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1.  An Oncogenic Virus Promotes Cell Survival and Cellular Transformation by Suppressing Glycolysis 
PLoS Pathogens  2016;12(5):e1005648.
Aerobic glycolysis is essential for supporting the fast growth of a variety of cancers. However, its role in the survival of cancer cells under stress conditions is unclear. We have previously reported an efficient model of gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV)-induced cellular transformation of rat primary mesenchymal stem cells. KSHV-transformed cells efficiently induce tumors in nude mice with pathological features reminiscent of Kaposi’s sarcoma tumors. Here, we report that KSHV promotes cell survival and cellular transformation by suppressing aerobic glycolysis and oxidative phosphorylation under nutrient stress. Specifically, KSHV microRNAs and vFLIP suppress glycolysis by activating the NF-κB pathway to downregulate glucose transporters GLUT1 and GLUT3. While overexpression of the transporters rescues the glycolytic activity, it induces apoptosis and reduces colony formation efficiency in softagar under glucose deprivation. Mechanistically, GLUT1 and GLUT3 inhibit constitutive activation of the AKT and NF-κB pro-survival pathways. Strikingly, GLUT1 and GLUT3 are significantly downregulated in KSHV-infected cells in human KS tumors. Furthermore, we have detected reduced levels of aerobic glycolysis in several KSHV-infected primary effusion lymphoma cell lines compared to a Burkitt’s lymphoma cell line BJAB, and KSHV infection of BJAB cells reduced aerobic glycolysis. These results reveal a novel mechanism by which an oncogenic virus regulates a key metabolic pathway to adapt to stress in tumor microenvironment, and illustrate the importance of fine-tuning the metabolic pathways for sustaining the proliferation and survival of cancer cells, particularly under stress conditions.
Author Summary
KSHV is causally associated with the development of Kaposi’s sarcoma and primary effusion lymphoma; however, the mechanism underlying KSHV-induced malignant transformation remains unclear. The recent development of an efficient KSHV-induced cellular transformation model of primary rat mesenchymal stem cells should facilitate the delineation of KSHV-induced oncogenesis. In this report, we have used this model to investigate the metabolic pathways mediating the proliferation and survival of KSHV-transformed cells. In contrast to most other cancers that depend on aerobic glycolysis for their fast growth, we demonstrate that KSHV suppresses aerobic glycolysis and oxidative phosphorylation in the transformed cells. Significantly, suppression of aerobic glycolysis enhances the survival of the KSHV-transformed cells under nutrient deprivation. Mechanistically, KSHV-encoded microRNAs and vFLIP suppress aerobic glycolysis by activating the NF-κB pathway to downregulate glucose transporters GLUT1 and GLUT3. We have further shown that GLUT1 and GLUT3 inhibit constitutive activation of the AKT and NF-κB pro-survival pathways. Strikingly, GLUT1 and GLUT3 are significantly downregulated in KSHV-infected cells in human KS tumors. Furthermore, we have detected reduced levels of aerobic glycolysis in several KSHV-infected primary effusion lymphoma cell lines and a KSHV-infected Burkitt’s lymphoma cell line BJAB. Our results reveal a novel mechanism by which an oncogenic virus regulates a key metabolic pathway to adapt to stress in tumor microenvironment, and illustrate the importance of fine-tuning the metabolic pathways for sustaining the proliferation and survival of cancer cells, particularly under nutrient stress microenvironment.
doi:10.1371/journal.ppat.1005648
PMCID: PMC4871371  PMID: 27187079
2.  LANA-Mediated Recruitment of Host Polycomb Repressive Complexes onto the KSHV Genome during De Novo Infection 
PLoS Pathogens  2016;12(9):e1005878.
One of the hallmarks of the latent phase of Kaposi’s sarcoma-associated herpesvirus (KSHV) infection is the global repression of lytic viral gene expression. Following de novo KSHV infection, the establishment of latency involves the chromatinization of the incoming viral genomes and recruitment of the host Polycomb repressive complexes (PRC1 and PRC2) to the promoters of lytic genes, which is accompanied by the inhibition of lytic genes. However, the mechanism of how PRCs are recruited to the KSHV episome is still unknown. Utilizing a genetic screen of latent genes in the context of KSHV genome, we identified the latency-associated nuclear antigen (LANA) to be responsible for the genome-wide recruitment of PRCs onto the lytic promoters following infection. We found that LANA initially bound to the KSHV genome right after infection and subsequently recruited PRCs onto the viral lytic promoters, thereby repressing lytic gene expression. Furthermore, both the DNA and chromatin binding activities of LANA were required for the binding of LANA to the KSHV promoters, which was necessary for the recruitment of PRC2 to the lytic promoters during de novo KSHV infection. Consequently, the LANA-knockout KSHV could not recruit PRCs to its viral genome upon de novo infection, resulting in aberrant lytic gene expression and dysregulation of expression of host genes involved in cell cycle and proliferation pathways. In this report, we demonstrate that KSHV LANA recruits host PRCs onto the lytic promoters to suppress lytic gene expression following de novo infection.
