Manchester Institute of Biotechnology, School of Chemistry, Faculty of Science and Engineering, The University of Manchester, Manchester M1 7DN, UK.
Biomolecular Analysis Core Facility, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK.
Nucleic Acids Res. 2019 Feb 28;47(4):1987-2001. doi: 10.1093/nar/gky1181.
Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.
卡波西肉瘤相关疱疹病毒 (KSHV) 转录出一种长的非编码多聚腺苷酸化核 (PAN) RNA，通过抑制宿主抗病毒反应相关基因以及支持潜伏状态的病毒蛋白，促进潜伏向裂解转变。KSHV 还表达几种早期蛋白，包括 ORF57（Mta），它是保守多功能 ICP27 蛋白家族的成员，对 productive replication 至关重要。ORF57/Mta 通过称为 Mta 反应元件 (MRE) 的区域与 PAN RNA 相互作用，稳定转录本并支持核积累。在这里，我们使用来自猴疱疹病毒 (HVS) 的 KSHV ORF57 的密切同源物，确定了与 PAN RNA MRE 复合物的球形结构域的晶体结构，揭示了一个也在 KSHV 中保守的尿嘧啶特异性结合位点。使用溶液 NMR，还在 KSHV ORF57 的无规卷曲 N 端结构域内映射了 RNA 结合，并且显示出对包含先前已知与 HVS ORF57 同源区域结合的 GAAGRG 基序的 RNA 片段的特异性。这些数据一起在疱疹病毒 ORF57 同源物的相邻结构域内定位了 novel differential RNA 识别位点，并揭示了它们与 PAN RNA 相互作用的高分辨率细节，从而深入了解对病毒功能至关重要的相互作用。
