Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral Interleukin-6 by X-Box Binding Protein 1

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman disease (MCD). The KSHV life cycle has two principal gene repertoires, latent and lytic. KSHV viral interleukin-6 (vIL-6), an analog of human IL-6, is usually lytic; production of vIL-6 by involved plasmablasts is a central feature of KSHV-MCD. vIL-6 also plays a role in PEL and KS. We show that a number of plasmablasts from lymph nodes of patients with KSHV-MCD express vIL-6 but not ORF45, a KSHV lytic gene. We further show that vIL-6 is directly induced by the spliced (active) X-box binding protein-1 (XBP-1s), a transcription factor activated by endoplasmic reticulum (ER) stress and differentiation of B cells in lymph nodes. The promoter region of vIL-6 contains several potential XBP-response elements (XREs), and two of these elements in particular mediate the effect of XBP-1s. Mutation of these elements abrogates the response to XBP-1s but not to the KSHV replication and transcription activator (RTA). Also, XBP-1s binds to the vIL-6 promoter in the region of these XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce vIL-6. Patient-derived PEL tumor cells that produce vIL-6 frequently coexpress XBP-1, and immunofluorescence staining of involved KSHV-MCD lymph nodes reveals that most plasmablasts expressing vIL-6 also coexpress XBP-1. These results provide evidence that XBP-1s is a direct activator of KSHV vIL-6 and that this is an important step in the pathogenesis of KSHV-MCD and PEL.

Importance: Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD) is characterized by severe inflammatory symptoms caused by an excess of cytokines, particularly KSHV-encoded viral interleukin-6 (vIL-6) produced by lymph node plasmablasts. vIL-6 is usually a lytic gene. We show that a number of KSHV-MCD lymph node plasmablasts express vIL-6 but do not have full lytic KSHV replication. Differentiating lymph node B cells express spliced (active) X-box binding protein-1 (XBP-1s). We show that XBP-1s binds to the promoter of vIL-6 and can directly induce production of vIL-6 through X-box protein response elements on the vIL-6 promoter region. We further show that chemical inducers of XBP-1s can upregulate production of vIL-6. Finally, we show that most vIL-6-producing plasmablasts from lymph nodes of KSHV-MCD patients coexpress XBP-1s. These results demonstrate that XBP-1s can directly induce vIL-6 and provide evidence that this is a key step in the pathogenesis of KSHV-MCD and other KSHV-induced diseases.

FIG 1

vIL-6 and ORF45 in KSHV-MCD Patients. (A) LANA expression in a germinal center involved with KSHV-MCD. LANA, stained in brown with DAB, appears as nuclear speckles in KSHV-infected cell nuclei. The majority of LANA-expressing plasmablasts are in the mantle zone. (B) vIL-6 expression in an adjacent cut from the same germinal center. There were fewer vIL-6-expressing cells, and these were less focused in the mantle zone. (C and D) ORF45 and vIL-6 coexpression in a germinal center involved with KSHV-MCD. ORF45 (a KSHV lytic gene) was stained in brown, while vIL-6 was stained in red. Panel C shows the whole germinal center while panel D shows a section at higher magnification. ORF45 and vIL-6 are colocalized in many cells (solid arrows). Some cells express only vIL-6 and not ORF45 (dashed arrows). (E) ORF45 and vIL-6 expression in a KSHV-MCD lymph node observed by IF and confocal microscopy. Each horizontal row shows a separate field from the same section in one of two patients examined. ORF45 was stained in red, and vIL-6 was stained in green. Some cells expressed only vIL-6 and not ORF45.

