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Metabolic enzymes fuel the synthesis of macromolecules that underpin the proliferation of cells and propagation of pathogens. NAD is a key cofactor for enzymes catalyzing metabolic reactions. We report that NAMPT, the rate-limiting enzyme of the salvage pathway of NAD synthesis, antagonizes tegument protein incorporation and restricts herpesvirus lytic replication in an enzyme-independent manner. We demonstrated that NAMPT was packaged into the virion and reduced lytic replication of human herpes simplex virus 1 (HSV-1). Mechanistically, NAMPT targets two core tegument proteins, UL36 and UL37 that mesh a scaffold for viral tegumentation during lytic replication. As such, NAMPT competitively antagonizes the incorporation of diverse tegument proteins into the virion, impairing viral tegumentation and infectivity of the progeny. Microscopy analysis indicates that NAMPT localizes to the TGN, which HSV-1 capsids bud into during tegumentation. Finally, NAMPT exhibited antiviral activity against multiple herpesviruses and loss of NAMPT rendered mice highly susceptible to HSV-1 infection. This study defines a new restriction factor targeting herpesvirus tegumentation, revealing a metabolism-independent antiviral activity of a metabolic enzyme.

Summary Herpes simplex virus 1 (HSV-1) establishes infections in humans and mice, but some non-human primates exhibit resistance via unknown mechanisms. Innate immune recognition pathways are highly conserved but are pivotal in determining susceptibility to DNA virus infections. We report that variation of a single amino acid residue in the innate immune sensor cGAS determines species-specific inactivation by HSV-1. The HSV-1 UL37 tegument protein deamidates human and mouse cGAS. Deamidation impairs the ability of cGAS to catalyze cGAMP synthesis, which activates innate immunity. HSV-1 with deamidase-deficient UL37 promotes robust antiviral responses and is attenuated in mice in a cGAS- and STING-dependent manner. Mutational analyses identified a single asparagine in human and mouse cGAS that is not conserved in many non-human primates. This residue underpins UL37-mediated cGAS deamidation and species permissiveness of HSV-1. Thus, HSV-1 mediates cGAS deamidation for immune evasion and exploits species sequence variation to disarm host defenses.

Aberrant expression of the MDR1-encoded P-glycoprotein (P-gp) is often associated with clinical multi-drug resistance (MDR) leading to poor prognosis and failure of chemotherapy. However, the precise and cooperative molecular mechanism responsible for MDR1 transcription and expression in acquired MDR remains elusive. We, herein, demonstrate that Wnt/β-catenin signal pathway is constitutively activated in Doxorubicin-induced MDR cancer cells, in which nuclear β -catenin specifically interacts with the transcriptional coactivator CBP in a MEK(1/2)/ERK(1/2) signal-dependent manner. Specific knockdown of both β-catenin and CBP by RNAi-mediated depletion abrogates MDR1 transcription and expression resulting in a complete reversal of P-gp-dependent efflux function and restoration of sensitivity to the Doxorubincin-induced cytotoxicity. Moreover, following pharmacological disruption of CBP and β - catenin interaction through inhibition of the MEK(1/2)/ERK(1/2) signal by the specific inhibitor PD98059, MDR1 transcription and its encoded P-gp-dependent function are abolished. These findings conclude that the CBP/β-catenin complex is a core component of the MDR1 transcriptional "enhancesome".

The viral Bcl-2 homolog (vBcl2) of Kaposi's sarcoma-associated herpesvirus (KSHV) displays efficient antiapoptotic and antiautophagic activity through its central BH3 domain, which functions to prolong the life span of virus-infected cells and ultimately enhances virus replication and latency. Independent of its antiapoptotic and antiautophagic activity, vBcl2 also plays an essential role in KSHV lytic replication through its amino-terminal amino acids (aa) 11 to 20. Here, we report a novel molecular mechanism of vBcl2-mediated regulation of KSHV lytic replication. vBcl2 specifically bound the tegument protein open reading frame 55 (ORF55) through its amino-terminal aa 11 to 20, allowing their association with virions. Consequently, the vBcl2 peptide derived from vBcl2 aa 11 to 20 effectively disrupted the interaction between vBcl2 and ORF55, inhibiting the incorporation of the ORF55 tegument protein into virions. This study provides new insight into vBcl2's function in KSHV virion assembly that is separable from its inhibitory role in host apoptosis and autophagy. IMPORTANCE KSHV, an important human pathogen accounting for a large percentage of virally caused cancers worldwide, has evolved a variety of stratagems for evading host immune responses to establish lifelong persistent infection. Upon viral infection, infected cells can go through programmed cell death, including apoptosis and autophagy, which plays an effective role in antiviral responses. To counter the host response, KSHV vBcl2 efficiently blocks apoptosis and autophagy to persist for the life span of virus-infected cells. Besides its anti-programmed-cell-death activity, vBcl2 also interacts with the ORF55 tegument protein for virion assembly in infected cells. Interestingly, the vBcl2 peptide disrupts the vBcl2-ORF55 interaction and effectively inhibits KSHV virion assembly. This study indicates that KSHV vBcl2 harbors at least three genetically separable functions to modulate both host cell death signaling and virion production and that the vBcl2 peptide can be developed as an anti-KSHV therapeutic application.

