in DRG neurons to promote pain hypersensitivity. Endogenous release of type I IFNs with the double stranded RNA mimetic poly(I:C) likewise produces pain hypersensitivity that is blunted in mice lacking MNK-eIF4E signaling. Our findings reveal mechanisms through which type I IFNs cause nociceptor sensitization with implications for understanding how viral infections promote pain and can lead to neuropathies.SIGNIFICANCE STATEMENTIt is increasingly understood that pathogens interact with nociceptors to alert organisms to infection as well as to mount early host defenses. While specific mechanisms have been discovered for diverse bacteria and fungal pathogens, mechanisms engaged by viruses have remained elusive. Here we show that type 1 interferons, one of the first mediators produced by viral infection, act directly on nociceptors to produce pain sensitization. Type I interferons act via a specific signaling pathway (MNK-eIF4E signaling) that is known to produce nociceptor sensitization in inflammatory and neuropathic pain conditions. Our work reveals a mechanism through which viral infections cause heightened pain sensitivity

scholarly journals Virus Multiplicity of Infection Affects Type I Interferon Subtype Induction Profiles and Interferon-Stimulated Genes

2015 ◽  
Vol 89 (22) ◽  
pp. 11534-11548 ◽  
Author(s):  
Luna A. Zaritsky ◽  
Jacquelyn R. Bedsaul ◽  
Kathryn C. Zoon
Keyword(s):  
Distinct Type ◽  
Gene Induction ◽  
Viral Diseases ◽  
Type I ◽  
Multiplicity Of Infection ◽  
The Novel ◽  
Type I Ifn ◽  
Independent Manner ◽  
Type I Ifns ◽  
In Cells

ABSTRACTType I interferons (IFNs) are induced upon viral infection and important mediators of innate immunity. While there is 1 beta interferon (IFN-β) protein, there are 12 different IFN-α subtypes. It has been reported extensively that different viruses induce distinct patterns of IFN subtypes, but it has not been previously shown how the viral multiplicity of infection (MOI) can affect IFN induction. In this study, we discovered the novel finding that human U937 cells infected with 2 different concentrations of Sendai virus (SeV) induce 2 distinct type I IFN subtype profiles. Cells infected at the lower MOI induced more subtypes than cells infected at the higher MOI. We found that this was due to the extent of signaling through the IFN receptor (IFNAR). The cells infected at the lower viral MOI induced the IFNAR2-dependent IFN-α subtypes 4, 6, 7, 10, and 17, which were not induced in cells infected at higher virus concentrations. IFN-β and IFN-α1, -2, and -8 were induced in an IFNAR-independent manner in cells infected at both virus concentrations. IFN-α5, -14, -16, and -21 were induced in an IFNAR-dependent manner in cells infected at lower virus concentrations and in an IFNAR-independent manner in cells infected at higher virus concentrations. These differences in IFN subtype profiles in the 2 virus concentrations also resulted in distinct interferon-stimulated gene induction. These results present the novel finding that different viral MOIs differentially activate JAK/STAT signaling through the IFNAR, which greatly affects the profile of IFN subtypes that are induced.IMPORTANCEType I IFNs are pleiotropic cytokines that are instrumental in combating viral diseases. Understanding how the individual subtypes are induced is important in developing strategies to block viral replication. Many studies have reported that different viruses induce distinct type I IFN subtype profiles due to differences in the way viruses are sensed in different cell types. However, we report in our study the novel finding that the amount of virus used to infect a system can also affect which type I IFN subtypes are induced due to the extent of activation of certain signaling pathways. These distinct IFN subtype profiles in cells infected at different MOIs are correlated with differences in interferon-stimulated gene induction, indicating that the same virus can induce distinct antiviral responses depending on the MOI. Because type I IFNs are used as therapeutic agents to treat viral diseases, understanding their antiviral mechanisms can enhance clinical treatments.

Targeting IRFs by ubiquitination: regulating antiviral responses

2008 ◽  
Vol 36 (3) ◽  
pp. 453-458 ◽  
Author(s):  
Rowan Higgs ◽  
Caroline A. Jefferies
Keyword(s):  
Transcription Factors ◽  
Viral Infection ◽  
Viral Proteins ◽  
Interferon Regulatory Factor ◽  
Type I ◽  
Regulatory Factor ◽  
Ubiquitin Pathway ◽  
Cellular Processes ◽  
Antiviral Responses ◽  
Type I Ifns

The IRF [IFN (interferon) regulatory factor] family of transcription factors control many cellular processes, including induction of key antiviral cytokines, type I IFNs, following viral infection. Recent studies have revealed several endogenous and viral proteins involved in ubiquitin-mediated regulation of IRF activity and thus having an impact on type I IFN signalling. Through the ubiquitin pathway, these proteins can manipulate the antiviral response either by initiating proteasomal degradation of the IRFs or, in contrast, by promoting activation of the IRFs.

scholarly journals Induction of USP25 by viral infection promotes innate antiviral responses by mediating the stabilization of TRAF3 and TRAF6