Author Summary
Persistent KSHV infection of humans requires the establishment of viral latency in B cells and endothelial cells following primary infection. This involves the spatially and temporally ordered recruitment of host epigenetic factors onto the viral DNA during de novo infection, resulting in the genome-wide repression of lytic (but not latent) gene expression. We have previously shown that the host epigenetic repressor Polycomb Repressive Complexes bind to the KSHV genome and play a role in the inhibition of lytic gene expression following de novo infection. Using reverse genetics approaches, we identified the latent KSHV protein LANA to be responsible for the recruitment of the Polycomb proteins onto the promoters of lytic genes following de novo infection. Importantly, our study revealed a novel role for LANA in KSHV infection. LANA initiates the recruitment of host repressive epigenetic factors onto the incoming viral genome to suppress lytic gene expression, which is ultimately required for the establishment of KSHV latency in the hosts and the development of KSHV-associated cancers.
doi:10.1371/journal.ppat.1005878
PMCID: PMC5015872  PMID: 27606464
3.  Suppression of Kaposi's Sarcoma-Associated Herpesvirus Infection and Replication by 5′-AMP-Activated Protein Kinase 
Journal of Virology  2016;90(14):6515-6525.
ABSTRACT
The host intracellular antiviral restriction factors inhibit viral infection and replication. The 5′-AMP-activated protein kinase (AMPK) is a cellular energy sensor regulating metabolic homeostasis. Activated AMPK inhibits the replication of numerous RNA viruses but enhances the entry of vaccinia virus. However, the role of AMPK in herpesvirus infection is unclear. In this study, we showed that the constitutive AMPK activity restricted Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication in primary human umbilical vein endothelial cells while KSHV infection did not markedly affect the endogenous AMPK activity. Knockdown of the AMPKα1 considerably enhanced the expression of viral lytic genes and the production of infectious virions, while overexpression of a constitutively active AMPK had the opposite effects. Accordingly, an AMPK inhibitor, compound C, augmented viral lytic gene expressions and virion productions but an AMPK agonist, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), suppressed both. Furthermore, a common diabetes drug, metformin, which carries an AMPK-agonistic activity, drastically inhibited the expression of viral lytic genes and the production of infectious virions, suggesting the use of metformin as a therapeutic agent for KSHV infection and replication. Together, these results identify the host AMPK as a KSHV restriction factor that can serve as a potential therapeutic target.
IMPORTANCE Host cells encode specific proteins to restrict viral infection and replication. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus associated with several cancers. In this study, we have identified 5′-AMP-activated protein kinase (AMPK), a cellular energy sensor, as a restriction factor of KSHV lytic replication during primary infection. Activation of AMPK suppresses, while inhibition of AMPK enhances, KSHV lytic replication by regulating the expression of viral genes. AICAR and metformin, both of which are AMPK agonists currently used in clinics for the treatment of conditions associated with metabolic disorders, inhibit KSHV lytic replication. Thus, our work has identified AMPK as a potential therapeutic target and AICAR and metformin as potential therapeutic agents for KSHV-associated cancers.
doi:10.1128/JVI.00624-16
PMCID: PMC4936134  PMID: 27147746
4.  Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene 
Journal of Virology  2015;90(2):1139-1143.
Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the β-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.
doi:10.1128/JVI.01897-15
PMCID: PMC4702683  PMID: 26491150
5.  Human Mesenchymal Stem Cells of Diverse Origins Support Persistent Infection with Kaposi’s Sarcoma-Associated Herpesvirus and Manifest Distinct Angiogenic, Invasive, and Transforming Phenotypes 
mBio  2016;7(1):e02109-15.
