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. 2020 Feb 14;94(5):e01555-19.
doi: 10.1128/JVI.01555-19. Print 2020 Feb 14.

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

Victoria Wang  1 , David A Davis  1 , Claire Deleage  2 , Catherine Brands  2 , Hong S Choi  1 , Muzammel Haque  1 , Robert Yarchoan  3
Affiliations

Induction of Kaposi's Sarcoma-Associated Herpesvirus-Encoded Thymidine Kinase (ORF21) by X-Box Binding Protein 1

Victoria Wang et al. J Virol. .

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). Like other herpesviruses, it has latent and lytic repertoires. However, there is evidence that some lytic genes can be directly activated by certain cellular factors. Cells undergoing endoplasmic reticulum stress express spliced X-box binding protein 1 (XBP-1s). XBP-1s is also present in large amounts in germinal center B cells. XBP-1s can activate the KSHV replication and transcription activator (RTA) and lytic replication. It can also directly activate KSHV-encoded viral interleukin-6 (vIL-6) and, thus, contribute to the pathogenesis of KSHV MCD. KSHV thymidine kinase (TK), the ORF21 gene product, can enhance the production of dTTP and is important for lytic replication. It can also phosphorylate zidovudine and ganciclovir to toxic moieties, enabling treatment of KSHV-MCD with these drugs. We show here that XBP-1s can directly activate ORF21 and that this activation is mediated primarily through two XBP-response elements (XRE) on the ORF21 promoter region. Deletion or mutation of these elements eliminated XBP-1s-induced upregulation of the promoter, and chromatin immunoprecipitation studies provide evidence that XBP-1s can bind to both XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce ORF21 within 4 hours, and ORF21 expression in the lymph nodes of patients with KSHV-MCD is predominantly found in cells with XBP-1. Thus, XBP-1s may directly upregulate KSHV ORF21 and, thus, contribute to the pathogenesis of KSHV-MCD and the activity of zidovudine and valganciclovir in this disease.IMPORTANCE Spliced X-box binding protein 1 (XBP-1s), part of the unfolded protein response and expressed in developing germinal center B cells, can induce Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and directly activate viral interleukin-6 (vIL-6). We show here that XBP-1s can also directly activate KSHV ORF21, a lytic gene. ORF21 encodes KSHV thymidine kinase (TK), which increases the pool of dTTP for viral replication and enhances lytic replication. Direct activation of ORF21 by XBP-1s can enhance viral replication in germinal center B cells and contribute to the pathogenesis of KSHV multicentric Castleman disease (MCD). KSHV-MCD is characterized by systemic inflammation caused, in part, by lytic replication and overproduction of KSHV vIL-6 in XBP-1s-expressing lymph node plasmablasts. KSHV thymidine kinase can phosphorylate zidovudine and ganciclovir to toxic moieties, and direct activation of ORF21 by XBP-1s may also help explain the effectiveness of zidovudine and valganciclovir in the treatment of KSHV-MCD.

Keywords: HHV-8; KSHV; Kaposi; ORF21; XBP-1; herpesvirus; thymidine kinase.

