BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis

Pathogenicity often differs dramatically among even closely related arenavirus species. For instance, Junin virus (JUNV), the causative agent of Argentine hemorrhagic fever (AHF), is closely related to Tacaribe virus (TCRV), which is normally avirulent in humans. While little is known about how host cell pathways are regulated in response to arenavirus infection, or how this contributes to virulence, these two viruses have been found to differ markedly in their ability to induce apoptosis. However, details of the mechanism(s) governing the apoptotic response to arenavirus infections are unknown. Here we confirm that TCRV-induced apoptosis is mitochondria-regulated, with associated canonical hallmarks of the intrinsic apoptotic pathway, and go on to identify the pro- and anti-apoptotic Bcl-2 factors responsible for regulating this process. In particular, levels of the pro-apoptotic BH3-only proteins Noxa and Puma, as well as their canonical transcription factor p53, were strongly increased. Interestingly, TCRV infection also led to the accumulation of the inactive phosphorylated form of another pro-apoptotic BH3-only protein, Bad (i.e. as phospho-Bad). Knockout of Noxa or Puma suppressed apoptosis in response to TCRV infection, whereas silencing of Bad increased apoptosis, confirming that these factors are key regulators of apoptosis induction in response to TCRV infection. Further, we found that while the highly pathogenic JUNV does not induce caspase activation, it still activated upstream pro-apoptotic factors, consistent with current models suggesting that JUNV evades apoptosis by interfering with caspase activation through a nucleoprotein-mediated decoy function. This new mechanistic insight into the role that individual BH3-only proteins and their regulation play in controlling apoptotic fate in arenavirus-infected cells provides an important experimental framework for future studies aimed at dissecting differences in the apoptotic responses between arenaviruses, their connection to other cell signaling events and ultimately the relationship of these processes to pathogenesis. Author summary Arenaviruses are important zoonotic pathogens that present a serious threat to human health. While some virus species cause severe disease, resulting in hemorrhagic fever and/or neurological symptoms, other closely related species exhibit little or no pathogenicity. The basis for these dramatically different outcomes is insufficiently understood, but investigations of host cell responses have suggested that apoptosis, i.e. non-inflammatory programmed cell death, is regulated differently between pathogenic and apathogenic arenaviruses. However, many questions remain regarding how these viruses interact with cell death pathways upon infection. Here we demonstrate that apoptosis induced by the avirulent Tacaribe virus (TCRV), proceeds via the mitochondria (i.e. the intrinsic apoptotic signaling pathway), and is regulated by a combination of factors that appear to balance activation (i.e. Noxa and Puma) and inactivation (i.e. Bad-P) of this cascade. During TCRV infection, the balance of these pro- and anti-apoptotic signals shifts the equilibrium late in the infection towards cell death. Importantly, we also found that the highly pathogenic Junin virus (JUNV), which does not trigger caspase activation or apoptotic cell death, nonetheless induces pro-apoptotic factors, thus supporting the existence of a specific mechanism by which this virus is able to evade apoptosis at late stages in this process.