Effect of Osmolytes and Chaperone-like Action of P-protein on Folding of Nucleocapsid Protein of Chandipura Virus

Amitabha Majumder,Soumen Basak,Tamal Raha,Santanu Pal Chowdhury,Dhrubajyoti Chattopadhyay,Siddhartha Roy

doi:10.1074/jbc.m011705200

Abstract

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses. The protein plays a common functional role in different RNA viruses by enwrapping the viral genomic RNA in an RNase-resistant form. Upon expression of the nucleocapsid protein alone in COS cells and in bacteria, it forms large insoluble aggregates. In this work, we have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents. The biological activity of the soluble recombinant protein has been tested, and it was found to possess efficient RNA-binding ability. The state of aggregation of the recombinant protein was monitored using light scattering. In the absence of nonionic detergents, it formed large aggregates. Aggregation was significantly reduced in the presence of osmolytes such as d-sorbitol. Aggregate formation was suppressed in the presence of another viral product, phosphoprotein P, in a chaperone-like manner. Both the osmolyte and phosphoprotein P also suppressed aggregation to a great extent during refolding from a guanidine hydrochloride-denatured form. The function of the phosphoprotein and osmolyte appears to be synergistic to keep the N-protein in a soluble biologically competent form in virus-infected cells.

Highlights

Amino acid sequences of nucleocapsid proteins are mostly conserved among different rhabdoviruses
We have reported for the first time the full-length cloning of the N gene of Chandipura virus and its expression in Escherichia coli in a soluble monomeric form and purification using nonionic detergents
In vesicular stomatitis virus (VSV), it was observed that interaction of the Nprotein with the P-protein keeps the N-protein in a soluble form in vivo that is capable of enwrapping the de novo synthesized genomic RNA

Summary

EXPERIMENTAL PROCEDURES

Materials—Pfu DNA polymerase and T4 DNA ligase were from New England Biolabs (Beverly, MA). The soluble and urea fractions were analyzed by 10% SDSpolyacrylamide gel electrophoresis, followed by Coomassie Blue staining Different parameters such as IPTG concentration, temperature, and duration of induction were varied to maximize expression of the N-protein in soluble form. Western Blot Analysis—Proteins were subjected to 10% SDS-polyacrylamide gel electrophoresis, and Western blotting was performed with mouse polyclonal anti-CHPV antibody as the primary antibody and alkaline phosphatase-conjugated goat anti-mouse IgG as the secondary antibody This was followed by color reaction with 4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate. For DLS experiments, the concentration of CHPV N-protein was kept at 1 mg/ml, and the concentration of D-sorbitol was kept at 250 mM In this experiment, the sample was illuminated with a 638.8 helium-neon solid-state laser, and the intensity of light scattered at an angle of 90° was measured. The hydrodynamic radius (RH) of the sample particles was derived from DT using Stokes-Einstein’s equation: DT ϭ kBT/6␲␩RH, where kB is the Boltzmann constant, T is the absolute temperature in degrees Kelvin, and ␩ is the solvent viscosity

RESULTS

DISCUSSION

Methods