Disruption of TLR3 Signaling Due to Cleavage of TRIF by the Hepatitis A Virus Protease-Polymerase Processing Intermediate, 3CD

Abstract

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3Cpro, that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3Cpro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3Cpro and downstream 3Dpol sequence, but not 3Dpol polymerase activity. Cleavage occurs at two non-canonical 3Cpro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3Dpol sequence modulates the substrate specificity of the upstream 3Cpro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3Cpro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

Human pDCs preferentially sense enveloped hepatitis A virions

“…Consistent with this, the TLR7-specific inhibitor, IRS-661, but not a control oligonucleotide, markedly blocked IFN-α production ( Figure 1D). We confirmed that pDCs were the responsible cell type by demonstrating a marked reduction in IFN-α production when peripheral mononuclear cells were depleted of BDCA4 + cells prior to cocultivation with infected cells type I IFNs (14)(15)(16)(17). Despite this, unlike the liver in acute hepatitis A, intrahepatic type I ISG expression is often robust in both acute and chronic hepatitis C (12,(18)(19)(20).…”

“…It is interesting to note, however, that the viral RNA present within the activating eHAV particles was predominantly if not exclusively of genome length ( Figure 2D), consistent with it being entirely encapsidated. The lack of a strong type I ISG response within the liver during acute hepatitis A (12) can be attributed to the absence of pDCs within the liver during the peak phase of the infection coupled with the cleavage of both MAVS and TRIF by HAV proteases (14,15). The signals that result in the initial recruitment of pDCs to the liver early in the course of the infection ( Figure 4) are unknown.…”

Acute hepatitis A virus infection is associated with a limited type I interferon response and persistence of intrahepatic viral RNA

“…S1A), the data presented here indicate that HAV must also possess potent mechanisms to disrupt IFN synthesis. This is supported by in vitro studies that show that HAV infection blocks the capacity of superinfecting viruses to induce type I IFN through the RIG-I/MDA5 pathway (36,37). We have shown that this is due to the HAV 3ABC protease, which is directed to the mitochondrial membrane where it cleaves MAVS (36).…”

“…We have shown that this is due to the HAV 3ABC protease, which is directed to the mitochondrial membrane where it cleaves MAVS (36). Recent data also indicate that the 3CD protease-polymerase processing intermediate disrupts TLR3 signaling by directing the cleavage of TRIF (37). HAV and HCV thus have evolved in a remarkably congruent fashion to target these signaling pathways and disrupt their ability to induce type I IFN synthesis.…”

TRIF-Mediated TLR3 and TLR4 Signaling Is Negatively Regulated by ADAM15

“…Recently, the 3CD protease-polymerase from hepatitis A virus was shown to disrupt TLR3-mediated activation of IRF3 and IFN-b promoter activation by targeting TRIF for degradation (48). Thus, it is clear that targeted degradation of TRIF, certainly by viruses, serves as a strategy to curtail antiviral immune signaling and as a more global mechanism to control inflammatory responses to pathogens.…”