Identification of specific structural element in the leader RNA of Chandipura virus and its implication in Phosphoprotein recognition

Abstract

Chandipura virus (CHPV) belongs to the mononegaloviridae family consisting of a nonsegmented RNA genome, tolling hundreds of children in India during 2002–05. Most crucial event in the life cycle of CHPV is the Transcription-Replication Switch; both being carried out by the same viral Polymerase, L. Viral Phosphoprotein phosphorylated at Ser62 (P1) is essential for Transcriptase activity of L whereas the unphosphorylated one (P0) promotes replication. 49-mer viral leader RNA (CHPl), transcribed from 3′-end of the genome, forms two complexes with P0 with nanomolar Kd, depending upon the protein concentration but fails to recognize P1. Here, we show by Dynamic Light Scattering that, P0 undergoes monomer to dimer to higher oligomer formation with increasing protein concentration. The 5′-terminal half of CHPl binds preferentially with the dimer of P0 whereas 3′-half interacts preferentially with the monomeric form as revealed by Tryptophan fluorescence quenching studies. NMR spectroscopic studies of CHPl and structure prediction suggest the presence of a stem-loop structure at the 3′-end of the RNA. Temperature dependence of 1H-1D NMR spectra confirms that CHPl contains a core structure that is stable at least up to 50°C. Effect of Mg2+ on folding of the RNA is also studied. Point mutants in the predicted stem-loop of CHPl indicate the role of different bases in recognizing P0 as evidenced by competitive RNA binding experiments using Gel Retardation Assay. Screening molecules to block this interaction may lead to antiviral agents. DST and CSIR, INDIA supported this study.