ABSTRACT
Kaposi’s sarcoma (KS), a highly angiogenic and invasive tumor often involving different organ sites, including the oral cavity, is caused by infection with Kaposi’s sarcoma-associated herpesvirus (KSHV). Diverse cell markers have been identified on KS tumor cells, but their origin remains an enigma. We previously showed that KSHV could efficiently infect, transform, and reprogram rat primary mesenchymal stem cells (MSCs) into KS-like tumor cells. In this study, we showed that human primary MSCs derived from diverse organs, including bone marrow (MSCbm), adipose tissue (MSCa), dental pulp, gingiva tissue (GMSC), and exfoliated deciduous teeth, were permissive to KSHV infection. We successfully established long-term cultures of KSHV-infected MSCa, MSCbm, and GMSC (LTC-KMSCs). While LTC-KMSCs had lower proliferation rates than the uninfected cells, they expressed mixtures of KS markers and displayed differential angiogenic, invasive, and transforming phenotypes. Genetic analysis identified KSHV-derived microRNAs that mediated KSHV-induced angiogenic activity by activating the AKT pathway. These results indicated that human MSCs could be the KSHV target cells in vivo and established valid models for delineating the mechanism of KSHV infection, replication, and malignant transformation in biologically relevant cell types.
IMPORTANCE
Kaposi’s sarcoma is the most common cancer in AIDS patients. While KSHV infection is required for the development of Kaposi’s sarcoma, the origin of KSHV target cells remains unclear. We show that KSHV can efficiently infect human primary mesenchymal stem cells of diverse origins and reprogram them to acquire various degrees of Kaposi’s sarcoma-like cell makers and angiogenic, invasive, and transforming phenotypes. These results indicate that human mesenchymal stem cells might be the KSHV target cells and establish models for delineating the mechanism of KSHV-induced malignant transformation.
doi:10.1128/mBio.02109-15
PMCID: PMC4742711  PMID: 26814175
6.  A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus 
mBio  2017;8(4):e01179-17.
ABSTRACT
While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi’s sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
IMPORTANCE
We have previously found that Kaposi’s sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
doi:10.1128/mBio.01179-17
PMCID: PMC5559638  PMID: 28811348
γ-nitrogen; asparagine; cancer; glutamine; KSHV
7.  Zika Virus NS4A and NS4B Proteins Deregulate Akt-mTOR Signaling in Human Fetal Neural Stem Cells to Inhibit Neurogenesis and Induce Autophagy 
Cell stem cell  2016;19(5):663-671.
SUMMARY
The current widespread outbreak of Zika virus (ZIKV) infection has been linked to severe clinical birth defects, particularly microcephaly, warranting urgent study of the molecular mechanisms underlying ZIKV pathogenesis. Akt-mTOR signaling is one of the key cellular pathways essential for brain development and autophagy regulation. Here, we show that ZIKV infection of human fetal neural stem cells (fNSCs) causes inhibition of the Akt-mTOR pathway, leading to defective neurogenesis and aberrant activation of autophagy. By screening the three structural proteins and seven nonstructural proteins present in ZIKV, we found that two, NS4A and NS4B, cooperatively suppress the Akt-mTOR pathway and lead to cellular dysregulation. Corresponding proteins from the closely related dengue virus do not have the same effect on neurogenesis. Thus, our study highlights ZIKV NS4A and NS4B as candidate determinants of viral pathogenesis and identifies a mechanism of action for their effects, suggesting potential targets for anti-ZIKV therapeutic intervention.
doi:10.1016/j.stem.2016.07.019
PMCID: PMC5144538  PMID: 27524440
8.  Infection-specific phosphorylation of glutamyl-prolyl tRNA synthetase induces antiviral immunity 
Nature immunology  2016;17(11):1252-1262.
The mammalian cytoplasmic multi-tRNA synthetase complex (MSC) is a depot system that regulates non-translational cellular functions. Here we found that the MSC component glutamyl-prolyl-tRNA synthetase (EPRS) switched its function following viral infection and exhibited potent antiviral activity. Infection-specific phosphorylation of EPRS at Ser990 induced its dissociation from the MSC, after which it was guided to the antiviral signaling pathway, where it interacted with PCBP2, a negative regulator of mitochondrial antiviral signaling protein (MAVS) that is critical for antiviral immunity. This interaction blocked PCBP2-mediated ubiquitination of MAVS and ultimately suppressed viral replication. EPRS-haploid (Eprs+/−) mice showed enhanced viremia and inflammation and delayed viral clearance. This stimulus-inducible activation of MAVS by EPRS suggests an unexpected role for the MSC as a regulator of immune responses to viral infection.
doi:10.1038/ni.3542
PMCID: PMC5173487  PMID: 27595231
9.  Association of Kaposi's Sarcoma-Associated Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene Expression 
Journal of Virology  2015;89(11):6148-6154.
