Author interview: Sanchita Mishra is a graduate student in Dr. Kesavardana Sannula’s group at the Department of Biochemistry, the Indian Institute of Science (IISc), Bangalore. She majored in Microbiology during her B.Sc. at the University of Delhi. Her current research focuses on understanding the mechanisms of host cell death regulation in response to respiratory RNA viral infections.
Lab PI name: Dr. Kesavardana Sannula, Indian Institute of Science (IISc)
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The study reveals how bat-originated zoonotic viruses, like SARS-CoV-2, manipulate host cell death responses by mimicking cell death-promoting RHIM signaling. In particular, SARS-CoV-2 Nsp13 promotes distinct mechanisms of cell death activation in bat and human cells, indicating possible events underlying cellular damage and inflammation in COVID-19.
What was the core problem you aimed to solve with this research?
Why are zoonotic viruses often highly pathogenic in humans? Do reservoir hosts (like bats) drive the evolution of pathogenic traits in zoonotic viruses? Research in our lab attempts to address these fundamental questions. We study innate immune responses against zoonotic RNA virus infections and viral evasion strategies. In severe cases of SARS-CoV-2 infection, cytokine storm and respiratory tissue damage are commonly observed. Host cell death activation after viral infection is a defense strategy to limit viral spread and mount a protective response. However, dysregulated cell death response can impair the tissue repair process and worsen the disease outcome. Since excessive respiratory cellular damage has been observed during SARS-CoV-2 infection, we aimed to understand how SARS-CoV-2 regulates host cell death signaling and its potential implications on pathogenesis.
Bats and birds are reservoirs for many pathogenic viruses with zoonotic potential. These reservoir hosts show less clinical symptoms despite hosting pathogenic viruses. However, zoonotic transmission of these viruses often causes severe disease in humans. Our lab is also interested in understanding what makes the reservoir hosts tolerant to pathogenic viruses. Through this study, we aimed to understand the differential regulation of host cell death in bats and humans to delineate mechanisms underlying dampened inflammation in bats.
How did you go about solving this problem?
The research in our lab is primarily focused on studying molecular details of the inflammatory cell death pathways (necroptosis and pyroptosis) and how viruses regulate them. RHIMs are protein motifs enabling protein-protein interactions and RHIM-signaling is crucial for cell death activation. Multiple DNA viruses have been reported to encode RHIM-mimics to inhibit RHIM-mediated cell death signaling in hosts. We had a simple question – Do RNA viruses encode RHIM mimics? Our preliminary analysis revealed multiple RNA viruses of zoonotic potential harboring putative RHIM mimicking sequences, and SARS-CoV-2 Nsp13 was one of them. We then employed state-of-the-art techniques to study the mechanism of cell death manipulation by this novel RNA viral RHIM.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Zoonotic respiratory RNA virus infections are often associated with cytokine storm and tissue damage, resulting in respiratory failure. The underlying intricate mechanisms of the pathogenesis (like in the case of COVID-19) and how these viruses manipulate host pathways are not completely understood.
RHIM (RIP Homotypic Interaction Motif) is a protein motif regulating host cell death and inflammation by promoting RHIM-mediated protein complex formation. Our study identified RHIM mimics in several bat viral proteins, including SARS-CoV-2 Nsp13. Since cell death is primarily activated as an antiviral strategy, we speculated that this Nsp13-RHIM inhibits host cell death. In contrast, Nsp13 promotes robust inflammatory human cell death activation, and mutating the RHIM in Nsp13 enhances cell survival. Mechanistically, Nsp13 was found to work in synergy with host RHIM proteins (ZBP1 & RIPK3) to promote cell death activation, possibly contributing to respiratory damage and pathogenesis in COVID-19. The study also identified SARS-CoV-2 genomic RNA segments in Z-conformation (Z-RNA) that enable the activation of Nsp13-mediated cell death.
