Monomer and dimer of Chandipura virus unphosphorylated p-protein binds leader RNA differently: implications for viral RNA synthesis

Abstract

Interaction of the leader RNA with the unphosphorylated P-protein has been proposed to play a key role in the transcription-replication transition of Chandipura virus, a model rhabdovirus. Electrophoretic mobility shift assay with the leader RNA and the unphosphorylated P-protein demonstrated existence of two distinct complexes in vitro. Measurements of stoichiometry indicate the protein monomer/RNA ratio to be 1 : 1 and 2 : 1 for faster and slower migrating bands, respectively. We have also observed a concentration-dependent oligomerization of the unphosphorylated P-protein, in sub-micromolar to low micromolar range. Sedimentation velocity, dynamic light scattering and large zone gel filtration experiments suggest a monomer-dimer-tetramer model of association. RNA binding experiments suggest that the two complexes assembled from one molecule of the leader RNA binding to either a protein monomer or a dimer. A truncated RNA consisting of a 3' region of the leader transcript exclusively formed the 1 : 1 complex, whereas a RNA consisting of only the 5' region forms the 2 : 1 complex exclusively. RNA binding experiments at different protein concentrations suggest that binding of the RNA comprising the 3' region weakens significantly at higher P₀ concentrations, whereas in contrast the binding of the RNA comprising the 5' region becomes modestly tighter. Implications of two different types of leader RNA-P-protein complexes in viral RNA synthesis are discussed.