Transcription of herpesvirus late genes depends on several virus-encoded proteins whose function is not completely understood. Here, we identify a viral trimeric complex of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 31 (ORF31), ORF24, and ORF34 that is required for late gene expression but not viral DNA replication. We found that (i) ORF34 bridges the interaction between ORF31 and ORF24, (ii) the amino-terminal cysteine-rich and carboxyl-terminal basic domains of ORF31 mediate the ORF31-ORF34 interaction required for late gene expression, and (iii) a complex consisting of ORF24, ORF31, and ORF34 specifically binds to the K8.1 late promoter. Together, our results support the model that a subset of lytic viral proteins assembles into a transcriptional activator complex to induce expression of late genes.
doi:10.1128/JVI.00272-15
PMCID: PMC4442431  PMID: 25810551
10.  Chloroquine, a FDA-approved Drug, Prevents Zika Virus Infection and its Associated Congenital Microcephaly in Mice 
EBioMedicine  2017;24:189-194.
Zika virus (ZIKV) has become a global public health emergency due to its rapidly expanding range and its ability to cause severe congenital defects such as microcephaly. However, there are no FDA-approved therapies or vaccines against ZIKV infection. Through our screening of viral entry inhibitors, we found that chloroquine (CQ), a commonly used antimalarial and a FDA-approved drug that has also been repurposed against other pathogens, could significantly inhibit ZIKV infection in vitro, by blocking virus internalization. We also demonstrated that CQ attenuates ZIKV-associated morbidity and mortality in mice. Finally, we proved that CQ protects fetal mice from microcephaly caused by ZIKV infection. Our methodology of focusing on previously identified antivirals in screens for effectiveness against ZIKV proved to be a rapid and efficient means of discovering new ZIKV therapeutics. Selecting drugs that were previously FDA-approved, such as CQ, also improves the likelihood that they may more quickly reach stages of clinical testing and use by the public.
Highlights
•5 out 16 tested Ebola virus entry inhibitors can inhibit ZIKV entry efficiently•Chloroquine can inhibit ZIKV internalization in vitro and reduce ZIKV-associated morbidity and mortality in mice•Chloroquine prevents ZIKV-associated congenital microcephaly in mice
Zika virus (ZIKV) is an emerging virus which can cause birth defects, however there are currently no effective treatments or vaccines. We tested the effects of 16 verified Ebola virus cell entry inhibitors on ZIKV infection, and found that chloroquine (CQ) could prevent ZIKV infection in cell cultures, consistent with results from a previous study. We then demonstrated that CQ can reduce ZIKV-associated morbidity and mortality in mice. Most importantly, it protects fetal mice from microcephaly caused by ZIKV infection. Therefore, CQ is a potential drug which would be used to treat ZIKV infection after clinical test.
doi:10.1016/j.ebiom.2017.09.034
PMCID: PMC5652284  PMID: 29033372
FDA-approved drug; Chloroquine; ZIKV entry; Microcephaly; Antiviral effects
11.  No TRIFling Matter on STING 
Cell host & microbe  2016;20(3):277-278.
STING is a crucial component of the mammalian innate immune response to microbial infection. In this issue of Cell Host & Microbe, Wang et al. (2016) report that TRIF, an adaptor of Toll-like receptors (TLRs), is essential for STING-mediated innate antiviral immunity as well as pro-protozoal responses.
doi:10.1016/j.chom.2016.08.009
PMCID: PMC5152617  PMID: 27631696
12.  Inhibition of highly pathogenic avian influenza (HPAI) virus by a peptide derived from vFLIP through its direct destabilization of viruses 
Scientific Reports  2017;7:4875.
