Michifumi Yamashita

Viral apoptosis is induced by IRF-3-mediated activation of Bax

Upon infection with many RNA viruses, the cytoplasmic retinoic acid inducible gene‐I (RIG‐I) pathway activates the latent transcription factor IRF‐3, causing its nuclear translocation and the induction of many antiviral genes, including those encoding interferons. Here, we report a novel and distinct activity of IRF‐3, in virus‐infected cells, that induces apoptosis. Using genetically defective mouse and human cell lines, we demonstrated that, although both pathways required the presence of RIG‐I, IPS1, TRAF3 and TBK1, only the apoptotic pathway required the presence of TRAF2 and TRAF6 in addition. More importantly, transcriptionally inactive IRF‐3 mutants, such as the one missing its DNA‐binding domain, could efficiently mediate apoptosis. Apoptosis was triggered by the direct interaction of IRF‐3, through a newly identified BH3 domain, with the pro‐apoptotic protein Bax, their co‐translocation to the mitochondria and the resulting activation of the mitochondrial apoptotic pathway. Thus, IRF‐3 is a dual‐action cytoplasmic protein that, upon activation, translocates to the nucleus or to the mitochondrion and triggers two complementary antiviral responses of the infected cell.

The IRF-3/Bax-Mediated Apoptotic Pathway, Activated by Viral Cytoplasmic RNA and DNA, Inhibits Virus Replication

ABSTRACT Induction of apoptosis in cells infected by Sendai virus (SeV), which triggers the cytosolic RIG-I pathway, requires the presence of interferon regulatory factor 3 (IRF-3). Independent of IRF-3s transcriptional role, a novel IRF-3 activation pathway causes its interaction with the proapoptotic protein Bax and its mitochondrial translocation to induce apoptosis. Here we report that two other RNA viruses, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV), may also activate the same pathway. Moreover, cytosolic DNA, produced by adenovirus or introduced by transfection, activated the pathway in an RNA polymerase III-dependent fashion. To evaluate the contribution of this newly discovered apoptotic pathway to the hosts overall antiviral response, we measured the efficiencies of replication of various viruses in vitro and viral pathogenesis in vivo, using cells and mice that are selectively deficient in components required for the apoptotic pathway of IRF-3. Our results clearly demonstrate that the IRF-3/Bax-mediated apoptotic signaling branch contributes significantly to the hosts protection from viral infection and consequent pathogenesis.

Epidermal Growth Factor Receptor Is Essential for Toll-Like Receptor 3 Signaling

TLR3-mediated antiviral responses require phosphorylation of TLR3 by a growth factor receptor and a nonreceptor tyrosine kinase. A Growth Factor Receptor Takes Its Toll Toll-like receptor 3 (TLR3) initiates innate immune responses to double-stranded RNA (dsRNA) released by viruses or apoptotic cells. Activation of TLR3 requires phosphorylation of two tyrosine residues in its cytoplasmic domain. Yamashita et al. identified the tyrosine kinases involved and dissected the temporal sequence of these phosphorylation events. In response to dsRNA, TLR3 was phosphorylated by the epidermal growth factor receptor ErbB1, a receptor tyrosine kinase that triggers cellular proliferation in response to ligand binding. This phosphorylation event was required to trigger phosphorylation of TLR3 at a different site by the tyrosine kinase Src. In cells lacking EGFR or treated with an inhibitor of EGFR, the TLR3-mediated antiviral response was impaired. These results indicate that TLR3-mediated antiviral responses require tyrosine kinases with established roles in regulating cell growth. Toll-like receptors (TLRs) recognize specific microbial products and elicit innate immune signals to activate specific transcription factors that induce protective proteins, such as interferon. TLR3 is localized to endosomes and recognizes double-stranded RNA (dsRNA), which is generated by virally infected or apoptotic cells. TLR3 has been genetically linked to several human diseases, including some without viral etiology. Unlike other TLRs, TLR3 requires phosphorylation of two specific tyrosine residues in its cytoplasmic domain to recruit the adaptor protein TRIF (Toll–interleukin-1 receptor domain–containing adaptor protein inducing interferon-β) and initiate the antiviral response. We showed that two protein tyrosine kinases, the epidermal growth factor receptor (EGFR) ErbB1 and Src, bound sequentially to dsRNA-activated TLR3 and phosphorylated the two tyrosine residues. In cells lacking EGFR or treated with an inhibitor of EGFR, viral replication was enhanced and induction of antiviral genes was impaired. Thus, these results reveal a connection between antiviral innate immunity and cell growth regulators.

