The novel mitochondria localization of influenza A virus NS1 visualized by FlAsH labeling

The nonstructural protein 1 (NS1) of the influenza A virus (IAV) is a multifunctional protein that counteracts host cell antiviral responses and inhibits host cell pre-mRNA processing. NS1 contains two nuclear localization signals that facilitate NS1 shuttling between cytoplasm and nucleus. In this study, we initially observed the novel mitochondria localization of NS1 in a subset of transfected cells. We then further monitored the localization dynamics of the NS1 protein in live cells infected with IAV expressing NS1 with insertion of a tetracysteine-tag. The resulting mutant virus showed similar levels of infectivity and expression pattern of NS1 to those of wild-type IAV. Pulse labeling using a biarsenical compound (fluorescein arsenical hairpin binder) allowed us to visualize the dynamic subcellular distribution of NS1 real time. We detected NS1 in mitochondria at a very early infection time point [1.5 h postinfection (hpi)] and observed the formation of a granular structure pattern in the nucleus at 4 hpi. This is the first identification of the novel mitochondria localization of NS1. The possible role of NS1 at an early infection time point is discussed.