The antiviral activities of synthesized Kα2-helix peptide, which was derived from the viral FLICE-like inhibitor protein (vFLIP) of Kaposi’s sarcoma-associated herpesvirus (KSHV), against influenza A virus (IAV) were investigated in vitro and in vivo, and mechanisms of action were suggested. In addition to the robust autophagy activity of the Kα2-helix peptide, the present study showed that treatment with the Kα2 peptide fused with the TAT peptide significantly inhibited IAV replication and transmission. Moreover, TAT-Kα2 peptide protected the mice, that were challenged with lethal doses of highly pathogenic influenza A H5N1 or H1N1 viruses. Mechanistically, we found that TAT-Kα2 peptide destabilized the viral membranes, depending on their lipid composition of the viral envelop. In addition to IAV, the Kα2 peptide inhibited infections with enveloped viruses, such as Vesicular Stomatitis Virus (VSV) and Respiratory Syncytial Virus (RSV), without cytotoxicity. These results suggest that TAT-Kα2 peptide is a potential antiviral agent for controlling emerging or re-emerging enveloped viruses, particularly diverse subtypes of IAVs.
doi:10.1038/s41598-017-04777-4
PMCID: PMC5501782  PMID: 28687749
13.  FAS-associated factor-1 positively regulates type I interferon response to RNA virus infection by targeting NLRX1 
PLoS Pathogens  2017;13(5):e1006398.
FAS-associated factor-1 (FAF1) is a component of the death-inducing signaling complex involved in Fas-mediated apoptosis. It regulates NF-κB activity, ubiquitination, and proteasomal degradation. Here, we found that FAF1 positively regulates the type I interferon pathway. FAF1gt/gt mice, which deficient in FAF1, and FAF1 knockdown immune cells were highly susceptible to RNA virus infection and showed low levels of inflammatory cytokines and type I interferon (IFN) production. FAF1 was bound competitively to NLRX1 and positively regulated type I IFN signaling by interfering with the interaction between NLRX1 and MAVS, thereby freeing MAVS to bind RIG-I, which switched on the MAVS-RIG-I-mediated antiviral signaling cascade. These results highlight a critical role of FAF1 in antiviral responses against RNA virus infection.
Author summary
Type I interferon-mediated antiviral response is critical for controlling virus infections. However, interferon-mediated immune responses need to be tightly regulated to maintain host immune homeostasis. Recently, molecules involved in regulating interferon-mediated innate immune response are the subject of much research. Among these, the first protein to be identified as a negative regulator of MAVS was the nucleotide-binding domain and leucine-rich repeat containing family member, NLRX1. NLRX1 associates with MAVS to inhibit antiviral signaling by interrupting virus-induced RLR-MAVS interactions. Interestingly, we found that FAF1 interacts with NLRX1 in response to RNA virus infection and this interaction inhibits binding of MAVS to NLRX1, which in turn switches on RIG-I mediated antiviral immune responses. As results, we showed that FAF1gt/gt mice, which deficient in FAF1, and FAF1 knockdown immune cells were highly susceptible to RNA virus infection and showed low levels of inflammatory cytokines and type I interferon (IFN) production. Our findings suggest that FAF1 is a crucial regulator that induces the antiviral innate immune responses against RNA virus infection.
doi:10.1371/journal.ppat.1006398
PMCID: PMC5456407  PMID: 28542569
14.  Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation 
mBio  2015;6(6):e01777-15.
ABSTRACT
Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation.
IMPORTANCE
Posttranslational modification of proteins enables cells to respond quickly to infections and immune stimuli in a tightly controlled manner. Specifically, covalent modification of proteins with the small protein ubiquitin is essential for cells to initiate and terminate immune signaling in response to bacterial and viral infection. This process is controlled by ubiquitin ligase enzymes, which themselves must be regulated to prevent persistent and deleterious immune signaling. However, how this regulation is achieved is poorly understood. This paper reports a novel ubiquitination event of the atypical ubiquitin ligase HOIP that is required to terminate bacterial lipopolysaccharide (LPS)-induced TLR4 immune signaling. Ubiquitination causes the HOIP ligase to undergo a conformational change, which blocks its enzymatic activity and ultimately terminates LPS-induced TLR4 signaling. These findings provide a new mechanism for controlling HOIP ligase activity that is vital to properly regulate a proinflammatory immune response.
doi:10.1128/mBio.01777-15
PMCID: PMC4659476  PMID: 26578682
15.  Akt kinase-mediated checkpoint of cGAS DNA sensing pathway 
Cell reports  2015;13(2):440-449.
SUMMARY
Upon DNA stimulation, cyclic GMP-AMP synthetase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-β (IFN-β) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-β production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-β production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-β production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts’ immune responses to DNA stimulation.
doi:10.1016/j.celrep.2015.09.007
PMCID: PMC4607670  PMID: 26440888
16.  Interplay between Kaposi’s sarcoma-associated herpesvirus and the innate immune system 
Cytokine & growth factor reviews  2014;25(5):597-609.