Host innate immune pathways are highly conserved in higher eukaryotes. Significance of the species-specific variation of innate immune components has not been explored with regard to DNA viruses. Here, we report the identification of a single site variation that defines the resistance of the cytosolic DNA sensor cGAS of nonhuman primates (NHPs) to deamidation mediated by human herpes simplex virus 1 (HSV-1). HSV-1 UL37, but not the deamidase-deficient UL37C819S mutant, deamidated human and mouse cGAS in vitro and in transfected cells. Biochemical analysis identified four sites of deamidation distributed throughout the Mab21 domain of cGAS. While deamidation did not significantly impair the DNA-binding and self-dimerizing activities of cGAS, deamidated cGAS failed to catalyze the synthesis of cGAMP that activates innate immune signaling to restrict DNA virus replication. Moreover, recombinant HSV-1 carrying the deamidase-inactivating C819S point mutation more robustly induced antiviral cytokine production and was highly attenuated in replication and pathogenesis in mice compared to HSV-1 wild-type, in a cGAS- and STING-dependent manner. Sequence comparison and mutational analyses defined a single asparagine within the activation loop of cGAS of human and mouse, but not most NHP cGASs, which underpins cGAS deamidation by UL37 and species permissiveness of HSV-1. This work uncovers a key role of protein deamidation in immune evasion whereby a human pathogen has evolved to exploit species sequence variation to disarm host immune defense.

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 K 5 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 K 3 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 EGFP high 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.

Interferon regulated factor 5 (IRF5) is a key regulator of inflammatory responses in human and mammals; however, its role in teleost remains largely unknown. In this study, IRF5 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized, which possesses conservation in structure and sequence to its mammalian counterparts. Black carp IRF5 (bcIRF5) was characterized as a predominantly cytosolic protein by immunofluorescent staining and showed little IFN promoter-inducing ability in reporter assay. The direct association between bcIRF5 and black carp TBK1 (bcTBK1) were identified through co-immunoprecipitation assay, and co-expressed bcIRF5 in EPC cells suppressed bcTBK1-mediated IFN promoter transcription in reporter assay. Surprisingly, the titer of grass carp reovirus (GCRV) in the media of EPC cells co-expressing bcIRF5 and bcTBK1 was obviously lower than that of EPC cells expressing bcTBK1 alone. It was interesting that expression of bcIRF5 and/or bcTBK1 in EPC cells showed little effect on cell growth; however, the survival ratio of EPC cells co-expressing bcTBK1 and bcIRF5 post GCRV infection was much lower than that of EPC cells expressing bcIRF5 or bcTBK1 alone. These results indicate that bcIRF5 negatively regulates bcTBK1-mediated IFN signaling in healthy cells; however, it correlates with bcTBK1 and triggers cell death to inhibit the virus replication during the innate immune activation.

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.

All herpesviruses share a remarkable propensity to establish latent infection. Human Kaposi's sarcoma-associated herpesvirus (KSHV) effectively enters latency after de novo infection, suggesting that KSHV has evolved with strategies to facilitate latent infection. NF-κB activation is imperative for latent infection of gammaherpesviruses. However, how NF-κB is activated during de novo herpesvirus infection is not fully understood. Here, we report that KSHV infection activates the inhibitor of κB kinase β (IKKβ) and the IKK-related kinase epsilon (IKKε) to enable host NF-κB activation and KSHV latent infection. Specifically, KSHV infection activated IKKβ and IKKε that were crucial for latent infection. Knockdown of IKKβ and IKKε caused aberrant lytic gene expression and impaired KSHV latent infection. Biochemical and genetic experiments identified RelA as a key player downstream of IKKβ and IKKε. Remarkably, IKKβ and IKKε were essential for phosphorylation of S 536 and S 468 of RelA, respectively. Phosphorylation of RelA S 536 was required for phosphorylation of S 468 , which activated NF-κB and promoted KSHV latent infection. Expression of the phosphorylation-resistant RelA S 536 A increased KSHV lytic gene expression and impaired latent infection. Our findings uncover a scheme wherein NF-κB activation is coordinated by IKKβ and IKKε, which sequentially phosphorylate RelA in a site-specific manner to enable latent infection after KSHV de novo infection.

Autophagy is an important innate safeguard mechanism for protecting an organism against invasion by pathogens. We have previously discovered that Kaposi's sarcoma-associated herpesvirus (KSHV) evades this host defense through tight suppression of autophagy by targeting multiple steps of autophagy signal transduction. Here, we report that KSHV K7 protein interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step by blocking Vps34 enzymatic activity, further highlighting how KSHV deregulates autophagy-mediated host immunity for its life cycle.

γ-Herpesviral immune evasion mechanisms are optimized to support the acute, lytic and the longterm, latent phase of infection. During acute infection, specific immune modulatory proteins limit, but also exploit, the antiviral activities of cell intrinsic innate immune responses as well as those of innate and adaptive immune cells. During latent infection, a restricted gene expression program limits immune targeting and cis-acting mechanisms to reduce the antigen presentation as well as antigenicity of latency-associated proteins. Here, we will review recent progress in our understanding of γ-herpesviral immune evasion strategies.

RIG-I detects double-stranded RNA (dsRNA) to trigger antiviral cytokine production. Protein deamidation is emerging as a post-translational modification that chiefly regulates protein function. We report here that UL37 of herpes simplex virus 1 (HSV-1) is a protein deamidase that targets RIG-I to block RNA-induced activation. Mass spectrometry analysis identified two asparagine residues in the helicase 2i domain of RIG-I that were deamidated upon UL37 expression or HSV-1 infection. Deamidation rendered RIG-I unable to sense viral dsRNA, thus blocking its ability to trigger antiviral immune responses and restrict viral replication. Purified full-length UL37 and its carboxyl-terminal fragment were sufficient to deamidate RIG-I in vitro. Uncoupling RIG-I deamidation from HSV-1 infection, by engineering deamidation-resistant RIG-I or introducing deamidase-deficient UL37 into the HSV-1 genome, restored RIG-I activation and antiviral immune signaling. Our work identifies a viral deamidase and extends the paradigm of deamidation-mediated suppression of innate immunity by microbial pathogens.

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.

