Limited impact of hepatitis A virus 3C protease-mediated cleavage on the functions of NEMO in human hepatocytes.

NF-κB essential modulator (NEMO) is critically involved in the induction of interferons (IFNs) and pro-inflammatory cytokines. Hepatitis A virus (HAV) 3C protease was recently identified to cleave NEMO in non-hepatic cells. This study aimed at understanding efficiency and function of HAV 3C-mediated NEMO cleavage in hepatocytes. HAV 3C protease and its precursor 3CD strongly affected NEMO abundance in ectopic expression models, which was not observed in HAV replicon cells and upon HAV infection. Using a cleavage-resistant NEMO mutant, we found that specific cleavage by 3C only marginally contributed to NEMO degradation, whereas the magnitude of the effect was due to cytotoxic effects induced by 3C activity. Cleavage efficiency generally did not suffice to disrupt the type I IFN or NF-κB signaling pathways. Knockout of NEMO indeed abrogated both pathways, whereas efficient knockdown had limited the impact on NEMO-mediated signaling, suggesting that low levels of NEMO are sufficient to maintain antiviral responses in hepatocytes. NEMO cleavage was barely detectable in a cell line harboring a persistent HAV replicon or in HAV-infected cells. HAV infection induced a robust innate immune response, which was not affected by efficient knockdown of NEMO, arguing for a limited potential contribution of NEMO cleavage to innate immune counteraction. Overall, our data suggest that HAV 3C is capable of partially cleaving NEMO as reported. However, since minute expression levels of NEMO were sufficient for induction of innate immunity, inefficient NEMO cleavage by HAV is unlikely to contribute to dampening of innate immune responses in hepatocytes.IMPORTANCEHepatitis A virus (HAV) establishes acute infections of the liver, which are always cleared, while a number of mechanisms have been identified contributing to immune escape. Among those, proteolytic cleavage of NF-κB essential modulator (NEMO) by HAV has been suggested to counteract innate immune responses. This study demonstrates that the HAV 3C protease cleaves NEMO inefficiently and does not result in substantial disruption of antiviral signaling. Importantly, NEMO remains capable of inducing an effective immune response in hepatocytes even at low expression levels. Our findings suggest a limited role for NEMO cleavage in HAV's interaction with host immunity and call for a revision of our understanding of HAV counteraction mechanisms.