Majerciak Vladimir, Alvarado-Hernandez Beatriz, Ma Yanping, Duduskar Shivalee, Lobanov Alexei, Cam Maggie, Zheng Zhi-Ming
Tumor Virus RNA Biology Section, HIV Dynamics and Replication Program, Center for Cancer Research, NCI/NIH, Frederick, Maryland, USA.
CCR Collaborative Bioinformatics Resource, Center for Cancer Research, NCI/NIH, Bethesda, Maryland, USA.
mBio. 2025 Jan 8;16(1):e0317224. doi: 10.1128/mbio.03172-24. Epub 2024 Dec 10.
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an RNA-binding protein ORF57 in lytic infection. Using an optimized CLIP-seq in this report, we identified ORF57-bound transcripts from 544 host protein-coding genes. By comparing with the RNA-seq profiles from BCBL-1 cells with latent and lytic KSHV infection and from HEK293T cells with and without ORF57 expression, we identified FOS RNA as one of the major ORF57-specific RNA targets. FOS dimerizes with JUN as a transcription factor AP-1 involved in cell proliferation, differentiation, and transformation. Knockout of the ORF57 gene from the KSHV genome led BAC16-iSLK cells incapable of FOS expression in KSHV lytic infection. The dysfunctional KSHV genome in FOS expression could be rescued by Lenti-ORF57 virus infection. ORF57 protein does not regulate FOS translation but binds to the 13-nt RNA motif near the FOS RNA 5' end and prolongs FOS mRNA half-life 7.7 times longer than it is in the absence of ORF57. This binding of ORF57 to FOS RNA is likely competitive to the binding of host nuclease AEN (ISG20L1) of which physiological RNase activity remains unknown. KSHV infection inhibits the expression of AEN, but not exosomal RNA helicase MTR4. FOS expression mediated by ORF57 inhibits transcription through FOS binding to promoter but transactivates , a regulator of G-protein-coupled receptors. FOS binds a conserved AP-1 site in the promoter and enhances RGS2 expression to phosphorylate AKT. Altogether, we have discovered that KSHV ORF57 specifically binds and stabilizes FOS RNA to increase FOS expression, thereby disturbing host gene expression and inducing pathogenesis during KSHV lytic infection.IMPORTANCEWe discovered that FOS, a heterodimer component of oncogenic transcription factor AP-1, is highly elevated in KSHV-infected cells by expression of a viral lytic RNA-binding protein, ORF57, which binds a 13-nt RNA motif near the FOS RNA 5' end to prolong FOS RNA half-life. This binding of ORF57 to FOS RNA is competitive to the binding of host RNA destabilizer(s). KSHV infection inhibits expression of host nuclease AEN, but not MTR4. FOS inhibits AEN transcription by binding to the promoter but transactivates RGS2 by binding to a conserved AP-1 site in the RGS2 promoter, thereby enhancing RGS2 expression and phosphorylation of AKT. Thus, KSHV lytic infection controls the expression of a subset of genes for signaling, cell cycle progression, and proliferation to potentially contribute to viral oncogenesis.
卡波西肉瘤相关疱疹病毒（KSHV）在裂解感染时编码一种RNA结合蛋白ORF57。在本报告中，我们使用优化的CLIP-seq技术，从544个宿主蛋白编码基因中鉴定出了与ORF57结合的转录本。通过比较潜伏和裂解性KSHV感染的BCBL-1细胞以及有或无ORF57表达的HEK293T细胞的RNA-seq图谱，我们确定FOS RNA是主要的ORF57特异性RNA靶标之一。FOS与JUN二聚化形成转录因子AP-1，参与细胞增殖、分化和转化。从KSHV基因组中敲除ORF57基因导致BAC16-iSLK细胞在KSHV裂解感染时无法表达FOS。Lenti-ORF57病毒感染可挽救FOS表达功能失调的KSHV基因组。ORF57蛋白不调节FOS的翻译，但与FOS RNA 5'端附近的13个核苷酸的RNA基序结合，使FOS mRNA半衰期延长7.7倍，比没有ORF57时长得多。ORF57与FOS RNA的这种结合可能与宿主核酸酶AEN（ISG20L1）的结合竞争，其生理核糖核酸酶活性尚不清楚。KSHV感染抑制AEN的表达，但不抑制外泌体RNA解旋酶MTR4的表达。由ORF57介导的FOS表达通过FOS与启动子结合抑制转录，但反式激活RGS2，RGS2是G蛋白偶联受体的调节剂。FOS结合RGS2启动子中的保守AP-1位点并增强RGS2表达以磷酸化AKT。总之，我们发现KSHV ORF57特异性结合并稳定FOS RNA以增加FOS表达，从而在KSHV裂解感染期间干扰宿主基因表达并诱导发病机制。
我们发现，致癌转录因子AP-1的异二聚体成分FOS在KSHV感染的细胞中因病毒裂解性RNA结合蛋白ORF57的表达而高度升高，ORF57与FOS RNA 5'端附近的13个核苷酸的RNA基序结合以延长FOS RNA半衰期。ORF57与FOS RNA的这种结合与宿主RNA去稳定剂的结合竞争。KSHV感染抑制宿主核酸酶AEN的表达，但不抑制MTR4的表达。FOS通过与启动子结合抑制AEN转录，但通过与RGS2启动子中的保守AP-1位点结合反式激活RGS2，从而增强RGS2表达和AKT磷酸化。因此，KSHV裂解感染控制信号传导、细胞周期进程和增殖相关的一组基因的表达，可能有助于病毒致癌作用。
