The Host Ubiquitin E3 Ligase ARIH2 Reduces Influenza A Virus Replication in the Airways and Mitigates Disease Progression in Mice

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Abstract Rationale: Influenza A virus (IAV) poses a major public health threat, causing severe lung inflammation. Due to frequent mutations, IAVs often develop resistance to current antiviral treatments. Hence, to identify potential alternative therapies, it is crucial to understand how IAVs interact with host proteins. Our previous research showed that IAV induces the host cellular protein Bcl-2 interacting killer (BIK), promoting viral replication in airway epithelial cells (AECs). However, the precise mechanism remains unclear. Here, we further investigated how IAV hijacks the host cellular proteins to promote viral replication and exacerbate lung inflammation. Methods: Human precision-cut lung slices (hPCLS) were transduced with adeno-associated viral (AAV) vectors expressing EGFP or ARIH2, followed by IAV infection. To explore the translational potential, ARIH2 was delivered intranasally via AAV6.2 vector in C57BL/6 mice followed by IAV-infection, and the impact on lung inflammation and survival was evaluated. Lung histology was assessed using hematoxylin and eosin (H&E) staining. Western blot, co-immunoprecipitation, ubiquitination assays, mass spectrometry (MS), and immunofluorescence were performed to assess protein expression. Luminex multiplex assays quantified inflammatory cytokines/chemokines in lung homogenates and supernatants of IAV-infected mice and hPCLS. Results: MS analysis identified ARIH2 as the primary BIK-interacting E3 ligase suppressed by IAV to stabilize BIK. This finding was supported by the observation that decreasing ARIH2 levels increased BIK protein levels in IAV-infected AECs. IAV nucleoprotein, distinct from other viral ribonucleoprotein components, specifically inhibited ARIH2 and induced BIK. Overexpression of ARIH2 reduced BIK levels and diminished IAV yield. Conversely, ARIH2 depletion led to increased BIK protein levels. Furthermore, ARIH2 showed a robust interaction with BIK. IAV inhibited ARIH2-mediated K48-linked BIK ubiquitination. On the contrary, ARIH2 overexpression in IAV-infected cells restored BIK ubiquitination and dampened IAV-induced pro-inflammatory cytokines, affirming that IAV interferes with ARIH2-mediated BIK degradation. In hPCLS, IAV infection suppressed ARIH2, thereby increasing BIK protein levels, while AAV-ARIH2 treatment significantly reduced viral load, underscoring its significant translational implications. In vivo, intranasal delivery of AAV-ARIH2 reduced lung viral load and inflammation, thereby reducing IAV-induced morbidity and mortality. Conclusion: ARIH2 emerges as a novel host cellular protein exploited by IAV to inhibit BIK degradation, thereby promoting viral replication. Enhancing ARIH2-mediated BIK ubiquitination presents a promising novel therapeutic strategy to reduce IAV replication and lung inflammation.

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