Structural Difference Recognized by a Monoclonal Antibody #404‐11 between the Rabies Virus Nucleocapsid (NC) Produced in Virus Infected Cells and the NC‐Like Structures Produced in the Nucleoprotein (N) cDNA‐Transfected Cells

Abstract

The negative-stranded RNA genome of the rabies virus encodes at least five species of structural proteins, nucleoprotein (N), nominal phosphoprotein (P), matrix protein (M), glycoprotein (G) and a large catalytic protein (L) in this order from its 3'-end to 5'-end. M and G proteins comprise the protein moiety of the viral envelope, while P and L proteins collaborate in viral RNA synthesis as non-catalytic and catalytic components of the viral RNA polymerase, respectively. The N protein is a virus-coded RNA-binding protein. Abstract: We investigated structural changes in the rabies virus (HEP-Flury strain) nucleocapsid (NC) during the virus replication, for which we used two anti-nucleoprotein (N) monoclonal antibodies (mAbs), #404-11 (specific for a conformation-dependently exposed linear epitope) and #1-7-11 (specific for a conformational epitope which is exposed after the nucleocapsid formation). Both mAbs recognized the N protein of the viral NC, but not of the RNA-free N-P complex. The 1-7-11 and 404-11 epitopes could be mapped to the N-terminal and the C-terminal regions of N protein, respectively. Immunoprecipitation studies demonstrated that treatment of the NC either with the alkaline phosphatase or sodium deoxycholate (DOC) resulted in dissociation of most P proteins from the NC and in the reduced reactivity to mAb #404-11, but not to mAb #1-7-11. NC-like structures produced in the N cDNA-transfected cells displayed strong reactivity to mAb #1-7-11; however, reactivity to mAb #404-11 was very weak. And, coexpression with viral phosphoprotein (P) resulted in little increase in reactivity to mAb #404-11 of the NClike structures, while the reactivity was significantly increased by cotransfection with P and the viral minigenome whose 3'-and 5'-end structures were derived from the viral genome. From these results, we assume that, although the 404-11 epitope is a linear one, the epitope-containing region is exposed only when N proteins encapsidate properly the viral RNA in collaboration with the P protein. Further, exposure of the 404-11 epitope region might be function-related, and be regulated by association and dissociation of the P protein.

Definition of the immune evasion-replication interface of rabies virus P protein

“…The affinity between N-pep and P CTD was made significantly stronger through cyclization. The idea of a conformational change and folding upon the N CTD binding with P CTD [60] is consistent with previous analysis showing that a monoclonal antibody against N bound exclusively in the context of an N-RNA complex with P, with the epitope mapped to the N CTD loop region [61]. The antibody did not recognize the epitope, mapped to residues 388-407, when P was removed from the complex, or in RNA-free N 0 protein, which binds to the N-terminal region (residues 23-50) of P protein, such that P protein provides a chaperone function for newly synthesized N protein prior to encapsidation of the viral RNA [61].…”

Structure of the vesicular stomatitis virus nucleocapsid in complex with the nucleocapsid-binding domain of the small polymerase cofactor, P

“…The contacts involve the elongated N terminus and an extended loop (C loop) within the C-terminal lobe, and they are required for RNA encapsidation (6). Residues within this loop have also been implicated in binding to the P (7,8). Recently, more insight into capsid formation was gained through crystallographic studies of an N protein with a serine-to-tryptophan mutation at residue 290 (called N290; ref.…”

Characterization of conformation-specific monoclonal antibodies against rabies virus nucleoprotein

“…Furthermore, anti-N Mabs have also been used to differentiate RABV strains isolated in different animal species and/or from different geographic locations [4] as well as to probe the structure–function relationship of the N protein [1, 21]. In the present study, we characterized three anti-N Mabs and found that two of them were directed against conformational epitopes.…”

“…Linear and conformational Mabs to RABV N have been developed previously [4, 12, 16, 21]. While Mabs to linear epitopes are suitable for diagnosis, Mabs to linear and conformational epitopes have been useful in probing the structure of the N protein, including identifying epitopes [3] and the site of phosphorylation [12].…”