To tackle these questions, we generated transgenic mice with hepatocyte-specific expression of HCV NS3/4A proteins. Immunohistochemistry shows the presence of NS3 protein in about 89% of the hepatocytes. The viral serine protease indeed cleaves MAVS, and only 21% hepatocytes in transgenic mice show the intact protein. Does this MAVS cleavage block the IFNα/β pathways and the innate immunity in the liver? We examined this question with an i.v. bolus of vesicular stomatitis virus (VSV), which elicits a rapid, RIG-I-dependent IFNα/β response in mice. To our surprise, NS3 transgenic mice mount strong IFNβ and IFN-stimulated gene (ISG) responses and promptly clear VSV in the liver. We also noticed that transgenic mice, when challenged, show strong ISG activation, which isn't necessarily proportional to their relative IFNβ induction. We speculate that the involvement of the IFN-positive feedback loop in the neighboring bystander hepatocytes and hepatic immune cells could partially compensate for NS3-mediated MAVS cleavage in the liver. With respect to HCV in patients, the hepatic distribution of HCV RNA has been reported to be focal, whereas ISG induction appears all across the liver.
The HCV genome contains the pathogen-associated molecular pattern (PAMP) sequence for RIG-I recognition. To further define the function of HCV-mediated MAVS cleavage, we hydrodynamically delivered HCV genomic RNA to the liver of our transgenic mice. Similar to VSV, HCV RNA results in a potent induction of IFNβ and ISG responses in the liver. Unexpectedly, however, the HCV RNA injection renders the NS3 protein undetectable. After ruling out the possibilities of a blockage in transcription and de novo protein synthesis, as well as immunoproteasome-mediated degradation, we hypothesized that an accelerated NS3 protein turnover is linked to the type I IFN response in the liver. To test this notion, we injected transgenic mice with recombinant IFNα or -β. IFNα infusion does not change the levels of NS3 expression, whereas IFNβ leads to a complete sequestration of NS3 in the liver. Protein blot analyses of LC3/Atg8 led us to the possibility that IFNα challenge induces an upregulation of autophagy. IFNβ injection results in decreased levels of both LC3-I and -II, indicative of enhanced autophagy as well as autolysosomal proteolysis. The blockade of this proteolysis by chloroquine prevents NS3 loss in the liver. Intriguingly, IFNβ or HCV RNA injection to a different strain of transgenic mice expressing the HCV structural proteins also renders the viral core protein undetectable; the same treatment, however, does not affect the levels of the Cre recombinase, a cytosolic transgenic protein, in the liver of the HCV mice. These results indicate that IFNβ targets HCV proteins to autophagic pathways, possibly due to their close association with the mitochondria or ER membranes.
HCV survives in an environment that is under constant immune pressure. To understand the mechanistic aspects of the virus-host interaction in such a hostile setting, we examined autophagic trafficking of HCV NS3 in response to IFN injection. Analysis of gradient centrifugation-derived vesicles from liver cells indicates that only the IFNβ-induced autophagosomes are directed to the lysosome for maturation and removal, whereas IFNα-induced subcellular vesicles are trafficked to the late endosomes. Interestingly, although HCV RNA can induce the lipidation of LC3 in the absence of MAVS, IFNβ cannot. These results indicate that IFNβ induced autophagy via a MAVS-dependent manner, which is different from that induced by HCV. Since MAVS is associated with mitochondria, IFNβ-induced autophagy likely involves mitochondria. Previously, we have shown that HCV induces incomplete autophagy via an unfolded protein response. The current data suggest possible mechanisms of IFNα/β in delivering viral components to the TLR3-containing endosomes, setting off a second wave of TLR3/TRIF-mediated antiviral response in vivo ().
In summary, our results demonstrate that the MAVS cleavage alone does not compromise global type I IFN and ISG responses in the liver. While HCV induces an incomplete autophagy for its replication and immune evasion, the IFNβ and -α responses target viral components to differential autophagic compartments for removal by lysosomal hydrolases and/or perhaps TLR3-mediated immune recognition. While several reports have revealed that HCV can subvert autophagy for its evolutionary advantage, our data have underscored a role of innate immune responses in wrestling this machinery and hampering viral replication. From this study, the autophagic apparatus appears to be a focal point for virus-host interaction in the liver. Viral replication clearly generates intracellular stress, which is detected by the host and has pathophysiological consequences. Short-term stress of the normal cellular processes can lead to production of chemokines (e.g., CXCL10) and cytokines (e.g., IL-7), as well as increased co-stimulatory molecule expression and antigen presentation; sustained strain and compensatory mechanisms, however, may result in fundamental deviations, leading to metabolic abnormalities and carcinogenesis. Additional studies focusing on the virus-autophagy interface may lead to new antivirals or novel therapeutics that shift the balance and result in viral clearance.
1 Mayura M Desai,1 Lynn Soong,1 and JH James Ou2
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