Research Article: Modulation of calcium signaling pathway by hepatitis C virus core protein stimulates NLRP3 inflammasome activation

Date Published: February 27, 2019

Publisher: Public Library of Science

Author(s): Amina A. Negash, Rebecca M. Olson, Stephen Griffin, Michael Gale, Christopher M. Walker.

http://doi.org/10.1371/journal.ppat.1007593

Abstract

Hepatitis C virus (HCV) infection remains a major cause of hepatic inflammation and liver disease. HCV triggers NLRP3 inflammasome activation and interleukin-1β (IL-1β) production from hepatic macrophages, or Kupffer cells, to drive the hepatic inflammatory response. Here we examined HCV activation of the NLRP3 inflammasome signaling cascade in primary human monocyte derived macrophages and THP-1 cell models of hepatic macrophages to define the HCV-specific agonist and cellular processes of inflammasome activation. We identified the HCV core protein as a virion-specific factor of inflammasome activation. The core protein was both necessary and sufficient for IL-1β production from macrophages exposed to HCV or soluble core protein alone. NLRP3 inflammasome activation by the HCV core protein required calcium mobilization linked with phospholipase-C activation. Our findings reveal a molecular basis of hepatic inflammasome activation and IL-1β release triggered by HCV core protein.

Partial Text

HCV continues as a global health problem causing chronic and progressive liver disease [1–5]. HCV is a major risk factor for hepatocellular carcinoma, and infection is a consistent cause of liver transplants. HCV is a small, enveloped, single-stranded RNA virus that belongs to the Flaviviridae family [6]. It is transmitted through parenteral routes and replicates primarily in the liver. Most often, exposure to HCV leads to chronic infection, which is characterized by persistent hepatic inflammation. The hallmark of chronic HCV infection is dysregulated and persistent inflammatory responses that are thought to serve as a platform for ongoing liver damage and the onset of cirrhosis and hepatocellular carcinoma [7]. While currently no vaccine for HCV is available for clinical use, the advent of direct acting antivirals (DAAs) has revolutionized patient care and these drugs are proven to be effective treatment options for HCV infected individuals beyond interferon (IFN)-based therapy [8, 9]. DAAs are oral regimens, well-tolerated and most patients achieve 80–90% sustained virologic responses (SVRs, defined as the absence of HCV RNA detection after cessation of treatment with DAAs). However, with DAAs there is a concern of the emergence of drug resistant HCV variants, the unknown effects of drug-to-drug interactions, and the expensive nature of these drugs [10, 11]. Most importantly, further prospective studies are needed to assess the effects of treatment with DAAs on preventing liver fibrosis and mitigating HCV-induced severe liver disease such as HCC [12, 13]. Therefore, understanding the complete molecular mechanism of HCV-induced hepatic inflammation is essential to design the best therapeutic regimen to treat hepatic inflammation and to reduce liver damage resulting from chronic HCV infection.

HCV is a potent inducer of IL-1β production in macrophages and our studies have shown that HCV itself contains all the factors needed to trigger both signal-one and signal-two of NLRP3 inflammasome activation [27, 33]. To determine which of the HCV virion component(s) is (are) essential for stimulating NLRP3 inflammasome activation, we prepared HCV and subjected it to ultraviolet (uv) light, resulting in inactivated HCV (uv-HCV) (Fig 1B). Uv-HCV retains the ability to bind and enter cells but is unable to replicate [27]. THP-1 macrophages produce active IL-1β after exposure to uv-HCV (Fig 1A and 1C) coincident with the rapid processing and activation of caspase-1 (Fig 1D) and the formation of ASC- specks indicative of inflammasome activation (Fig 1E). These results collectively reveal that the components of the inactivated HCV virion, containing the viral RNA and structural proteins, but not the viral non-structural protein(s) [44], serve as the NLRP3 inflammasome agonist to drive caspase-1 processing, ASC-speck formation and IL-1β release from macrophages exposed to HCV. Thus, while viral replication is not required for inflammasome activation, components of the incoming virion must deliver the necessary signals, both to prime (signal-one) and activate the NLRP3 inflammasome (signal-two).

Our findings defines the molecular mechanism by which HCV triggers IL-1β production in macrophages (Fig 8). Hepatic macrophages effectively phagocytose macromolecules in the liver to continually survey the hepatic environment and respond to microbial threats [16]. Within the HCV-infected liver, virus-derived antigens, viral RNA and/or viral protein and other inflammatory mediators such as TNF-α can serve as priming factors in hepatic macrophages leading to NFκB activation and proIL-1β production [27, 69]. Our observations demonstrate that the resulting signal-one primed macrophages are then responsive to HCV core protein both within the virion and as a cell-free protein present in patient blood [70]. HCV virion or core protein uptake then deposits the viral core protein in the cell cytoplasm where it induces phospholipase C-mediated calcium flux leading to NLRP3 inflammasome activation, thus establishing the hepatic inflammatory environment.

 

Source:

http://doi.org/10.1371/journal.ppat.1007593

 

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