Human β-Coronavirus OC43 Infection Alters Expression of Tight Junction Protein

Mondal, Debanjana (2025) Human β-Coronavirus OC43 Infection Alters Expression of Tight Junction Protein. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS Dissertation of Debanjana Mondal (20MS186)) 20MS186_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (2MB)

Abstract

The COVID-19 pandemic highlights the necessity for a deeper understanding of coronavirus pathobiology to aid in therapeutic development. SARS-CoV-2, classified as a β-coronavirus, necessitates BSL-3 facilities due to its high infective potential. In contrast, the human β-coronavirus OC43, which induces mild symptoms, serves as a safer BSL-2 model for investigating coronavirus pathobiology and devising therapeutic strategies. Researchers worldwide are focused on studying various viral and host cell factors, exploring their roles in pathogenesis and replication within host cells. Identifying these factors may unveil new therapeutic targets to inhibit viral transmission. One such host cell factor is intercellular junctions. Prior investigations in our laboratory revealed that upon OC43 infection, connexin 43, a gap junction protein, is significantly downregulated. The infection by OC43 hampers Golgi function, disrupting the trafficking of Cx43 and modifying gap junction intercellular communication (GJIC). This disruption affects the formation of hemichannels and gap junctions, contributing to the pathogenic effects of the virus by disrupting cellular homeostasis. Current studies are underway to assess the status of another type of intercellular junction, tight junctions, following OC43 infection. Two crucial and widely expressed tight junction proteins, Occludin and ZO-1, are also downregulated in human lung epithelial cells after infection, A549. Previous research in our lab has already indicated that the disruption of the Golgi apparatus following HCoV-OC43 infection is a primary mechanism leading to the downregulation of the gap junction protein connexin 43. Therefore, we aimed to investigate whether this mechanism similarly influences the alteration of tight junction proteins. By treating the cells with Brefeldin A, we disrupted the Golgi apparatus, creating conditions akin to those observed post-OC43 infection to validate our hypothesis. Our findings indicate that virus-induced Golgi disruption is indeed responsible for the impairment of tight junction protein expression. Additionally, we aimed to determine whether the downregulation of tight junction proteins resulted solely from active viral replication or if there were any secreted factors from the virus or host cells that could also contribute to these changes. Experiments utilizing UV-inactivated conditioned media revealed that even in the absence of active viral replication, soluble factors released by infected cells were enough to reduce TJ protein expression. To further explore potential molecular drivers of this effect, we investigated the involvement of reactive oxygen species (ROS) and associated signaling pathways. The DCFDA assay and RT-PCR evaluation of NRF2 indicated no significant rise in ROS production or antioxidant responses in infected cells. In agreement with these observations, MAPK protein 10 | P a g e levels—often linked to ROS-mediated signaling—demonstrated a notable decrease following infection, implying that the MAPK pathway is not activated under these circumstances. Interestingly, western blot analysis of the NF-κB subunit P65/RelA revealed a significant increase in infected cells, suggesting activation of the NF-κB signaling pathway. Although matrix metalloproteinases (MMP2 and MMP9), typically regulated downstream of NF-κB and known to compromise tight junctions, did not show changes in expression at the mRNA level, it is plausible that other NF-κB-regulated effectors contribute to this effect. We propose that proinflammatory cytokines and chemokines, which are well-established transcriptional targets of NF-κB, may mediate the observed TJ disruption by affecting epithelial barrier function and contributing to cellular stress responses. Our results demonstrate that infection with HCoV-OC43 in A549 cells disrupts tight junction integrity, likely via an NF-κB-mediated inflammatory mechanism induced by the virus, rather than through ROS or MMP activation. These insights improve our understanding of how coronaviruses compromise epithelial barriers and could have implications for disease pathogenesis.. Future research will focus on profiling cytokine responses and investigating TJ gene expression at the mRNA level to further clarify the regulatory mechanisms involved.

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