Investigating the role of necroptosis in MERS-CoV pathogenesis

Professor Chu, Hin   (Principal Investigator (PI))

12

2018-05-01

2019-04-30

63460

Investigating the role of necroptosis in MERS-CoV pathogenesis

Apoptosis, MERS-CoV, MLKL, Necroptosis, RIPK1, RIPK3

VirologyMicrobiology

201711159220

Seed Fund for PI Research – Basic Research

2017

Completed

The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 [1] and has since caused 2103 laboratory-confirmed cases with at least 733 deaths in 27 countries as of September 2017 [2]. Importantly, MERS-CoV is highly pathogenic and has a case-mortality rate of approximately 35%, which is the highest among all identified human coronaviruses including the severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus HKU1 (HCoV-HKU1), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), and human coronavirus 229E (HCoV-229E). The clinical features of severe MERS include high fever, pneumonia, acute respiratory distress syndrome (ARDS), as well as extrapulmonary manifestations including gastrointestinal symptoms, lymphopenia, acute kidney injury, hepatic inflammation, and pericarditis [3]. Despite the high mortality rate of MERS-CoV infection, the detailed pathogenic mechanisms of MERS-CoV remain largely unknown. In this regard, studies from us and other groups have discovered several lines of evidence that partly addressed the high pathogenicity of MERS-CoV. First, MERS-CoV has an extraordinarily broad spectrum of human cell tropism that might contribute to the extensive systemic dissemination of the virus in vivo [4]. Second, MERS-CoV is capable of infecting and replicating in human primary macrophages and dendritic cells, which results in the aberrant expression of cytokines and pro-inflammatory cytokines [5,6]. Third, MERS-CoV efficiently antagonizes multiple host anti-viral responses through its viral proteins [7-11]. However, despite the discoveries over the past five years, a lot of questions on MERS-CoV-induced pathogenicity remain largely unanswered. Histopathological examination on the lung tissue of an autopsy from a fatal case of MERS-CoV infection demonstrated substantial alveolar damage, suggesting that direct cytopathic effects of the type II pneumocytes contributed to the pathogenesis of severe and fatal MERS-CoV infection [12]. In parallel, an ex vivo study that examined MERS-CoV-infected human lung tissue found evidence of chromatin condensation, nuclear fragmentation, and membrane blebbing of infected type II pneumocytes coming off of the alveolar wall, which suggested the presence of apoptosis [13]. Importantly, analysis of BAL from a MERS-CoV-infected patient similarly demonstrated hallmarks of apoptosis in both infected lung epithelial cells and infected leukocytes [13]. On the other hand, acute necrosis was observed in the kidney tubular epithelium from a case report of a MERS survivor [14]. Take together, the available clinical evidence suggested that MERS-CoV-induced tissue damage could be orchestrated by an intricate combination of different cell death pathways. Recently, we have demonstrated that MERS-CoV could induce apoptosis in kidney cells by modulating the function of Smad7 and FGF2, which might explain the kidney-related pathology observed in MERS cases [15]. In a parallel line of study, we illustrated the capacity of MERS-CoV to efficiently infect primary human T lymphocytes, which involved the activation of both the intrinsic and extrinsic apoptosis pathways [16]. However, in subsequent experiments, our data suggested that inhibition of apoptosis was not sufficient in preventing MERS-CoV-induced cell death, which hinted the involved of other forms of cell death upon MERS-CoV infection. Necroptosis, also known as programmed or regulated necrosis, is a highly regulated form of caspase-independent cell death. Cells undergo necroptosis are characterized by cell swelling, loss of plasma membrane integrity, and release of cytosolic contents into the extracellular space [17]. In stark contrast to apoptosis, necroptosis is highly inflammatory. Our preliminary data suggested that the viability of MERS-CoV-infected cells could only be rescued by the combined treatment of apoptosis and necroptosis inhibitors, which suggested the involvement of apoptosis and necroptosis in MERS-CoV-infected cells. Given the importance of cell death and tissue damage in MERS-CoV pathogenesis, we aim to further investigate the involvement of necroptosis in MERS-CoV infections. First, we will verify the induction of necroptosis in MERS-CoV-infected cells. Second, we will determine the effect of necroptosis induction on MERS-CoV replication. Finally, using the hDPP4 mouse, we will investigate the outcome of necroptosis inhibition in MERS-CoV-infected animals and evaluate if necroptosis inhibition can be applied independently or simultaneously with other anti-viral regimens as an anti-viral therapy in the combat against MERS-CoV. Reference: 1. Zaki AM, et al. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814-1820. 2. WHO. http://www.who.int/emergencies/mers-cov/en/ 3. Assiri A, et al. 2013. Epidemiological, demographic, and clinical characteristics of 47 cases of Middle East respiratory syndrome coronavirus disease from Saudi Arabia: a descriptive study. Lancet Infect Dis 13:752-761. 4. Chan JF, et al. Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation. J Infect Dis. 2013;207(11):1743-52. 5. Zhou J, et al. Active replication of Middle East respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis. J Infect Dis. 2014;209(9):1331-42. 6. Chu H, et al. Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response. Virology. 2014;454-455:197-205. 7. Rabouw HH, et al. Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses. PLoS Pathog. 2016;12(10):e1005982. 8. Thornbrough JM, et al. Middle East Respiratory Syndrome Coronavirus NS4b Protein Inhibits Host RNase L Activation. MBio. 2016;7(2):e00258. 9. Niemeyer D, et al. Middle East respiratory syndrome coronavirus accessory protein 4a is a type I interferon antagonist. J Virol. 2013;87(22):12489-95. 10. Siu KL, et al. Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response. J Virol. 2014;88(9):4866-76. 11. Lokugamage KG, et al. Middle East Respiratory Syndrome Coronavirus nsp1 Inhibits Host Gene Expression by Selectively Targeting mRNAs Transcribed in the Nucleus while Sparing mRNAs of Cytoplasmic Origin. J Virol. 2015;89(21):10970-81. 12. Ng DL, et al. Clinicopathologic, Immunohistochemical, and Ultrastructural Findings of a Fatal Case of Middle East Respiratory Syndrome Coronavirus Infection in the United Arab Emirates, April 2014. Am J Pathol. 2016;186(3):652-8. 13. Hocke AC, et al. Emerging human middle East respiratory syndrome coronavirus causes widespread infection and alveolar damage in human lungs. Am J Respir Crit Care Med. 2013;188(7):882-6. 14. Cha RH, et al. A Case Report of a Middle East Respiratory Syndrome Survivor with Kidney Biopsy Results. J Korean Med Sci. 2016 Apr;31(4):635-40. 15. Yeung ML, Yao Y, Jia L, Chan JF, Chan KH, Cheung KF, et al. MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2. Nat Microbiol. 2016;1:16004. 16. Chu H, et al. Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways. J Infect Dis. 2016;213(6):904-14. 17. Orzalli MH, et al. Apoptosis and Necroptosis as Host Defense Strategies to Prevent Viral Infection. Trends Cell Biol. 2017 Nov;27(11):800-809.