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La protein binds the predicted loop structures in 3'-noncoding region of Japanese encephalitis virus genome: role in virus replication

National Institute of Immunology

¹ E-mail: vrati{at}nii.res.in

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. We have previously shown that three proteins of 32, 35 and 50 kDa bind the 3'-stem-loop (SL) structure of JEV 3'-NCR, and one of these identified as 36-kDa Mov34 protein. Using electrophoretic mobility-shift and UV-cross-linking assays, and yeast three-hybrid system, we now show that La protein binds the 3'-SL of JEV. The binding was stable under high salt condition (300 mM KCl) and affinity of RNA protein interaction was high; dissociation constant (Kd) for La binding with 3'-SL was 12 nM, indicating that this RNA-protein interaction is physiological plausible. Only N-terminal half of La protein containing RNA-recognition motifs 1 and 2 interacted with JEV RNA. RNA toe-printing assay followed by deletion mutagenesis showed that La protein bound to predicted loop structures in 3'-SL RNA. Further, we show that siRNA-mediated down regulation of La protein resulted in repression of JEV replication in cultured cells.

Received 4 February 2009; accepted 26 February 2009.