Evasion of RIG-I/MDA5 and TLR3-mediated innate immunity by hepatitis A virus
Since the identification of several families of pattern recognition receptors (PRRs), their roles in the innate immune system and how they are regulated by the invading pathogens have been the subjects of extensive research. Cellular helicases RIG-I and MDA5, and Toll-like receptor 3 (TLR3) are PRRs that detect virus-specific double stranded RNA (dsRNA). Activation of these PRRs by dsRNA lead to their interaction with adaptor proteins, which engage downstream kinases to activate two critical transcription factors, NF-kB and IRF3, in the induction of type I interferons (IFNs) and IFN-stimulated genes (ISGs) that ultimately establish an antiviral state. These signaling pathways are central to host antiviral defense and thus become targets for viral interference. Hepatitis A virus (HAV), a hepatotropic picornavirus, is capable of blocking IRF3 activation and type I IFN expression in cell culture, but the exact mechanism(s) remains undefined. Our studies revealed that HAV disrupts RIG-I/MDA5-mediated induction of type I IFN through proteolysis of MAVS, a mitochondrial-localized adaptor of RIG-I and MDA5, by the viral 3ABC protease precursor. The 3ABC cleavage of MAVS requires both the protease activity of 3Cpro and a transmembrane domain in 3A that directs 3ABC to mitochondria. The signaling ability of MAVS depends on its mitochondrial localization; cleavage of MAVS by 3ABC removes MAVS from mitochondia, thus abolishing its adaptor function. We also demonstrated that the parallel, yet independent, TLR3 signaling pathway is also inhibited by HAV through cleavage of the adaptor protein TRIF by the 3CD protease-polymerase precursor. Cleavage of TRIF by 3CD requires both the protease activity of 3Cpro and the 3Dpol moiety, but not the 3Dpol polymerase activity, in an “in cis” manner. This research also revealed a unique order of processing in the 3CD cleavage of TRIF, and an unexpected role of the 3Dpol domain in modulating the substrate specificity of 3CD that allows it to cleave non-canonical 3Cpro cleavage sites within TRIF. The data generated in this dissertation provide two major mechanisms by which HAV evades innate immune responses, and extend our understanding of the signaling pathways of the innate immune system.