Direct and biologically significant interactions of human herpesvirus 8 interferon regulatory factor 1 with STAT3 and Janus kinase TYK2.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198-222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1.

STAT and Janus kinase targeting by human herpesvirus 8 interferon regulatory factor in the suppression of type-I interferon signaling.

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is involved etiologically in AIDS-associated KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease, in which both viral latent and lytic functions are important. HHV-8 encodes four viral interferon regulatory factors (vIRFs) that are believed to contribute to viral latency (in PEL cells, at least) and/or to productive replication via suppression of cellular antiviral and stress signaling. Here, we identify vIRF-1 interactions with signal transducer and activator of transcription (STAT) factors 1 and 2, interferon (IFN)-stimulated gene factor 3 (ISGF3) cofactor IRF9, and associated signal transducing Janus kinases JAK1 and TYK2. In naturally infected PEL cells and in iSLK epithelial cells infected experimentally with genetically engineered HHV-8, vIRF-1 depletion or ablation, respectively, led to increased levels of active (phosphorylated) STAT1 and STAT2 in IFNß-treated, and untreated, cells during lytic replication and to associated cellular-gene induction. In transfected 293T cells, used for mechanistic studies, suppression by vIRF-1 of IFNß-induced phospho-STAT1 (pSTAT1) was found to be highly dependent on STAT2, indicating vIRF-1-mediated inhibition and/or dissociation of ISGF3-complexing, resulting in susceptibility of pSTAT1 to inactivating dephosphorylation. Indeed, coprecipitation experiments involving targeted precipitation of ISGF3 components identified suppression of mutual interactions by vIRF-1. In contrast, suppression of IFNß-induced pSTAT2 was effected by regulation of STAT2 activation, likely via detected inhibition of TYK2 and its interactions with STAT2 and IFN type-I receptor (IFNAR). Our identified vIRF-1 interactions with IFN-signaling mediators STATs 1 and 2, co-interacting ISGF3 component IRF9, and STAT-activating TYK2 and the suppression of IFN signaling via ISGF3, TYK2-STAT2 and TYK2-IFNAR disruption and TYK2 inhibition represent novel mechanisms of vIRF function and HHV-8 evasion from host-cell defenses.

USP7-Dependent Regulation of TRAF Activation and Signaling by a Viral Interferon Regulatory Factor Homologue.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs 1 to 4), all of which are expressed during lytic replication and inhibit a variety of antiviral signaling pathways. Viral IRFs 1, 2, and 3 are also expressed during latency in primary effusion lymphoma (PEL) cells, and vIRF-1 and vIRF-3 have been reported to promote PEL cell viability. Viral IRFs 1, 3, and 4 are known to interact with ubiquitin-specific protease 7 (USP7); interactions of vIRF-1 and vIRF-3 with USP7 promote PEL cell viability and regulate productive replication. Here, we report that vIRF-2 also targets USP7, utilizing a PSTS motif matching the USP7 N-terminal domain-binding A/PxxS consensus, but uniquely requires catalytic domain residues for intracellular interaction. In functional and mechanistic analyses, tumor necrosis factor receptor-associated factor (TRAF)-mediated signaling and associated polyubiquitination of TRAFs 3 and 6, specifically, were regulated negatively by USP7 and positively by vIRF-2-USP7 interaction, the latter competing for USP7-TRAF association. Using depletion, depletion-complementation, and targeted mutagenesis approaches, vIRF-2 was determined to promote latent PEL cell viability, likely independently of USP7 interaction, while lytic replication was inhibited by vIRF-2, in part or in whole via USP7 interaction. Together, our data identify a new molecular determinant of USP7 recognition, TRAF3/6-specific targeting by the deubiquitinase, associated activation of these TRAFs by vIRF-2, and activities of vIRF-2 and vIRF-2-USP7 interaction in HHV-8 latent and lytic biology.IMPORTANCE Human herpesvirus 8-encoded IRF homologues were the first to be identified in a virus. Through inhibitory interactions with cellular IRFs and other mediators of antiviral signaling, the vIRFs are believed to be essential for productive replication and also for latency in particular cell types. The deubiquitinase USP7 is a regulator of key cellular pathways, modulates HHV-8 latent and lytic infection, and is targeted by vIRFs 1, 3, and 4. Here, we report that vIRF-2 also interacts with USP7, via a means distinguishable from USP7 interactions with other vIRFs and other proteins, that this interaction modulates antiviral signaling via disruption of USP7 interactions with innate immune signaling proteins TRAF3 and TRAF6, and that vIRF-2 targeting of USP7 regulates HHV-8 productive replication. The presented data are the first to identify vIRF-2 targeting of USP7 and its role in HHV-8 biology, expanding our understanding of the repertoire and importance of virus-host interactions.

Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication.

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and localized largely to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 proinflammatory and/or angiogenic viral proteins, in HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), in addition to an MCD-related disorder involving systemic elevation of proinflammatory cytokines, including vIL-6 and human IL-6 (hIL-6). In these diseases, lytic (productive) replication, in addition to viral latency, is believed to play a critical role. Proreplication activity of vIL-6 has been identified experimentally in PEL and endothelial cells, but the relative contributions of different vIL-6 interactions have not been established. Productive interactions of vIL-6 with the IL-6 signal transducer, gp130, can occur within the ER, but vIL-6 also interacts in the ER with a nonsignaling receptor called vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2), calnexin, and VKORC1v2- and calnexin-associated proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII). Here, we report the systematic characterization of interaction-altered vIL-6 variants and the lytic phenotypes of recombinant viruses expressing selected variants. Our data identify the critical importance of vIL-6 and its ER-localized activity via gp130 to productive replication in inducible SLK (epithelial) cells, absence of detectable involvement of vIL-6 interactions with VKORC1v2, GlucII, or UGGT1, and the insufficiency and lack of direct contributory effects of extracellular signaling by vIL-6 or hIL-6. These findings, obtained through genetics-based approaches, complement and extend previous analyses of vIL-6 activity.IMPORTANCE Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) was the first viral IL-6 homologue to be identified. Experimental and clinical evidence suggests that vIL-6 is important for the onset and/or progression of HHV-8-associated endothelial-cell and B-cell pathologies, including AIDS-associated Kaposi's sarcoma and multicentric Castleman's disease. The protein is unusual in its poor secretion from cells and its intracellular activity; it interacts, directly or indirectly, with a number of proteins beyond the IL-6 signal transducer, gp130, and can mediate activities through these interactions in the endoplasmic reticulum. Here, we report the characterization with respect to protein interactions and signal-transducing activity of a panel of vIL-6 variants and utilization of HHV-8 mutant viruses expressing selected variants in phenotypic analyses. Our findings establish the importance of vIL-6 in HHV-8 productive replication and the contributions of individual vIL-6-protein interactions to HHV-8 lytic biology. This work furthers understanding of the biological significance of vIL-6 and its unique intracellular interactions.

Insulin-Like Growth Factor 2 Receptor Expression Is Promoted by Human Herpesvirus 8-Encoded Interleukin-6 and Contributes to Viral Latency and Productive Replication.

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) localizes largely to the endoplasmic reticulum (ER) and here associates functionally with both the gp130 signal transducer and the novel ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). The latter interaction contributes to the viability of latently infected primary effusion lymphoma (PEL) cells and to HHV-8 productive replication, in part via promotion of ER-associated degradation (ERAD) of nascent pro-cathepsin D (pCatD) and consequent suppression of lysosome-localized proapoptotic mature CatD. Here we report that VKORC1v2 associates with insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, which is involved in trafficking of mannose-6-phosphate-conjugated glycoproteins to lysosomes. VKORC1v2 effected reduced IGF2R expression in a manner dependent on VKORC1v2-IGF2R interaction, while vIL-6, which could inhibit VKORC1v2-IGF2R interaction, effected increased expression of IGF2R. These effects were independent of changes in IGF2R mRNA levels, indicating likely posttranslational mechanisms. In kinetic analyses involving labeling of either newly synthesized or preexisting IGF2R, vIL-6 promoted accumulation of the former while having no detectable effect on the latter. Furthermore, vIL-6 led to decreased K48-linked ubiquitination of IGF2R and suppression of ERAD proteins effected increased IGF2R expression and loss of IGF2R regulation by vIL-6. Depletion-based experiments identified IGF2R as a promoter of PEL cell viability and virus yields from lytically reactivated cultures. Our findings identify ER-transiting nascent IGF2R as an interaction partner of VKORC1v2 and target of vIL-6 regulation and IGF2R as a positive contributor to HHV-8 biology, thereby extending understanding of the mechanisms of VKORC1v2-associated vIL-6 function.IMPORTANCE HHV-8 vIL-6 promotes productive replication in the context of reactivated lytic replication in primary effusion lymphoma (PEL) and endothelial cells and sustains latently infected PEL cell viability. Viral IL-6 is also considered to contribute significantly to HHV-8-associated pathogenesis, since vIL-6 can promote cell proliferation, cell survival, and angiogenesis that are characteristic of HHV-8-associated Kaposi's sarcoma, PEL and multicentric Castleman's disease (MCD), in addition to proinflammatory activities observed in MCD-like "Kaposi's sarcoma-associated herpesvirus-induced cytokine syndrome." We show in the present study that vIL-6 can promote productive replication and latent PEL cell viability through upregulation of the mannose-6-phosphate- and peptide hormone-interacting receptor IGF2R, which is a positive factor in HHV-8 biology via these activities. VKORC1v2-enhanced ER-associated degradation of IGF2R and vIL-6 promotion of IGF2R expression through prevention of its interaction with VKORC1v2 and consequent rescue from degradation represent newly recognized activities of VKOCR1v2 and vIL-6.

