C19-06 Targeting The Arih2-bik Axis: A Host-directed Therapeutic Strategy Against Influenza A Virus

Background Influenza A virus (IAV) remains a serious global health threat requiring host-directed therapeutics due to rising antiviral resistance. We previously identified the pro-apoptotic protein Bcl-2-interacting killer (BIK) as a proviral factor; Bik-deficient mice are protected from severe IAV outcomes. Furthermore, a genetic variation (rs738276) in BIK, which influences its expression, correlates with altered viral replication and human influenza severity. We found that IAV stabilizes BIK by inhibiting its proteasome-mediated degradation. We hypothesized that IAV targets specific E3 ubiquitin ligase responsible for BIK ubiquitination to promote replication. This study identifies the BIK-binding E3 ubiquitin ligase targeted by IAV and evaluates its therapeutic potential against IAV infection in vitro, in vivo, and ex vivo. Methods C57BL/6 wild-type (WT) and Bik knockout (Bik-/-) mice were used for in vivo studies. We employed mass spectrometry (MS), Western blot, co-immunoprecipitation, and ubiquitination assays to identify BIK-regulating E3 ligase and assess protein interactions. The proposed regulatory axis was confirmed using loss- and gain-of-function experiments in cell models. The therapeutic potential of ARIH2 was tested ex vivo using human precision-cut lung slices (hPCLS) transduced with AAV-ARIH2 and subsequently infected with IAV. In vivo, ARIH2 was delivered intranasally via AAV6.2 to mice prior to IAV infection. Therapeutic impact was evaluated by assessing viral load, lung inflammation (H&E histology and Luminex multiplex assay), and overall mouse survival. Results Compared to WT controls, bik-/- mice infected with IAV displayed significantly higher survival, less weight loss, markedly lower lung viral loads and attenuated lung inflammation. Mass spectrometry identified the RBR E3 ubiquitin ligase, ARIH2, as the primary IAV-suppressed BIK-interacting E3 ligase. This was supported by decreased ARIH2 and increased BIK protein levels in IAV-infected cells. We found that IAV nucleoprotein (NP) specifically suppresses ARIH2 expression, leading to BIK stabilization. This antagonism impairs ARIH2-mediated K48-linked ubiquitination of BIK. Loss- and gain-of-function experiments confirmed this regulatory axis: ARIH2 depletion stabilized BIK, while its overexpression promoted BIK degradation, reducing viral titers, and dampening inflammatory cytokine release. Therapeutically, AAV-mediated ARIH2 delivery significantly reduced BIK accumulation and lowered viral load in hPCLS. In vivo, intranasal AAV-ARIH2 administration reduced lung viral load and inflammation, resulting in improved survival against lethal IAV infection. Conclusion ARIH2 is a novel host antiviral factor restraining IAV by targeting BIK for proteasomal degradation. This study reveals a previously unrecognized viral strategy where IAV NP disables an E3 ligase and highlights the ARIH2-BIK axis as a promising target for host-based influenza therapeutics. This abstract is funded by: NIH (R01AI187330; R01AI148180)