Differential Modulation of Junctional Proteins in Lung Epithelial Cells and Astrocytes Following Human Coronavirus OC43 Infection

Karmakar, Souvik (2026) Differential Modulation of Junctional Proteins in Lung Epithelial Cells and Astrocytes Following Human Coronavirus OC43 Infection. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Souvik Karmakar (20RS069)) 20RS069.pdf - Submitted Version Restricted to Repository staff only Download (14MB)

Abstract

The recent COVID-19 pandemic, caused by the human β-coronavirus SARS-CoV-2, has had a lasting impact worldwide due to its high disease severity and zoonotic potential. This pandemic led to a surge in research on human β-coronaviruses, but was severely hindered due to the risks involved and the requirement for a biosafety level 3 (BSL-3) laboratory. An alternative human β-coronavirus with less severe symptoms can be considered a model for studying SARS-CoV-2 pathogenesis. Among several other human β-coronaviruses, HCoV-OC43, a BSL-2 pathogen, can be considered a valuable model, owing to its structural and functional similarities to highly virulent coronaviruses. Research on limiting coronavirus infection is primarily focused on viral factors, such as entry, replication, and spread. However, identifying host factors to restrict infection has not been explored extensively. Recent studies on coronaviruses have reported their ability to alter several host cellular pathways, facilitating their replication and spread. Dysregulation of these host cellular pathways by virulent coronaviruses, such as SARS-CoV-2, in neuronal and epithelial tissues can lead to persistent symptoms even after the acute infection has resolved. This clinical term is known as Long COVID, indicating that the damage to cellular pathways may have long-term clinical consequences. A key feature of coronavirus infection is the disruption of membrane organisation and damage to cellular junctions. The cellular junctions mainly affected are gap junctions, tight junctions, and adherens junctions. This thesis investigates the effect of HCoV-OC43 infection on junction proteins, which are crucial for intercellular communication and maintaining cellular barrier integrity. The first aim primarily focused on the alterations of gap junctions (Connexin 43), tight junctions (Occludin and Zonula occludens-1), and adherens junctions (β-catenin and E-cadherin) in the human lung epithelial cell line, A549. HCoV-OC43 infection resulted in significant reductions in protein expression and altered trafficking of these proteins to the plasma membrane. The loss of Cx43 on the cell surface led to reduced gap junction intercellular communication and decreased hemichannel activity. Similarly, Occludin, ZO-1, β-catenin, and E-cadherin showed reduced expression and disrupted membrane localization. These changes in the expression and localization of junctional proteins were associated with increased expression of endoplasmic reticulum (ER) stress markers, suggesting that virus-induced cellular stress might contribute to the disruption of cellular junction proteins. The second aim extends the study to examine the impact of HCoV-OC43 infection on the junctional proteins of the central nervous system (CNS), as several reports suggest that HCoV-OC43 can infect CNS cells in both humans and mice. Among different neuroglial cells, primary astrocytes isolated from neonatal mice were chosen due to their crucial roles in providing metabolic support, maintaining CNS homeostasis, and ensuring blood-brain barrier integrity through the expression of several cellular junction proteins. Immunofluorescence, immunoblotting, and qRT-PCR results revealed significant downregulation of gap junction, tight junction, and adherens junction proteins following HCoV-OC43 infection in primary astrocytes. These results highlight astrocytes as a critical target for coronavirus-induced disruption of CNS homeostasis and also offer insights into the potential mechanisms underlying the neurological complications observed in Long COVID. The third aim investigates the mechanisms contributing to the disruptions of these cellular junction proteins. Infection with HCoV-OC43 led to the activation of the NF-κB signaling pathway, triggering the production of several pro-inflammatory cytokines and chemokines, as well as type I interferon responses. The inflammatory response also upregulated the expression of matrix metalloproteinases (MMP) in both A549 cells and astrocytes. In parallel, the infection led to a reduction in the expression of the trans-Golgi apparatus marker TGN46, indicating the disruption of this central organelle. The upregulation of MMPs and loss of TGN46 suggest potential mechanisms behind the disruptions and impaired trafficking of junctional proteins. Interestingly, reactive oxygen species (ROS) levels were reduced, and the ERK/MAPK signaling pathway was not activated post-HCoV-OC43 infection. These findings indicate that HCoV-OC43 disrupts intercellular junctions in lung and CNS cell models through NF-κB-mediated inflammation, MMP9 induction, and Golgi fragmentation. This work provides insights into coronavirus pathogenesis and identifies the NF-κB pathway and the Golgi apparatus as potential therapeutic targets, thereby enhancing our preparedness for future coronavirus threats.

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