2015 ◽  
Vol 112 (36) ◽  
pp. 11324-11329 ◽  
Author(s):  
Dandan Lin ◽  
Man Zhang ◽  
Meng-Xin Zhang ◽  
Yujie Ren ◽  
Jie Jin ◽  
...  
Keyword(s):  
Viral Infection ◽  
Proinflammatory Cytokines ◽  
Feedback Regulation ◽  
Type I Interferons ◽  
Type I ◽  
Wild Type ◽  
Dna Viruses ◽  
Type I Ifns ◽  
Specific Protease ◽  
Rna And Dna

Host pathogen-recognition receptors detect nucleic acid from invading viruses and initiate a series of signaling pathways that lead to the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we found that a viral infection-induced deubiquitinase (DUB), ubiquitin-specific protease 25 (USP25) was required for host defense against RNA and DNA viruses. The activation of transcription factors IRF3 and NF-κB was impaired and the production of type I IFNs and proinflammatory cytokines was inhibited in Usp25−/− cells compared with the wild-type counterparts after RNA or DNA viruses infection. Consistently, USP25 deficient mice were more susceptible to H5N1 or HSV-1 infection compared with the wild-type mice. USP25 was associated with TRAF3 and TRAF6 after infection by RNA or DNA viruses and protected virus-induced proteasome-dependent or independent degradation of TRAF3 and TRAF6, respectively. Moreover, reconstitution of TRAF3 and TRAF6 into Usp25−/− MEFs restored virus-triggered production of type I IFNs and proinflammatory cytokines. Our findings thus reveal a previously uncovered positive feedback regulation of innate immune responses against RNA and DNA viruses by USP25.

scholarly journals UBXN3B Restricts Viral Pathogenesis by Maintaining Hematopoietic Homeostasis

2021 ◽  
Author(s):  
Tingting Geng ◽  
Duomeng Yang ◽  
Tao Lin ◽  
Andrew Harrison ◽  
Binsheng Wang ◽  
...  
Keyword(s):  
Viral Infection ◽  
Type I ◽  
Lung Pathology ◽  
Viral Loads ◽  
Type I Ifns ◽  
Multipotent Progenitors ◽  
Specific Immunoglobulin ◽  
The Right ◽  
Severe Lung

ABSTRACTHematopoiesis is finely regulated to enable timely production of the right number and type of mature immune cells to maintain tissue homeostasis. Dysregulated hematopoiesis may compromise antiviral immunity and/or exacerbate immunopathogenesis. Herein, we report an essential and new role of ubiquitin X domain containing gene 3B (UBXN3B) in balancing myelopoiesis and lymphopoiesis. Ubxn3b deficiency (Ubxn3b-/-) results in a remarkable increase in myeloid cells and neutrophil-to-lymphocyte ratio, along with a reduction in lymphocytes in steady-state mice. This dysregulation is exacerbated during viral infection and renders mice highly vulnerable to severe lung pathology induced by severe acute respiratory syndrome coronavirus 2 and arthritis by arthritogenic alphaviruses. Ubxn3b-/- mice present normal type I IFNs, higher viral loads and inflammatory mediators, lower virus-specific immunoglobulin G and slower resolution of disease, when compared to Ubxn3b+/+ littermates. Mechanistically, Ubxn3b-/- mice have fewer multipotent progenitors and common lymphoid progenitors, but more common myeloid progenitors. In particular, the precursor and immature B cell numbers are dramatically decreased in the bone marrow of Ubxn3b-/- mice. These data demonstrate that UBXN3B signaling is essential for restricting viral infection and immunopathogenesis by maintaining hematopoietic homeostasis.

scholarly journals USP22 promotes IRF3 nuclear translocation and antiviral responses by deubiquitinating the importin protein KPNA2

2020 ◽  
Vol 217 (5) ◽  
Author(s):  
Zeng Cai ◽  
Meng-Xin Zhang ◽  
Zhen Tang ◽  
Qiang Zhang ◽  
Jing Ye ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Viral Infection ◽  
Unknown Function ◽  
Nuclear Translocation ◽  
Therapeutic Strategies ◽  
Deubiquitinating Enzyme ◽  
Type I ◽  
Antiviral Responses ◽  
Type I Ifns ◽  
Increased Susceptibility

USP22 is a cytoplasmic and nuclear deubiquitinating enzyme, and the functions of cytoplasmic USP22 are unclear. Here, we discovered that cytoplasmic USP22 promoted nuclear translocation of IRF3 by deubiquitianting and stabilizing KPNA2 after viral infection. Viral infection induced USP22-IRF3 association in the cytoplasm in a KPNA2-depedent manner, and knockdown or knockout of USP22 or KPNA2 impaired IRF3 nuclear translocation and expression of downstream genes after viral infection. Consistently, Cre-ER Usp22fl/fl or Lyz2-Cre Usp22fl/fl mice produced decreased levels of type I IFNs after viral infection and exhibited increased susceptibility to lethal viral infection compared with the respective control littermates. Mechanistically, USP22 deubiquitinated and stabilized KPNA2 after viral infection to facilitate efficient nuclear translocation of IRF3. Reconstitution of KPNA2 into USP22 knockout cells restored virus-triggered nuclear translocation of IRF3 and cellular antiviral responses. These findings define a previously unknown function of cytoplasmic USP22 and establish a mechanistic link between USP22 and IRF3 nuclear translocation that expands potential therapeutic strategies for infectious diseases.

