Lubyova Barbora, Hodek Jan, Zabransky Ales, Prouzova Hana, Hubalek Martin, Hirsch Ivan, Weber Jan
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, IOCB & Gilead Research Center, Prague, Czech Republic.
Dept. Genetics and Microbiology, Faculty of Sciences, Charles University, Prague, Czech Republic.
PLoS One. 2017 Oct 24;12(10):e0186982. doi: 10.1371/journal.pone.0186982. eCollection 2017.
In mammals, protein arginine methyltransferase 5, PRMT5, is the main type II enzyme responsible for the majority of symmetric dimethylarginine formation in polypeptides. Recent study reported that PRMT5 restricts Hepatitis B virus (HBV) replication through epigenetic repression of HBV DNA transcription and interference with encapsidation of pregenomic RNA. Here we demonstrate that PRMT5 interacts with the HBV core (HBc) protein and dimethylates arginine residues within the arginine-rich domain (ARD) of the carboxyl-terminus. ARD consists of four arginine rich subdomains, ARDI, ARDII, ARDIII and ARDIV. Mutation analysis of ARDs revealed that arginine methylation of HBc required the wild-type status of both ARDI and ARDII. Mass spectrometry analysis of HBc identified multiple potential ubiquitination, methylation and phosphorylation sites, out of which lysine K7 and arginines R150 (within ARDI) and R156 (outside ARDs) were shown to be modified by ubiquitination and methylation, respectively. The HBc symmetric dimethylation appeared to be linked to serine phosphorylation and nuclear import of HBc protein. Conversely, the monomethylated HBc retained in the cytoplasm. Thus, overexpression of PRMT5 led to increased nuclear accumulation of HBc, and vice versa, down-regulation of PRMT5 resulted in reduced levels of HBc in nuclei of transfected cells. In summary, we identified PRMT5 as a potent controller of HBc cell trafficking and function and described two novel types of HBc post-translational modifications (PTMs), arginine methylation and ubiquitination.
在哺乳动物中，蛋白质精氨酸甲基转移酶5（PRMT5）是负责多肽中大部分对称二甲基精氨酸形成的主要II型酶。最近的研究报道，PRMT5通过对乙肝病毒（HBV）DNA转录的表观遗传抑制和干扰前基因组RNA的衣壳化来限制HBV复制。在此，我们证明PRMT5与HBV核心（HBc）蛋白相互作用，并使羧基末端富含精氨酸结构域（ARD）内的精氨酸残基发生二甲基化。ARD由四个富含精氨酸的亚结构域ARD I、ARD II、ARD III和ARD IV组成。ARD的突变分析表明，HBc的精氨酸甲基化需要ARD I和ARD II的野生型状态。对HBc的质谱分析确定了多个潜在的泛素化、甲基化和磷酸化位点，其中赖氨酸K7以及精氨酸R150（在ARD I内）和R156（在ARDs外）分别被证明发生了泛素化和甲基化修饰。HBc的对称二甲基化似乎与丝氨酸磷酸化和HBc蛋白的核输入有关。相反，单甲基化的HBc保留在细胞质中。因此，PRMT5的过表达导致HBc的核积累增加，反之亦然，PRMT5的下调导致转染细胞细胞核中HBc水平降低。总之，我们确定PRMT5是HBc细胞转运和功能的有效调控因子，并描述了两种新型的HBc翻译后修饰（PTM），即精氨酸甲基化和泛素化。