Cytokines play crucial roles in curtailing the propagation and spread of pathogens within the host. As obligate pathogens, gammaherpesviruses have evolved a plethora of mechanisms to evade host immune responses. We have previously shown that murine gammaherpesvirus 68 (γHV68) induces the degradation of RelA, an essential subunit of the transcriptionally active NF-κB dimer, to evade cytokine production. Here, we report that the immediately early gene product of γHV68, r eplication t rans a ctivator (RTA), functions as a ubiquitin E3 ligase to promote RelA degradation and abrogate cytokine production. A targeted genomic screen identified that RTA, out of 24 candidates, induces RelA degradation and abolishes NF-κB activation. Biochemical analyses indicated that RTA interacts with RelA and promotes RelA ubiquitination, thereby facilitating RelA degradation. Mutations within a conserved cysteine/histidine-rich, putative E3 ligase domain impaired the ability of RTA to induce RelA ubiquitination and degradation. Moreover, infection by recombinant γHV68 carrying mutations that diminish the E3 ligase activity of RTA resulted in more robust NF-κB activation and cytokine induction than did infection by wild-type γHV68. These findings support the conclusion that γHV68 subverts early NF-κB activation and cytokine production through RTA-induced RelA degradation, uncovering a key function of RTA that antagonizes the intrinsic cytokine production during gammaherpesvirus infection.

Host innate immunity represents the first line of defense against invading pathogens and shapes the course and outcome of pathogen infection. Mammals have evolved an array of highly conserved pattern recognition receptors (PRRs) that monitor the presence of “non-self” components or danger signals (Akira et al., 2006; Medzhitov, 2007). The innate immune signal transduction and viral regulation have been extensively reviewed elsewhere (Zhang et al., 2010), we therefore briefly summarize the signaling cascades that upregulate the transcription of antiviral inflammatory cytokines in response to viral infection. In higher eukaryotes, PRRs are classified into three categories according to their structural and functional conservation, including membrane-anchored C-type lectin, toll-like receptors (TLRs), and cytosolic NOD-like or RIGI-like receptors (NLRs and RLRs). Upon infection, PRRs sense pathogen-associated molecular patterns (PAMPs) that are usually pathogen structural components or replication intermediates (Akira et al., 2006; Medzhitov, 2007). While TLRs patrol extracellular and endosomal compartments, NLRs and RLRs monitor the cytosolic environment for foreign or self danger entities. Upon association with PAMPs, PRRs dimerize with their cognate adaptors (such as MyD88, TRIF, MAVS [also known as VISA, IPS-1, and CARDIF], or RIP2) that trigger the activation of two closely-related innate immune kinase complexes, IKKαβγ and TBK1-IKKe (also known as IKKi) (Sun et al., 2010). Through phosphorylation, these kinase complexes induce the activation of NFκB and interferon regulatory factors (IRFs), transcription factors that, in turn, upregulate the expression of antiviral proinflammatory cytokines and interferons (Chen et al., 1996; Mercurio et al., 1997; Fitzgerald et al., 2003; Sharma et al., 2003), thereby constituting potent antiviral innate immunity. The critical roles of these innate immune pathways have been elucidated by studies using gene knockout mice and conversely, investigations of pathogen evasion strategies that antagonize host innate immune responses. Within this short article, we will summarize our recent findings and introduce a new concept, innate immune exploitation whereby host innate immune activation is usurped by viruses to benefit their infection, for example, to promote viral replication.

The Kaposi's sarcoma-associated herpesvirus (KSHV) G protein-coupled receptor (vGPCR) is a bona fide signaling molecule that is implicated in KSHV-associated malignancies. Whereas vGPCR activates specific cellular signaling pathways in a chemokine-independent fashion, vGPCR binds a broad spectrum of CC and CXC chemokines, and the roles of chemokines in vGPCR tumorigenesis remain poorly understood. We report here that vGPCR is posttranslationally modified by sulfate groups at tyrosine residues within its N-terminal extracellular domain. A chemokine-binding assay demonstrated that the tyrosine sulfate moieties were critical for vGPCR association with GRO-α (an agonist) but not with IP-10 (an inverse agonist). A sulfated peptide corresponding to residues 12 through 33 of vGPCR, but not the unsulfated equivalent, partially inhibited vGPCR association with GRO-α. Although the vGPCR variant lacking sulfotyrosines activated downstream signaling pathways, the ability of the unsulfated vGPCR variant to induce tumor growth in nude mice was significantly diminished. Furthermore, the unsulfated vGPCR variant was unable to induce the secretion of proliferative cytokines, some of which serve as vGPCR agonists. This implies that autocrine activation by agonist chemokines is critical for vGPCR tumorigenesis. Indeed, GRO-α increased vGPCR-mediated AKT phosphorylation and vGPCR tumorigenesis in a sulfotyrosine-dependent manner. Our findings support the conclusion that autocrine activation triggered by chemokine agonists via sulfotyrosines is necessary for vGPCR tumorigenesis, thereby providing a rationale for future therapeutic design targeting the tumorigenic vGPCR.

Protein deamidation has been considered a nonenzymatic process associated with protein functional decay or “aging.” Recent studies implicate protein deamidation in regulating signal transduction in fundamental biological processes, such as innate immune responses. Work investigating gammaherpesviruses and bacterial pathogens indicates that microbial pathogens deploy deamidases or enzyme-deficient homologues (pseudoenzymes) to induce deamidation of key signaling components and evade host immune responses. Here, we review studies on protein deamidation in innate immune signaling and present several imminent questions concerning the roles of protein deamidation in infection and immunity.

G protein-coupled receptors (GPCRs) constitute the largest family of molecules that transduce signals across the plasma membrane. Herpesviruses are successful pathogens that evolved diverse mechanisms to benefit their infection. Several human herpesviruses express GPCRs to exploit cellular signaling cascades during infection. These viral GPCRs demonstrate distinct biochemical and biophysical properties that result in the activation of a broad spectrum of signaling pathways. In immune-deficient individuals, human herpesvirus infection and the expression of their GPCRs are implicated in virus-associated diseases and pathologies. Emerging studies also uncover diverse mutations in components, particularly GPCRs and small G proteins, of GPCR signaling pathways that render the constitutive activation of proliferative and survival signal, which contributes to the oncogenesis of various human cancers. Hijacking GPCR-mediated signaling is a signature shared by diseases associated with constitutively active viral GPCRs and cellular mutations activating GPCR signaling, exposing key molecules that can be targeted for anti-viral and anti-tumor therapy.

