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.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.

Keywords: UDP-glucose:glycoprotein glucosyltransferase 1; VKORC1v2; calnexin cycle; endoplasmic reticulum; glucosidase II; human herpesvirus 8; protein folding; viral interleukin-6.

FIG 1

Interactions of vIL-6 and VKORC1v2 with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out using lysates of cells cotransfected with expression plasmids for CBD-fused vIL-6 and S-tag-fused GlucIIα or CBD-fused VKORC1v2 (v2-CBD) and StrepII-tagged UGGT1 to test the interactions between these protein pairs. CBD- and KDEL ER retention motif-linked hIL-6 (hIL-6-CBD.K) and CBD-tagged VKORC1v1 (v1-CBD) were used as negative controls in the respective experiments. Protein complexes were precipitated with CBD-binding chitin beads, and SDS-PAGE-fractionated and membrane-transferred proteins in precipitates and cell lysates were identified by immunoblotting with CBD antibody to visualize “baits” vIL-6 and VKORC1v2 or with S-peptide or StrepII antibody for detection of their candidate binding partners. β-actin probing of cell lysates provided a loading control. (B) Further precipitations were undertaken to test whether vIL-6 and VKORC1v2 could interact with UGGT1 and GlucIIα, respectively. In this experiment, GlucIIα-S and StrepII-UGGT1 were coexpressed with CBD-tagged vIL-6 or VKORC1v2, or negative controls hIL-6-CBD.K (hIL-6-C′.K) or VKORC1v1-CBD (v1-CBD). Chitin bead precipitates and cell lysates were analyzed as described previously. (C) Immunoprecipitation (IP) of vIL-6 from BCBL-1 PEL cell lysates and subsequent immunoblotting for GlucIIα and UGGT1. A sample of the lysate (10% of amount used for IP) was analyzed alongside material precipitated with vIL-6 antiserum (vIL-6) or control rabbit serum.

FIG 2

Identification of large protein complexes containing VKORC1v2, vIL-6, GlucIIα, and UGGT1. Flag-tagged VKORC1v2, coexpressed with epitope-tagged vIL-6 (twin-StrepII, TS), GlucIIα (S-peptide), and UGGT1 (StrepII), was precipitated from cotransfected HEK293T cell lysates using Flag-immunobeads, and the washed precipitate was layered onto a 5 to 50% iodixanol gradient and ultracentrifuged at 107,000 × g for 3 h. Fractions of the gradient were extracted sequentially from the top of the column (the “light” fraction, fraction 1) to the bottom (fraction 14). Samples of each fraction were then subjected to SDS-PAGE, membrane transfer, and immunoblotting for the detection of each of the vector-expressed proteins. Horizontal arrows indicate continuous fractions in which VKORC1v2 and vIL-6 were detected; GlucIIα and UGGT1 were detected in particular fractions (+); copeaks (*) of all four proteins were observed in fraction 10 (vertical arrow).

FIG 3

Cocomplexing between VKORC1v2, vIL-6, GlucIIα and UGGT1. Flag- and StrepII-tagged VKORC1v2 HA-tagged vIL-6, and S-tagged GlucIIα were coexpressed with StrepII-UGGT1 in expression vector-cotransfected HEK293T cells. The first three proteins were targeted for serial precipitation with Flag-antibody, HA-antibody, and S-protein beads, respectively. After the first and second precipitations, 3×Flag and HA peptides were used for competitive release of proteins from the beads (see Materials and Methods). Samples from each precipitation and input lysates were denatured, fractionated by SDS-PAGE, blotted onto membranes, and probed with appropriate epitope-tag antibodies (as indicated on the blots) for detection of the respective proteins. The test (all proteins) and various control transfections are tabulated (top left), and the precipitation sequence and targeted proteins are illustrated above the associated blots. Immunoblot band identities are indicated by arrows, asterisks and associated labels; note that detection of S-peptide (*GlucIIα) preceded probing for StrepII (UGGT1 and VKORC1v2), accounting for codetection on the upper blots. The detected StrepII-UGGT1 band in the final precipitate is indicated by an arrowhead (lane 1, rightmost blot).

