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.

A 2019, 'The mediating role of early maladaptive schemas in the relationship between childhood traumatic events and complex posttraumatic stress disorder symptoms in older adults (>64 years)',

It is well-known that viruses activate various inflammasomes, which can initiate the programmed cell death pathway known as pyroptosis, subsequently leading to cell lysis and release of inflammatory cytokines IL-1 and IL-18. This pathway can be triggered by various sensors, including, but not limited to, NLRP3, AIM2, IFI16, RIG-I, and NLRC4. Many viruses are known either to activate or inhibit inflammasomes as a part of the innate immune response or as a mechanism of pathogenesis. Early research in the field of virus-induced pyroptosis suggested a dichotomy, with RNA viruses activating the NLRP3 inflammasome and DNA viruses activating the AIM2 inflammasome. More recent research has shown that this dichotomy may not be as distinct as once thought. It seems many viruses activate multiple inflammasome sensors. Here, we detail which viruses fit the dichotomy as well as many that appear to defy this clearly false dichotomy. It seems likely that most, if not all, viruses activate multiple inflammasome sensors, and future research should focus on expanding our understanding of inflammasome activation in a variety of tissue types as well as virus activation of multiple inflammasomes, challenging biases that stemmed from early literature in this field. Here, we review primarily research performed on human viruses but also include details regarding animal viruses whenever possible.

Introduction: Unnatural amino acids (UAAs) share the same basic structure as proteinogenic amino acids. However, UAAs permit additional functions and applications to proteins due to their different side chains. Recent UAA applications include using fluorescent UAAs to label proteins. The UAA system provides an alternative method to traditional protein labeling mechanisms (antibodies, GFP, and tags, such as HA and HIS), which can affect protein functionality and topology. The purpose of this study was to visualize the hepatitis C virus (HCV) core protein using the fluorescent UAA Anap (3-[(6-acetyl-2-naphthalenyl)amino]-L-alanine). Methods: Huh-7.5 cells were co-transfected with HCV core plasmids containing amber stop codons at various positions throughout the coding sequence and a second plasmid encoding the orthogonal tRNA/synthetase pair that facilitates Anap incorporation. Three days post transfection, cells were stained for core protein and lipid droplets (LDs) and visualized using immunofluorescence or confocal microscopy. Results: We have optimized transfection protocols for the efficient expression of the tRNA/synthetase pair required for Anap incorporation and are able to visualize our core mutant proteins containing Anap. We have successfully substituted Anap into 11 different positions within the core, including substitutions for tryptophan, tyrosine, and phenylalanine residues. In addition, we have shown that our core mutants associate with cellular LDs, suggesting that the incorporation of the UAA did not disrupt core protein expression, stability, or cellular localization. Conclusions: We have demonstrated the establishment of a UAA incorporation system in an HCV protein without any obvious impact on core protein function. The ability to label viral proteins using fluorescent UAAs eliminates the requirement of antibodies or tags for protein visualization. In conclusion, the UAA system is a useful method to study HCV proteins and can potentially be used to label viruses for live cell and animal studies.

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