Understanding of the innate immune response to viral infections is rapidly progressing, especially with regards to the detection of DNA viruses. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a large dsDNA virus that is responsible for three human diseases: Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. The major target cells of KSHV (B cells and endothelial cells) express a wide range of pattern recognition receptors (PRRs) and play a central role in mobilizing inflammatory responses. On the other hand, KSHV encodes an array of immune evasion genes, including several pirated host genes, which interfere with multiple aspects of the immune response. This review summarizes current understanding of innate immune recognition of KSHV and the role of immune evasion genes that shape the antiviral and inflammatory responses.
doi:10.1016/j.cytogfr.2014.06.001
PMCID: PMC4252609  PMID: 25037686
KSHV; Innate Immunity
17.  Lack of Autophagy Induces Steroid Resistant Airway inflammation 
Background
Neutrophilic corticosteroid-resistance asthma accounts for a significant proportion of asthma however, little is known about mechanisms that underlie the pathogenesis of the disease.
Objective
To address the role of autophagy in lung inflammation and the pathogenesis of corticosteroid-resistant Neutrophilic asthma.
Methods
We developed CD11c-specific Atg5-/- mice and used several murine models to investigate the role of autophagy in asthma.
Results
We found, for the first time, that deletion of Atg5 gene specifically in CD11c+ cells which leads to the impairment of autophagy pathway, causes unprovoked spontaneous airway hyperreactivity and severe neutrophilic lung inflammation in mice. We found that severe lung inflammation impairs the autophagy pathway particularly in pulmonary CD11c+ cells in wild type (WT) mice. We further found that adoptive transfer of Atg5-/- but not WT bone marrow derived DCs augments lung inflammation with elevated IL-17A in the lungs. Our data indicates that neutrophilic asthma in Atg5-/- mice is glucocorticoid-resistance and IL-17A dependent.
Conclusion
Our results suggest that lack of autophagy in pulmonary CD11c+ cell induces neutrophilic airway inflammation and hyperreactivity.
doi:10.1016/j.jaci.2015.09.033
PMCID: PMC4860134  PMID: 26589586
Autophagy; asthma; lung inflammation; neutrophilic asthma; corticosteroid-resistance asthma
18.  Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication 
Journal of Virology  2015;89(10):5308-5317.
ABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle.
IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication.
doi:10.1128/JVI.00102-15
PMCID: PMC4442505  PMID: 25740994
19.  CD95 Signaling Inhibits B Cell Receptor-Mediated Gammaherpesvirus Replication in Apoptosis-Resistant B Lymphoma Cells 
Journal of Virology  2016;90(21):9782-9796.
ABSTRACT
While CD95 is an apoptosis-inducing receptor and has emerged as a potential anticancer therapy target, mounting evidence shows that CD95 is also emerging as a tumor promoter by activating nonapoptotic signaling pathways. Gammaherpesviral infection is closely associated with lymphoproliferative diseases, including B cell lymphomas. The nonapoptotic function of CD95 in gammaherpesvirus-associated lymphomas is largely unknown. Here, we show that stimulation of CD95 agonist antibody drives the majority of sensitive gammaherpesvirus-transformed B cells to undergo caspase-dependent apoptosis and promotes the survival and proliferation of a subpopulation of apoptosis-resistant B cells. Surprisingly, CD95-mediated nonapoptotic signaling induced beta interferon (IFN-β) expression and correlatively inhibited B cell receptor (BCR)-mediated gammaherpesviral replication in the apoptosis-resistant lymphoma cells without influencing BCR signaling. Further analysis showed that IFN-β alone or synergizing with CD95 blocked the activation of lytic switch proteins and the gene expression of gammaherpesviruses. Our findings indicate that, independent of its apoptotic activity, CD95 signaling activity plays an important role in blocking viral replication in apoptosis-resistant, gammaherpesvirus-associated B lymphoma cells, suggesting a novel mechanism that indicates how host CD95 prototype death receptor controls the life cycle of gammaherpesviruses independent of its apoptotic activity.