Novel Characteristics of the Function and Induction of Murine p56 Family Proteins

ABSTRACT The interferon-stimulated gene 56 (ISG56) family is induced strongly in response to virus infection, interferons (IFNs) and double-stranded RNA (dsRNA). In the mouse, this family comprises three members, ISG56, ISG54, and ISG49, which are clustered on chromosome 19 and encode the corresponding proteins p56, p54, and p49. Here, we report differential properties of these proteins and their distinct induction patterns in different cell types. All three murine proteins bound to the c-subunit of the translation initiation factor eIF3, but unlike the other members, p49 did not inhibit protein synthesis. Using a newly raised antibody, we demonstrated that both in vitro and in vivo, p49 expression was strongly induced by IFN, dsRNA, and Sendai virus. However, in kidney mesangial cells, as opposed to podocytes, encephalomyocarditis virus, vesicular stomatitis virus, or extracellular dsRNA did not induce any of the p56 family proteins, although they were robustly expressed after Sendai virus infection or dsRNA transfection. Furthermore, protein-specific differences in the regulation of p56 family members became evident in various leukocyte types: all three proteins were induced by IFN in T cells, but in B cells p56 and ISG56 mRNA could not be detected. Similarly, p56 was selectively uninducible in plasmacytoid dendritic cells, whereas in myeloid dendritic cells, all three family members were expressed. These results revealed novel cell type-, inducer-, and gene-specific regulation of the ISG56 family of genes.

Role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by Sendai virus.

ABSTRACT Infection of cultured cells by paramyxoviruses causes cell death, mediated by a newly discovered apoptotic pathway activated by virus infection. The key proapoptotic protein in this pathway is interferon regulatory factor 3 (IRF-3), which upon activation by virus infection binds BAX, translocates it to mitochondria, and triggers apoptosis. When IRF-3-knockdown cells were infected with Sendai virus (SeV), persistent infection (PI) was established. The PI cells produced infectious SeV continuously and constitutively expressed many innate immune genes. Interferon signaling was blocked in these cells. The elevated levels of IRF-3-driven genes in the PI cells indicated that the amount of residual IRF-3 activated by endogenous SeV was high enough to drive the transcriptional effects of IRF-3 but too low to trigger its apoptotic activity. We confirmed this IRF-3 threshold idea by generating a tetracycline (Tet)-inducible cell line for IRF-3 expression, which enabled us to express various levels of IRF-3. PI could be established in the Tet-off cell line, and as expected, when doxycycline was withdrawn, the cells underwent apoptosis. Finally, we tested for PI establishment in 12 mouse embryo fibroblasts by natural selection. Eleven lines became persistently infected; although seven out of them had low IRF-3 levels, four did not. When one of the latter four was further analyzed, we observed that it expressed a very low level of caspase 3, the final executor protease of the apoptotic pathway. These results demonstrated that SeV PI can arise from infection of normal wild-type cells, but only if they can find a way to impair the IRF-3-dependent apoptotic pathway.

