Live-cell single-vRNP imaging identifies viral gene expression signatures that shape influenza infection heterogeneity

Cell-to-cell heterogeneity in infection outcome is a general feature of most viruses, but the underlying mechanisms are poorly understood. Here, we developed a live-cell single-molecule imaging technology to visualize infection by unmodified influenza A viruses (IAVs) with unprecedented resolution. Using this approach, we generated a detailed kinetic map of IAV infection, which identified viral ribonucleoprotein (vRNP) replication, nuclear export, and virion budding as important sources of heterogeneity. Mechanistically, we show that infection heterogeneity is caused by differential viral gene expression signatures, resulting from widespread transcriptional defects and loss of viral genome segments. For example, loss of NS, but surprisingly not polymerase subunits, severely delays replication onset, and loss of M and NS, but not HA, underlies vRNP nuclear export defects. In summary, our work identifies the origin and consequences of infection heterogeneity and provides a broadly applicable technology that allows high-resolution phenotyping of unmodified IAVs and other negative-strand RNA viruses.