Summary Activation of nuclear factor of activated T cells (NFAT) is crucial for immune responses. IKKe is an IκB kinase (IKK)-related kinase, and the function of IKKe remains obscure in T cells, despite its abundant expression. We report that IKKe inhibits NFAT activation and T cell responses by promoting NFATc1 phosphorylation. During T cell activation, IKKe was transiently activated to phosphorylate NFATc1. Loss of IKKe elevated T cell antitumor and antiviral immunity and, therefore, reduced tumor development and persistent viral infection. IKKe was activated in CD8 + T cells of mice bearing melanoma or persistently infected with a model herpesvirus. These results collectively show that IKKe promotes NFATc1 phosphorylation and inhibits T cell responses, identifying IKKe as a crucial negative regulator of T cell activation and a potential target for immunotherapy.

NF-κB transcription factors regulate the expression of hundreds of genes primarily involved in immune responses. Signaling events leading to NF-κB activation constitute a major antiviral immune pathway. To replicate and persist within their hosts, viruses have evolved diverse strategies to evade and exploit cellular NF-κB immune signaling cascades for their benefit. We summarize recent studies concerning viral manipulation of the NF-κB signaling pathway downstream of pattern recognition receptors. Signal transduction mediated by pattern recognition receptors is a research frontier for both infectious disease and innate immunology.

Autophagy, the degradation of cytoplasmic components, is an evolutionarily conserved homeostatic process involved in environmental adaptation, lifespan determination and tumour development. The tumor suppressor Beclin1 is part of the PI(3) kinase class III (PI(3)KC3) lipid-kinase complex that induces autophagy. The autophagic activity of the Beclin1-PI(3)KC3 complex, however, is suppressed by Bcl-2. Here, we report the identification of a novel coiled-coil UV irradiation resistance-associated gene (UVRAG) as a positive regulator of the Beclin1-PI(3)KC3 complex. UVRAG, a tumour suppressor candidate that is monoallelically mutated at high frequency in human colon cancers, associates with the Beclin1-Bcl-2-PI(3)KC3 multiprotein complex, where UVRAG and Beclin1 interdependently induce autophagy. UVRAG-mediated activation of the Beclin1-PI(3)KC3 complex promotes autophagy and also suppresses the proliferation and tumorigenicity of human colon cancer cells. These results identify UVRAG as an essential component of the Beclin1-PI(3)KC3 lipid kinase complex that is an important signalling checkpoint for autophagy and tumour-cell growth.

Infected cells recognize viral replication as a DNA damage stress and elicit the ataxia telangiectasia-mutated (ATM)/p53-mediated DNA damage response signal transduction pathway as part of the host surveillance mechanisms, which ultimately induces the irreversible cell cycle arrest and apoptosis. Viruses have evolved a variety of mechanisms to counteract this host intracellular innate immunity. Kaposi's sarcoma-associated herpesvirus (KSHV) viral interferon regulatory factor 1 (vIRF1) interacts with the cellular p53 tumor suppressor through its central DNA binding domain, and this interaction inhibits transcriptional activation of p53. Here, we further demonstrate that KSHV vIRF1 downregulates the total p53 protein level by facilitating its proteasome-mediated degradation. Detailed biochemical study showed that vIRF1 interacted with cellular ATM kinase through its carboxyl-terminal transactivation domain and that this interaction blocked the activation of ATM kinase activity induced by DNA damage stress. As a consequence, vIRF1 expression greatly reduced the level of serine 15 phosphorylation of p53, resulting in an increase of p53 ubiquitination and thereby a decrease of its protein stability. These results indicate that KSHV vIRF1 comprehensively compromises an ATM/p53-mediated DNA damage response checkpoint by targeting both upstream ATM kinase and downstream p53 tumor suppressor, which might circumvent host growth surveillance and facilitate viral replication in infected cells.

During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathioneS-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.

T cells play a central role in orchestrating immunity against pathogens, particularly viruses. Thus, impairing T cell activation is an important strategy employed by viruses to escape host immune control. The tyrosine kinase–interacting protein (Tip) of the T lymphotropic Herpesvirus saimiri (HVS) is constitutively present in lipid rafts and interacts with cellular Lck tyrosine kinase and p80 endosomal protein. Here we demonstrate that, due to the sequestration of Lck by HVS Tip, T cell receptor (TCR) stimulation fails to activate ZAP70 tyrosine kinase and to initiate downstream signaling events. TCR ζ chains in Tip-expressing T cells were initially phosphorylated to recruit ZAP70 molecule upon TCR stimulation, but the recruited ZAP70 kinase was not subsequently phosphorylated, resulting in TCR complexes that were stably associated with inactive ZAP70 kinase. Consequently, Tip expression not only markedly inhibited TCR-mediated intracellular signal transduction but also blocked TCR engagement with major histocompatibility complexes on the antigen-presenting cells and immunological synapse formation. These results demonstrate that a lymphotropic herpesvirus has evolved a novel mechanism to deregulate T cell activation to disarm host immune surveillance. This process contributes to the establishment and maintenance of viral latency.

