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, 9 (1), 2372

Cell Type-Specific Interferon-γ-mediated Antagonism of KSHV Lytic Replication

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Cell Type-Specific Interferon-γ-mediated Antagonism of KSHV Lytic Replication

Mi-Kyung Park et al. Sci Rep.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally associated with several malignant tumors: Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). KS remains the most common AIDS-related malignancy since the AIDS epidemic and thus has been extensively studied. KS is characterized as an angioproliferative disease with massive immune cell infiltration at the early stage. High levels of proinflammatory cytokines and growth factors are found in KS lesions, and their involvement in the survival and growth of tumor cells has been well characterized. However, little is known about the role of the inflammatory microenvironment in the regulation of KSHV gene expression and/or viral replication. In the present study, we demonstrated that IFN-γ and TNF-α profoundly inhibited KSHV progeny production in primary human lymphatic endothelial cells (LECs) as well as induced KSHV-producer cells (iSLK.219) with doxycycline. Of note, IFN-γ inhibited overall KSHV gene expression, while the effects of TNF-α were confined to a selected set of genes, which were also downregulated by IFN-γ. The addition of IFN-γ up to 36 hr after induction of viral lytic replication was effective in terms of the inhibition of infectious virion production, suggesting that its inhibitory effect is exerted at the early stages of KSHV life cycle. We believe these data have potentially important implications for rationalizing a therapeutic agent to treat KSHV-induced tumors in which lytic replication plays a critical role in their pathogenesis: KS and MCD.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
IFN-γ and TNF-α greatly inhibit KSHV progeny production. (A) iSLK.219 cells were induced with Dox (0.2 μg/ml), and at the time of induction, each indicated cytokine/chemokine/growth factor was added to the culture. RANTES: 20 units/ml; IP-10, IFN-γ, TNFs, PDGF isotypes: 200 units/ml. At 3 days of treatment, infectious virus titer in the culture supernatant was determined. The means ± SEM of 5 independent experiments performed in duplicate are plotted. For the analysis of KSHV genome replication, iSLK.219 cells were induced as described in (A). At the time of induction, IFN-γ (B) or TNF-α (C) was added into the culture at 20 or 200 units/ml. At the indicated time points, cells were harvested and genomic DNA was prepared for quantitation of the number of the KSHV genomes by real-time PCR. Data are presented as means ± SEM from three independent experiments. *P < 0.01 by one-way ANOVA Tukey Method.
Figure 2
Figure 2
IFN-γ inhibits RFP expression, a lytic marker of KSHV lytic replication, in a dose-dependent manner. iSLK.219 cells were induced without or with Dox (0.2 μg/ml) in the absence/presence of IFN-γ or TNF-α (final 8 to 5000 units/ml) for 2 days before flow cytometry. Live cells were gated for RFP, whose expression was driven by the polyadenylated nuclear (PAN) RNA promoter. Data are presented as mean ± SEM from three independent experiments. *P < 0.01 by one-way ANOVA Tukey.
Figure 3
Figure 3
IFN-γ and TNF-α impair the production of infectious viruses in LECs. Human primary cells (LECs, HUVECs, TIME) were infected with rKSHV.219 at MOI = 3 in the absence/presence of IFN-γ (A) or TNF-α (B) (0 to 5000 units/ml) for the indicated time period. Infectious viruses in the culture supernatant were determined. The means ± SEM of three independent experiments is plotted. *P < 0.01 by one-way ANOVA Tukey Method.
Figure 4
Figure 4
Levels of the surface expression of IFN-γ and TNF-α receptors are comparable between primary and transformed human cells used. The expression of IFNGR (CD119) and TNFRα (CD120a) was assessed by flow cytometry in the indicated cell type using specific antibodies. Filled and open histograms represent isotype and each receptor-specific antibody staining, respectively. One representative result is shown from 3 independent experiments.
Figure 5
Figure 5
IFN-γ inhibits KSHV lytic replication in a time- and dose-dependent manner. iSLK.219 cells were induced without or with Dox (0.2 μg/ml), and IFN-γ (0, 20, or 200 units/ml) was added into the culture at the indicated time. Infectious virus titer in the culture supernatant was determined at 72 hr post-induction and normalized to the medium-treated control. The means ± SEM of triplicated samples is plotted. One representative result is shown from 3 independent experiments. *P < 0.01 by one-way ANOVA Tukey method.
Figure 6
Figure 6
IFN-γ and TNF-α have different effects on KSHV gene expression in a dose-dependent manner. (A) SLK, iSLK, and iSLK.219 cells were left untreated or induced with Dox (0.2 μg/ml), and at the time of induction, where indicated, IFN-γ or TNF-α (0, 20 or 200 units/ml) was added to the culture. At 2 days post-induction, cell lysates were prepared for Western blot analysis. Temporal expression of KSHV genes was examined: latent, immediate-early (IE), and delayed-early (DE). Arrowhead indicates the specific ORF59 band. β-Actin was included as a loading control. One representative result is shown from 3 independent experiments. The intensity of each specific band was determined using ImageJ software and was normalized to that of the Dox-treated sample. The numbers indicate the relative intensity compared to the Dox-alone control. (B) The means ± SEM of 3 independent experiments are plotted. *P < 0.01 by one-way ANOVA Tukey Method.
Figure 7
Figure 7
IFN-γ globally down-regulates viral gene expression, but TNF-α does not. (A) iSLK.219 cells were induced to lytic replication by Dox (0.2 μg/ml) for 36 hr before the addition of IFN-γ or TNF-α. The concentration of each cytokine is indicated (units/ml). Four hours after treatment with the cytokines, total RNA was extracted and processed for the custom KSHV tiling array. KSHV genes downregulated by more than 50% by both IFN-γ and TNF-α are indicated on the right. Green bands with strong signal, which are not annotated in the middle of the array, are transcripts from the repeated regions of the one end of the KSHV genome. The scale color bar is shown on the left. (B) Summary of KSHV genes that were significantly downregulated by IFN-γ compared to the Dox-alone control. Viral genes that were inhibited by more than 2-fold are shown on the left, and those that were between 1.5- and 2-fold are on the right. Viral genes are grouped according to their temporal regulation. IE: immediate-early genes; DE: delayed-early genes.

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References

    1. Ensoli B, et al. Biology of Kaposi’s sarcoma. Eur J Cancer. 2001;37:1251–1269. doi: 10.1016/S0959-8049(01)00121-6. - DOI - PubMed
    1. Nickoloff BJ, Griffiths CE. The spindle-shaped cells in cutaneous Kaposi’s sarcoma. Histologic simulators include factor XIIIa dermal dendrocytes. Am J Pathol. 1989;135:793–800. - PMC - PubMed
    1. Nickoloff BJ, Griffiths CE. Factor XIIIa-expressing dermal dendrocytes in AIDS-associated cutaneous Kaposi’s sarcomas. Science. 1989;243:1736–1737. doi: 10.1126/science.2564703. - DOI - PubMed
    1. Herndier B, Ganem D. The biology of Kaposi’s sarcoma. Cancer Treat Res. 2001;104:89–126. doi: 10.1007/978-1-4615-1601-9_4. - DOI - PubMed
    1. Kang S, Myoung J. Primary lymphocyte infection models for KSHV and its putative tumorigenesis mechanisms in B cell lymphomas. J Microbiol. 2017;55:319–329. doi: 10.1007/s12275-017-7075-2. - DOI - PubMed
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