Tumor Virus RNA Biology Section, HIV Dynamics and Replication Program, Center for Cancer Research (CCR), National Cancer Institute, NIH, Frederick, Maryland, Unites States of America.
CCR Collaborative Bioinformatics Resource, National Cancer Institute, NIH, Bethesda, Maryland, Unites States of America.
PLoS Pathog. 2022 Jul 14;18(7):e1010311. doi: 10.1371/journal.ppat.1010311. eCollection 2022 Jul.
RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.
RNA 剪接在真核基因的表达中起着至关重要的作用。我们之前曾表明，KSHV ORF57 是一种促进病毒裂解基因表达的病毒剪接因子。在本报告中，我们比较了携带野生型（WT）和 ORF57 敲除（57KO）KSHV 基因组的 BCBL-1 细胞在病毒裂解感染过程中病毒 RNA 的剪接谱。我们对来自 RNA-seq 的病毒 RNA 剪接接头的分析，在 WT 中鉴定出 269 个 RNA 剪接事件，在 57KO 基因组中鉴定出 255 个 RNA 剪接事件，包括跨越病毒基因组大部分区域的剪接事件和 vIRF4 circRNA 的产生。从 PAN 区域检测不到 circRNA。我们发现 57KO 改变了靶向病毒 RNA 的 RNA 剪接效率。对 ORF57 剪接调节最敏感的两种 RNA 是具有 8 个外显子和 7 个内含子的 K15 RNA 和编码病毒胸苷酸合成酶（ORF70）和膜相关 E3-泛素连接酶（K3）的双顺反子 RNA。ORF57 抑制 K15 内含子 1 和 2 的剪接。ORF70/K3 RNA 具有两个内含子，其中第一个内含子位于 ORF70 编码区内，作为替代内含子，第二个内含子位于 ORF70 和 K3 之间的基因间区，作为组成性内含子。在表达 ORF57 的 WT 细胞中，大多数 ORF70/K3 转录物保留第一个内含子以维持完整的 ORF70 编码区。相比之下，在 57KO 细胞中，第一个内含子被大量剪接。使用包含 ORF70/K3 基因座的小基因，我们进一步证实了 ORF57 对 ORF70/K3 RNA 剪接的调节作用，独立于其他病毒因子。通过监测蛋白表达，我们表明 ORF57 介导的第一个内含子保留导致全长 ORF70 蛋白的表达。ORF57 的缺失促进了第一个内含子的剪接和 K3 蛋白的表达。总的来说，我们得出结论，ORF57 调节 ORF70/K3 双顺反子 RNA 的可变剪接，以控制 K3 介导的免疫逃逸和 ORF70 参与病毒裂解感染中的病毒 DNA 复制。
