ADP-riboxanation upregulates the E3 ISG15 ligase activity of HERC5
To determine the biological function of HERC5 ADP-riboxanation catalyzed by S. flexneri effector OspC1, we overexpressed the ISG15-congjugating system together with WT or MUT OspC1 in 293T cells. As reviewed in the previous publication and observed in our experiment, only free ISG15, but not ISGylation, was induced upon type I IFN stimulation (Zhang et al., 2011; Figure 4a). That being said, the ISGylation observed in the overexpression experiments were all caused by exogenous expression of ISG15-congjugating system (Figure 4b). Comparing to the MUT group, the ISGylation generated in the WT group were much stronger (Figure 4c), which indicated that ADP-riboxanation of HERC5 catalyzed by S. flexneri effector OspC1 promoted the activity of its E3 ISG15 activity.
To further verify this conclusion, we tested the ISGylation of HERC5 itself and RIG-I in the overexpression system in the presence of WT or MUT OspC1, and found that the ISGylation of HERC5 itself was enhanced slightly, whereas the ISGylation of RIG-I was promoted by S. flexneri effector OspC1 significantly. Given that the ISGylation of RIG-I negatively regulates its activation as a feedback mechanism to fine-tune host innate immune response (Kim et al., 2008),Shigella may subvert host innate anti-bacterial defence by hijacking this negative mechanism via OspC1-catalyzed HERC5 ADP-riboxanation, which results in the accumulation of non-functional ISGylated RIG-I in the host cells.