IMPORTANCE Gammaherpesviruses are closely associated with lymphoid malignancies and other cancers. Viral replication and persistence strategies leading to cancer involve the activation of antiapoptotic and proliferation programs, as well as evasion of the host immune response. Here, we provide evidence that the stimulation of CD95 agonist antibody, mimicking one of the major mechanisms of cytotoxic T cell killing, inhibits B cell receptor-mediated gammaherpesviral replication in CD95 apoptosis-resistant lymphoma cells. CD95-induced type I interferon (IFN-β) contributes to the inhibition of gammaherpesviral replication. This finding sheds new light on the CD95 nonapoptotic function and provides a novel mechanism for gammaherpesviruses that helps them to escape host immune surveillance.
doi:10.1128/JVI.00668-16
PMCID: PMC5068529  PMID: 27558422
20.  Autophagy side of MB21D1/cGAS DNA sensor 
Autophagy  2014;10(6):1146-1147.
The MB21D1/cGAS (Mab-21 domain-containing 1/cyclic GMP-AMP [cGAMP] synthetase), acts as an intracellular pattern recognition receptor (PPR) to sense cytosolic pathogen DNAs and subsequently generates the second messenger cGAMP to initiate the TMEM173/STING pathway for interferon (IFN) production. Intriguingly, we have recently demonstrated crosstalk between the intracellular DNA sensing pathway and autophagy machinery by demonstrating a direct interaction between the MB21D1 DNA sensor and the BECN1/Beclin 1 autophagy protein. This interaction not only suppresses MB21D1 enzymatic activity to halt cGAMP production, but also enhances the autophagy-mediated degradation of cytosolic microbial DNAs. This demonstrates that MB21D1 is the molecular link between the intracellular DNA sensing pathway and the autophagy pathway, ultimately developing well-balanced immune responses against pathogens.
doi:10.4161/auto.28769
PMCID: PMC4091176  PMID: 24879161
cGAS; Beclin 1; autophagy; interferon
21.  Crosstalk between cGAS DNA sensor and Beclin-1 autophagy protein shapes innate anti-microbial immune responses 
Cell host & microbe  2014;15(2):228-238.
Robust immune responses are essential for eliminating pathogens, but must be metered to avoid prolonged immune activation and potential host damage. Upon recognition of microbial DNA, the cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthetase, or cGAS, produces the second messenger cGAMP to initiate the STING pathway and subsequent interferon (IFN) production. We report that the direct interaction between cGAS and the Beclin-1 autophagy protein not only suppresses cGAMP synthesis to halt IFN production upon double stranded (ds)DNA stimulation or herpes simplex virus-1 infection, but also enhances autophagy-mediated degradation of cytosolic pathogen DNAs to prevent excessive cGAS activation and persistent immune stimulation. Specifically, this interaction releases Rubicon, a negative autophagy regulator, from the Beclin-1 complex, activating phosphatidylinositol 3-kinase class III activity and thereby inducing autophagy to remove cytosolic pathogen DNAs. Thus, the cGAS-Beclin-1 interaction shapes innate immune responses by regulating both cGAMP production and autophagy, resulting in well-balanced anti-microbial immune responses.
doi:10.1016/j.chom.2014.01.009
PMCID: PMC3950946  PMID: 24528868
22.  Kaposi's Sarcoma-Associated Herpesvirus K3 and K5 Ubiquitin E3 Ligases Have Stage-Specific Immune Evasion Roles during Lytic Replication 
Journal of Virology  2014;88(16):9335-9349.
ABSTRACT
The downregulation of immune synapse components such as major histocompatibility complex class I (MHC-I) and ICAM-1 is a common viral immune evasion strategy that protects infected cells from targeted elimination by cytolytic effector functions of the immune system. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two membrane-bound ubiquitin E3 ligases, called K3 and K5, which share the ability to induce internalization and degradation of MHC-I molecules. Although individual functions of K3 and K5 outside the viral genome are well characterized, their roles during the KSHV life cycle are still unclear. In this study, we individually introduced the amino acid-coding sequences of K3 or K5 into a ΔK3 ΔK5 recombinant virus, at either original or interchanged genomic positions. Recombinants harboring coding sequences within the K5 locus showed higher K3 and K5 protein expression levels and more rapid surface receptor downregulation than cognate recombinants in which coding sequences were introduced into the K3 locus. To identify infected cells undergoing K3-mediated downregulation of MHC-I, we employed a novel reporter virus, called red-green-blue-BAC16 (RGB-BAC16), which was engineered to harbor three fluorescent protein expression cassettes: EF1α-monomeric red fluorescent protein 1 (mRFP1), polyadenylated nuclear RNA promoter (pPAN)-enhanced green fluorescent protein (EGFP), and pK8.1-monomeric blue fluorescent protein (tagBFP), marking latent, immediate early, and late viral gene expression, respectively. Analysis of RGB-derived K3 and K5 deletion mutants showed that while the K5-mediated downregulation of MHC-I was concomitant with pPAN induction, the reduction of MHC-I surface expression by K3 was evident in cells that were enriched for pPAN-driven EGFPhigh and pK8.1-driven blue fluorescent protein-positive (BFP+) populations. These data support the notion that immunoreceptor downregulation occurs by a sequential process wherein K5 is critical during the immediately early phase and K3 plays a significant role during later stages.
