卡波西肉瘤相关疱疹病毒 (KSHV) 通过编码有助于逃避免疫的庞大蛋白质网络，在宿主体内建立持续感染。这些靶向先天免疫途径之一是 cGAS-STING 途径，它抑制 KSHV 的潜伏期重新激活。此前，我们鉴定了 KSHV 编码的多种 cGAS/STING 抑制剂，这表明病毒蛋白对该途径的反作用对于维持成功的 KSHV 生命周期至关重要。然而，这些病毒蛋白如何阻断先天免疫并促进 KSHV 裂解复制的详细机制仍然很大程度上未知。在这项研究中，我们报告了 ORF48（之前确定的 cGAS/STING 途径的负调节因子）是最佳 KSHV 裂解复制所必需的。我们使用 siRNA 和基于缺失的系统来评估完整 ORF48 在 KSHV 裂解周期中的重要性。在这两个系统中，ORF48的缺失导致裂解基因转录、裂解蛋白表达、病毒基因组复制和感染性病毒粒子产生的缺陷。 ORF48 基因组缺失导致 KSHV 转录组更加强大和全面的抑制，这可能是由于 RTA 启动子活性的破坏。从机制上讲，过表达的 ORF48 被发现与 HEK293 细胞中的内源性 STING 共定位并相互作用。在重新激活的 iSLK.219 细胞中也检测到内源性 ORF48 和 STING 相互作用。与对照细胞系相比，稳定表达 ORF48 的 HUVEC 细胞在 ISD 或 diABZI 处理后表现出抑制 STING 依赖性先天免疫信号传导。然而，在我们基于 iSLK 的裂解系统中 ORF48 的丢失未能诱导 IFNβ 的产生，这表明 ORF48 在 KSHV 裂解阶段对 STING 信号传导具有冗余作用。因此，ORF48 是通过需要进一步探索的其他机制实现最佳 KSHV 裂解复制所必需的。版权所有：© 2024 Veronese 等人。这是一篇根据知识共享署名许可条款分发的开放获取文章，允许在任何媒体上不受限制地使用、分发和复制，前提是注明原始作者和来源。

Kaposi's sarcoma-associated herpesvirus (KSHV) establishes persistent infection in the host by encoding a vast network of proteins that aid immune evasion. One of these targeted innate immunity pathways is the cGAS-STING pathway, which inhibits the reactivation of KSHV from latency. Previously, we identified multiple cGAS/STING inhibitors encoded by KSHV, suggesting that the counteractions of this pathway by viral proteins are critical for maintaining a successful KSHV life cycle. However, the detailed mechanisms of how these viral proteins block innate immunity and facilitate KSHV lytic replication remain largely unknown. In this study, we report that ORF48, a previously identified negative regulator of the cGAS/STING pathway, is required for optimal KSHV lytic replication. We used both siRNA and deletion-based systems to evaluate the importance of intact ORF48 in the KSHV lytic cycle. In both systems, loss of ORF48 resulted in defects in lytic gene transcription, lytic protein expression, viral genome replication and infectious virion production. ORF48 genome deletion caused more robust and global repression of the KSHV transcriptome, possibly due to the disruption of RTA promoter activity. Mechanistically, overexpressed ORF48 was found to colocalize and interact with endogenous STING in HEK293 cells. Endogenous ORF48 and STING interactions were also detected in reactivated iSLK.219 cells. Compared with the control cell line, HUVEC cells stably expressing ORF48 exhibited repressed STING-dependent innate immune signaling upon ISD or diABZI treatment. However, the loss of ORF48 in our iSLK-based lytic system failed to induce IFNβ production, suggesting a redundant role of ORF48 on STING signaling during the KSHV lytic phase. Thus, ORF48 is required for optimal KSHV lytic replication through additional mechanisms that need to be further explored.Copyright: © 2024 Veronese et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.