FIG 4

VKORC1v2-independence of vIL-6 interactions with GlucIIα and UGGT1. (A) Coprecipitation assays were carried out essentially as outlined in the legend to Fig. 1, but genetically engineered VKORC1v2-deficient (KO) cells were included along with native HEK293T cells. Chitin beads were used to precipitate vIL-6-CBD from the lysates of both cell types; coprecipitated GlucIIα-S and StrepII-UGGT1 (left and right panels, respectively) were detected by immunoblotting. Empty vector (vec) was used as a negative control to check for nonspecific matrix binding by the proteins. (B) Serial precipitations of vIL-6 (Flag-tagged) and GlucIIα (S-tagged) from appropriately transfected wild-type and VKORC1v2-KO cell lysates was carried out, followed by immunoblotting to detect coprecipitated UGGT1 (StrepII-tagged). StrepII/Flag-VKORC1v1 (S/F-VKORC1v1) was included in some of the transfection mixes to provide a negative control.

FIG 5

Profolding activity of vIL-6. (A) HEK293T cells were cotransfected with vectors expressing a GFP-fused version of the NHK folding variant of α1-antitrypsin (NHK-GFP) and vIL-6 or with NHK-GFP expression plasmid and empty vector (vec). At 48 h posttransfection, cell lysates were generated using NP-40-containing buffer, material microcentrifuged, and pellet (insoluble [insol.]) and supernatant (soluble [sol.]) fractions were denatured, SDS-PAGE fractionated, and immunoblotted for detection of GFP and also vIL-6 (to confirm expression) and ER luminal soluble protein BiP (to ensure appropriate separation of soluble and pellet fractions). The relative amounts of soluble to insoluble NHK-GFP are shown in the chart. (B) An independent assay was undertaken using secreted HHV-8 glycoprotein L (gL), fused to CBD, as the readout for folding. CBD-tagged HHV-8 gH was coexpressed with gL-CBD to enable secretory trafficking, and either vIL-6 expression plasmid or empty vector (vec) was cotransfected into HEK293T cells with the glycoprotein expression plasmids. After 48 h, cells and media were harvested for preparation of lysates and chitin bead precipitates (for concentration of secreted gL-CBD). Cell lysates (1% of total) and media precipitates (10%) were analyzed by immunoblotting for detection of gL-CBD; gH, vIL-6, and β-actin (loading control) were also analyzed in the lysate fractions. (C) An experiment equivalent to that of panel A was carried out in native HEK293T cells or VKORC1v2- or UGGT1-knockout (KO) derivatives. CBD- and KDEL motif-tagged hIL-6 (h6-C′-K) was included as a negative control along with empty vector (vec) for comparison with vIL-6 (CBD-fused, v6-CBD). Calculated ratios of NHK-GFP in the soluble and insoluble fractions of cell lysates are shown below each corresponding set of immunoblots. Digitally captured data from all immunoblots (panels A to C) were quantified using GeneTools (Syngene) analysis software.

FIG 6

GlucIIα and VKORC1v2 binding properties of vIL-6 domain substitution variants. (A) Diagrammatic representation of vIL-6 variants containing domain-based substitutions of hIL-6 residues for colinear vIL-6 sequences; plasmid vectors expressing each protein fused C-terminally to CBD were reported previously (35). SS, signal sequence; SP+N, signal peptide plus N-terminal residues; D1 and D2, N- and C-terminal halves of the helix-D region, respectively. (B and C) Coprecipitation-based binding assays equivalent to those of Fig. 1 were carried out to determine the GlucIIα (S-tagged) and VKORC1v2 (SF-tagged) binding activities of each of the CBD-fused vIL-6 variants. Negative controls were provided by KDEL motif-linked hIL-6-CBD (hIL-6-KDEL), empty vector (vec) in place of GlucIIα-S (B) and SF-VKORC1v1 (Flag-v1) as a substitute for VKORC1v2-SF (VKORC1v2-Flag) (C). The results from repeat experiments for vIL-6.hSP (B and C) and vIL-6.hD1 (B) are shown to the right of the “screening” blots. In panel C, arrowheads “1” and “2” indicate Flag-tagged VKORC1v1 and VKORC1v2 protein bands.

FIG 7

Interactions of selected vIL-6 variants. (A) Coprecipitation binding assays were carried out essentially as outlined for Fig. 6 to test the effects of grouped point mutations within the D1 region of vIL-6 for their effects on GlucIIα and gp130 interactions. Immunoblotting analysis of coprecipitates of variant vIL-6.D1m11 is shown alongside data for binding-competent vIL-6. Dotted lines indicate the juxtaposition of lanes that were discontiguous on the original blot (i.e., deletion of intervening lanes). (B) Similar coprecipitation assays were carried out to test the binding of vIL-6.hSP, vIL-6.hD1, and vIL-6.D1m11 to StrepII-tagged UGGT1 (U1). (C) Immunoblotting analysis of coprecipitates from an analogous experiment to test the binding of vIL-6.D1m11, along with vIL-6.hSP and vIL-6.hD1, to VKORC1v2 (v2, Flag-tagged).

