Genetic modification of primary airway epithelial cell cultures defines MX1 as a barrier for zoonotic influenza A viruses in the human respiratory epithelium

Genetic modification of primary human airway epithelial cell cultures has identified MX1 as a barrier to zoonotic influenza A viruses. In humans, the interferon-stimulated MX1 gene produces MX1 protein, which hinders the replication of zoonotic IAVs. Resistance to MX1 is seen as a key trait of human-adapted IAVs and a factor in zoonotic and pandemic potential.

However, studies on MX1-mediated restriction and viral escape have mostly been done in cell lines and animal models, which don't fully represent the human airway epithelium—the primary site of IAV replication.

Primary human airway epithelial cell cultures, like airway organoids and air-liquid interface cultures, offer a more realistic model for studying MX1 restriction. Yet, challenges in efficiently modifying these cultures have hindered research in this area. To address this, researchers employed engineered virus-like particles (eVLPs) to introduce Cas9-sgRNA ribonucleoprotein complexes into primary hAECs, effectively knocking out MX1 expression.

This method achieved a 90% genomic-level editing efficiency and near-complete loss of MX1 protein, while maintaining epithelial differentiation at the air-liquid interface.

When MX1 was knocked out in hAEC ALI cultures, the replication of the human-adapted H1N1pdm09 strain showed only a slight increase. However, the zoonotic bovine H5N1/Texas and avian H7N9/Anhui strains exhibited significantly impaired interferon lambda-mediated restriction, with viral titers up to 3-4 logs TCID50 higher in MX1-knockout cultures compared to MX1-competent cultures.

These findings provide direct evidence that MX1 restricts zoonotic IAVs in primary human airway epithelium, highlighting the importance of this gene in assessing the sensitivity of emerging IAVs to antiviral host factors and informing evaluations of their zoonotic and pandemic risk.