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

Abstract

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. 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.

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

“…Activities of vIL-6 are mediated via interactions with the gp130 (IL-6) signaling receptor and also the ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2) (1)(2)(3). The former interaction promotes gp130-mediated mitogen-activated protein kinase and signal transducer and activator of transcription (STAT) signaling; the latter interaction affects protein folding through associations with calnexin cycle enzymes and also regulates ER-associated degradation (ERAD) via interactions with ERAD chaperone and translocon proteins (4,5). Although vIL-6 is produced in abundance during productive (lytic) replication, it is also expressed at low, functional levels during latency in primary effusion lymphoma (PEL) B cells and is important for maintenance of cell viability in examined PEL cell lines (6).…”

“…Proviability and proreplication activities of vIL-6 via VKORC1v2 in PEL cells involve the suppression of proapoptotic cathepsin D (CatD) expression, via vIL-6/VKORC1v2-enhanced ERAD of ER-transiting pro-CatD (4,7). Pro-folding activities of vIL-6 via VKORC1v2 and calnexin cycle protein interactions (5) could conceivably also contribute to virus latent and/or lytic biology. These activities of VKORC1v2 are the first to be described and represent novel means of vIL-6 function.…”

“…4B). Whether this effect of vIL-6 on IGF2R levels involved VKORC1v2 was tested by comparing vIL-6 activity in genetically engineered VKORC1v2-null versus native HEK293T cells (5). Regulation of IGF2R expression by vIL-6 was not apparent in the VKORC1v2-deficient cells, whereas vIL-6 again led to elevated IGF2R levels in wild-type HEK293T cells ( Fig.…”

“…(C) Chitin bead-based precipitation analyses of (endogenous) IGF2R interactions with VKORC1v2-CBD (v2-CBD) and vIL-6-CBD; VKORC1v1-CBD (v1-CBD) and empty vector were transfected into parallel cultures to provide negative controls. ment (1,6), we reasoned that these proteins were likely to act on nascent, ER-transiting IGF2R to mediate its regulation, for example through effects on protein folding and/or ERAD, known to be affected by vIL-6 and VKORC1v2 (4,5). To test this, we generated a plasmid vector expressing IGF2R protein fused to HaloTag (21), allowing fluorescence pulse-labeling of the protein in transfected cells.…”

Herpesviruses and the Unfolded Protein Response

“…vIL-6 may interfere with HYOU1-BiP interactions, resulting in increased protein misfolding and UPR activation. However, there is also evidence that vIL-6 may promote protein folding by binding components of the calnexin cycle, including UDP-glucose: glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII) [156]. Therefore, vIL-6 may play a role in fine-tuning UPR signaling by directly controlling proteostasis.…”