MeV Phosphoprotein

The measles virus (MeV) phosphoprotein (P) tethers the polymerase to the nucleocapsid template for transcription and genome replication. The binding of P to nucleocapsid is mediated by the X domain of P (XD) and the conserved sequence (Box-2) within the C-terminal domain of the nucleoprotein (N (TAIL)). XD binding induces N (TAIL) α-helical folding, which in turn has been proposed to stabilize the polymerase-nucleocapsid complex, with cycles of binding and release required for transcription and genome replication. The current work directly assesses the relationships among XD-induced N (TAIL) folding, XD-N (TAIL) binding affinity, and polymerase activity. Amino acid substitutions that abolished XD-induced N (TAIL) α-helical folding were performed within the Box-2 of Edmonston MeV N (TAIL). Polymerase activity in minireplicons was maintained despite a 35-fold decrease in XD-N (TAIL) binding affinity or a reduction in or loss of XD-induced N (TAIL) alpha-helical folding. Recombinant infectious viruses were recovered for all mutants, and transcriptase elongation rates remained within a 1.7-fold range of parent viruses. However, Box-2 mutations did impose a significant challenge to infectivity, reflected in an increase in the amount of input genomes required to match the infectivity of parent viruses. Diminished infectivity could not be attributed to changes in virion protein composition or to the production of defective interfering particles, where changes from parent viruses were within a 3-fold range. The results indicated that MeV polymerase activity, which is not infectious, tolerates amino acid changes in the XD-binding region of the nucleoprotein. Selectional pressure for conservation of the Box-2 sequence may thus reflect a role in assuring the fidelity of polymerase functions or the assembly of viral particles required for optimal infectivity.