The Role of Cell Death in Viral Infection
The regulation of cell death during viral infection is an important factor in the balance of virus and host survival. In this series, we look at pathways that are regulated through cellular responses to virus, as well as in response to virally encoded proteins. Infection of the coronaviruses SARS-CoV and SARS-CoV-2 causes severe lung injury with extensive damages to alveolar and bronchial epithelial cells, and produces extra-pulmonary damage.
To learn more about the role ER stress and autophagy in viral infection, specifically SARS-CoV-2, check out this post.
In response to viral infection, cell death occurs in both infected cells and neighboring cells via an inflammatory cytokine storm. Programmed cell death manifests in morphologically distinct processes of apoptosis and necrosis that have a high degree of crosstalk. Apoptosis can be induced by several of the coronavirus encoded proteins, as well through the unfolded protein response (UPR). This is a tightly controlled process that has been studied for decades and is generally associated with the activation of Caspase-3, which is produced as a zymogen and activated by proteolytic cleavage by upstream regulatory caspases. Caspase-3 cleaves a number of proteins (such as PARP) involved in the disassembly of the cell. Intrinsic apoptosis is controlled by members of the Bcl-2 family, which trigger mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c and the subsequent activation of Caspase-9 and Caspase-3. The Bcl-2 family consists of a large number of associated proteins that include executioner members (Bax, Bak), anti-apoptotic members (Bcl-2, Bcl-xL, Mcl-1, A1/Bfl-1, Bcl-w), and “BH-3 only” proteins (Bim, Bid, Bad, Bik, Puma, Noxa, Hrk, BMF) that are highly regulated and trigger Bax/Bak activation. SARS coronaviruses have acquired mechanisms to directly regulate the Bcl-2 family to induce apoptosis. For example, SARS-CoV protein 7a induces apoptosis via binding to Bcl-xL. Also, SARS-CoV and SARS-CoV-2 envelope protein (E) has a conserved BH3 domain that enables apoptosis and is important for viral virulence. Apoptosis can also be triggered by an extrinsic pathway through death receptors in the TNFR superfamily that lead to the recruitment of Caspase-8, the death-inducing signaling complex (DISC), and subsequent activation of Caspase-3. Apoptosis can be monitored through a number of established assays, including activation of caspases such as Caspase-3, monitoring expression of Bcl-2 family members, and cytochrome c release, as well as Annexin V staining.
More recently it has been found that necrotic cell death can occur as part of the inflammatory response, which likely contributes to tissue damage and fibrosis. Necroptosis and pyroptosis represent two of the pathways that result in the morphological features of necrosis, including cell swelling, plasma membrane pore formation, and release of damage associated molecular patterns (DAMPs) such as HMGB1 and inflammatory cytokines. Necroptosis is a cell defense pathway that is activated when apoptosis is inhibited. Indeed, many viruses have adopted mechanisms to subvert apoptosis that can result in activation of this lytic response. It requires the activation of the RIPK3 kinase that phosphorylates the MLKL at Ser358 (Ser345 in mouse). Phosphorylation of MLKL leads to oligomerization and formation of pore complex. RIPK3 activation is triggered through several RIP homotypic interaction motif (RHIM) domain interactions, including RIPK1, TRIF, and ZBP1, and results in the phosphorylation of RIPK3 at Ser227 (Thr231/Ser232 in mouse). Canonical necroptosis signaling mediated by RIPK1 is associated with autophosphorylation of RIPK1 at Ser166 and can be inhibited by necrostatins. Alternatively, RIPK3 can be activated by innate immune responses, including Toll-like receptor (TLR) recruitment of TRIF or by DNA virus included activation of ZPB1. Apoptosis inhibits necroptosis through cleavage of RIPK1 and RIPK3 by Caspase-8. Necroptosis has, in fact, been observed in neuronal cells by the neuroinvasive coronavirus OC43. The SARS-CoV accessory protein 3a interacts with RIPK3 to help drive necrotic cell death.
Another pathway resulting in necrotic cell death, pyroptosis, is characterized by the pore forming ability of members of the gasdermin family. Pyroptosis is induced in cells of the innate immune system upon activation of TLRs and DAMPs. Canonical activation of this pathway involves the cleavage of Gasdermin D by Caspase-1 in which the N-terminal fragment of Gasdermin D forms a membrane pore. Caspase-1 also cleaves and activates inflammatory cytokines like IL-1b and IL-18, which are secreted through Gasdermin D pores. Caspase-1 can be activated via pathogen sensing complexes termed inflammasome. The inflammasome complex containing NLRP3, ASC, and Caspase-1 are activated as part of a 2-step process involving microbial pathogens, potassium efflux, and lysosomal damaging agents. Importantly, Caspase-3 can cleave Gasdermin D at an additional N-terminal site and inactivate its pore forming ability. In SARS 3a, which as stated above activates necroptosis, but can also activate Capase-1 either directly or through the NRLP3 inflammasome. Studies have shown that SARS-CoV induces NLRP3 inflammasome activation and pyroptosis and promotes an increase in serum levels of IL-1b.
The response to infection can include activation of pathways of apoptosis, necrosis, and pyroptosis that can play a critical role in viral virulence and damage to host tissues. These pathways have a high degree of crosstalk, but the emergence of new regents to tease apart these pathways should enable a much better understanding. The role for emerging therapeutics that regulate these responses is an area of intense interest.
- Fung TS, Liu DX. Coronavirus infection, ER stress, apoptosis and innate immunity. Front Microbiol. 2014 Jun 17;5:296. doi: 10.3389/fmicb.2014.00296. PMID: 24987391; PMCID: PMC4060729.
- Tan YX, Tan TH, Lee MJ, Tham PY, Gunalan V, Druce J, Birch C, Catton M, Fu NY, Yu VC, Tan YJ. Induction of apoptosis by the severe acute respiratory syndrome coronavirus 7a protein is dependent on its interaction with the Bcl-XL protein. J Virol. 2007 Jun;81(12):6346-55. doi: 10.1128/JVI.00090-07. Epub 2007 Apr 11. PMID: 17428862; PMCID: PMC1900074.
- Yue Y, Nabar NR, Shi CS, Kamenyeva O, Xiao X, Hwang IY, Wang M, Kehrl JH. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death. Cell Death Dis. 2018 Sep 5;9(9):904. doi: 10.1038/s41419-018-0917-y. PMID: 30185776; PMCID: PMC6125346.
- Siu KL, Yuen KS, Castaño-Rodriguez C, Ye ZW, Yeung ML, Fung SY, Yuan S, Chan CP, Yuen KY, Enjuanes L, Jin DY. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC. FASEB J. 2019 Aug;33(8):8865-8877. doi: 10.1096/fj.201802418R. Epub 2019 Apr 29. PMID: 31034780; PMCID: PMC6662968.
- Meessen-Pinard M, Le Coupanec A, Desforges M, Talbot PJ. Pivotal Role of Receptor-Interacting Protein Kinase 1 and Mixed Lineage Kinase Domain-Like in Neuronal Cell Death Induced by the Human Neuroinvasive Coronavirus OC43. J Virol. 2016 Dec 16;91(1):e01513-16. doi: 10.1128/JVI.01513-16. PMID: 27795420; PMCID: PMC5165216.
- Miller K, McGrath ME, Hu Z, Ariannejad S, Weston S, Frieman M, Jackson WT. Coronavirus interactions with the cellular autophagy machinery. Autophagy. 2020 Sep 23:1-9. doi: 10.1080/15548627.2020.1817280. Epub ahead of print. PMID: 32964796.