FIG 2

Schematic of vIL-6 luciferase (LUC) promoter and constructs showing the location of the potential XBP response elements (XRE) and activation of the vIL-6 promoter constructs by spliced XBP-1. (A) The sequence of the vIL-6 promoter contains two TATA boxes. Ten XRE core sequences, ACGT (47), are found within 1.7 kb upstream of the vIL-6 start codon (positions 17871 to 19567 of KSHV-BAC36; GenBank accession number HQ404500.1): XRE1, −163 to −160; XRE2, −245 to −242; XRE3, −447 to −444; XRE4, −769 to −766; XRE5, −991 to −988; XRE6, −1262 to −1259; XRE7, −1331 to −1328; XRE8, −1352 to −1349; XRE9, −1401 to −1398; and XRE10, −1607 to −1604. Each potential XRE sequence is denoted as a square. Consensus XREs are indicated in black, and other (core-only) XREs are shown in gray. The direction of each consensus XRE is indicated with an arrow (core XRE sequence only, 5′-ACGT-3′; the core sequence is underlined in the consensus XRE, 5′-NNGNTGACGTGKNNNWT-3′). Constructs pvIL6-2, pvIL6-3, and pvIL6-4 were made by sequential deletions, as shown. (B) Comparison of the activation of the vIL-6 promoter luciferase reporter by the XBP-1 unspliced (XBP-1u) or spliced (XBP-1s) form. Hep3B cells were cotransfected with 300 ng of a vIL-6 promoter luciferase construct and 50 ng of an internal β-Gal control plasmid (pGL3-basic [pGL3b]) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or the pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the value for the pGL3-basic reporter transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations (*, P ≤ 0.01; **, P ≤ 0.005, for the comparisons shown, normalized in each case for the results with the pGL3-basic control). The comparison between results for pvIL6-1/XBP-1u and pvIL6-1/pcDNA was not significant (P > 0.05). (C) Comparison of the activation of truncated forms of the vIL-6 luciferase reporter by XBP-1s or the pcDNA3.1 plasmid control. Hep3B cells were cotransfected with 300 ng of each vIL-6 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or the pcDNA3.1 control. Values are expressed as fold increase over the value for pvIL6-1 transfected with an empty expression vector (pcDNA3.1) and represent the means of three independent experiments. Error bars denote the standard deviations.

FIG 3

Site-directed mutagenesis of XRE2 and XRE3 of the pvIL6-2 promoter and response to XBP-1s, RTA, and HIF-1. (A) Constructs of the wild-type pvIL6-2 and mutant reporter plasmids. Three different mutant reporters (pvIL6-M2, pvIL6-M3, and pvIL6-M2/3) were constructed, each containing a 4-base substitution within the core XRE sequence or sequences. All mutations were made in the pvIL6-2 promoter (HRE core sequence, 5′-RCGTG-3′; RTA response element [RRE] sequence in the vIL-6 promoter, 5′-GTGGTTCTAAGTCGCACGTTAGAAACCCCGCCCCCTGGTGCTCACTTT-3′; the core XRE sequence is underlined). Consensus XREs including XRE2 and XRE3 are indicated in black, and XRE1 is indicated in gray. (B) Comparison of the activation of a wild-type pvIL6-2 luciferase reporter with that of the three mutant luciferase reporters by XBP-1s. Hep3B cells were cotransfected with 300 ng of pvIL6-2 or mutant promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s or a pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the value for the pvIL6-2 reporter transfected with an empty expression vector (pcDNA3.1) and represent the means of three independent experiments. Error bars denote the standard deviations. After values were normalized to the results with the pcDNA3.1 expression plasmid control for each promoter, the activity of the pvIL6-M2/3 reporter was significantly lower than that of pvIL6-2 (P ≤ 0.05), while comparisons of results of the other reporters were not significant. (C) Comparison of the activation by RTA of the wild-type pvIL6-2 luciferase reporter with that of the three mutant luciferase reporters. Hep3B cells were cotransfected with 300 ng of a pvIL6-2 promoter luciferase reporter or the mutant promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding RTA or a pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value for the pvIL6-2 reporter transfected with an empty expression vector (pcDNA3.1) and represent the means of three independent experiments. Error bars denote the standard deviations. After normalization to the results with the pcDNA3.1 expression plasmid control for each promoter, the activity of the pvIL6-M2 reporter was significantly greater than that of pvIL6-2 (P ≤ 0.05), while the results with the other reporters in comparison to those with pvIL6-2 were not significant. (D) Comparison of the activation of the wild-type vIL6-1 (1,696 bp) luciferase reporter or the HIF-responsive PTPRZ-1 reporter in response to degradation-resistant HIF-1α or HIF-2α. Hep3B cells were cotransfected with 300 ng of the pvIL6-1 luciferase reporter or PTPRZ-1 promoter and 50 ng of an internal β-Gal control plasmid in the presence of 250 ng of an expression plasmid encoding degradation-resistant HIF-1α, HIF-2α, or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pcDNA3.1 expression vector-treated control for each reporter construct in a representative experiment; error bars denote the standard deviations of triplicate determinations.

