Novel mechanisms of antiviral innate immune regulation by the hepatitis C virus NS3-NS4A protease
Hepatitis C virus (HCV) evasion of the host immune system is largely mediated by the actions of the HCV NS3-NS4A protease complex, which consists of the serine protease and RNA helicase NS3 and its membrane targeting co-factor NS4A. NS3-NS4A has multiple functions in the HCV life cycle, with roles in both HCV replication and regulation of innate immune signaling. To regulate innate immune signaling, NS3-NS4A inactivates multiple signaling proteins, including MAVS, an adaptor protein in the RIG-I antiviral signaling pathway, and Riplet, an E3 ubiquitin ligase that activates RIG-I. Inactivation of these host proteins results in an inhibition of downstream signaling through the transcription factor IRF3 and inhibition of the subsequent induction of IFN-β. What directs the multiple functions of NS3-NS4A throughout the HCV life cycle is largely undetermined. Here, we identify a tyrosine residue within the transmembrane domain of NS4A that uncouples the various function of NS3-NS4A.
First, to uncouple the roles of NS3-NS4A in replication and immune evasion, I focused on the NS4A transmembrane domain and generated an NS4A mutant (Y16F) in a full-length HCV infectious clone, a subgenomic replicon, and an over-expression construct. I then assessed viral replication of HCV wild-type (WT) and Y16F viruses by measuring replication of a subgenomic HCV replicon in two related liver hepatoma cell lines: Huh7, which have functional RIG-I signaling, and Huh-7.5 cells, which lack functional RIG-I signaling. The HCV Y16F virus replicated to similar levels as WT HCV in Huh-7.5 cells. However, in Huh7 cells, replication of HCV Y16F was decreased compared to the HCV WT. I used CRISPR-Cas9 gene editing to delete proteins in the RIG-I pathway, including RIG-I, MAVS, and IRF3, in Huh7 cells, infected these cells with HCV WT or Y16F viruses, and then measured virus replication. I found that Y16F viral replication was not restored to the levels of WT in Huh7-RIG-I KO cells or the Huh7-MAVS KO cells, but it was restored to the levels of WT in the Huh7-IRF3 KO cells. I also found that the HCV NS3-NS4A Y16F mutation reduced the ability of over-expressed NS3-NS4A to block IRF3 activation, as measured through nuclear translocation via immunofluorescence microscopy. Further the NS3-NS4A Y16F mutation also had a reduced ability to block the induction of interferon-stimulated genes during both HCV replication and infection. This reveals that HCV NS4A Y16 can regulate a RIG-I-independent, yet IRF3-dependent, signaling pathway that limits viral replication.
Second, to further characterize this RIG-I-independent, IRF3-dependent signaling pathway, I examined the interactions of HCV NS3-NS4A with two of its known host substrates, MAVS and Riplet. To test whether the Y16F mutation prevented NS3-NS4A cleavage of MAVS, I performed a MAVS cleavage assay during both overexpression of NS3-NS4A and MAVS and also during infection with HCV WT and Y16F viruses. NS3-NS4A Y16F was able to cleave MAVS just like WT during both conditions. Next, I found that over-expression of NS3-NS4A WT changed Riplet intracellular localization and that NS4A interacted with Riplet. However, the NS4A Y16F mutation prevented NS4A-Riplet interactions in both of these contexts. Interestingly, I found that Huh-7.5 cells express lower levels of Riplet protein and mRNA compared to Huh7 cells. When full-length Riplet was added exogenously to Huh-7.5 cells, HCV Y16F virus replication was reduced compared to WT. However, when a Riplet construct missing the RING domain, which is essential for Riplet signaling, was added exogenously to Huh-7.5, both WT and Y16F viruses now replicated similarly. Taken together, these data identify NS3-NS4A Y16 as important for regulating a previously uncharacterized Riplet-mediated signaling pathway that limits HCV infection.
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