Human Herpesvirus 8 Interferon Regulatory Factors 1 and 3 Mediate Replication and Latency Activities via Interactions with USP7 Deubiquitinase.

Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRF-1 to -4) that likely function to suppress innate immune and cellular stress responses through inhibitory interactions with various cellular proteins involved in these activities. It is notable that vIRF-1 and -4 have been reported to interact with the deubiquitinase ubiquitin-specific protease 7 (USP7), substrates of which include p53 and the p53-targeting and -destabilizing ubiquitin E3 ligase MDM2. Structural studies of vIRF-1 and vIRF-4 USP7 binding sequences in association with USP7 have been reported; both involve interactions with N-terminal-domain residues of USP7 via EGPS and ASTS motifs in vIRF-1 and vIRF-4, respectively, but vIRF-4 residues also contact the catalytic site. However, the biological activities of vIRF-1 and vIRF-4 via USP7 interactions are unknown. Here, we report that vIRF-3, which is latently, as well as lytically, expressed in HHV-8-infected primary effusion lymphoma (PEL) cells, also interacts with USP7-via duplicated EGPS motifs-and that this interaction is important for PEL cell growth and viability. The interaction also contributes to suppression of productive virus replication by vIRF-3, which we identify here. We further show that vIRF-1, which is expressed at low levels in PEL latency, promotes latent PEL cell viability and that this activity and vIRF-1-promoted productive replication (reported previously) involve EGPS motif-mediated USP7 targeting by vIRF-1. This study is the first to identify latent and lytic functions of vIRF-1 and vIRF-3, respectively, and to address the biological activities of these vIRFs through their interactions with USP7.IMPORTANCE HHV-8 is associated with Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease; both latent and lytic viral functions are believed to contribute. Viral interferon regulatory factors specified by HHV-8 are thought to be critically important for successful productive replication through suppression of innate immune and stress responses triggered by the lytic cycle. Latently expressed vIRF-3 contributes significantly to PEL cell survival. Here, we identify ubiquitin-specific protease 7 (USP7) deubiquitinase targeting by vIRF-3 (in addition to previously reported USP7 binding by vIRF-1 and vIRF-4); the importance of vIRF-1 and vIRF-3 interactions with USP7 for latent PEL cell growth and viability; and the positive and negative contributions, respectively, of USP7 targeting by vIRF-1 and vIRF-3 to HHV-8 productive replication. This is the first report of the biological importance of vIRF-1 in PEL cell latency, the modulation of productive replication by vIRF-3, and the contributions of vIRF-USP7 interactions to HHV-8 biology.

Human Herpesvirus 8 Interleukin-6 Interacts with Calnexin Cycle Components and Promotes Protein Folding.

