title: Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.
abstract: 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.
authors.affiliation: National Institute of Immunology, New Delhi, India.
authors.forename: Deepika
authors.initials: D
authors.lastname: Bhullar
authors.affiliation: National Institute of Immunology, New Delhi, India.
authors.forename: Richa
authors.initials: R
authors.lastname: Jalodia
authors.affiliation: Vaccine and Infectious Disease Research Centre, Translational Health Science & Technology Institute, Gurgaon, India.
authors.forename: Manjula
authors.initials: M
authors.lastname: Kalia
authors.affiliation: National Institute of Immunology, New Delhi, India; Vaccine and Infectious Disease Research Centre, Translational Health Science & Technology Institute, Gurgaon, India.
authors.forename: Sudhanshu
authors.initials: S
authors.lastname: Vrati
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004672:Encephalitis, Japanese / Q000821:virology*; D006801:Humans; D010766:Phosphorylation; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism*; D021381:Protein Transport; D012367:RNA, Viral / Q000378:metabolism*; D034421:Sus scrofa / Q000821:virology; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
chemical_list: C096041:NS5 protein, flavivirus; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D038941:Polypyrimidine Tract-Binding Protein; D000242:Cyclic AMP