NS1 binding protein regulates stress granule dynamics and clearance by inhibiting p62 ubiquitination

Abstract NS1 binding protein (NS1-BP), a non-structural NS1-binding protein of influenza A virus, regulates viral or host RNA processing/export, cancer progression, or neurite/dendritic spine regulation. However, its precise roles in stress-induced responses without viral infection are largely unknown. Therefore, this study aims to investigate the novel roles of NS1-BP, which interact with GABARAP subfamily proteins, including LC3-interacting region-containing proteins, in regulating stress granules (SGs) during oxidative stress. NS1-BP interacts with core SG components and localizes to GABARAP-containing SGs during oxidative stress. Moreover, it associates with p62, acting as an adaptor for selective autophagy via its Kelch-motif and ubiquitin-associated domain in p62 in a stress-dependent manner. NS1-BP knockout (KO) HeLa cells demonstrated altered SG dynamics, mirroring observation in p62 KO or GABARAP triple KO cells, indicating impaired autophagic SG degradation. NS1-BP KO cells, compared to wild-type (WT) cells, showed increased p62 ubiquitination, leading to autophagic p62 degradation, while NS1-BP overexpression reduces p62 ubiquitination. In NS1-BP KO cells, overexpression of p62 WT, not p62 K420R or K435R, restored SGs size and number. Additionally, amyotrophic lateral sclerosis (ALS)-induced pluripotent stem cell-derived motor neurons showed reduced NS1-BP levels, resulting in SG morphology dysregulation. Our findings reveal the novel role of NS1-BP in negatively regulating p62 ubiquitination, influencing SG dynamics and clearance during oxidative stress. This highlights its relevance to ALS pathogenesis associated with SGs.