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1.  The Cytosolic DNA Sensor cGAS Forms An Oligomeric Complex with DNA and Undergoes Switch-like Conformational Changes in the Activation Loop 
Cell reports  2014;6(3):421-430.
The presence of DNA in the cytoplasm is a danger signal that triggers immune and inflammatory responses. Cytosolic DNA binds to and activates cyclic GMP-AMP (cGAMP) synthase (cGAS), which produces the second messenger cGAMP. cGAMP binds to the adaptor protein STING and activates a signaling cascade that leads to the production of type-I interferons and other cytokines. Here we report the crystal structures of human cGAS in its apo form, representing its auto-inhibited conformation, as well as cGAMP-bound and sulfate-bound forms. These structures reveal switch-like conformational changes of an activation loop that result in the rearrangement of the catalytic site. The structure of DNA-bound cGAS reveals a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the protein-protein interface and the two DNA binding surfaces are critical for cGAS activation. These results provide new insights into the mechanism of DNA sensing by cGAS.
PMCID: PMC3969844  PMID: 24462292
2.  A Novel Missense Mutation in the Nuclear Factor-κB Essential Modulator (NEMO) Gene Resulting in Impaired Activation of the NF-κB Pathway and a Unique Clinical Phenotype Presenting as MRSA Subdural Empyema 
Journal of clinical immunology  2010;30(6):881-885.
We describe a previously unreported 437 T→G missense mutation producing a V146G substitution in the first coiled-coil (CC1) domain of nuclear factor-κB essential modulator (NEMO) in a 9-month-old boy with ectodermal dysplasia with immunodeficiency who presented with methicillin-resistant Staphylococcus aureus subdural empyema. We performed in vitro experiments to determine if this novel mutation resulted in impaired NF-κB signaling.
IκBα phosphorylation experiments were performed using a Jurkat T cell line lacking endogenous NEMO expression that was transfected with vectors containing either the wild type or the patient’s V146G mutation. The cells were stimulated with TNF-α to activate the NF-κB pathway. Phosphorylated IκBα was detected by immunoblotting with anti-phospho-IκBα antibodies. Peripheral blood mononuclear cells from the patient were stimulated with TNF-α or anti-CD3 and anti-CD28. Impaired IκBα degradation was detected using antibodies against the IκBα protein.
While TNF-α stimulation resulted in IκBα phosphorylation in NEMO-deficient Jurkat cells reconstituted with wild-type NEMO, cell transfected with the V146G mutant exhibited a 75% reduction in phospho-IκBα. Peripheral blood mononuclear cells from the patient showed impaired degradation of IκBα after stimulation when compared with normal controls.
The patient’s V146G mutation results in impaired NF-κB activation in vitro. The mutation extends the known N-terminal boundary within the CC1 domain that produces an ectodermal dysplasia phenotype, and defines an infectious susceptibility previously unappreciated in ectodermal dysplasia with immunodeficiency (methicillin-resistant S. aureus subdural empyema), broadening the clinical spectrum associated with the disease.
PMCID: PMC3951109  PMID: 20652730
NEMO; immunodeficiency; ectodermal; dysplasia; mutation; MRSA
3.  Structural basis for the prion-like MAVS filaments in antiviral innate immunity 
eLife  2014;3:e01489.
Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments.
eLife digest
When infected by a virus, the body will generally launch an immune response to eliminate the infectious agent. Activation of the innate immune system–the first line of defense against infection—requires the host cells to recognize the presence of a pathogen and to sound the alarm once the invader is detected.
Viruses can contain DNA or RNA, and when a virus containing double stranded RNA enters a cell, or starts replicating within the cytoplasm, proteins called RIG-I-like receptors (RLRs) will detect these RNA molecules. This will trigger a signaling cascade that results in the production of type I interferons, the proteins that activate cells of the innate immune system.