scholarly journals IWS1 phosphorylation promotes cell proliferation and predicts poor prognosis in EGFR mutant lung adenocarcinoma patients, through the cell cycle-regulated U2AF2 RNA splicing

2020 ◽  
Author(s):  
Georgios I. Laliotis ◽  
Evangelia Chavdoula ◽  
Maria D. Paraskevopoulou ◽  
Abdul D. Kaba ◽  
Alessandro La Ferlita ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Cycle ◽  
Cell Proliferation ◽  
Lung Adenocarcinoma ◽  
Rna Splicing ◽  
Cell Cycle Progression ◽  
Phosphorylation Site ◽  
Tumor Stage ◽  
Exon 2

AbstractOur previous studies have shown that IWS1 (Interacts with Spt6) is a phosphorylation target of AKT and regulates the alternative RNA splicing of FGFR2, linking IWS1 with human Non-Small Cell Lung Cancer. To further address the role of IWS1 in alternative RNA splicing in lung cancer, we performed an RNA-seq study using lung adenocarcinoma cells in which IWS1 was knocked down or replaced by its phosphorylation site mutant. The results identified a novel, exon 2 deficient splice variant of the splicing factor U2 Associated-Factor 2 (U2AF2), whose abundance increases, upon the loss of phosphorylated IWS1. This exon encodes part of the U2AF65 Serine-Rich (RS) Domain, which is required for its binding with pre-mRNA Processing factor 19 (Prp19). Here, we show that U2AF2 exon 2 inclusion depends on phosphorylated IWS1, by promoting Histone H3K36 trimethylation and the assembly of LEDGF/SRSF1 splicing complexes, in a cell-cycle specific manner. Inhibition of the pathway results in the downregulation of cell cycle division associated 5 (CDCA5), a phosphorylation target and regulator of ERK, leading to G2/M phase arrest, impaired cell proliferation and tumor growth in mouse xenografts models, an effect more pronounced in EGFR mutant cells. Analysis of lung adenocarcinoma samples revealed strong correlations between IWS1 phosphorylation, U2AF2 RNA splicing, and Sororin/p-ERK levels, especially in EGFR, as opposed to KRAS mutant patients. More importantly, IWS1 phosphorylation and U2AF2 RNA splicing pattern are positively correlated with tumor stage, grade, relapse and metastasis, and associated with poor survival in lung adenocarcinoma patients, harboring EGFR, but not KRAS, mutations. This work highlights the instrumental role of the AKT/p-IWS1 axis to alternative RNA splicing in governing cell cycle progression and tumorigenesis, and proposes this axis as a novel drug target in EGFR mutant lung adenocarcinoma, by concomitantly affecting the epigenetic regulation of RNA processing and oncogenic signals.

scholarly journals Overexpression of the SETD2 WW domain inhibits the phosphor-IWS1/SETD2 interaction and the oncogenic AKT/IWS1 RNA splicing program.

2021 ◽  
Author(s):  
Georgios I. Laliotis ◽  
Evangelia Chavdoula ◽  
Vollter Anastas ◽  
Satishkumar Singh ◽  
Adam I. Kenney ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Target Genes ◽  
Therapeutic Potential ◽  
Human Cancer ◽  
Metastatic Potential ◽  
Egfr Mutations ◽  
Type I ◽  
Transcriptional Elongation ◽  
Ww Domain ◽  
Intronless Genes

Our earlier studies had shown that AKT phosphorylates IWS1, and that following phosphorylation, IWS1 recruits the histone methyltransferase SETD2 to an SPT6/IWS1/ALY complex, which assembles on the Ser2-phosphorylated CTD of RNA Pol II. Recruited SETD2 methylates histone H3 at K36, during transcriptional elongation of target genes, and this regulates multiple steps in RNA metabolism. By regulating the RNA splicing of U2AF2, it controls cell proliferation. Importantly, pathway activity correlates with grade, stage and metastatic potential of lung adenocarcinomas, especially those with EGFR mutations. By regulating nucleocytoplasmic mRNA transport of intronless genes, including those encoding type I IFNs, it regulates sensitivity to viral infection. Here, we show that SETD2 interacts with IWS1 via its WW domain, that the interaction is IWS1 phosphorylation-dependent and that WW domain overexpression blocks the interaction and inhibits the pathway and its biological outcomes. We conclude that blocking the phosphor-IWS1/SETD2 interaction is feasible and has significant therapeutic potential in human cancer.

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