FIG 4

CHIP assay showing binding of XBP-1s to the vIL-6 promoter. BCBL-1 endogenous XBP-1s was induced by TM treatment (2 μg/ml) for 48 h and then cross-linked. Chromatin IP of fragmented DNA was performed with XBP-1 antibody or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2 or XRE3 of the vIL-6 promoter, and the data were quantitated as described in Materials and Methods. Results shown are the means ± standard deviations of triplicate determinations from a typical experiment of two experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not with XBP-1 antibody or a control IgG, was enriched for RPL30 exon 3.

FIG 5

vIL-6 upregulation mediated by chemical inducers of XBP-1s in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of tunicamycin (TM) to induce ER stress; cells were also treated with 15 nM 12-O-tetra-dodecanoyl-phorbol-13-acetate (TPA) as an inducer of RTA. (A) RT-PCR showing expression at of total XBP-1, XBP-1s, XBP-1u, vIL-6, RTA, and 18S ribosomal RNA as a loading control in BCBL-1 cells treated with various doses of TM, a DMSO control, or 15 nM TPA. Cells treated with TM or the DMSO control were harvested at 24 h, while those treated with TPA were harvested at 48 h. (B) Real-time quantitative PCR showing expression of vIL-6 and RTA mRNA in BCBL-1 cells cultured in the same way as described for panel A and harvested at 8 h and 24 h, as well as a TPA control harvested at 48 h. Shown are the means ± the standard deviations of triplicate determinations from one representative experiment expressed as the fold change compared to the level of the DMSO control. (C) Time course (24 h) of the fold increase of vIL-6 and RTA mRNA over baseline in cells exposed to 0.25 μg/ml TM. Shown are the means ± the standard deviations of triplicate determinations from one representative experiment expressed as the fold change compared to the level of the DMSO control. (D) Western blot showing XBP-1s (55 kDa), vIL-6 (22 kDa and 28 kDa), and RTA (85 kDa) expression in BCBL-1 cells 24 h after treatment with 0.5 μg/ml TM, 2 μg/ml TM, or 15 nM TPA. DMSO was used as the control.

FIG 6

Expression of XBP-1 and vIL-6 in BCBL-1 cells exposed to chemical inducers of ER stress and in primary PEL cells. (A) BCBL-1 cells were treated with BFA for 24 h or TM for 48 h to induce ER stress or with TPA for 48 h to induce KSHV lytic replication. Cells expressing XBP-1s were stained in red, and cells expressing vIL-6 were stained in green. BFA-treated and TM-treated cells showed coexpression of XBP-1s and vIL-6. TPA-treated cells expressed vIL-6 but not XBP-1s. (B) Staining of primary PEL cells from a plural effusion for XBP and vIL-6. Cells expressing vIL-6 were stained in red while cells expressing XBP-1 were stained in green. A second patient tested gave similar results.

FIG 7

Detection of XBP-1- and vIL-6-positive cells in an MCD lymph node. Fluorescence immunostaining of a KSHV-MCD lymph node shows the distribution of vIL-6 (green) and XBP-1 (red) proteins in nucleated cells (marked by DAPI, blue. Arrowheads in images stained for vIL-6 point to XBP-1 and vIL-6 double-positive cells. Lower-magnification images (upper and left lower quadrants) show that the vIL-6-positive cells are also XBP-1 positive; higher-magnification images (lower right panels) show the predominantly nuclear localization of XBP-1 and cytoplasmic localization of vIL-6.