Members of the RLR family of receptors, including RIG-I and MDA5, initiate the signaling cascade by interacting with the mitochondrial antiviral-signaling (MAVS) protein. Recent work revealed that upon activation by RIG-I or MDA5, MAVS proteins aggregate on the surface of mitochondria and form protein filaments. These filaments then activate inactive MAVS proteins, leading to the formation of more filaments. While a region of the MAVS protein called caspase activation and recruitment domain (CARD) is known to be involved in the formation of the filaments, the chemical interactions that govern the formation process have yet to be described.
Now, using cryo-electron microscopy, Xu et al. have shown that these filaments are comprised of three-stranded helixes. This came as something of a surprise because other similar filaments known as prions are made of tightly packed beta sheets. Xu et al. went on to visualize full-length MAVS filaments in virus-infected cells, and to verify that mutations that impair the assembly of MAVS filaments also prevent RNA viruses from triggering the production of interferon. These results have the potential to inform future studies of the innate immune response, as well as investigations into the assembly of proteins to form prion-like filaments.
PMCID: PMC3932521  PMID: 24569476
MAVS; innate immunity; prion-like filaments; cryoEM reconstruction; three-stranded filaments; human; viruses
4.  Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses 
Science (New York, N.Y.)  2013;341(6148):10.1126/science.1240933.
Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus (MLV) and Simian immunodeficiency virus (SIV). These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.
PMCID: PMC3860819  PMID: 23929945
5.  Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells 
eLife  2014;3:e01535.
The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells.
eLife digest
A virus is basically molecules of DNA or RNA inside a protein shell, and in order to reproduce, it must infect a living cell and take control of it. However, the attacked cell will fight back and try to eliminate the invader. Activation of this so-called innate immune response requires the host cells to recognize that they have been infected, which they do by detecting the tell-tale molecules that indicate the presence of the virus.
When an RNA virus infects a cell, the tell-tale molecules are often atypical RNA molecules carried by the virus or produced as the virus replicates. Recognition of this ‘foreign’ material by receptor proteins inside the cell triggers the production of molecules called interferons, which activate the innate defence systems that eliminate the virus.
Different receptor proteins recognize different RNA viruses. For example, a receptor called MDA5 is known to respond to the picornaviruses, some of which can cause inflammation of the brain and heart muscle. However, the identities of the specific RNA molecules that are recognized by the MDA5 receptor have not been known. Deddouche et al. have now identified one such RNA molecule with the help of a second receptor protein, called LGP2.
The LGP2 receptor is not able to give the signal to produce interferons, so it is thought to work by binding to the RNA molecule to form a complex that is then relayed to MDA5 to give this signal. By isolating the complexes of LGP2 receptor from picornavirus-infected cells and sequencing the associated RNA, it was possible to identify the mystery RNA trigger. Deddouche et al. then tested picornaviruses in which this piece of RNA had been deleted from the genome, and found that the mutant viruses triggered a much weaker interferon response. By providing insight into the ways that some viruses are detected by the innate immune system, this research may inform future work on the development of new treatments to control viral infection.
PMCID: PMC3967861  PMID: 24550253
RIG-I-like receptors; MDA5; LGP2; picornavirus; EMCV; antiviral immunity; human; mouse; viruses
6.  Regulation of NF-κB by Ubiquitination 
Current opinion in immunology  2013;25(1):4-12.
The nuclear factor κ enhancer binding protein (NF-κB) family of transcription factors regulates the expression of a large array of genes involved in diverse cellular processes including inflammation, immunity and cell survival. Activation of NF-κB requires ubiquitination, a highly conserved and versatile modification that can regulate cell signaling through both proteasome dependent and independent mechanisms. Studies in the past few years have provided new insights into the mechanisms underlying regulation of NF-κB by ubiquitination, including the involvement of multiple linkages of ubiquitin, the essential role of ubiquitin binding, and the function of unanchored polyubiquitin chains. In this review, we will focus on recent advances in understanding the role of ubiquitination in NF-κB regulation in various pathways.
