Plasticity in structural and functional interactions between the phosphoprotein and nucleoprotein of measles virus

Area of Science:

• Virology
• Molecular Biology
• Structural Biology

Background:

• Measles virus (MeV) phosphoprotein (P) is essential for viral transcription and replication by linking the polymerase to the nucleocapsid template.
• P protein binding to the nucleoprotein (N) is mediated by the P's X domain (XD) and a conserved sequence (Box-2) in N's C-terminal domain (N(TAIL)).
• XD binding induces N(TAIL) alpha-helical folding, proposed to stabilize the polymerase-nucleocapsid complex for efficient viral RNA synthesis.

Purpose of the Study:

• To directly investigate the relationship between XD-induced N(TAIL) folding, binding affinity, and measles virus polymerase activity.
• To assess the impact of mutations in the Box-2 region of N(TAIL) on viral replication and infectivity.

Main Methods:

• Introduction of amino acid substitutions within the Box-2 region of Edmonston MeV N(TAIL) to abolish or reduce XD-induced N(TAIL) alpha-helical folding.
• Assessment of polymerase activity using minireplicon assays.
• Recovery of recombinant infectious MeV and analysis of transcriptase elongation rates, virion protein composition, and defective interfering particle production.

Main Results:

• Mutations in Box-2 that disrupted N(TAIL) folding or reduced binding affinity (up to 35-fold) did not significantly impair polymerase activity or transcriptase elongation rates.
• Recombinant viruses with Box-2 mutations were recovered, but exhibited significantly reduced infectivity, requiring more input genome for comparable spread.
• Diminished infectivity was not attributable to altered virion protein composition or defective interfering particle production.

Conclusions:

• Measles virus polymerase activity tolerates significant alterations in the XD-binding region of the nucleoprotein, including loss of induced N(TAIL) folding.
• The conserved Box-2 sequence is critical for optimal viral infectivity, suggesting a role beyond polymerase activity, potentially in viral particle assembly or stability.
• Selectional pressure for Box-2 conservation likely ensures efficient viral particle formation for high-level infectivity.