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

2018 ◽  
Vol 93 (5) ◽  
Author(s):  
Qian Li ◽  
Daming Chen ◽  
Qiwang Xiang ◽  
John Nicholas
Keyword(s):  
Growth Factor ◽  
Cell Viability ◽  
Interleukin 6 ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Latently Infected ◽  
Mannose 6 Phosphate ◽  
Productive Replication

ABSTRACTHuman 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.IMPORTANCEHHV-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.

scholarly journals Promotion of Endoplasmic Reticulum-Associated Degradation of Procathepsin D by Human Herpesvirus 8-Encoded Viral Interleukin-6

2015 ◽  
Vol 89 (15) ◽  
pp. 7979-7990 ◽  
Author(s):  
Daming Chen ◽  
John Nicholas
Keyword(s):  
Endoplasmic Reticulum ◽  
Interleukin 6 ◽  
Cathepsin D ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Gp130 Receptor ◽  
Epoxide Reductase ◽  
Productive Replication

ABSTRACTThe interleukin-6 homologue (viral interleukin-6 [vIL-6]) of human herpesvirus 8 is implicated in viral pathogenesis due to its proproliferative, inflammatory, and angiogenic properties, effected through gp130 receptor signaling. In primary effusion lymphoma (PEL) cells, vIL-6 is expressed latently and is essential for normal cell growth and viability. This is mediated partly via suppression of proapoptotic cathepsin D (CatD) via cocomplexing of the endoplasmic reticulum (ER)-localized CatD precursor, pro-CatD (pCatD), and vIL-6 with the previously uncharacterized ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). vIL-6 suppression of CatD occurs also during reactivated productive replication in PEL cells and is likely to contribute to proreplication functions of vIL-6. Here, we report that vIL-6 suppresses CatD through vIL-6, VKORC1v2, and pCatD association with components of the ER-associated degradation (ERAD) machinery. In transfected cells, expression of vIL-6 along with CatD led to proteasome-dependent (inhibitor-sensitive) decreases in CatD levels and the promotion of pCatD polyubiquitination. Depletion of particular ERAD-associated isomerases, lectins, and translocon components, including ERAD E3 ubiquitin ligase HRD1, diminished suppression of CatD by vIL-6. Coprecipitation assays identified direct or indirect interactions of VKORC1v2, vIL-6, and pCatD with translocon proteins (SEL1L and/or HRD1) and ERAD-associated lectins OS9 and XTP3-B. Endogenous CatD expression in PEL cells was increased by depletion of ERAD components, and suppression of CatD by vIL-6 overexpression in PEL cells was dependent on HRD1. Our data reveal a new mechanism of ER-localized vIL-6 activity and further characterize VKORC1v2 function.IMPORTANCEHuman herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6), unlike cellular IL-6 proteins, is secreted inefficiently and sequestered mainly in the endoplasmic reticulum (ER), from where it can signal through the gp130 receptor. We have recently reported that vIL-6 also associates with a novel membrane protein termed vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2) and mediates suppression of VKORC1v2-cointeracting cathepsin D, a stress-released proapoptotic protein negatively impacting HHV-8 latently infected primary effusion lymphoma (PEL) cell viability and reactivated virus productive replication. Here, we have examined the mechanistic basis of the VKORC1v2–vIL-6 interaction-dependent suppression of cathepsin D and have found that this novel activity of vIL-6 is mediated through coassociation of VKORC1v2, procathepsin D, and vIL-6 with components of the ER-associated degradation (ERAD) machinery. Our findings provide information of significance for potential antiviral and therapeutic targeting of VKORC1v2-mediated vIL-6 activities and also indicate the nature of VKORC1v2 function in normal cell biology.

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

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Qiwang Xiang ◽  
Hyunwoo Ju ◽  
John Nicholas
Keyword(s):  
Cell Viability ◽  
Catalytic Domain ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Lytic Replication ◽  
Targeted Mutagenesis ◽  
Human Herpesvirus 8 ◽  
Antiviral Signaling ◽  
Herpesvirus 8 ◽  
Productive Replication

ABSTRACT 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.

scholarly journals Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6

2009 ◽  
Vol 83 (13) ◽  
pp. 6874-6882 ◽  
Author(s):  
Daming Chen ◽  
Young Bong Choi ◽  
Gordon Sandford ◽  
John Nicholas
Keyword(s):  
Interleukin 6 ◽  
Primary Effusion Lymphoma ◽  
Intracellular Localization ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Detectable Effect ◽  
Herpesvirus 8 ◽  
Chaperone Protein ◽  
Latently Infected ◽  
Dependent Interaction

ABSTRACT Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling α-receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.