PMCID: PMC3594545  PMID: 23312890
7.  Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING 
Molecular cell  2013;51(2):10.1016/j.molcel.2013.05.022.
The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.
PMCID: PMC3808999  PMID: 23747010
8.  microRNA-21 promotes cardiac fibrosis and development of heart failure with preserved left ventricular ejection fraction by up-regulating Bcl-2 
The morbidity and mortality of heart failure with preserved left ventricular ejection fraction (HFpEF) were similar to those of systolic heart failure, but the pathogenesis of HFpEF remains poorly understood. It was demonstrated that, in systolic heart failure, microRNA-21 (miR-21) could inhibit the apoptosis of cardiac fibroblasts, leading to cardiac hypertrophy and myocardial fibrosis, but the role of miR-21 in HFpEF remains unknown. By employing cell culture technique, rat myocardiocytes and cardiac fibroblasts were obtained. The expression of miR-21 in the two cell types under different conditions was compared and we found that the miR-21 expression was significantly higher in cardiac fibroblasts than in myocardiocytes. We established a rat HFpEF model and harvested the tissues of cardiac apex for pathological examination, Northern blotting and so forth. We found that miR-21 expression was significantly higher in model rats than in sham-operated rats, and the model rats developed the cardiac atrophy and cardiac fibrosis. After injection of miR-21 antagonist, the the cardiac atrophy and cardiac fibrosis were conspicuously ameliorated. Both in vivo and in vitro, inhibition of miR-21 expression resulted in reduced Bcl-2 expression while over-expression of miR-21 led to elevation of Bcl-2 expression. Our study suggested that miR-21 promoted the development of HFpEF by up-regulating the expression of anti-apoptotic gene Bcl-2 and thereby suppressing the apoptosis of cardiac fibrosis.
PMCID: PMC3925900  PMID: 24551276
Cardiac fibrosis; miR-21; Bcl-2; HFpEF
9.  The role of ubiquitylation in immune defence and pathogen evasion 
Nature reviews. Immunology  2011;12(1):10.1038/nri3111.
Ubiquitylation is a widely used post-translational protein modification that regulates many biological processes, including immune responses. The role of ubiquitin in immune regulation was originally uncovered through studies of antigen presentation and the nuclear factor-κB family of transcription factors, which orchestrate host defence against microorganisms. Recent studies have revealed crucial roles of ubiquitylation in many aspects of the immune system, including innate and adaptive immunity and antimicrobial autophagy. In addition, mounting evidence indicates that microbial pathogens exploit the ubiquitin pathway to evade the host immune system. Here, we review recent advances on the role of ubiquitylation in host defence and pathogen evasion.
PMCID: PMC3864900  PMID: 22158412
10.  Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway 
Science (New York, N.Y.)  2012;339(6121):10.1126/science.1232458.
The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.
PMCID: PMC3863629  PMID: 23258413
11.  Cyclic-GMP-AMP Is An Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA 
Science (New York, N.Y.)  2012;339(6121):10.1126/science.1229963.
Cytosolic DNA induces type-I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA.
PMCID: PMC3855410  PMID: 23258412
12.  RNA Helicase Signaling Is Critical for Type I Interferon Production and Protection against Rift Valley Fever Virus during Mucosal Challenge 
Journal of Virology  2013;87(9):4846-4860.
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
PMCID: PMC3624317  PMID: 23408632
13.  Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination 
Cell  2013;152(5):1051-1064.
Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a novel role for ubiquitination in this process. We find that the novel E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the Retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired Retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of Retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and Retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 and reveal novel aspects of retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
PMCID: PMC3640276  PMID: 23452853
14.  Competing E3 Ubiquitin Ligases Determine Circadian Period by Regulated Degradation of CRY in Nucleus and Cytoplasm 
Cell  2013;152(5):1091-1105.
