Abstract
Extensive inflammation in the liver is known to contribute to the pathogenesis of hepatitis C virus (HCV) infection. Apoptosis has, for a long time, been known to act as a mechanism of hepatocyte death, but our previous research also identified inflammasome-mediated pyroptosis in infected and uninfected bystander cells as an additional mechanism of HCV-induced cytopathicity. The purpose of this study was to investigate the mechanism of HCV-induced cell death and to determine the timing and relative contributions of apoptosis and pyroptosis during HCV infection. In a model employing a cell culture-adapted strain of JFH-1 HCV and Huh-7.5 hepatocyte-like cells, we found that pyroptosis occurred earlier than did apoptosis during infection. CRISPR knockout of NLRP3 resulted in decreased caspase-1 activation, but not complete elimination, indicating multiple sensors are likely involved in HCV-induced pyroptosis. Knockout of gasdermin-D resulted in increased activation of apoptosis-related caspase-3, suggesting potential crosstalk between the two cell death pathways. An unexpected decrease in activated caspase-1 levels was observed when caspase-3 was knocked out, implying that caspase-3 may have a role in the initiation of pyroptosis, at least in the context of HCV infection. Lower viral titres in culture fluids and increased ratios of intracellular to extracellular levels of infectious virus were observed in knockout versus wild-type Huh-7.5 cells, suggesting that HCV may induce programmed cell death in order to enhance virus release from infected cells. These results contribute to the understanding of HCV pathogenesis and add to the increasing volume of literature suggesting various programmed cell death pathways are not mutually exclusive.
Highlights
Multiple forms of programmed cell death (PCD) have been demonstrated in the context of virus infection and it is generally believed that viruses utilize these mechanisms to induce disease [1,2,3,4]
To follow up on these findings, the current study aimed to identify the relative timing of these forms of programmed cell death during hepatitis C virus (HCV) infection, to confirm the involvement of pyroptosisassociated proteins NLRP3 and GSDM-D and apoptosisassociated caspase-3, and to determine whether programmed cell death has a role in viral spread during HCV infection in vitro
To confirm this result and to elucidate the relative timing by which apoptosis and pyroptosis occur during HCV infection, infected (MOI=1) and uninfected cells, and culture fluids were compared for the presence of cleaved caspase-1, indicative of pyroptosis, and cleaved caspase-3, indicative of apoptosis, using Western blot analysis
Summary
Multiple forms of programmed cell death (PCD) have been demonstrated in the context of virus infection and it is generally believed that viruses utilize these mechanisms to induce disease [1,2,3,4]. Some reports detail how PCD is employed as a host response to contain the virus [reviewed in reference [5]]. Both apoptosis and pyroptosis have been implicated in both contexts. The extrinsic pathway depends on initiation by ligand-binding of a death receptor on the cell surface, leading to cleavage of caspase-8. Both pathways result in activation of executioner caspase-3 which leads to cell shrinkage, condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies that are cleared by circulating macrophages [both pathways extensively reviewed in reference [6]]. Apoptosis has been described as immunologically silent while still having a role in pathogenesis induced by some viruses [7, 8]
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call