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Figures

FIG 1
FIG 1
Schematic of ORF21 luciferase (LUC) promoter constructs and results of activation of various promoter constructs by XBP-1u and XBP-1s. (A) ORF21 promoter contains 4 XBP-1 response element (XRE) core sequences (5ʹ-ACGT-3ʹ) (44), including 2 consensus (5ʹ-NNGNTGACGTGKNNNWT-3ʹ) XRE sequences (3 and 4) within 1,239 bp upstream of the ORF21 start codon (nucleotide positions 34144 to 35382 of KSHV-BAC36; GenBank accession number HQ404500). Consensus XREs are indicated as black squares and the other two (core only) XREs as gray squares. XRE1, −8 to −5; XRE2, −262 to −259; XRE3, −321 to −318; XRE4, −629 to −626. Direction of each XRE is indicated with an arrow. Consensus core HREs are shown as black triangles. Constructs of promoters pORF21-624, pORF21-316, and pORF21-256 were made by sequential deletions as shown. (B) Comparison of the activation of the ORF21-1239, vIL-6, and ORF36 promoter luciferase reporter constructs by XBP-1 unspliced (XBP-1u) or spliced (XBP-1s). HEK-293T cells were cotransfected with 300 ng of different promoter luciferase plasmids and 50 ng of an internal β-Gal control plasmid (pGL3 basic empty vector) in the presence of 100 ng of an expression plasmid encoding XBP-1u, XBP-1s, or pcDNA3.1 expression plasmid control. Values are expressed as fold increase over the respective control reporter plasmid transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values (*P ≤ 0.05, **P ≤ 0.01) for the comparison shown with the pGL3B control. (C) Comparison of the activation of ORF21 and truncated forms of the ORF21 luciferase reporter by XBP-1s or pcDNA3.1 plasmid control. 293T cells were cotransfected with 300 ng of each ORF21 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 pcDNA3.1 control. Values are expressed as fold increase over pGL3basic transfected with an empty expression vector (pcDNA3.1) and represent the mean of three independent experiments. Error bars denote the standard deviations, and asterisks show the P values as in (B).
FIG 2
FIG 2
Effect of XRE3 and XRE4 mutations in the ORF21 promoter on the response to XBP1s. (A) Construct of the wild-type ORF21 and mutant reporter plasmids. Three different mutant reporters for each XRE were constructed in the pORF21-1239 full-length promoter, containing a 2- to 4-bp substitution within core XRE sequences. DNA sequences for the XRE3, XRE4 wild type, and mutant plasmids are shown. The underlined regions and bold letters indicate the mutations from wild type for each mutant construct. (B) Comparison of the activation of wild-type pORF21-1239 luciferase reporter with the XRE3 or XRE4 mutant luciferase reporters by XBP-1s. HEK-293T cells were cotransfected with 300 ng of pORF21-1239WT or X3 or X4 M1, 2, or 3 promoters and 50 ng of an internal β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 expression plasmid control (gray bars). Values are expressed as fold increase over the pGL3basic 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 and ***P < 0.005 for the comparisons shown; none of the comparisons between the X4 mutations and wild type (WT) were significant (P > 0.05).
FIG 3
FIG 3
The ORF21 promoter contains potential HREs but does not respond to hypoxia in the absence of XBP-1s. (A). Comparison of the activation of wild-type pORF21 and pORF36 luciferase reporter in response to hypoxia. HEK-293T cells were cotransfected with 300 ng of pORF21 or 36 luciferase promoter, 100 ng of an expression plasmid encoding XBP-1s (black bars) or pcDNA3.1 control (gray bars), and 50 ng of an internal β-Gal control plasmid, cultured in normoxia for 32 hours, and then cells were treated in normoxic or hypoxic (1% oxygen) conditions for 16 hours. Values are expressed as the fold increase over the value for the pGL3 basic reporter transfected with an empty expression vector (pcDNA3.1) in normoxia and represent the mean of three independent experiments. Error bars denote the standard deviations. (*P ≤ 0.05; NS, not significant). (B). Comparison of the activation of the wild-type pORF21-1239 luciferase reporter or the HIF-responsive pORF36 reporter in response to degradation-resistant HIF-1 (dr-HIF-1). HEK-293T cells were cotransfected with 300 ng of the pORF21-1239 luciferase reporter or pORF36 promoter and 50 ng of an internal-β-Gal control plasmid in the presence of 100 ng of an expression plasmid encoding dr-HIF-1 or the pcDNA3.1 expression plasmid control. Values are expressed as the fold increase over the value of the pGL3basic reporter transfected with an empty expression vector pcDNA3.1 for each reporter construct and represent the