A TRIF-Independent Branch of TLR3 Signaling

dsRNA is a common pathogen-associated molecular pattern that is recognized by cellular TLR3 and used by virus-infected cells to activate specific transcription factors and trigger induction of antiviral genes. In this article, we report a new branch of TLR3 signaling that does not lead to gene induction but affects many cellular properties, such as cell migration, adhesion, and proliferation. We demonstrated that the migration of multiple cell lineages was affected by dsRNA treatment or influenza virus infection in a TLR3-dependent fashion. Surprisingly, for this effect of TLR3 signaling, the adaptor proteins, TRIF and MyD88, were not required. The effects of the new pathway were mediated by the proto-oncoprotein c-Src, which bound to TLR3 after dsRNA stimulation of cells. The response was biphasic: upon dsRNA treatment, we observed an immediate increase in cell motility followed by its strong inhibition. Our results indicate that the first phase was mediated by dsRNA-induced phosphorylation and activation of Src, whereas the second phase resulted from the sequestration of activated Src in lipid rafts, thus decreasing its active cytoplasmic pool. As expected, two other functions of Src, its effect on cell adhesion and cell proliferation, were also inhibited by dsRNA treatment. These results demonstrate that activated TLR3 can engage Src to trigger multiple cellular effects and reveal a possible link between innate immune response and cell growth regulation. This study also provides a rare example of TLR-mediated cellular effects that do not require gene induction and the first example, to our knowledge, of an adaptor-independent effect of any TLR.

The Dual Functions of IL-1 Receptor-Associated Kinase 2 in TLR9-Mediated IFN and Proinflammatory Cytokine Production

Bone marrow-derived plasmacytoid dendritic cells (pDCs) from IL-1R–associated kinase (IRAK)2-deficient mice produced more IFNs than did wild-type pDCs upon stimulation with the TLR9 ligand CpG. Furthermore, in CpG-stimulated IRAK2-deficient pDCs there was increased nuclear translocation of IFN regulatory factor 7, the key transcription factor for IFN gene transcription in these cells. In IRAK2-deficient macrophages, enhanced NF-κB activation and increased expression of CpG-induced genes were detected within 2 h after treatment. However, at later times, NF-κB activation was decreased and, in contrast to the results with IFN, there was less secretion of other proinflammatory cytokines (such as TNF-α) and chemokines in CpG-stimulated IRAK2-deficient pDCs and macrophages. Therefore, although IRAK2 is a negative regulator of TLR9-mediated IFN production through its modulation of the transcriptional activity of IFN regulatory factor 7, it is also a positive regulator of TLR9-mediated proinflammatory cytokine and chemokine production at some level subsequent to transcription.

Constitutively Bound EGFR–Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal

Mammalian TLRs recognize microbial infection or cell death–associated danger signals and trigger the appropriate cellular response. These responses determine the strength and the outcome of the host–microbe interaction. TLRs are transmembrane proteins located on the plasma or the endosomal membrane. Their ectodomains recognize specific microbial or endogenous ligands, and the cytoplasmic domains interact with specific proteins to activate intracellular signaling pathways. TLR9, an endosomal TLR, is activated by endocytosed DNA. Activated TLR9 recruits the cytoplasmic adapter MyD88 and other signaling proteins to induce the synthesis of inflammatory cytokines and IFN. Uncontrolled activation of TLR9 leads to the undesired overproduction of inflammatory cytokines and consequent pathogenesis. Therefore, appropriate activation and the regulation of TLR9 signaling are critical. Tyrosine (Tyr) phosphorylation of TLR9 is essential for its activation; however, the role of specific Tyr kinases is not clear. In this article, we report that epidermal growth factor receptor (EGFR), a membrane-bound protein Tyr kinase, is essential for TLR9 signaling. Genetic ablation of EGFR or pharmacological inhibition of its kinase activity attenuates TLR9-mediated induction of genes in myeloid and nonmyeloid cell types. EGFR is constitutively bound to TLR9; upon ligand stimulation, it mediates TLR9 Tyr phosphorylation, which leads to the recruitment of MyD88, activation of the signaling kinases and transcription factors, and gene induction. In mice, TLR9-mediated liver injury and death are blocked by an EGFR inhibitor or deletion of the EGFR gene from myeloid cells, which are the major producers of inflammatory cytokines.

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.