# Mitochondrial targeting by measles virus nucleoprotein modulates viral spread in human airway epithelium

## TL;DR

The study reveals that the measles virus uses a specific part of its nucleoprotein to target mitochondria in human airway cells, which helps the virus replicate while avoiding strong immune responses.

## Contribution

The discovery of a novel mitochondrial localization signal in the measles virus nucleoprotein that influences replication and immune evasion.

## Key findings

- Measles virus replication centers form near mitochondria in human airway epithelial cells.
- The nucleoprotein of measles virus contains a mitochondrial localization signal in its amino-terminal region.
- Mutations in specific arginine residues of the nucleoprotein affect viral replication and infectious center formation.

## Abstract

Measles is the most infectious human respiratory virus: on average, one individual with measles infects 12–18 susceptible people in a population without immunity. However, how measles virus (MeV) establishes infection in the human respiratory epithelium is insufficiently understood. Since our analyses of MeV infections of well-differentiated primary human airway epithelial cells (HAE) revealed perturbations of mitochondrial gene expression, we tested mitochondrial function. MeV replication disrupted mitochondrial membrane potential and induced superoxide production. This resulted in cGAS-dependent interferon-stimulated gene expression without interferon induction. We then assessed by differential centrifugation whether MeV replicates in mitochondrial proximity. Indeed, MeV proteins and genome were enriched in mitochondrial fractions. We identified a previously unrecognized potential mitochondrial localization signal (MLS) in the MeV nucleoprotein (N), the first protein expressed during infection and showed that the first 70 amino acids of N are sufficient to deliver a GFP reporter to mitochondria. Mutational analyses revealed that arginine 6 and arginine 13 of the N protein are critical for targeting. Recombinant MeV mutants harboring single MLS amino acid substitutions exhibited altered replication kinetics and infectious center formation in HAE, despite similar ISG expression profiles to wild-type MeV. Thus, the MeV N protein amino-terminal arm, previously known only to promote formation of the helical ribonucleocapsid protecting the viral genome, also codes for an MLS. In newly infected cells, this signal may target the formation of MeV replication factories near mitochondria without provoking a canonical RNA sensing pathway. Notably, the MLS appears unique to Morbillivirus N proteins within the Paramyxoviridae family, which are also distinguished by the unique ability to form infectious centers in HAE. Our findings reveal a novel mechanism by which MeV exploits mitochondrial proximity to coordinate replication and modulate host responses, offering new insights into virus-host interactions at the organelle level.

Measles is the most infectious human respiratory virus but the mechanisms favoring its spread in the human respiratory epithelium are incompletely understood. Prompted by our observation of altered mitochondrial gene expression after measles virus (MeV) infection of well-differentiated primary human epithelial cells, we characterized the mechanisms by which MeV interacts with mitochondira in this ex vivo system. We discovered that MeV replication centers form near mitochondria and that the virus’s nucleoprotein (N) contains a previously unrecognized mitochondrial localization signal (MLS) in its amino-terminal arm. Mutational analysis revealed that specific arginine residues are essential for mitochondrial targeting and may influence viral replication dynamics. Despite proximity to mitochondria, MeV induces only a limited innate immune response. Our findings provide new insights into how MeV may exploit mitochondrial membranes to facilitate early replication and potentially modulate host immunity and cell-to-cell spread. This work highlights a novel function of the MeV N protein and discloses a new process by which a virus interacts with mitochondria to balance replication and immune evasion.