mean of three independent experiments. Error bars denote standard deviation; ****P ≤ 0.001; NS, not significant. (C) Western blot showing XBP-1s (55 kDa) and RTA (85 kDa) expression in BCBL-1 cells 48 hours after treatment with 0.5 μg/ml to 2.5 μg/ml of tunicamycin. As seen, TM induces XBP-1s and RTA but does not induce HIF-1α production in BCBL-1 cells. However, HIF-1α protein expression is seen in BCBCl-1 cells 48 hours after treatment with CoCl2 at 75 μM. Actin was used as the loading control, and TPA was a control for KSHV activation. (D) Comparison of the activation of the pORF21-1239 luciferase reporter by RTA, XBP-1s, or both. HEK-293T cells were cotransfected with 300 ng of pGL3b control reporter plasmid DNA or the pORF21-1239 promoter luciferase reporter and 50 ng of an internal β-Gal control plasmid in the presence of 10 ng of a DNA expression plasmid encoding RTA and 100 ng pcDNA3.1, 100 ng of an expression plasmid for XBP-1s and 10 ng pcDNA3.1, or both RTA and XBP-1s expression plasmids. Values are expressed as the fold increase over the value for the pGL3b basic reporter transfected with pcDNA3.1. Shown are the mean ± standard deviation of triplicate determinations from one representative experiment expressed as the fold change compared with the level of nontreated cells.
FIG 4
FIG 4
ChIP showing binding of XBP-1s to the ORF21 promoter in TM-treated cells. BCBL-1 cells were treated with DMSO (A) or TM treatment (0.5 μg/ml) (B) for 48 hours to induce XBP1s and then cross-linked. Chromatin IP of fragmented DNA was performed with anti-XBP-1 antibody, CHIP positive-control anti-histone H3 antibody, or control IgG. Precipitated DNA was assayed by qPCR with specific primers for amplification of XRE2, 3, or 4 of the ORF21 promoter and with primers for an ORF21, ORF36 non-XRE region as a negative control. The data were quantitated as described in the Materials and Methods. Results shown are the mean ± standard deviation of triplicate determinations from a typical experiment of three experiments performed. In controls performed at the same time, DNA immunoprecipitated with histone H3 antibody, but not XBP-1 antibody or control IgG, was enriched for RPL30 exon 3.
FIG 5
FIG 5
XBP-1, ORF21, RTA ,and vIL-6 mRNA upregulation mediated by TM, a chemical inducer of XBP-1s, in the BCBL-1 PEL line. BCBL-1 cells were treated with increasing doses of TM to induce ER stress; cells were also treated with TPA as an inducer of RTA or a DMSO control. Real-time quantitative PCR showing expression of spliced XBP-1 (A) and total XBP-1 (B) in BCBL-1 cells treated with the compounds shown for 4 h, 8 h, and 24 h. (C, D, and E) Real-time quantitative PCR showing expression of ORF21, vIL-6, and RTA mRNA in BCBL-1 cells cultured in the same way and harvested at 4 hours (C), 8 hours (D), and 24 hours (E). Shown are the mean ± the standard deviation of triplicate determinations from one representative experiment out of three expressed as the fold change compared with the DMSO control.
FIG 6
FIG 6
Toxicity of TM, AZT, and GCV in the BCBL-1 cell line after exposure to 0.25 μg/ml TM. BCBL-1 cells were put into culture in 200 μl wells at 100,000 cells ml−1 and incubated for 24 h. The cells were then treated with the indicated concentrations of AZT (10 μM) and GCV (250 μM or 500 μM) in the absence (gray bar) or presence (black bar) of TM at 0.25 μg/ml for 72 h. Cell viability was then determined using the ATP viability assay, and the relative live cell number was calculated against the PBS controls exposed or not exposed to TM. Bars show the average of treatments done in triplicate. * denotes a P value of <0.05 and ** denotes a P value of <0.01 comparing the relative decrease in control versus TM-treated cells. Note that in the TM control, the relative live cell number was decreased by 23% compared with the non-TM control.
FIG 7
FIG 7
RNAscope analysis of ORF21 and XBP-1 in representative sections of a lymph node from a patient with KSHV-MCD. (A and B) A paraformaldehyde-fixed paraffin-embedded lymph node from a patient with KSHV-MCD was analyzed for ORF21 (green) and XBP-1 (red) mRNA as described in the Materials and Methods. The white arrows denote cells that express both ORF21 and XBP-1, while the pink arrow denotes a cell that expresses ORF21 only. In addition, CD20 protein expression is identified by immunohistofluorescence (blue), and nuclei identified by 4′,6-diamidino-2-phenylindole (DAPI) is shown in gray. (C) A probe-control section stained for CD20. It is worth noting that most KSHV plasmablasts do not express CD20. The scale bar is 100 μm.
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