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.
Subject(s)
Molecular Chaperones/physiology , Nucleocapsid/chemistry , Phosphoproteins/physiology , Protein Folding , Rhabdoviridae/chemistry , Viral Structural Proteins/physiology , Light , Protein Conformation , Recombinant Proteins/pharmacology , Scattering, Radiation , Sorbitol/pharmacologyABSTRACT
The phosphoprotein P of Chandipura (CHP) virus, an Indian isolate of rhabdovirus, was found to support transcription upon phosphorylation by casein kinase II (CKII). A phosphorylation-induced change in the protein conformation was found to occur at the N-terminal region of the protein. Biochemical studies for further characterization of this phosphorylation-based conformational alteration demonstrated that phosphorylation leads to the transition from an 'open' to 'closed' structure of the protein. The phosphate group introduced by CKII was found to be resistant to phosphatases. This phosphorylation-based structural alteration changes the accessible hydrophobic surface area of the protein and also the available digestion sites of different proteases. The phosphorylated form of P protein was found to be a dimer by His-tag dilution assay. Using the same approach it was found that the N-terminal 46 amino acids are responsible for P-P dimerization, only after phosphorylation.
Subject(s)
Phosphoproteins/chemistry , Vesiculovirus/physiology , Viral Structural Proteins/chemistry , Binding Sites/genetics , Casein Kinase II , Chymotrypsin/chemistry , Dimerization , Molecular Chaperones , Mutagenesis, Site-Directed , Peptide Mapping , Phosphoproteins/genetics , Phosphoric Monoester Hydrolases/chemistry , Phosphorylation , Protein Binding/genetics , Protein Conformation , Protein Serine-Threonine Kinases/chemistry , Trypsin/chemistry , Viral Structural Proteins/geneticsABSTRACT
It has previously been shown that phosphorylation of P protein of vesicular stomatitis virus as well as Chandipura (CHP) virus is required for transcription activation and replication switch. The structural nature of this crucial conformational change, however, is largely unknown. We have studied the phosphorylation-associated conformational change in the P protein of Chandipura (CHP) virus using chemical modification, fluorescence, and circular dichroism spectroscopy. Sulfhydryl groups of unphosphorylated CHP-P protein are unreactive to DTNB under nondenaturing conditions. Upon phosphorylation, one sulfhydryl group becomes reactive. We have identified this sulfhydryl group as cysteine 57. The two tryptophan residues (105 and 135) become significantly more buried in the phosphorylated protein. Circular dichroism spectra show significant enhancement in the far-UV region upon phosphorylation. Anisotropy decay of AEDANS-labeled C57 CHP-P protein shows rapid rotation of the probe, suggesting significant mobility of the N-terminal domain in the phosphorylated P protein. The results suggest a global conformational change in the N-terminal domain of the P protein is induced by phosphorylation and yet the phosphorylated N-terminal domain shows significant flexibility.
Subject(s)
Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phosphoproteins , Viral Structural Proteins/chemistry , Anilino Naphthalenesulfonates/chemistry , Circular Dichroism , Fluorescent Dyes/chemistry , Humans , Naphthalenesulfonates/chemistry , Phosphorylation , Protein Conformation , Rhabdoviridae/chemistry , Spectrometry, Fluorescence , Sulfhydryl Reagents , Tryptophan/chemistry , Vesicular stomatitis Indiana virus , Viral Structural Proteins/metabolismABSTRACT
Bacterially expressed unphosphorylated P protein of Chandipura Virus was found to be efficiently phosphorylated in vitro by casein kinase II (CKII). The phosphorylated form of the P protein supported the transcription in vitro but the unphosphorylated form could not. Kinetic data suggests that CKII incorporates one molecule of phosphate. Western blotting with monoclonal antibody against phosphoserine and phosphoaminoacid analysis confirmed that the phosphate accepting residue was serine. Comparison with P protein of other viruses and tryptic digest of the phosphorylated protein predicted the ser62 was the probable site for phosphorylation. This was further confirmed by substituating ser62 with alanine by site-directed mutagenesis. CKII was unable to phosphorylate the mutated P protein which in turn could not support the transcription in vitro. The phosphorylated P protein eluted from the gel filtration at the position of its dimer in contrast to the unphosphorylated protein which eluted as monomer.
