Enhanced SARS-CoV-2 entry via UPR-dependent AMPK-related kinase NUAK2

Severe acquired respiratory syndrome coronavirus 2 (SARS-CoV-2) induces strong remodelling of host cell endoplasmic reticulum (ER) for the formation of its repli-cation organelles, causing ER stress and initiating an unfolded protein response (UPR). Though SARS-CoV-2-induced host cell transcriptional changes mimic UPR-induced changes, yet general UPR is detrimental for infection, highlighting the need to investi-gate the contribution of single UPR pathways to infection. Here, by screening the downstream signalling factors differentially regulated by inhibiting IRE1a-XBP1 during SARS-CoV-2 infection, we discovered novel IRE1a-XBP1 regulated host-dependency factor for SARS-CoV-2 entry, NUAK2. Using biochemical and correlative light and elec-tron microscopy approach, we showed that SARS-CoV-2 infection causes marginal ac-tivation of signalling sensor IRE1a leading to its phosphorylation, clustering, and XBP1 splicing. Ultrastructure of IRE1a clusters showed dense ER membrane rearrangements with embedded cytoplasmic fenestrations, highlighting distinct ultrastructure features under full IRE1a activation during chemically induced UPR versus marginal activation during SARS-CoV-2 infection. Blunting NUAK2 levels with RNA interference or its pharmacological inhibition reduced SARS-CoV-2 infection, binding, and internalisation by decreasing cell surface ACE2 levels. In addition, NUAK2 inhibited cells showed SARS-CoV-2 particles dispersed in the cytoplasm early in infection, indicating a defect in viral trafficking. Since NUAK2 contributes to the maturation of actin cytoskeleton and hence endocytosis and cargo trafficking, collectively our data suggest that NUAK2 pro-motes SARS-CoV-2 binding to cells by maintaining cell surface ACE2 levels and regu-lating viral trafficking.