HITS-CLIP analysis uncovers a link between the Kaposi's sarcoma-associated herpesvirus ORF57 protein and host pre-mRNA metabolism.
HITS-CLIP analysis uncovers a link between the Kaposi's sarcoma-associated herpesvirus ORF57 protein and host pre-mRNA metabolism.
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DOI：
10.1371/journal.ppat.1004652
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发表时间：
2015-02
期刊：
影响因子：
6.7
通讯作者：
Conrad NK
中科院分区：
文献类型：
--
作者：
Sei E;Wang T;Hunter OV;Xie Y;Conrad NK
卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌病毒，可引起卡波西肉瘤、原发性渗出性淋巴瘤（PEL）和某些形式的多中心Castleman病。KSHV ORF 57蛋白是一种保守的基因表达转录后调节因子，对病毒复制至关重要。ORF 57是多功能的，但其大部分活性与其结合RNA的能力直接相关。我们使用通过交联免疫沉淀（HITS-CLIP）分离的RNA的高通量测序，在BEL细胞中裂解再激活期间全面鉴定了与ORF 57结合的病毒和宿主RNA。正如预期的那样，ORF 57结合的RNA片段在整个KSHV基因组中定位，包括已知的ORF 57配体PAN RNA。与先前发表的ChIP结果一致，我们观察到ORF 57结合基因组的oriLyt区域附近的RNA。对宿主RNA片段的检查显示，ORF 57结合的RNA的子集来自转录本5 ′末端。这些5 ′结合片段的位置与前体mRNA的最5 ′外显子-内含子连接密切相关。我们选择了四种候选物（BTG 1、EGR 1、ZFP 36和TNFSF 9），并分析了它们在裂解期的前mRNA和mRNA水平。对稳态和新产生的RNA的分析表明，这些候选ORF 57结合的前mRNA在整个感染过程中比对照RNA持续更长的时间，这与ORF 57在前mRNA代谢中的作用一致。此外，ORF 57的外源性表达足以增加前mRNA水平，并且在一种情况下，增加推定的ORF 57靶标的mRNA水平。这些结果表明，ORF 57在裂解再活化过程中与特定宿主前mRNA相互作用，并可能通过稳定前mRNA来改变其加工。这些数据表明，ORF 57参与调节宿主基因的表达，除了KSHV基因的表达在裂解再活化。在病毒复制过程中，致癌的卡波西肉瘤相关疱疹病毒（KSHV）调节宿主和病毒基因表达。KSHV ORF 57是一种多功能转录后调节因子，对病毒复制和稳定病毒RNA至关重要。以前的研究表明，ORF 57 RNA结合对其活性至关重要，但ORF 57靶标的全谱尚不清楚。在这里，我们采用了高通量分析，以确定在裂解再活化过程中结合的ORF 57的RNA片段。正如预期的那样，我们发现了映射到病毒基因组的靶标，并且我们进一步发现了新的宿主靶标，其中一个子集在其5 '端附近结合了ORF 57。对该子集的进一步检查表明，ORF 57优先结合在最5 ′的外显子-内含子边界。ORF 57影响来自这些基因的前mRNA丰度，最有可能是通过稳定否则不稳定的低效剪接的前mRNA。在至少一种情况下，这种稳定导致宿主基因的mRNA表达增加。我们认为，KSHV采用相同的机制来稳定无内含子病毒RNA和细胞未剪接的前mRNA，以调节病毒和宿主基因的表达在裂解再活化。
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. During viral replication, the oncogenic Kaposi’s sarcoma-associated herpesvirus (KSHV) modulates both host and viral gene expression. KSHV ORF57 is a multifunctional posttranscriptional regulator that is essential for viral replication and stabilizes viral RNAs. Previous studies demonstrated that ORF57 RNA-binding is essential for its activity, but the full spectrum of ORF57 targets are unknown. Here we employed a high-throughput analysis to identify RNA fragments bound by ORF57 during lytic reactivation. As expected, we found targets that mapped to the viral genome, and we further uncovered novel host targets, a subset of which had ORF57 bound near their 5´ ends. Further examination of this subset demonstrated that ORF57 bound preferentially at the 5´-most exon-intron boundary. ORF57 affected the pre-mRNA abundance from these genes, most likely by stabilizing otherwise unstable inefficiently spliced pre-mRNAs. In at least one case, this stabilization led to increases in mRNA expression of the host gene. We suggest that KSHV employs the same mechanism to stabilize intronless viral RNAs and cellular unspliced pre-mRNAs to modulate viral and host gene expression during lytic reactivation.
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