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Abstract:
Coronaviruses manipulate host cellular pathways to support their replication and
spread. During infection, both virus-host cell fusion and virus-induced cell-cell fusion can
disrupt membrane organization and damage junctional proteins. The main junctional complexes
affected are gap junctions, tight junctions , and adherens junctions. Understanding how these
proteins are regulated during HCoV O C43 infection is essential, as they play crucial roles in
intercellular communication and barrier integrity. This thesis investigates how Human
coronavirus OC43 (HCoV OC43), a β coronavirus with neurotropic potential, alters junctional
protein expression a nd localization in human lung epithelial cells A549 and primary astrocytes
isolated from neonatal pups of C57BL/6 mice , and explores the molecular mechanisms driving
these changes. The first aim primarily examined gap junctions ( Cx43), adherens junctions β
catenin, E cadherin )), and supervised a BSMS thesis investigating tight junction proteins
(Occludin, ZO 1) HCoV OC43 infection led to significant reductions in protein expressions and
altered trafficking of these proteins to the cell surface . Connexin 43 was reduced from the plasma
membrane, with loss of gap junction communication and decreased hemichannel activity.
Similarly, Occludin, ZO 1, β catenin, and E cadherin showed reduced expression and disrupted
membrane localization . These effects were associ ated with increased expression of ER and
oxidative stress markers, suggesting that virus induced cellular stress contributes to junctional
dysregulation. The second aim extended the study to primary astrocytes isolated from neonatal
mice to determine the i mpact on central nervous system (CNS) relevant junctional proteins , key
in maintaining CNS homeostasis . Immunofluorescence, Western blotting, and qRT PCR
revealed substantial downregulation of gap junction (Cx43), tight junction (Occludin, ZO 1), and
adher ens junction (β catenin, E cadherin) proteins following HCoV OC43 infection, highlighting
astrocytes as a critical target for coronavirus induced disruption of CNS homeostasis The studies
in primary astrocytes provide insights into the potential mechanism s contributing to neurological
complications observed in Long COVID. The third aim explored mechanisms contributing to
junctional disruption. HCoV OC43 infection led to strong activation of the NF κ B signaling
pathway, which triggered the production of several pro inflammatory cytokines and chemokines,
along with type I interferon responses. This inflammatory signaling als o drove the upregulation
of matrix metalloproteinases in both A549 cells and astrocytes, providing a potential mechanism
behind the di sruptions of junctional proteins . In parallel, infection caused loss of the Golgi
apparatus, highlighted by the reduction of TGN46, suggesting impaired trafficking of junctional
proteins. Interestingly, reactive oxygen species (ROS) levels were reduced, an d 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 may inform future therapeutic strategies targeting junctional
stability and inflammatory signaling. |