La protein binds the predicted loop structures in the 3′ non-coding region of Japanese encephalitis virus genome: role in virus replication

Abstract

Japanese encephalitis virus (JEV) genome is a single-stranded, positive-sense RNA with non-coding regions (NCRs) of 95 and 585 bases at its 5′ and 3′ ends, respectively. These may bind to viral or host proteins important for viral replication. It has been shown previously that three proteins of 32, 35 and 50 kDa bind the 3′ stem–loop (SL) structure of the JEV 3′ NCR, and one of these was identified as 36 kDa Mov34 protein. Using electrophoretic mobility-shift and UV cross-linking assays, as well as a yeast three-hybrid system, it was shown here that La protein binds to the 3′ SL of JEV. The binding was stable under high-salt conditions (300 mM KCl) and the affinity of the RNA–protein interaction was high; the dissociation constant (Kd) for binding of La protein to the 3′ SL was 12 nM, indicating that this RNA–protein interaction is physiologically plausible. Only the N-terminal half of La protein containing RNA recognition motifs 1 and 2 interacted with JEV RNA. An RNA toe-printing assay followed by deletion mutagenesis showed that La protein bound to predicted loop structures in the 3′ SL RNA. Furthermore, it was shown that small interfering RNA-mediated downregulation of La protein resulted in repression of JEV replication in cultured cells.