Our study suggests bats to be the likely source of the evolution of RHIM mimics in bat-originated viruses, as bats express host RHIM proteins like humans. Interestingly, bats show mild clinical symptoms and tissue damage compared to humans despite harboring viruses with RHIM mimics. To understand this conundrum, we tested whether and how Nsp13-RHIM regulates bat cell death. Nsp13 activates cell death in bat cells, albeit in an RHIM-independent manner. Interestingly, Nsp13-induced bat cell death is primarily non-inflammatory and lower in magnitude than human cell death. This host response in bats might be enough to clear the viral replication niche without causing severe inflammation.
“What piqued our interest is that a viral replication-transcription complex protein (Nsp13) decided to regulate host cell death. Nsp13 has been considered a therapeutic target for restricting SARS-CoV-2 replication but with little success. In my opinion, the novel RHIM-cell death regulating function of Nsp13 could be a better strategy to limit SARS-CoV-2 induced tissue damage and inflammation. We are currently exploring this. Broadly, my group is curious to study bats as reservoir hosts and the trajectories of RNA virus evolution in them. There lie fundamental clues of zoonotic virus emergence that might eventually instruct pandemic preparedness, and we are actively working towards identifying these clues.” — Dr. Kesavardana Sannula
What are the potential implications of your findings for the field and society?
Disease outbreaks caused by zoonotic RNA viruses are a serious concern for human health and the economy. Multiple bat viruses of zoonotic potential are just an unfortunate spillover away from causing the next outbreak. The fundamental discovery of this study is not just limited to SARS-CoV-2 infection biology and potentially has a broader application. We have identified a novel cell death activating RNA viral RHIM in SARS-CoV-2 Nsp13. Targeting Nsp13-RHIM-induced host cell death would potentially be an effective therapeutic strategy against COVID-19. Additionally, since Nsp13 RHIM is highly conserved among pathogenic human coronaviruses, the cell death-inducing function of Nsp13 can be a pan-β-coronaviral target for drug development. Our findings have also shed light on how reservoir hosts, like bats, drive the evolution of new viral traits associated with tissue damage during human infections. Moreover, the study touches upon the fundamental differences in the cell death regulation of bats and humans that provide some clues on why pathogenic viruses are tolerated in bats but cause severe pathology in humans. Understanding the differential host responses can be an excellent reference point for developing novel and unique therapeutic strategies. These findings are crucial for predicting the pathogenic potential of viruses with zoonotic ability. This will further enable pandemic preparedness and help us fight future disease outbreaks.
What was the exciting moment during your research?
As we set out to understand the role of RNA viruses in regulating host cell death, we started looking for RHIM-mimicking sequences in RNA viral proteins. We were absolutely elated to identify RHIM sequences, not only in SARS-CoV-2 proteins but several other pathogenic RNA viruses of bat origin. The reported DNA viral RHIM mimics could restrict host cell death activation. When we initially identified the RHIM mimic in SARS-CoV-2 proteins, we assumed Nsp13-RHIM would inhibit host cell death. The cell death-promoting function of Nsp-13 RHIM came as a surprise to us. This was a rather unexpected phenotype, and exploring the new aspect of the hypothesis was tough. But as subsequent experiments worked, things started to make sense, and we gradually established the mechanism. We were quite happy to see the story unfold.
One of the most fascinating aspects of this study was our efforts to understand the RHIM-mediated cell death regulation in bat cells. The absence of bat-specific resources made our assays challenging, but we eventually figured it out. Working towards establishing a bat-specific mechanism of cell death regulation was quite thought-provoking. This was my favourite part of the journey.
Seeing our work receive great recognition from the print media and being featured in multiple news articles was exciting. While I have always been curious about viruses and infection biology, the wide recognition of our research and its potential impact has inspired me to pursue the upcoming projects with greater motivation.
Reference: Mishra S, Jain D, Dey AA, Nagaraja S, Srivastava M, Khatun O, Balamurugan K, Anand M, Ashok AK, Tripathi S, Ganji M, Kesavardhana S, Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling, iScience (2024). https://doi.org/10.1016/j.isci.2024.111444
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