IMPORTANCE Although the roles of K3 and K5 outside the viral genome are well characterized, the function of these proteins in the context of the KSHV life cycle has remained unclear, particularly in the case of K3. This study examined the relative contributions of K3 and K5 to the downregulation of MHC-I during the lytic replication of KSHV. We show that while K5 acts immediately upon entry into the lytic phase, K3-mediated downregulation of MHC-I was evident during later stages of lytic replication. The identification of distinctly timed K3 and K5 activities significantly advances our understanding of KSHV-mediated immune evasion. Crucial to this study was the development of a novel recombinant KSHV, called RGB-BAC16, which facilitated the delineation of stage-specific phenotypes.
doi:10.1128/JVI.00873-14
PMCID: PMC4136276  PMID: 24899205
23.  The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation 
The Journal of Experimental Medicine  2014;211(7):1333-1347.
Independent of its known role in NF-κB transcription, the HOIL-1L containing LUBAC is required for assembly and activation of the NLRP3 inflammasome via linear ubiquitination of ASC.
Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-κB–mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow–derived macrophages (BMDMs) independently of NF-κB activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L−/− mice have reduced IL-1β secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients.
doi:10.1084/jem.20132486
PMCID: PMC4076580  PMID: 24958845
24.  Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection 
The Journal of General Virology  2016;97(Pt 2):422-434.
Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70 % reduction of SLPI mRNA and a >60 % decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.
doi:10.1099/jgv.0.000341
PMCID: PMC4804641  PMID: 26555393
25.  Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Targets Expression of Cellular IRF4 and the Myc Gene To Facilitate Lytic Replication 
Journal of Virology  2014;88(4):2183-2194.
Besides an essential transcriptional factor for B cell development and function, cellular interferon regulatory factor 4 (c-IRF4) directly regulates expression of the c-Myc gene, which is not only associated with various B cell lymphomas but also required for herpesvirus latency and pathogenesis. Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent of Kaposi's sarcoma and primary effusion lymphoma, has developed a unique mechanism to deregulate host antiviral innate immunity and growth control by incorporating four viral homologs (vIRF1 to -4) of cellular IRFs into its genome. Previous studies have shown that several KSHV latent proteins, including vIRF3, vFLIP, and LANA, target the expression, function, and stability of c-Myc to establish and maintain viral latency. Here we report that the KSHV vIRF4 lytic protein robustly suppresses expression of c-IRF4 and c-Myc, reshaping host gene expression profiles to facilitate viral lytic replication. Genomewide gene expression analysis revealed that KSHV vIRF4 grossly affects host gene expression by upregulating and downregulating 118 genes and 166 genes, respectively, by at least 2-fold. Remarkably, vIRF4 suppressed c-Myc expression by 11-fold, which was directed primarily by the deregulation of c-IRF4 expression. Real-time quantitative PCR (RT-qPCR), single-molecule in situ hybridization, and chromatin immunoprecipitation assays showed that vIRF4 not only reduces c-IRF4 expression but also competes with c-IRF4 for binding to the specific promoter region of the c-Myc gene, resulting in drastic suppression of c-Myc expression. Consequently, the loss of vIRF4 function in the suppression of c-IRF4 and c-Myc expression ultimately led to a reduction of KSHV lytic replication capacity. These results indicate that the KSHV vIRF4 lytic protein comprehensively targets the expression and function of c-IRF4 to downregulate c-Myc expression, generating a favorable environment for viral lytic replication. Finally, this study further reinforces the important role of the c-Myc gene in KSHV lytic replication and latency.
doi:10.1128/JVI.02106-13
PMCID: PMC3911525  PMID: 24335298

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