Davis David A, Astter Yana, Treco Emma N, Shrestha Prabha, Stream Alexandra, Haque Muzammel, Mulugeta Naomi, Yarchoan Robert
HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA.
Division of Biotechnology and Molecular Medicine, Department of Pathobiological Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, Louisiana, USA.
J Virol. 2025 Aug 29:e0062225. doi: 10.1128/jvi.00622-25.
Viral infections lead to caspase activation as a cellular defense response. Some viruses overcome this response by encoding viral proteins that undergo caspase cleavage and, by various mechanisms, aid in cell survival. Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen acts as a pseudo-substrate for caspases-1 and -3, thereby interfering with the inflammatory and apoptotic response. Here, we screened the KSHV proteome for additional caspase cleavage sites. Using SitePrediction, many KSHV proteins with potential caspase cleavage sites were identified. Among the highest-scoring proteins was the early lytic protein, K5. Treatment of BJAB-K5-FLAG-expressing cells with ⍺Fas led to caspase processing of K5-FLAG. Using mass spectrometry, we determined that caspase cleavage of K5 occurs at D222. K5 was also cleaved by caspases when KSHV was induced to lytic replication. Interestingly, the expression of K5-FLAG significantly inhibited ⍺Fas-induced caspase-mediated cell death. To determine if K5 plays a protective role in KSHV-infected cells, iSLK cells infected with wild-type or K5-knockout BAC16 virus were induced to lytic replication. Although lytic induction showed no significant effect on the viability of wild-type-infected cells, the viability of K5-knockout cells decreased by 25%. Thus, K5 may protect KSHV-infected cells from caspase-mediated cell death during lytic replication. Interestingly, cleavage of K5 by caspases did not affect its previously reported ability to downregulate immune surface markers. These data suggest that K5 not only downregulates immune surface markers to help avoid immune recognition but plays an additional role in mitigating caspase-mediated cell death during lytic replication.IMPORTANCEKaposi sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman's disease, and KSHV inflammatory cytokine syndrome. Understanding how KSHV thwarts host defense responses is necessary to help develop strategies to treat these rare, yet deadly, diseases. We profiled potential caspase cleavage sites in the viral proteome and found many viral proteins with high-scoring caspase cleavage sites. We follow up on this by demonstrating that K5 is, in fact, a substrate for caspases and and provide data to suggest that K5 plays a role in obviating caspase-mediated cell death during lytic replication. The work described here furthers our understanding of the roles that KSHV proteins, like K5, play to prevent or divert the host apoptotic defense responses that involve host caspase activation that normally would lead to cell death.
病毒感染会导致半胱天冬酶激活，这是一种细胞防御反应。一些病毒通过编码可被半胱天冬酶切割的病毒蛋白来克服这种反应，并通过各种机制帮助细胞存活。卡波西肉瘤相关疱疹病毒（KSHV）的潜伏相关核抗原作为半胱天冬酶-1和-3的假底物，从而干扰炎症和凋亡反应。在此，我们在KSHV蛋白质组中筛选了其他半胱天冬酶切割位点。使用位点预测软件，鉴定出许多具有潜在半胱天冬酶切割位点的KSHV蛋白。得分最高的蛋白之一是早期裂解蛋白K5。用αFas处理表达BJAB-K5-FLAG的细胞会导致K5-FLAG的半胱天冬酶加工。通过质谱分析，我们确定K5的半胱天冬酶切割发生在D222处。当KSHV被诱导进行裂解复制时，K5也会被半胱天冬酶切割。有趣的是，K5-FLAG的表达显著抑制了αFas诱导的半胱天冬酶介导的细胞死亡。为了确定K5在KSHV感染的细胞中是否发挥保护作用，用野生型或K5基因敲除的BAC16病毒感染iSLK细胞并诱导其进行裂解复制。尽管裂解诱导对野生型感染细胞的活力没有显著影响，但K5基因敲除细胞的活力下降了25%。因此，K5可能在裂解复制期间保护KSHV感染的细胞免受半胱天冬酶介导的细胞死亡。有趣的是，半胱天冬酶对K5的切割并不影响其先前报道的下调免疫表面标志物的能力。这些数据表明，K5不仅下调免疫表面标志物以帮助避免免疫识别，而且在减轻裂解复制期间半胱天冬酶介导的细胞死亡中发挥额外作用。重要性卡波西肉瘤相关疱疹病毒（KSHV）是卡波西肉瘤、原发性渗出性淋巴瘤、多中心Castleman病和KSHV炎性细胞因子综合征的病原体。了解KSHV如何挫败宿主防御反应对于帮助制定治疗这些罕见但致命疾病的策略是必要的。我们分析了病毒蛋白质组中的潜在半胱天冬酶切割位点，发现许多病毒蛋白具有高分的半胱天冬酶切割位点。我们通过证明K5实际上是半胱天冬酶的底物来跟进这一发现，并提供数据表明K5在裂解复制期间在避免半胱天冬酶介导的细胞死亡中发挥作用。此处描述的工作进一步加深了我们对K5等KSHV蛋白在预防或转移宿主凋亡防御反应中所起作用的理解，这些反应涉及通常会导致细胞死亡的宿主半胱天冬酶激活。
