Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
PLoS Pathog. 2023 Nov 20;19(11):e1011806. doi: 10.1371/journal.ppat.1011806. eCollection 2023 Nov.
Human herpesvirus 8 (HHV-8) encodes four viral interferon regulatory factors (vIRFs) that target cellular IRFs and/or other innate-immune and stress signaling regulators and suppress the cellular response to viral infection and replication. For vIRF-1, cellular protein targets include IRFs, p53, p53-activating ATM kinase, BH3-only proteins, and antiviral signaling effectors MAVS and STING; vIRF-1 inhibits each, with demonstrated or likely promotion of HHV-8 de novo infection and productive replication. Here, we identify direct interactions of vIRF-1 with STAT3 and STAT-activating Janus kinase TYK2 (the latter reported previously by us to be inhibited by vIRF-1) and suppression by vIRF-1 of cytokine-induced STAT3 activation. Suppression of active, phosphorylated STAT3 (pSTAT3) by vIRF-1 was evident in transfected cells and vIRF-1 ablation in lytically-reactivated recombinant-HHV-8-infected cells led to increased levels of pSTAT3. Using a panel of vIRF-1 deletion variants, regions of vIRF-1 required for interactions with STAT3 and TYK2 were identified, which enabled correlation of STAT3 signaling inhibition by vIRF-1 with TYK2 binding, independently of STAT3 interaction. A viral mutant expressing vIRF-1 deletion-variant Δ198-222 refractory for TYK2 interaction and pSTAT3 suppression was severely compromised for productive replication. Conversely, expression of phosphatase-resistant, protractedly-active STAT3 led to impaired HHV-8 replication. Cells infected with HHV-8 mutants expressing STAT3-refractory vIRF-1 deletion variants or depleted of STAT3 displayed reduced vIRF-1 expression, while custom-peptide-promoted STAT3 interaction could effect increased vIRF-1 expression and enhanced virus replication. Taken together, our data identify vIRF-1 targeting and inhibition of TYK2 as a mechanism of STAT3-signaling suppression and critical for HHV-8 productive replication, the importance of specific pSTAT3 levels for replication, positive roles of STAT3 and vIRF-1-STAT3 interaction in vIRF-1 expression, and significant contributions to lytic replication of STAT3 targeting by vIRF-1.
人类疱疹病毒 8（HHV-8）编码四种病毒干扰素调节因子（vIRFs），它们靶向细胞 IRFs 和/或其他先天免疫和应激信号调节剂，并抑制细胞对病毒感染和复制的反应。对于 vIRF-1，细胞蛋白靶标包括 IRFs、p53、p53 激活 ATM 激酶、BH3 仅蛋白和抗病毒信号效应物 MAVS 和 STING；vIRF-1 抑制每个蛋白，并且已经证明或可能促进 HHV-8 的从头感染和有性复制。在这里，我们鉴定了 vIRF-1 与 STAT3 和 STAT 激活的 Janus 激酶 TYK2 的直接相互作用（后者先前被我们报道为被 vIRF-1 抑制），并鉴定了 vIRF-1 对细胞因子诱导的 STAT3 激活的抑制作用。在转染细胞中明显观察到 vIRF-1 对活性磷酸化 STAT3（pSTAT3）的抑制作用，并且在溶酶体激活的重组 HHV-8 感染细胞中 vIRF-1 的缺失导致 pSTAT3 水平升高。使用 vIRF-1 缺失变体的面板，鉴定了 vIRF-1 与 STAT3 和 TYK2 相互作用所需的区域，这使得 vIRF-1 通过 TYK2 结合而不是 STAT3 相互作用来抑制 STAT3 信号。表达对 TYK2 相互作用和 pSTAT3 抑制具有抗性的 vIRF-1 缺失变体 Δ198-222 的病毒突变体在有性复制中严重受损。相反，表达磷酸酶抗性、持续激活的 STAT3 导致 HHV-8 复制受损。感染表达对 STAT3 有抗性的 vIRF-1 缺失变体的 HHV-8 突变体或耗尽 STAT3 的细胞显示 vIRF-1 表达降低，而定制肽促进 STAT3 相互作用可增加 vIRF-1 表达并增强病毒复制。总之，我们的数据表明，vIRF-1 靶向并抑制 TYK2 是 STAT3 信号抑制的机制，对 HHV-8 的有性复制至关重要，特定 pSTAT3 水平对复制的重要性，STAT3 和 vIRF-1-STAT3 相互作用在 vIRF-1 表达中的积极作用，以及 vIRF-1 对 STAT3 靶向的溶酶体复制的重要贡献。