Signal transducer and activator of transcription 3 (Stat3) protein belongs to a seven-member family of latent cytoplasmic transcription factors that contribute to signal transduction initiated by cytokines, hormones, and growth factors (13). Stat3 proteins control fundamental cellular processes, including survival, proliferation, and differentiation. Upon stimulation with interferons, interleukin-6, granulocyte-macrophage colony-stimulating factor, epidermal growth factor, or platelet-derived growth factor, Stat3 protein becomes activated by phosphorylation on a single tyrosine (Y705) and dimerizes through a reciprocal interaction between the SH2 domain of one monomer and the phosphorylated tyrosine of the other (29). The dimers accumulate in the nucleus, recognize specific DNA elements, and activate transcription. Given the critical roles of Stat3 protein, it is hypothesized that inappropriate or aberrant activation of Stat3 contributes to cellular transformation and, in particular, leukemogenesis (6, 19, 20). In fact, the constitutive activation of Stat3 has been detected in many cancer cells and tissues, including those of multiple myeloma, leukemia, lymphoma, breast, lung, and head and neck cancer (6, 19, 20). Furthermore, it has been shown that Stat3 exerts its growth-deregulating activity by activating the expression of cellular genes that are involved in cell cycle progression such as fos, cyclin D1, myc, and pim-1 and by activating antiapoptotic processes such as Bcl-2 and Bcl-XL (3, 5, 7, 17, 35). Thus, activation of the Stat3 signal transduction pathway is likely a common strategy used by various viruses to transform a normal cell to a cancerous cell (8, 38). In fact, viral Src has been shown to interact with Stat3 and to activate Stat3 to induce cell growth transformation (4, 34, 37). Herpesvirus saimiri (HVS) belongs to the gamma subfamily of herpesviruses (Gammaherpesvirinae). HVS naturally infects the squirrel monkey (Saimiri sciureus), a common South American primate, but with no apparent disease association. However, HVS infection of marmosets, owl monkeys, and other species of New World primates results in rapidly progressing fulminant lymphomas, lymphosarcomas, and leukemias of T-cell origin (18, 26). HVS can be further subclassified into three subgroups (subgroups A, B, and C) on the basis of the extent of DNA sequence divergence at the left end of coding DNA (32). Subgroups A and C are highly oncogenic and are able to immortalize common marmoset T lymphocytes to interleukin 2-independent growth in vitro (16, 36). Subgroup-C strains are further capable of immortalizing human, rabbit, and rhesus monkey lymphocytes into continuously proliferating T-cell lines (1, 2). Mutational analyses have demonstrated that the leftmost open reading frame in the coding sequence of subgroup A strain 11 is not required for viral replication but is required for immortalization of common marmoset T lymphocytes in vitro and for lymphoma induction in vivo (14, 15). This open reading frame is termed STP-A11, for saimiri transformation-associated protein of subgroup A strain 11 (33). At a position and an orientation equivalent to those of the STP-A11 reading frame, the highly oncogenic HVS subgroup C strain 488 contains a distantly related reading frame termed STP-C488 (2, 28). Despite limited sequence similarity, STP-A11 and STP-C488 seem to be organized similarly in terms of the presence and localization of basic structural elements (2, 28). Both proteins are predicted to have a highly acidic amino terminus and collagen-like repeats in the central region. The primary amino acid sequence of STP-A11 has 9 repeats of a collagen-like motif (Gly-X-Y, where X and/or Y is proline), and in STP-C488 this motif is directly repeated 18 times (2, 28). The STP-A11 and STP-C488 proteins also contain a hydrophobic stretch at their carboxyl termini sufficient for a membrane-spanning domain (27). Both STPs have transforming and tumor-inducing activities independent of the rest of the herpesvirus genome (28). Specifically, both can transform rodent fibroblast cells, resulting in apparent loss of contact inhibition, formation of foci, growth at reduced serum concentrations, and formation of invasive tumors in nude mice (28). To understand the structural and functional properties of STP-A, we analyzed the primary amino acid sequences of six different subgroup-A isolates (31). This analysis revealed that STP-A contains interesting structural and functional elements, including the 60PVQES64 binding motif for tumor necrosis factor (TNF) receptor-associated factors (TRAFs) and the 115YAEV118 SH2 binding motif for Src family kinases. Indeed, biochemical analysis has demonstrated that STP-A is capable of interacting with TRAF and Src kinase through the 60PVQES64 and 115YAEV118 motifs, respectively. While the role of TRAF interaction has not been well characterized, Src interaction has been shown to induce the tyrosine phosphorylation of STP-A11 as well as of other cellular proteins (30, 31). In this report, we further demonstrate that STP-A11 interacts with Stat3 independently of TRAF and Src, and that Src kinase associated with STP-A11 phosphorylates Stat3, resulting in its nuclear localization and transcriptional activation. Consequently, the constitutive activation of Stat3 induced by STP-A11 leads to cell survival and proliferation upon serum deprivation. Thus, the STP-A11 oncoprotein targets multiple cellular signaling molecules to elicit cell growth transformation, which ultimately contributes to T-cell transformation induced by HVS subgroup-A strains.

Pathogens exploit host machinery to establish an environment that favors their propagation. Because of their pivotal roles in cellular physiology, protein degradation pathways are common targets for viral proteins. Protein-linking integrin-associated protein and cytoskeleton 1 (PLIC1), also called ubiquilin, contains an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain. PLIC1 is proposed to function as a regulator of the ubiquitination complex and proteasome machinery. Kaposi's sarcoma-associated herpesvirus (KSHV) contains a small membrane protein, K7, that protects cells from apoptosis induced by various stimuli. We report here that cellular PLIC1 is a K7-interacting protein and that the central hydrophobic region of K7 and the carboxy-terminal UBA domain of PLIC1 are responsible for their interaction. Cellular PLIC1 formed a dimer and bound efficiently to polyubiquitinated proteins through its carboxy-terminal UBA domain, and this activity correlated with its ability to stabilize cellular I kappa B protein. In contrast, K7 interaction prevented PLIC1 from forming a dimer and binding to polyubiquitinated proteins, leading to the rapid degradation of I kappa B. Furthermore, K7 expression promoted efficient degradation of the p53 tumor suppressor, resulting in inhibition of p53-mediated apoptosis. These results indicate that KSHV K7 targets a regulator of the ubiquitin- and proteasome-mediated degradation machinery to deregulate cellular protein turnover, which potentially provides a favorable environment for viral reproduction.

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.

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 (E 14 ) of vBcl-2 is critical for KSHV lytic replication. Mutating E 14 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.