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

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Qian Li ◽  
Qiwang Xiang ◽  
Daming Chen ◽  
John Nicholas
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Interactions ◽  
Interleukin 6 ◽  
Kaposi's Sarcoma ◽  
Castleman’S Disease ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Signal Transducer ◽  
Herpesvirus 8 ◽  
Productive Replication

ABSTRACT 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.

scholarly journals Intracellular Signaling Mechanisms and Activities of Human Herpesvirus 8 Interleukin-6

2008 ◽  
Vol 83 (2) ◽  
pp. 722-733 ◽  
Author(s):  
Daming Chen ◽  
Gordon Sandford ◽  
John Nicholas
Keyword(s):  
Signal Transduction ◽  
Interleukin 6 ◽  
Human Herpesvirus ◽  
Human Herpesvirus 8 ◽  
Α Subunit ◽  
Lytic Gene ◽  
Growth And Survival ◽  
Herpesvirus 8 ◽  
Latently Infected ◽  
Infected Cells

ABSTRACT Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) has been implicated as a key factor in virus-associated neoplasia because of its proproliferative and survival effects and also in view of its angiogenic properties. A major difference between vIL-6 and human IL-6 (hIL-6) is that vIL-6, uniquely, is largely retained and can signal intracellularly. While vIL-6 is generally considered to be a lytic gene, several reports have noted its low-level expression in latently infected primary effusion lymphoma (PEL) cultures, in the absence of other lytic gene expression. Thus, intracellular autocrine signal transduction by the viral cytokine may be of particular relevance to the growth and survival of latently infected cells and to pathogenesis. Here we report that most intracellular vIL-6 is located in the endoplasmic reticulum (ER), signals via the gp130 signal transducer in this compartment, and does so independently of the gp80 α-subunit of the IL-6 receptor, required for hIL-6 signal transduction. Signaling and biological assays incorporating ER-retained vIL-6 and hIL-6 confirmed vIL-6 activity, specifically, in this compartment. Knockdown of vIL-6 expression in PEL cells led to markedly reduced cell growth in normal culture, independently of extracellular cytokines. This could be reversed by reintroduction via virus vector of exclusively ER-retained vIL-6. These data indicate that in virus biology vIL-6 may act to support the growth and survival of cells latently infected with HHV-8 in an autocrine manner via intracrine signaling and that these activities may contribute to the maintenance of latently infected cells and to virus-induced neoplasia.

scholarly journals Detection of Direct Binding of Human Herpesvirus 8-Encoded Interleukin-6 (vIL-6) to both gp130 and IL-6 Receptor (IL-6R) and Identification of Amino Acid Residues of vIL-6 Important for IL-6R-Dependent and -Independent Signaling

2001 ◽  
Vol 75 (7) ◽  
pp. 3325-3334 ◽  
Author(s):  
Hong Li ◽  
Hailin Wang ◽  
John Nicholas
Keyword(s):  
Complex Formation ◽  
Interleukin 6 ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Biologically Active ◽  
Angiogenic Factor ◽  
Human Herpesvirus 8 ◽  
Signal Transducer ◽  
Herpesvirus 8 ◽  
Direct Binding

ABSTRACT Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease; in all of these diseases, interleukin-6 (IL-6) has been implicated as a likely mitogenic and/or angiogenic factor. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]) that has been shown to be biologically active in several assays and whose activities mirror those of its mammalian counterparts. Like these proteins, vIL-6 mediates its effects through the gp130 signal transducer, but signaling is not dependent on the structurally related IL-6 receptor (IL-6R; gp80) subunit of the receptor-signal transducer complex. However, as we have shown previously, IL-6R can enhance vIL-6 signal transduction and can enable signaling through a gp130 variant (gp130.PM5) that is itself unable to support vIL-6 activity, indicating that IL-6R can form part of the signaling complex. Also, our analysis of a panel of vIL-6 mutants in transfection experiments in Hep3B cells (that express IL-6R and gp130) showed that most were able to function normally in this system. Here, we have used in vitro vIL-6–receptor binding assays to demonstrate direct binding of vIL-6 to both gp130 and IL-6R and vIL-6-induced gp130–IL-6R complex formation, and we have extended our functional analyses of the vIL-6 variants to identify residues important for IL-6R-independent and IL-6R-dependent signaling through native gp130 and gp130.PM5, respectively. These studies have identified residues in vIL-6 that are important for IL-6R-independent and IL-6R-mediated functional complex formation between vIL-6 and gp130 and that may be involved directly in binding to gp130 and IL-6R.

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