Generation of recombinant vaccinia virus and analysis of virus-induced cell death.

Vaccinia virus is a large double-stranded DNA virus that is widely used to express foreign genes from different origins. We generated recombinant vaccinia virus that expresses a viral inhibitor to examine its effect on virus-induced necroptosis. We provide a detailed protocol to describe the generation of recombinant vaccinia virus, validation of protein expression, and determination of necroptosis using live cell imaging. This approach can be adapted to examine the effect of other cell death regulators on virus-induced cell death. For complete details on the use and execution of this protocol, please refer to Liu et al. (2021).

The death-inducing activity of RIPK1 is regulated by the pH environment.

Receptor-interacting protein kinase 1 (RIPK1) is a serine/threonine kinase that dictates whether cells survive or die in response to the cytokine tumor necrosis factor (TNF) and other inflammatory stimuli. The activity of RIPK1 is tightly controlled by multiple posttranslational modification mechanisms, including ubiquitination and phosphorylation. Here, we report that sensitivity to TNF-induced, RIPK1-dependent cell death was tunable by the pH environment. We found that an acidic extracellular pH, which led to a concomitant decrease in intracellular pH, impaired the kinase activation of RIPK1 and autophosphorylation at Ser166 Consequently, formation of the cytosolic death-inducing complex II and subsequent RIPK1-dependent necroptosis and apoptosis were inhibited. By contrast, low pH did not affect the formation of membrane-anchored TNFR1-containing signaling complex (complex I), RIPK1 ubiquitination, and NF-κB activation. TNF-induced cell death in Ripk1 -/- cells was not sensitive to pH changes. Furthermore, mutation of the conserved His151 abolished the pH dependence of RIPK1 activation, suggesting that this histidine residue functions as a proton acceptor to modulate RIPK1 activity in response to pH changes. These results revealed an unexpected environmental factor that controls the death-inducing activity of RIPK1.