On viral infection, infected cells can become the target of host immune responses or can go through a programmed cell death process, called apoptosis, as a defense mechanism to limit the ability of the virus to replicate. To prevent this, viruses have evolved elaborate mechanisms to subvert the apoptotic process. Here, we report the identification of a novel antiapoptotic K7 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) which expresses during lytic replication. The KSHV K7 gene encodes a small mitochondrial membrane protein, and its expression efficiently inhibits apoptosis induced by a variety of apoptogenic agents. The yeast two-hybrid screen has demonstrated that K7 targets cellular calcium-modulating cyclophilin ligand (CAML), a protein that regulates the intracellular Ca 2+ concentration. Similar to CAML, K7 expression significantly enhances the kinetics and amplitudes of the increase in intracellular Ca 2+ concentration on apoptotic stimulus. Mutational analysis showed that K7 interaction with CAML is required for its function in the inhibition of apoptosis. This indicates that K7 targets cellular CAML to increase the cytosolic Ca 2+ response, which consequently protects cells from mitochondrial damage and apoptosis. This is a novel viral antiapoptosis strategy where the KSHV mitochondrial K7 protein targets a cellular Ca 2+ -modulating protein to confer resistance to apoptosis, which allows completion of the viral lytic replication and, eventually, maintenance of persistent infection in infected host.

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs., Author Summary G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal across plasma membrane. Herpesviral GPCRs (vGPCRs) activate diverse signaling cascades and are implicated in viral pathogenesis (e.g., tumor development). In contrast to cellular GPCRs that are chiefly regulated via cognate ligand-association, vGPCRs are constitutively active independent of ligand-binding. vGPCRs provide useful tools to dissect signal transduction from plasma membrane receptors to nuclear transcription factors. To probe the activation of nuclear factor of T cells (NFAT), we demonstrate that vGPCRs target the ER calcium ATPase to increase cytosolic calcium concentration and activate NFAT. Inhibition of NFAT activation impairs tumor formation induced by vGPCRs, implying the antitumor therapeutic potential via disabling NFAT activation.

RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here, we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologs of phosphoribosylformylglycinamidine synthetase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homolog thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme.

Aberrant expression of the MDR1-encoded P-glycoprotein (P-gp) is often associated with clinical multi-drug resistance (MDR) leading to poor prognosis and failure of chemotherapy. However, the precise and cooperative molecular mechanism responsible for MDR1 transcription and expression in acquired MDR remains elusive. We, herein, demonstrate that Wnt/β-catenin signal pathway is constitutively activated in Doxorubicin-induced MDR cancer cells, in which nuclear β -catenin specifically interacts with the transcriptional coactivator CBP in a MEK(1/2)/ERK(1/2) signal-dependent manner. Specific knockdown of both β-catenin and CBP by RNAi-mediated depletion abrogates MDR1 transcription and expression resulting in a complete reversal of P-gp-dependent efflux function and restoration of sensitivity to the Doxorubincin-induced cytotoxicity. Moreover, following pharmacological disruption of CBP and β - catenin interaction through inhibition of the MEK(1/2)/ERK(1/2) signal by the specific inhibitor PD98059, MDR1 transcription and its encoded P-gp-dependent function are abolished. These findings conclude that the CBP/β-catenin complex is a core component of the MDR1 transcriptional "enhancesome".

Bacillus thuringiensis is a kind of insecticidal microorganism which can produce a variety of toxin proteins, it is particularly important to find an effective strategy to identify novel toxin proteins rapidly and comprehensively with the discovery of the wild-type strains. Multi-dimensional high-performance liquid chromatography combined with mass spectrometry has become one of the main methods to detect and identify toxin proteins and proteome of B. thuringiensis. In this study, protein samples from B. thuringiensis strain 4.0718 were analyzed on the basis of two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC–MS/MS), and tryptic peptides of whole cell from the late sporulation phase were eluted at different concentration gradients of ammonium chloride and followed by secondary mass spectrum identification. 831 and 894 proteins were identified from two biological replicates, respectively, while 1,770 and 1,859 peptides were detected correspondingly. Among the identified proteins and peptides, 606 proteins and 1,259 peptides were detected in both replicates, which mean that 1,119 proteins and 2,370 peptides were unique to the proteome of this strain. A total of 15 toxins have been identified successfully, and seven of them were firstly discovered in B. thuringiensis strain 4.0718 that were Crystal protein (A1E259), pesticidal protein (U5KS09), Cry2Af1 (A4GVF0), Cry2Ad (Q9RM89), Cry1 (K4HMB5), Cry1Bc (Q45774), and Cry1Ga (Q45746). The proteomic strategy employed in the present study has provided quick and exhaustive identification of toxins produced by B. thuringiensis.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies. Murine γHV68 is a natural pathogen of murine rodents. γHV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways. Recently, we have discovered that γHV68 hijacks MAVS and IKKβ, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NFΚB activation and antiviral cytokine production. Specifically, γHV68 infection activates IKKβ and that activated IKKβ phosphorylates RelA to accelerate RelA degradation. As such, γHV68 efficiently uncouples NFΚB activation from its upstream activated IKKβ, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.

