Institute of Human Virology and Ministry of Education Key Laboratory of Tropical Disease Control, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
Department of Microbiology, University of Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA.
J Virol. 2018 Mar 14;92(7). doi: 10.1128/JVI.02177-17. Print 2018 Apr 1.
Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication and constant primary infection of fresh cells are crucial for viral tumorigenicity. The virus-encoded bZIP family protein K8 plays an important role in viral DNA replication in both viral reactivation and infection. The mechanism underlying the functional role of K8 in the viral life cycle is elusive. Here, we report that K8 is an RNA binding protein that also associates with many other proteins, including other RNA binding proteins. Many protein-protein interactions involving K8 are mediated by RNA. Using a UV rossinking and mmunorecipitation (CLIP) procedure combined with high-throughput sequencing, RNAs that are associated with K8 in BCBL-1 cells were identified, including both viral (PAN, T1.4, T0.7, etc.) and cellular (MALAT-1, MRP, 7SK, etc.) RNAs. An RNA binding motif in K8 was defined, and mutation of the motif abolished the ability of K8 to bind to many noncoding RNAs, as well as viral DNA replication during infection, suggesting that the K8 functions in viral replication are carried out through RNA association. The functions of K8 and associated T1.4 RNA were investigated in detail, and the results showed that T1.4 mediates the binding of K8 to ori-Lyt DNA. The T1.4-K8 complex physically bound to KSHV ori-Lyt DNA and recruited other proteins and cofactors to assemble a replication complex. Depletion of T1.4 abolished DNA replication in primary infection. These findings provide mechanistic insights into the role of K8 in coordination with T1.4 RNA in regulating KSHV DNA replication during infection. Genomewide analyses of the mammalian transcriptome revealed that a large proportion of sequence previously annotated as noncoding regions is actually transcribed and gives rise to stable RNAs. The emergence of a large number of noncoding RNAs suggests that functional RNA-protein complexes, e.g., ribosomes or spliceosomes, are not ancient relics of the last ribo-organism but would be well adapted to a regulatory role in biology. K8 has been puzzling because of its unique characteristics, such as multiple regulatory roles in gene expression and DNA replication without DNA binding capability. This study reveals the mechanism underlying its regulatory role by demonstrating that K8 is an RNA binding protein that binds to DNA and initiates DNA replication in coordination with a noncoding RNA. It is suggested that many K8 functions, if not all, are carried out through its associated RNAs.
卡波西肉瘤相关疱疹病毒 (KSHV) 的裂解复制和新鲜细胞的持续原发性感染对病毒的致瘤性至关重要。病毒编码的 bZIP 家族蛋白 K8 在病毒再激活和感染过程中的病毒 DNA 复制中发挥重要作用。K8 在病毒生命周期中的功能作用的机制尚不清楚。在这里，我们报告 K8 是一种 RNA 结合蛋白，它还与许多其他蛋白质（包括其他 RNA 结合蛋白）相关。涉及 K8 的许多蛋白质-蛋白质相互作用是由 RNA 介导的。使用紫外线交联和免疫沉淀（CLIP）程序结合高通量测序，鉴定了与 BCBL-1 细胞中的 K8 相关的 RNA，包括病毒（PAN、T1.4、T0.7 等）和细胞（MALAT-1、MRP、7SK 等）RNA。K8 中定义了一个 RNA 结合基序，并且该基序的突变使 K8 丧失了与许多非编码 RNA 以及感染期间病毒 DNA 复制的结合能力，这表明 K8 在病毒复制中的功能是通过 RNA 结合来实现的。详细研究了 K8 和相关的 T1.4 RNA 的功能，结果表明 T1.4 介导 K8 与 ori-Lyt DNA 的结合。T1.4-K8 复合物物理结合到 KSHV ori-Lyt DNA 上，并招募其他蛋白质和辅助因子组装复制复合物。T1.4 的耗竭消除了原发性感染中的 DNA 复制。这些发现为 K8 在与 T1.4 RNA 协调调节感染期间 KSHV DNA 复制中的作用提供了机制见解。对哺乳动物转录组的全基因组分析表明，以前注释为非编码区的大部分序列实际上是转录的，并产生稳定的 RNA。大量非编码 RNA 的出现表明，功能 RNA-蛋白质复合物（例如核糖体或剪接体）不是最后一个核糖体生物的古老遗物，而是非常适应生物学中的调节作用。K8 因其独特的特性（例如在基因表达和 DNA 复制中具有多种调节作用而没有 DNA 结合能力）而令人困惑。本研究通过证明 K8 是一种 RNA 结合蛋白，它与 DNA 结合并与非编码 RNA 协调启动 DNA 复制，揭示了其调节作用的机制。表明 K8 的许多功能（如果不是全部）都是通过与其相关的 RNA 来实现的。