Period determination in the mammalian circadian clock involves the turnover rate of the repressors, CRY and PER. Here we show that CRY ubiquitination engages two competing E3 ligase complexes that either lengthen or shorten circadian period in mice. Cloning of a short-period circadian mutant, Past-time, revealed a glycine to glutamate (G149E) missense mutation in Fbxl21, an F-box protein gene that is a paralog of Fbxl3 that targets the CRY proteins for degradation. While loss-of-function of FBXL3 leads to period lengthening, mutation of Fbxl21 causes period shortening. FBXL21 forms an SCF E3 ligase complex that slowly degrades CRY in the cytoplasm, but antagonizes the stronger E3 ligase activity of FBXL3 in the nucleus. FBXL21 plays a dual role: protecting CRY from FBXL3 degradation in the nucleus and promoting CRY degradation within the cytoplasm. Thus, the balance and cellular compartmentalization of competing E3 ligases for CRY determine circadian period of the clock in mammals.
PMCID: PMC3694781  PMID: 23452855
15.  MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades 
eLife  2013;2:e00785.
RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-κB and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-κB activation, also play a crucial role in IRF3 activation in antiviral immune responses.
eLife digest
The innate immune system can detect and destroy viruses, bacteria and other pathogens that enter the human body. In particular, inside cells, viral RNA can bind to and activate a protein called RIG-I. This protein switches on another protein, called MAVS, which can activate other copies of itself. These MAVS molecules then aggregate together on the membrane of mitochondria and send a signal that leads to the production of small proteins, called cytokines, which stimulate an inflammatory response and ultimately neutralize the virus.
Although many of the proteins that are activated by MAVS in the innate immunity signaling pathway have been identified, precisely how MAVS transmits this signal is unknown. Now, Liu et al. explore how this protein can propagate signals in the innate immune response by monitoring activation of the transcription factors IRF3 and NF-κB, which transcribe cytokine genes.
Previous studies have suggested that a protein known as ubiquitin is needed to activate RIG-I, and that this protein collaborates with MAVS to signal through the innate immunity pathway. Liu et al. found that a group of proteins including TRAF2, TRAF5, TRAF6 and LUBAC relay the antiviral signal by binding to MAVS. These so-called ‘E3 ligases’ string ubiquitin together in chains called polyubiquitin, which is essential for activating signaling after, or downstream of, MAVS; however, the association of these E3 ligases with MAVS also requires that multiple copies of MAVS cluster together.
MAVS, the TRAF proteins and LUBAC collectively recruit other innate immunity pathway proteins to activate IRF3 and NF-κB, and thus transcription of the genes that control the innate immunity response. Together, these results show the intricate interplay of proteins needed to eliminate viruses from the body.
PMCID: PMC3743401  PMID: 23951545
MAVS; innate immunity; virus; ubiquitin; signaling; mitochondria; Human; Mouse; Viruses
16.  Cyclic di-GMP Sensing via the Innate Immune Signaling Protein STING 
Molecular cell  2012;46(6):735-745.
Detection of foreign materials is the first step of successful immune responses. Stimulator of interferon genes (STING) was shown to directly bind cyclic diguanylate monophosphate (c-di-GMP), a bacterial second messenger, and to elicit strong interferon responses. Here we elucidate the structural features of the cytosolic c-di-GMP binding domain (CBD) of STING and its complex with c-di-GMP. The CBD exhibits an α + β fold and is a dimer in the crystal and in solution. Surprisingly, one c-di-GMP molecule binds to the central crevice of a STING dimer, using a series of stacking and hydrogen bonding interactions. We show that STING is autoinhibited by an intramolecular interaction between the CBD and the C-terminal tail (CTT) and that c-di-GMP releases STING from this autoinhibition by displacing the CTT. The structures provide a remarkable example of pathogen-host interactions in which a unique microbial molecule directly engages the innate immune system.
