Bhullar Deepika, Jalodia Richa, Kalia Manjula, Vrati Sudhanshu
National Institute of Immunology, New Delhi, India.
Vaccine and Infectious Disease Research Centre, Translational Health Science & Technology Institute, Gurgaon, India.
PLoS One. 2014 Dec 29;9(12):e114931. doi: 10.1371/journal.pone.0114931. eCollection 2014.
Japanese encephalitis virus (JEV) has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs). The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB) interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+), and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-). The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(-) RNA with viral RNA-dependent RNA polymerase (NS5 protein), an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA)-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.
日本脑炎病毒（JEV）具有单链正义RNA基因组，包含一个单一的开放阅读框，两侧为5'-和3'-非编码区（NCR）。该病毒基因组通过负义RNA中间体进行复制。NCR及其在负义RNA中的互补序列是RNA复制酶复合物组装的位点，从而调节RNA合成和病毒复制。在本研究中，我们表明55 kDa的多嘧啶序列结合蛋白（PTB）在体外与正义基因组RNA的5'-NCR（5NCR(+)）及其在负义复制中间体RNA中的互补序列（3NCR(-)）相互作用。通过JEV RNA共免疫沉淀和JEV RNA-PTB共定位实验，在感染细胞中验证了病毒RNA与PTB的相互作用。有趣的是，我们观察到在JEV感染早期，核PTB发生磷酸化偶联的易位，转移至与JEV RNA共定位的细胞质灶。我们在培养细胞中进行的PTB沉默和过表达研究确定了PTB在JEV复制中的抑制作用。使用RNA-蛋白质结合试验，我们表明PTB竞争性抑制JEV 3NCR(-) RNA与病毒RNA依赖性RNA聚合酶（NS5蛋白）的结合，而这是正义基因组RNA合成所必需的事件。已知cAMP可促进蛋白激酶A（PKA）介导的PTB磷酸化。我们表明，用cAMP类似物处理的细胞在细胞质中磷酸化PTB水平升高，且JEV复制显著受到抑制。此处呈现的数据显示了一种新型的、cAMP诱导的、PTB介导的先天性宿主反应，可有效抑制哺乳动物细胞中的JEV复制。
