Abstract

The nucleocapsid (N) protein and the phosphoprotein (P) of VSV play critical roles in viral gene expression. The N protein encapsidates the viral RNA to form N-RNA or nucleocapsid (NC) complex that serves as the template for genome transcription and replication by the viral RNA-dependent RNA polymerase (RdRp), a complex of the viral P and L proteins. To examine the NCs functions in infected cells, the NCs were fluorescently tagged. This was attempted by replacing the coding regions of VSV N and P proteins with those for enhanced green fluorescence protein-fused N (NeGFP) or eGFP-fused P (PeGFP) proteins. Although multiple attempts to recover recombinant VSV encoding NeGFP were unsuccessful, recombinant VSV encoding PeGFP (VSV-PeGFP) was readily recovered. The NCs of VSV-PeGFP could be visualized as green in infected cell cytoplasm since PeGFP was associated with the NCs. Using VSV-PeGFP, we demonstrated that the cellular microtubules are involved in intracellular trafficking of newly synthesized viral NCs. To examine the role of the N protein in template functions of N-RNA complex, we identified a highly conserved region spanning residues (282)GLSSKSPYSS(291) in the N protein of Rhabdoviruses and subjected it to alanine-scanning mutagenesis. Analysis of the mutants demonstrated that the tyrosine residue at position 289 (Y289) is critical for replication functions of the protein. To determine the molecular basis of the defect in Y289A mutant in replication, we examined the interaction of the N protein with itself, with the P protein, and its ability to encapsidate the viral RNA. The mutant Y289A was found to interact with the N protein efficiently but its interaction with the P protein or with the viral RNA was defective. We have also observed that several single amino acid substitutions in this highly conserved region of N, rendered the resulting nucleocapsid template nonfunctional in transcription without adversely affecting replication functions. These results suggest that the structure of the N protein and the resulting N-RNA complex regulate the viral template functions in transcription and replication.