PMCID: PMC3697849  PMID: 22705373
17.  Volumetric modulation arc radiotherapy with flattening filter-free beams compared with conventional beams for nasopharyngeal carcinoma: a feasibility study 
Chinese Journal of Cancer  2013;32(7):397-402.
There is increasing interest in the clinical use of flattening filter-free (FFF) beams. In this study, we aimed to investigate the dosimetric characteristics of volumetric modulated arc radiotherapy (VMAT) with FFF beams for nasopharyngeal carcinoma (NPC). Ten NPC patients were randomly selected to undergo a RapidArc plan with either FFF beams (RA-FFF) or conventional beams (RA-C). The doses to the planning target volumes (PTVs), organs at risk (OARs), and normal tissues were compared. The technical delivery parameters for RapidArc plans were also assessed to compare the characteristics of FFF and conventional beams. Both techniques delivered adequate doses to PTVs. For PTVs, RA-C delivered lower maximum and mean doses and improved conformity and homogeneity compared with RA-FFF. Both techniques provided similar maximum doses to the optic nerves and lenses. For the brain stem, spinal cord, larynx, parotid glands, oral cavity, and skin, RA-FFF showed significant dose increases compared to RA-C. The dose to normal tissue was lower in RA-FFF. The monitor units (MUs) were (536 ± 46) MU for RA-FFF and (501 ± 25) MU for RA-C. The treatment duration did not significantly differ between plans. Although both treatment plans could meet clinical needs, RA-C is dosimetrically superior to RA-FFF for NPC radiotherapy.
PMCID: PMC3845599  PMID: 23237224
Flattening filter-free beams; RapidArc; nasopharyngeal carcinoma; volumetric modulated arc therapy; TrueBeam
19.  Human Metapneumovirus M2-2 Protein Inhibits Innate Cellular Signaling by Targeting MAVS 
Journal of Virology  2012;86(23):13049-13061.
Human metapneumovirus (hMPV) is a leading cause of respiratory infections in pediatric populations globally, with no prophylactic or therapeutic measures. Recently, a recombinant hMPV lacking the M2-2 protein (rhMPV-ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making it a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein and host cells that regulates viral infection/propagation is largely unknown. By taking advantage of the available reverse genetics system and ectopic expression system for viral protein, we found that M2-2 not only promotes viral gene transcription and replication but subverts host innate immunity, therefore identifying M2-2 as a novel virulence factor, in addition to the previously described hMPV G protein. Since we have shown that the RIG-I/MAVS pathway plays an important role in hMPV-induced signaling in airway epithelial cells, we investigated whether M2-2 antagonizes the host cellular responses by targeting this pathway. Reporter gene assays and coimmunoprecipitation studies indicated that M2-2 targets MAVS, an inhibitory mechanism different from what we previously reported for hMPV G, which affects RIG-I- but not MAVS-dependent gene transcription. In addition, we found that the domains of M2-2 responsible for the regulation of viral gene transcription and antiviral signaling are different. Our findings collectively demonstrate that M2-2 contributes to hMPV immune evasion through the inhibition of MAVS-dependent cellular responses.
PMCID: PMC3497653  PMID: 23015697
20.  Advanced nasopharyngeal carcinoma radiotherapy with volumetric modulated arcs and the potential role of flattening filter-free beams 
The purpose of this study is to investigate the dosimetric characteristics of volumetric modulated arc therapy (VMAT) with flattening filter-free (FFF) beams and assess the role of VMAT in the treatment of advanced nasopharyngeal carcinoma (NPC).
Ten cases of CT data were randomly selected from advanced NPC patients. Three treatment plans were optimized for each patient, RapidArc with FFF beams (RA-FFF), conventional beams (RA) and static gantry intensity-modulated radiation therapy (IMRT). The doses to the planning target volumes (PTVs), organs at risk (OARs), skin and normal tissue were compared. All the plans were delivered on a Varian TrueBeam linear accelerator and verified using the Delta4 phantom. Technical delivery parameters including the mean gamma score, treatment delivery time and monitor units (MUs) were also analyzed.