Viral interleukin-6 (vIL-6) encoded by human herpesvirus 8 (HHV-8) is believed to contribute via mitogenic, survival, and angiogenic activities to HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease through autocrine or paracrine mechanisms during latency or productive replication. There is direct evidence that vIL-6 promotes latently infected PEL cell viability and proliferation and also viral productive replication in PEL and endothelial cells. These activities are mediated largely through endoplasmic reticulum (ER)-localized vIL-6, which can induce signal transduction via the gp130 signaling receptor, activating mitogen-activated protein kinase and signal transducer and activator of transcription signaling, and interactions of vIL-6 with the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). The latter functional axis involves suppression of proapoptotic lysosomal protein cathepsin D by promotion of the ER-associated degradation of ER-transiting, preproteolytically processed procathepsin D. Other interactions of VKORC1v2 and activities of vIL-6 via the receptor have not been reported. We show here that both vIL-6 and VKORC1v2 interact with calnexin cycle proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1), which catalyzes monoglucosylation of N-glycans, and oppositely acting glucosidase II (GlucII), and that vIL-6 can promote protein folding. This activity was found to require VKORC1v2 and UGGT1, to involve vIL-6 associations with VKORC1v2, UGGT1, and GlucII, and to operate in the context of productively infected cells. These findings document new VKORC1v2-associated interactions and activities of vIL-6, revealing novel mechanisms of vIL-6 function within the ER compartment.IMPORTANCE HHV-8 vIL-6 prosurvival (latent) and proreplication functions are mediated from the ER compartment through both gp130 receptor-mediated signal transduction and interaction of vIL-6 with the ER membrane protein VKORC1v2. This report identifies interactions of vIL-6 and VKORC1v2 with calnexin cycle enzymes GlucII and UGGT1, which are involved in glycan processing and nascent protein folding. The presented data show that vIL-6 and VKORC1v2 can cocomplex with GlucII and UGGT1, that vIL-6 promotes protein folding, and that VKORC1v2, UGGT1, and vIL-6 interactions with GlucII and UGGT1 are important for the profolding activity of vIL-6, which can be detected in the context of infected cells. This newly identified ER activity of vIL-6 involving VKORC1v2 may promote viral latency (in PEL cells) and productive replication by limiting the damaging effects of unfolded protein response signaling in addition to enhancing viral protein folding. This is the first report of such a function for a cytokine.

The type 3 effector NopL of Sinorhizobium sp. strain NGR234 is a mitogen-activated protein kinase substrate.

Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.

Proteotoxic stress impairs cellular homeostasis and underlies the pathogenesis of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, the loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in the tissues of ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

NopD of Bradyrhizobium sp. XS1150 Possesses SUMO Protease Activity.

Effectors secreted by the type III protein secretion system (T3SS) of rhizobia are host-specific determinants of the nodule symbiosis. Here, we have characterized NopD, a putative type III effector of Bradyrhizobium sp. XS1150. NopD was found to possess a functional N-terminal secretion signal sequence that could replace that of the NopL effector secreted by Sinorhizobium sp. NGR234. Recombinant NopD and the C-terminal domain of NopD alone can process small ubiquitin-related modifier (SUMO) proteins and cleave SUMO-conjugated proteins. Activity was abolished in a NopD variant with a cysteine-to-alanine substitution in the catalytic core (NopD-C972A). NopD recognizes specific plant SUMO proteins (AtSUMO1 and AtSUMO2 of Arabidopsis thaliana; GmSUMO of Glycine max; PvSUMO of Phaseolus vulgaris). Subcellular localization analysis with A. thaliana protoplasts showed that NopD accumulates in nuclear bodies. NopD, but not NopD-C972A, induces cell death when expressed in Nicotiana tabacum. Likewise, inoculation tests with constructed mutant strains of XS1150 indicated that nodulation of Tephrosia vogelii is negatively affected by the protease activity of NopD. In conclusion, our findings show that NopD is a symbiosis-related protein that can process specific SUMO proteins and desumoylate SUMO-conjugated proteins.

Identification of two functional nuclear localization signals in the capsid protein of duck circovirus.

The capsid protein (CP) of duck circovirus (DuCV) is the major immunogenic protein and has a high proportion of arginine residues concentrated at the N terminus of the protein, which inhibits efficient mRNA translation in prokaryotic expression systems. In this study, we investigated the subcellular distribution of DuCV CP expressed via recombinant baculoviruses in Sf9 cells and the DNA binding activities of the truncated recombinant DuCV CPs. The results showed that two independent bipartite nuclear localization signals (NLSs) situated at N-terminal 1-17 and 18-36 amino acid residue of the CP. Moreover, two expression level regulatory signals (ELRSs) and two DNA binding signals (DBSs) were also mapped to the N terminus of the protein and overlapped with the two NLSs. The ability of CP to bind DNA, coupled with the karyophilic nature of this protein, strongly suggests that it may be responsible for nuclear targeting of the viral genome.

ORF3 of duck circovirus: a novel protein with apoptotic activity.

Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P<0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity.