La protein can simultaneously bind to both 3'- and 5'-noncoding regions of Japanese encephalitis virus genome

Abstract

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with noncoding regions (NCRs) of 95 and 585 bases at its 5' and 3' ends, respectively. These may interact with viral or host proteins important for viral replication. We have previously shown that La protein binds the 3'-stem-loop (SL) structure of JEV 3'-NCR. Using electrophoretic mobility shift and ultraviolet crosslinking assays, we now show that La protein binds both 3'-SL and 5'-NCR of JEV. La protein binding to 5'-NCR RNA was stable under high salt condition (300 mM KCl) and the affinity of RNA protein interaction was high; the dissociation constant (K_d) for La binding with 5'-NCR RNA was 8.8 nM, indicating the physiological relevance of the interaction. RNA toe-printing assays showed that La protein interacted with nucleotides located in the top loop of the predicted structure of 5'-NCR RNA. Using competitive binding studies and 5'-3' coprecipitation assay, we have demonstrated that La protein could simultaneously bind both JEV 3'- and 5'-NCRs. This may help circularize the viral genome for its efficient transcription and translation.