All the techniques delivered adequate doses to the PTVs. RA-FFF gave the highest D1% (dose received by 1% of the volume), but the poorest conformity index (CI) and homogeneity index (HI) among the PTVs except for the planning target volume of involved regional lymph nodes (PTV66) CI, which showed no significant difference among three techniques. For the planning target volume of the primary nasopharyngeal tumor (PTV70), RA-FFF provided for higher mean dose than other techniques. For the planning target volume receiving 60 Gy (PTV60) and PTV66, RA delivered the lowest mean doses whereas IMRT delivered the highest mean doses. IMRT demonstrated the highest percentage of target coverage and D99% for PTV60. RA-FFF provided for the highest doses to the brain stem, skin and oral cavity. RA gave the highest D1% to the right optic nerve among three techniques while no significant differences were found between each other. IMRT delivered the highest mean doses to the parotid glands and larynx while RA delivered the lowest mean doses. Gamma analysis showed an excellent agreement for all the techniques at 3%/3mm. Significant differences in the MUs were observed among the three techniques (p < 0.001). Delivery times for RA-FFF and RA were 152 ± 7s and 153 ± 7s, respectively, nearly 70% lower than the 493 ± 24s mean time for IMRT.
All treatment plans met the planning objectives. The dose measurements also showed good agreement with computed doses. RapidArc technique can treat patients with advanced NPC effectively, with good target coverage and sparing of critical structures. RA has a greater dosimetric superiority than RA-FFF.
PMCID: PMC3720531  PMID: 23672519
21.  Differential roles for RIG-I-like receptors and nucleic acid-sensing TLR pathways in controlling a chronic viral infection 
The necessity for pathogen recognition of viral infection by the innate immune system in initiating early innate and adaptive host defenses is well documented. However, little is known about the role these receptors play in the maintenance of adaptive immune responses and their contribution to resolution of persistent viral infections. Here, we demonstrate a non-redundant functional requirement for both nucleic acid-sensing Toll-like receptors (TLR) and RIG-I-like receptors (RLR) in the control of a mouse model of chronic viral infection. Whereas the RLR pathway was important for production of type I interferons and optimal CD8+ T cell responses, nucleic acid-sensing TLRs were largely dispensable. In contrast, optimal anti-viral antibody responses required intact signaling through nucleic acid-sensing TLRs, and the absence of this pathway correlated with less virus-specific antibody and deficient long-term virus control of a chronic infection. Surprisingly, absence of the TLR pathway had only modest effects on antibody production in an acute infection with a closely related virus strain, suggesting that persistent TLR stimulation may be necessary for optimal antibody responses in a chronic infection. These results indicate that innate virus recognition pathways may play critical roles in the outcome of chronic viral infections through distinct mechanisms.
PMCID: PMC3331923  PMID: 22447976
22.  Both K63 and K48 ubiquitin linkages signal lysosomal degradation of the LDL receptor 
Journal of Lipid Research  2013;54(5):1410-1420.
Linkage-specific ubiquitination often leads to distinct cellular events. It has been difficult to establish definitively the requirement for a particular linkage in mammalian degradation pathways due to the inability to deplete endogenous ubiquitin while maintaining cell viability. The E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL) targets the low density lipoprotein receptor (LDLR) for degradation. The nature of the linkages employed to signal lysosomal degradation of the LDLR, and to signal proteasomal autodegradation of IDOL, have not been determined. We used an inducible RNAi strategy to replace endogenous ubiquitin with mutants lacking K48 or K63. We found that IDOL catalyzes the transfer of ubiquitin chains to itself and to the LDLR that do not contain exclusively K48 or K63 linkages. Thus, LDLR can be targeted to the lysosome by either K48 or K63 linkages. We further demonstrate that although both ubiquitin conjugating enzyme E2 (UBE2)Ds and UBE2N/V1 can catalyze LDLR ubiquitination in a cell-free system, UBE2Ds appear to be the major E2 enzymes employed by IDOL in cells, consistent with their ability to catalyze both K48 and K63 linkages. The results reveal mechanistic insight into the posttranscriptional control of lipoprotein uptake and provide a test of the requirement of linkage-specific ubiquitination for specific lysosomal and proteasomal degradation pathways in mammalian cells.
