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Ubiquitin and Viral Signaling
Summary: Zhijian "James" Chen investigates the biochemical mechanism underlying the function of ubiquitin in activating protein kinases. He is also interested in how viruses trigger the host immune responses, and he is using this as a model system to understand how animal cells defend against invading foreign genomes. These two avenues of research have recently converged, pointing to ubiquitin's key role in antiviral signaling.
We are taking biochemical and genetic approaches to dissect two signaling pathways: ubiquitin-mediated activation of protein kinases and antiviral innate immunity. We have uncovered a novel function of ubiquitin in activating protein kinases in the nuclear factor κB (NF-κB) signaling cascade through a proteasome-independent mechanism. Our current effort is focused on elucidating the biochemical mechanism underlying the regulatory function of ubiquitin. In viral signaling, we are particularly interested in dissecting the signaling pathway by which a host cell mounts an immune response to RNA virus infection. We are also interested in how some viruses evade the host immune system.
Ubiquitin Signaling
The transcription factor NF-κB is activated by a large variety of agents, including proinflammatory cytokines and microbial pathogens. NF-κB is normally retained in the cytosol through its association with the inhibitory proteins of the IκB family. Stimulation of cells leads to the rapid activation of the IκB kinase (IKK) complex, which phosphorylates IκB and targets this inhibitor for polyubiquitination and subsequent degradation by the proteasome. This allows NF-κB to enter the nucleus to activate a battery of genes involved in inflammation, immunity, and survival of cells.
In the course of studying the regulation of IKK by TRAF6, a protein essential for NF-κB activation by interleukin-1 receptor (IL-1R) and Toll-like receptors (TLRs), we identified two factors that mediate IKK activation by TRAF6. One of these factors is a ubiquitin-conjugating enzyme (E2) complex consisting of Ubc13 and Uev1A, and the other is a protein kinase complex containing TAK1, TAB1, and TAB2. We found that TRAF6 functions as a ubiquitin ligase (E3) that, in conjunction with the Ubc13-Uev1A complex, catalyzes the synthesis of a unique polyubiquitin chain linked through lysine-63 of ubiquitin. This K63 polyubiquitination activates the TAK1 kinase complex through a proteasome-independent mechanism.
Further studies showed that TAB2 and its homolog TAB3 contain a highly conserved zinc finger domain known as the NZF domain, which binds preferentially to K63 polyubiquitin chains, and that this binding is required for TAK1 activation. After TAK1 is activated, it phosphorylates IKKβ at two serine residues within the activation loop, leading to activation of the IKK complex. We also identified a novel ubiquitin-binding domain (NUB) in NEMO, the essential regulatory subunit of the IKK complex. This domain binds to K63 polyubiquitin chains and recruits IKK to the TAK1 complex, facilitating the phosphorylation of IKK by TAK1. The activated TAK1 also phosphorylates MAP kinase kinases (MKKs), leading to the activation of Jun N-terminal kinase (JNK) and p38 kinase. Our results support a model in which K63 polyubiquitin chains serve as a scaffold to recruit and assemble protein kinase complexes, leading to their activation.
Ubiquitin-mediated activation of protein kinases plays a pervasive role in multiple signaling pathways, including those emanating from the tumor necrosis factor receptors and T cell receptors. In addition, recent studies suggest that K63 polyubiquitination is important for signaling by intracellular pathogen sensors, including the NOD-like receptors (NLRs) and retinoic acid–induced gene-I (RIG-I)-like receptors (RLRs) that detect cytosolic bacteria and viruses, respectively (see below).
Antiviral Signaling Pathways
Viral infection is detected by the host through two distinct pathways. One of these pathways is mediated by a subfamily of TLRs, such as TLR3, -7, -8, and -9, which are located on the endosomal membrane. These TLRs detect viral DNA or RNA in the lumen of the endosome and transmit signals to the cytosolic adapter MyD88, which activates NF-κB and interferon regulatory factors (IRFs) through TRAF6. NF-κB and IRFs induce type I interferons and other cytokines to suppress viral replication and assembly. The other antiviral pathway detects viral RNA in the cytosol through the RNA helicase proteins RIG-I and MDA5 (melanoma differentiation antigen). These proteins bind to viral double-stranded RNA or single-stranded RNA containing 5'-triphosphate, which is distinguished from the host RNA that is normally 5'-capped. The binding of the viral RNA induces a conformational change that exposes the amino-terminal CARD domains of RIG-I and MDA5. These CARD domains interact with the CARD domain of another protein that we termed MAVS (mitochondrial antiviral signaling). We found that MAVS is anchored to the mitochondrial outer membrane through a carboxyl-terminal transmembrane domain and that this mitochondrial localization is essential for its signaling function. MAVS activates IKK and TBK1 in the cytosol, which in turn activate NF-κB and IRF3, respectively. Recently, using a combination of biochemical and genetic approaches, we found that MAVS activates IKK and TBK1 though a mechanism that depends on K63 polyubiquitination.
The importance of the mitochondrial localization of MAVS is exploited by some viruses that manage to escape the host immune system. For example, hepatitis C virus (HCV) employs the viral protease NS3/4A to cleave MAVS at a cysteine residue (Cys-508) adjacent to the mitochondrial transmembrane domain, thereby dislodging MAVS from the mitochondria and rendering it inactive in inducing interferons. This allows the virus to evade the host immune system to establish persistent infection in more than 100 million people worldwide.
To investigate the role of MAVS in vivo, we have engineered mouse strains lacking the Mavs gene. In most of the cells we have examined, including fibroblasts, macrophages, and conventional dendritic cells, deletion of MAVS abolished the induction of interferons and cytokines by RNA viruses such as Sendai virus. However, in plasmacytoid dendritic cells, MyD88, but not MAVS, is required for interferon induction by Sendai virus. Both MyD88 and MAVS contribute to effective antiviral immunity in vivo, as mice lacking either gene can still produce interferons in response to infection by certain RNA viruses, whereas mice lacking both genes are completely defective in producing interferons. These results indicate that effective antiviral immune responses require cooperation of different cell types that utilize distinct virus-sensing pathways.
We have used the mice lacking Mavs or Myd88, or both, to investigate innate and adaptive immune responses against respiratory syncytial virus (RSV), an RNA virus that causes respiratory diseases, especially in infants and the elderly. The Mavs-deficient mice were completely defective in producing type I interferons and proinflammatory cytokines in response to RSV infection, whereas the Myd88-deficient mice produced these cytokines normally. Surprisingly, because of an intact CD8 T cell response, mice lacking Mavs alone, or both Mavs and Myd88, were still able to clear RSV. We found that, unlike conventional dendritic cells and alveolar macrophages that require Mavs for expression of costimulatory molecules, a small subset of pulmonary dendritic cells were activated in the absence of Mavs and Myd88, and these cells could traffic to the draining lymph node to activate CD8 T cells. These results provide an example of effective adaptive immune responses in the absence of innate immunity triggered by MAVS and MyD88. Work is in progress to characterize the pulmonary dendritic cell subset that can be activated independently of innate immune cytokines.
This research is supported in part by grants from the National Institutes of Health and the Robert Welch Foundation.
Last updated March 19, 2009
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