Endoplasmic Reticulum Chaperone ERp29 Regulates Murine β-Coronavirus Induced Gap Junction Intercellular Communication

Bose, Abhishek (2020) Endoplasmic Reticulum Chaperone ERp29 Regulates Murine β-Coronavirus Induced Gap Junction Intercellular Communication. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Abhishek Bose (13IP041)) 13IP041.pdf - Submitted Version Restricted to Repository staff only Download (10MB)

Abstract

In the Central nervous system (CNS), gap-junctions (GJs) are critical to establish “panglial syncytium” by metabolically coupling glial cells. One of the ways to maintain metabolic coupling is through gap junction intercellular coupling (GJIC). For several years RNA viruses have been reported to decrease gap-junctional intercellular communication (GJIC) among infected cells. Recently, we showed in a mouse model of neuroinflammatory demyelination that Mouse Hepatitis Virus (MHV) infection disrupts Connexin43 (Cx43)-containing astrocytic GJs. However, the underlying mechanism remained unelucidated. In our study in primary astrocytes and DBT astrocytoma cells, MHV infection was found to upregulate ER stress protein GRP78 (Bip) and downregulate ERp29, an ER resident PDI family protein known to promote Cx43 assembly and trafficking to the plasma membrane, with increasing multiplicity of virus infection. Stable transfection of exogenous ERp29 in DBT cells, also rescued the trafficking of Cx43 to cell surface. Corroborating these findings, treatment of infected primary astrocytes or DBT cells with 4-phenylbutyrate (4-PBA), known to upregulate native ERp29, facilitated Cx43 trafficking to the cell surface and re-established GJIC. Further studies showed increasing exogenous ERp29 expression and 4-PBA treatment decreased viral infectivity, fusogenicity and replication. TMT labeling and label free preparation of pull-down fraction followed by LC-MS Mass spectrometric analysis revealed differential regulation of binding partners of Cx43 like PDIA3/A4 and heat shock proteins HSP-90/70 suggesting that several chaperones of Cx43 besides ERp29 might also have important role in reducing GJIC and can be potential molecular targets against virus induced astroglial dystrophy. Therefore, our study with a prototypic murine β-CoV (m-CoV) mouse hepatitis virus A59 (MHV-A59) infection suggesting a potential retention of Cx43 as could be one of the putative mechanisms of altered ER function and gap junctions in CoV induced cellular pathogenesis, which can be an important target to develop cell based therapeutic strategies.

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