G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that transmit diverse extracellular signals across a membrane. Herpesvirus genomes encode multiple GPCRs implicated in viral pathogenesis. Kaposi sarcoma-associated herpesvirus GPCR (kGPCR) activates proliferative pathways and, when expressed in endothelium in mice, sufficiently induces angiogenic tumor resembling human Kaposi’s sarcoma. IKKε, an IκB kinase (IKK)-related kinase, is implicated in inflammation-driven tumorigenesis. We report here that IKKε is critically required for kGPCR tumorigenesis and links kGPCR to NF-κB activation. Using kGPCR-induced tumor models, we found that IKKε expression was drastically up-regulated in Kaposi sarcoma-like lesions and that loss of IKKε abolished tumor formation. Moreover, kGPCR interacted with and activated IKKε. Activated IKKε promoted NF-κB subunit RelA (also known as p65) phosphorylation, which correlated with NF-κB activation and inflammatory cytokine expression. The robust expression of IKKε and phosphorylated RelA was observed in human Kaposi sarcoma. Finally, a kinase-defective mutant of IKKε effectively abrogated NF-κB activation and tumorigenesis induced by kGPCR. Collectively, our findings uncover a critical IKKε in promoting NF-κB activation and tumorigenesis induced by a viral GPCR.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection. Murine γHV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. γHV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in γHV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during γHV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during γHV68 lytic replication ex vivo. Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant γHV68 carrying these mutations can be used to recapitulate the phenotypes of γHV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo. Recently, we have discovered that γHV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, γHV68 de novo infection activates the immune kinase IKKβ and activated IKKβ phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, γHV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

Upon viral infection, the mitochondrial antiviral signaling (MAVS)-IKKβ pathway is activated to restrict viral replication. Manipulation of immune signaling events by pathogens has been an outstanding theme of host-pathogen interaction. Here we report that the loss of MAVS or IKKβ impaired the lytic replication of gamma-herpesvirus 68 (γHV68), a model herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus. γHV68 infection activated IKKβ in a MAVS-dependent manner; however, IKKβ phosphorylated and promoted the transcriptional activation of the γHV68 replication and transcription activator (RTA). Mutational analyses identified IKKβ phosphorylation sites, through which RTA-mediated transcription was increased by IKKβ, within the transactivation domain of RTA. Moreover, the lytic replication of recombinant γHV68 carrying mutations within the IKKβ phosphorylation sites was greatly impaired. These findings support the conclusion that γHV68 hijacks the antiviral MAVS-IKKβ pathway to promote viral transcription and lytic infection, representing an example whereby viral replication is coupled to host immune activation., Author Summary Innate immunity represents the first line of defense against pathogen infection. Recent studies uncovered an array of sensors that detect pathogen-associated molecular patterns and induce antiviral cytokine production via two closely related kinase complexes, i.e., the IKKα/β/γ and TBK-1/IKKε. To counteract host immune defense, herpesviruses have evolved diverse strategies to evade, manipulate, and exploit host immune responses. Here we report that infection by murine gamma-herpesvirus 68 (γHV68), a model gamma-herpesvirus for human Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus, activated the IKKβ kinase and IKKβ was usurped to promote viral transcriptional activation. As such, uncoupling IKKβ from transcriptional activation by biochemical and genetic approaches impaired γHV68 lytic replication. Our study represents an example whereby viral lytic replication is coupled to host innate immune activation and sheds light on herpesvirus exploitation of immune responses.

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been consistently implicated in the pathogenesis of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). The hallmark of KSHV infection is to establish life-long persistency that has imposed enormous pressure on this virus to escape host immune recognition. Although many of the mechanistic details require further elucidation, KSHV devotes a significant portion of its genome to gene products that together sabotage almost every aspect of host’s immune system. A more thorough understanding of these strategies helps to unravel the complexities of viral pathogenesis and may reveal targets for novel therapeutic strategies. This review provides an overview of the current understanding of how KSHV evades or mitigates the host immune response.

The Kaposi's sarcoma-associated herpesvirus (KSHV) genome encodes a G protein-coupled receptor (vGPCR). vGPCR is a ligand-independent, constitutively active signaling molecule that promotes cell growth and proliferation; however, it is not clear how vGPCR is negatively regulated. We report here that the KSHV K7 small membrane protein interacts with vGPCR and induces its degradation, thereby dampening vGPCR signaling. K7 interaction with vGPCR is readily detected in transiently transfected human cells. Mutational analyses reveal that the K7 transmembrane domain is necessary and sufficient for this interaction. Biochemical and confocal microscopy studies indicate that K7 retains vGPCR in the endoplasmic reticulum (ER) and induces vGPCR proteasomeal degradation. Indeed, the knockdown of K7 by shRNA-mediated silencing increases vGPCR protein expression in BCBL-1 cells that are induced for KSHV lytic replication. Interestingly, K7 expression significantly reduces vGPCR tumorigenicity in nude mice. These findings define a viral factor that negatively regulates vGPCR protein expression and reveal a post-translational event that modulates GPCR-dependent transformation and tumorigenicity., Author Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma. KSHV is also found in primary effusion lymphoma and multicentric Castleman's disease, rare lymphoproliferative diorders associated with immuno-suppression. The KSHV genome encodes a G protein-coupled receptor (vGPCR) that is believed to contribute to the KSHV-associated malignancies. vGPCR is a ligand-independent, constitutively active signaling molecule. It is not clear how vGPCR is negatively regulated. Here, we report that the KSHV small membrane K7 protein interacts with vGPCR through its putative transmembrane domain. Interaction with K7 retains vGPCR in the ER and facilitates its degradation by the proteasome, thereby reducing vGPCR protein expression. Consequently, K7 significantly reduces vGPCR-mediated transformation in vitro and tumor formation in nude mice. Our findings reveal that K7 functions as a viral factor to dampen vGPCR protein expression and negatively modulate the tumor-inducing capacity of vGPCR, implying that KSHV has evolved mechanisms to avoid deleterious effects and to permit persistent infection within its host.

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome–endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG–Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy — autophagosome formation and maturation — but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies1 have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, underscoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3- mediated autophagy and tumor cell control.Addendum to:Autophagic and Tumor Suppressor Activity of a Novel Beclin 1-Binding Protein UVRA...

