HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

The Kaposi’s sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi’s sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman’s disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exon-intron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation.卡波西肉瘤相关疱疹病毒（Kaposi’s sarcoma associated herpesvirus, KSHV）是一种致癌病毒，可引发卡波西肉瘤、原发性渗出性淋巴瘤（primary effusion lymphoma, PEL）以及部分多中心Castleman病。KSHV编码的ORF57蛋白是一类保守的基因表达转录后调控因子，对病毒复制至关重要。ORF57具有多种生物学功能，但其绝大多数活性均与其结合RNA的能力直接相关。本研究在PEL细胞的裂解激活过程中，采用交联免疫沉淀结合RNA高通量测序（high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation, HITS-CLIP）技术，在全基因组范围内鉴定了ORF57结合的病毒与宿主RNA。正如预期，ORF57结合的RNA片段全域匹配KSHV基因组，包括已知的ORF57配体PAN RNA。与既往发表的染色质免疫沉淀（ChIP）结果一致，我们观察到ORF57结合的RNA位于基因组裂解复制起点（oriLyt）区域附近。对宿主RNA片段的分析显示，部分ORF57结合的RNA来源于转录本的5'端。这些5'端结合片段的位置与前体mRNA（pre-mRNA）最远端的外显子-内含子接头高度吻合。我们选取了BTG1、EGR1、ZFP36及TNFSF9四个候选靶标，分析了它们在裂解期的前体mRNA与mRNA水平。对稳态RNA及新合成RNA的分析结果表明，这些候选ORF57结合的前体mRNA在感染过程中的持续时长显著长于对照RNA，这与ORF57在前体mRNA代谢中发挥的作用相符。此外，外源表达ORF57即可提升推定的ORF57靶标的前体mRNA水平，其中一个靶标的mRNA水平也得到了上调。上述结果证实，ORF57在裂解激活过程中可与特定宿主前体mRNA结合，并可能通过稳定前体mRNA来改变其加工过程。这些数据表明，在裂解激活阶段，ORF57除调控KSHV的基因表达外，还参与调控宿主的基因表达。