The Role of Ariadne RBR E3 ubiquitin protein ligase 2 (ARIH2) in influenza A virus replication and pathogenesis in the airway epithelial cells - PROJECT SUMMARY Influenza A viruses (IAV) pose significant global health threats, causing annual epidemics with substantial morbidity and mortality. The prevailing resistance of the majority of H1N1 and H3N2 strains to antiviral drugs, coupled with the emergence of the highly pathogenic avian H5N1 IAV that has breached species barriers to infect humans, underscores the urgent need for a paradigm shift towards identifying drugs effective across a spectrum of viral strains. The airway epithelium is the primary site for IAV replication. Our prior investigations have highlighted the host cellular protein BIK as a pivotal player in IAV replication within airway epithelial cells (AECs), influencing disease severity in humans. BIK deficiency impedes, while its overexpression exacerbates viral replication, virus-induced lung inflammation, and mortality. Additionally, we have identified a single nucleotide polymorphism in the BIK gene associated with influenza severity. IAVs facilitate their replication in AECs by stabilizing BIK protein through inhibiting proteasomal BIK degradation. Our mass spectrometry analysis has unveiled a novel BIK-interacting E3 ligase, Ariadne RBR E3 ubiquitin protein ligase 2 (ARIH2), which is inhibited by IAV to stabilize BIK. ARIH2 depletion enhances BIK levels and viral replication, while its expression reduces both, identifying ARIH2 as a novel IAV restriction factor exploited by the virus. Our proposal aims to elucidate how IAV manipulates ARIH2 to inhibit proteasome-mediated degradation of BIK, thereby promoting viral replication. We will test the central hypothesis that IAV nucleoprotein (NP) inhibits ARIH2 to block the ubiquitin-dependent degradation of BIK, thereby promoting viral replication. Conversely, ARIH2 mitigates viral burden in AECs by directing BIK towards proteasome-mediated degradation. To test this hypothesis, we proposed three Specific Aims. Aim 1 investigates how IAVs exploit ARIH2 to Inhibit BIK degradation and promote viral replication in the AECs. This Aim focuses on identifying crucial ARIH2 domains and BIK lysine residues for ubiquitination, exploring the impact of ARIH2-mediated BIK degradation on viral replication and inflammatory responses. Aim 2 explores ARIH2's role in mitigating influenza disease progression in vivo. Aim 3 evaluates ARIH2’s therapeutic potential in mitigating viral load and lung inflammation in normal human bronchial epithelial cells and human Precision-Cut Lung Slices. The proposed studies may uncover ARIH2 as a universal and novel target for treating influenza infections, offering therapeutic promise due to its enzymatic nature and potential selectivity towards BIK. These studies will have profound translational implications and hold the potential to enhance prevention and treatment strategies for emerging and reemerging influenza strains.