PMCID: PMC3653405  PMID: 23419260
low density lipoprotein receptor; lysosomal protein degradation; proteasomal protein degradation; E3 ubiquitin ligase
23.  SnapShot: Pathways of Antiviral Innate Immunity 
Cell  2010;140(3):436-436.e2.
PMCID: PMC3586550  PMID: 20144765
24.  A20 Ubiquitin Ligase-Mediated Polyubiquitination of RIP1 Inhibits Caspase-8 Cleavage and TRAIL-Induced Apoptosis in Glioblastoma 
Cancer Discovery  2012;2(2):140-155.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a cancer therapeutic target. However, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL-targeted therapies. We show here that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through two signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 (DR5) and receptor-interacting protein 1 (RIP1), forms a plasma membrane bound preligand assembly complex (PLAC) under physiologic conditions. TRAIL treatment leads to the recruitment of caspase-8 to the PLAC for the assembly of a death-inducing signaling complex (DISC). In the DISC, the C-terminal Zinc finger (Znf) domain of A20 ubiquitin ligase mediates RIP1 ubiquitination through lysine (K)-63-linked polyubiquitin chains that bind the protease domain of caspase-8 and inhibits its dimerization, cleavage and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells.
PMCID: PMC3354650  PMID: 22585859
A20; apoptosis; caspase-8; TRAIL; ubiquitination
25.  XRCC1 codon 399Gln polymorphism is associated with radiotherapy-induced acute dermatitis and mucositis in nasopharyngeal carcinoma patients 
To evaluate the association between single nucleotide polymorphisms (SNPs) at the 194 and 399 codons of XRCC1, and the risk of severe acute skin and oral mucosa reactions in nasopharyngeal carcinoma patients in China.
114 patients with nasopharyngeal carcinoma were sequentially recruited in this study. Heparinized peripheral blood samples were taken for SNPs analysis before the start of radiation treatment. SNPs in XRCC1 (194Arg/Trp and 399Arg/Gln) gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Dermatitis at upper neck and oral mucositis were clinically recorded according to the Common Terminology Criteria for Adverse Events v.3.0.
The variant allele frequencies were 0.289 for XRCC1 194Trp and 0.263 for XRCC1 399Gln. Of the 114 patients, 24 experienced grade 3 acute dermatitis and 48 had grade 3 acute mucositis. The XRCC1 399Arg/Gln was significantly associated with the development of grade 3 dermatitis (Odds Ratio, 2.65; 95% CI, 1.04–6.73; p = 0.037, χ2 = 4.357). In addition, it was also associated with higher incidence of grade 3 mucositis with a borderline statistical significance (Odds Ratio, 2.11; 95% CI, 0.951–4.66; p = 0.065, χ2 = 3.411). The relationship between XRCC1 194Arg/Trp and acute dermatitis, and mucositis was not found.
Our investigation shows, for the first time, that patients with the XRCC1 399Arg/Gln genotype were more likely to experience severe acute dermatitis and oral mucositis. With further validation, the information can be used to determine personalized radiotherapy strategy.
PMCID: PMC3570437  PMID: 23375119
Single nucleotide polymorphisms (SNPs); X-ray cross-complementing group 1 (XRCC1); Nasopharyngeal carcinoma (NPC); Radiotherapy; Acute skin reactions; Acute mucosa reactions

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