FIG 8

Analysis of profolding activities of binding-abrogated vIL-6 variants. (A) Diagram of firefly luciferase (FLuc)-based reporter vector constructed to enable quantitation of profolding activity of vIL-6 in transfected cells. FLuc was coexpressed with Renilla luciferase (RLuc, for normalization) via internal ribosome entry site (IRES)-mediated internal translational initiation of the former. CAG, CMV enhancer-chicken β-actin promoter; H, hairpin-loop preventing ribosome read-through. (B) Validation of the reporter vector in transfected HEK293T cells, comparing profolding effects of vIL-6 as assessed by immunoblotting-determined levels of soluble versus insoluble NHK-GFP (analogous to assays shown in Fig. 5) with coexpressed FLuc activities in the soluble fraction (FLuc lysate). FLuc activities in the culture media, reflecting levels of properly folded and secreted NHK-FLuc, were also measured. FLuc activities are shown relative to those in vIL-6-expressing cells (set at 100%). CBD-fused vIL-6 and hIL-6 (KDEL-tagged) are indicated by v6-CBD/v6 and h6-CBD-K/h6, respectively. (C) Determination of vIL-6 variant relative to wild-type vIL-6 profolding activities, as determined by analysis of secreted NHK-FLuc activities in transfected HEK293T cultures. (D) Assessment of vIL-6 influence on NHK-FLuc folding (relative expression of secreted protein) as a function of gp130 depletion, mediated via gp130 mRNA-directed shRNA vector cotransfection with vIL-6-encoding or empty (vec) expression vectors. Nonsilencing (NS), untargeted shRNA expression vector was used to transfect parallel cultures. Depletion of gp130 was confirmed by immunoblotting, to detect both gp130 and vIL-6/gp130-activated STAT3 (pSTAT3), relative to total STAT3 and actin (middle panel). Functional gp130 depletion was also verified by assaying for activity of secreted Cypridina luciferase expressed from a cotransfected gp130 signaling-responsive reporter (see Materials and Methods) (chart, right). For all reporter experiments (panels B to D), transfections were conducted in duplicate, and the FLuc activities (normalized to lysate-derived RLuc activities) from these samples were averaged. Error bars show deviations of these values from the means.

FIG 9

Effects of vIL-6 on protein folding in the context of infection. (A) The BAC16 HHV-8 genome was mutated at the vIL-6 ORF translational initiation codon (ATG→TTG) to generate a vIL-6-knockout virus. Reversion of this mutation to ATG (TTG→ATG) was done to generate a repaired virus (Rep) for use as a control in phenotypic analyses. The restriction profiles of the wild-type (WT) BAC16 and engineered genomes were indistinguishable, shown here for BamHI-HindIII digestions, demonstrating overall genome integrity following targeted recombination. The arrow indicates the position of the ORFK2-containing restriction fragment. The sizes of marker (m) bands are indicated. (B) Verification of knockout and repair of the vIL-6 ORF in BAC16-vIL-6.TTG (TTG) and BAC16-vIL-6.ATG^rep (Rep), respectively. Cultures of iSLK cells infected with wild-type (WT) or each of the engineered viruses were harvested 48 h after lytic induction with doxycycline (Dox; 1.9 nM) and sodium butyrate (NaB; 1 mM) for preparation of cell extracts for immunoblotting. (C) BAC16-infected iSLK cells were transfected with the RLuc/NHK-FLuc reporter vector (see Fig. 8A), and the cultures were treated with doxycycline and sodium butyrate to induce lytic replication. Cells and media were harvested after 48 h for measurement of secreted (correctly folded) HNK-FLuc (measured from 20 μl, 1%, of media) and lysate-contained RLuc (using 20 μl, 10%, of lysates). Average FLuc activities (normalized to RLuc values) from duplicate cultures infected with each engineered virus are shown relative to average FLuc activity (set at 100%) from duplicate wild-type BAC16-infected cultures. Error bars indicate deviations of the duplicate values from the means. (D) Analogous experiments were carried out using BAC16-vIL-6.hSP (hSP) and BAC16-vIL-6.hSP^rep (hSP^rep) in addition to BAC16 (WT) and BAC16-vIL-6.TTG (TTG). Data, derived from biological duplicates, are presented as outlined for panel C.