Kaposi's sarcoma (KS) is a multifocal angiogenic tumor and appears to be a hyperplastic disorder caused, in part, by local production of inflammatory cytokines. The K1 lymphocyte receptor-like protein of KS-associated herpesvirus (KSHV) efficiently transduces extracellular signals to elicit cellular activation events through its cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). To further delineate K1-mediated signal transduction, we purified K1 signaling complexes and identified its cellular components. Upon stimulation, the K1 ITAM was efficiently tyrosine phosphorylated and subsequently interacted with cellular Src homology 2 (SH2)-containing signaling proteins Lyn, Syk, p85, PLCγ2, RasGAP, Vav, SH2 domain-containing protein tyrosine phosphatase 1/2, and Grab2 through its phosphorylated tyrosine residues. Mutational analysis demonstrated that each tyrosine residue of K1 ITAM contributed to the interactions with cellular signaling proteins in distinctive ways. Consequently, these interactions led to the marked augmentation of cellular signal transduction activity, evidenced by the increase of cellular tyrosine phosphorylation and intracellular calcium mobilization, the activation of NF-AT and AP-1 transcription factor activities, and the production of inflammatory cytokines. These results demonstrate that KSHV K1 effectively recruits a set of cellular SH2-containing signaling molecules to form the K1 signalosome, which elicits downstream signal transduction and induces inflammatory cytokine production.

Because of its central regulatory role, T-cell receptor (TCR) signal transduction is a common target of viruses. We report here the identification of a small signaling protein, ORF5, of the T-lymphotropic tumor virus herpesvirus saimiri (HVS). ORF5 is predicted to contain 89 to 91 amino acids with an amino-terminal myristoylation site and six SH2 binding motifs, showing structural similarity to cellular LAT (linker for activation of T cells). Sequence analysis showed that, despite extensive sequence variation, the myristoylation site and SH2 binding motifs were completely conserved among 13 different ORF5 isolates. Upon TCR stimulation, ORF5 was efficiently tyrosine phosphorylated and subsequently interacted with cellular SH2-containing signaling proteins Lck, Fyn, SLP-76, and p85 through its tyrosine residues. ORF5 expression resulted in the marked augmentation of TCR signal transduction activity, evidenced by increased cellular tyrosine phosphorylation, intracellular calcium mobilization, CD69 surface expression, interleukin-2 production, and activation of the NF-AT, NF-kappa B, and AP-1 transcription factors. Despite its structural similarity to cellular LAT, however, ORF5 could only partially substitute for LAT function in TCR signal transduction. These results demonstrate that HVS utilizes a novel signaling protein, ORF5, to activate TCR signal transduction. This activation probably facilitates viral gene expression and, thereby, persistent infection.

The initial description of Kaposi’s sarcoma can be traced back to 1872, when a Hungarian dermatologist, Moritz Kaposi, published a case of skin cancer with clinical presentation of so-called pigmented sarcomas. This disease was designated Kaposi’s sarcoma (KS) in 1891. There are four distinct epidemiological forms of KS: classic KS occurring predominantly in Europe and the Mediterranean; endemic KS of the human immunodeficiency virus (HIV-1) negative patients in Africa; posttransplant or iatrogenic KS; and HIV-associated KS. The speculation of a viral etiology for KS was confirmed by the discovery of KSHV from HIV-associated KS lesions in 1994 (Chang et al. 1994). Subsequent epidemiological studies have demonstrated that KSHV DNA is present in all forms of KS tumors, suggesting that KSHV is the etiology agent for the development of KS (Sarid et al. 1999). In addition to KS, KSHV also associates with primary effusion lymphoma (PEL) and an immunoblast variant of Castleman’s disease (CD), which are of B cell origin (Cesarman et al. 1995; Soulier et al. 1995).

Lipid rafts are proposed to function as platforms for both receptor signaling and trafficking. Following interaction with antigenic peptides, the T-cell receptor (TCR) rapidly translocates to lipid rafts, where it transmits signals and subsequently undergoes endocytosis. The Tip protein of herpesvirus saimiri (HVS), which is a T-lymphotropic tumor virus, interacts with cellular Lck tyrosine kinase and p80, a WD domain-containing endosomal protein. Interaction of Tip with p80 induces enlarged vesicles and recruits Lck and TCR complex into these vesicles for trafficking. We report here that Tip is constitutively present in lipid rafts and that Tip interaction with p80 but not with Lck is necessary for its efficient localization in lipid rafts. The Tip-Lck interaction was required for recruitment of the TCR complex to lipid rafts, and the Tip-p80 interaction was critical for the aggregation and internalization of lipid rafts. These results suggest the potential mechanism for Tip-mediated TCR downregulation: Tip interacts with Lck to recruit TCR complex to lipid rafts, and it subsequently interacts with p80 to initiate the aggregation and internalization of the lipid raft domain and thereby downregulate the TCR complex. Thus, the signaling and targeting functions of HVS Tip rely on two functionally and genetically separable mechanisms that independently target cellular Lck tyrosine kinase and p80 endosomal protein.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating the decay of all mRNAs, it helps redirect the cell from host to viral gene expression and facilitates the sequential expression of different classes of viral genes. While it is clear that Vhs induces mRNA degradation, it is uncertain whether it is itself an RNase or somehow activates a cellular enzyme. This question was addressed by using a combination of genetic and biochemical approaches. The Vhs homologues of alphaherpesviruses share sequence similarities with a family of mammalian, yeast, bacterial, and phage nucleases. To test the functional significance of these similarities, Vhs was mutated to alter residues corresponding to amino acids known to be critical to the nuclease activity of cellular homologues. In every instance, mutations that inactivated the nuclease activity of cellular homologues also abolished Vhs activity. Recent experiments showed that Vhs interacts with the cellular translation initiation factor eIF4H. In this study, the coexpression of Vhs and a glutathioneS-transferase (GST)-eIF4H fusion protein in bacteria resulted in the formation of a complex of the proteins. The wild-type Vhs/GST-eIF4H complex was isolated and shown to have RNase activity. In contrast, Vhs mutations that altered key residues in the nuclease motif abolished the nuclease activity of the recombinant Vhs/GST-eIF4H complex. The results provide genetic and biochemical evidence that Vhs is an RNase, either alone or as a complex with eIF4H.

During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using the Saccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathione S -transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.