Mouse Hepatitis Virus Infection Remodels Connexin43-Mediated Gap Junction Intercellular Communication In Vitro and In Vivo

Abstract

Gap junctions (GJs) form intercellular channels which directly connect the cytoplasm between neighboring cells to facilitate the transfer of ions and small molecules. GJs play a major role in the pathogenesis of infection-associated inflammation. Mutations of gap junction proteins, connexins (Cxs), cause dysmyelination and leukoencephalopathy. In multiple sclerosis (MS) patients and its animal model experimental autoimmune encephalitis (EAE), Cx43 was shown to be modulated in the central nervous system (CNS). The mechanism behind Cx43 alteration and its role in MS remains unexplored. Mouse hepatitis virus (MHV) infection-induced demyelination is one of the best-studied experimental animal models for MS. Our studies demonstrated that MHV infection downregulated Cx43 expression at protein and mRNA levels in vitro in primary astrocytes obtained from neonatal mouse brains. After infection, a significant amount of Cx43 was retained in endoplasmic reticulum/endoplasmic reticulum Golgi intermediate complex (ER/ERGIC) and GJ plaque formation was impaired at the cell surface, as evidenced by a reduction of the Triton X-100 insoluble fraction of Cx43. Altered trafficking and impairment of GJ plaque formation may cause the loss of functional channel formation in MHV-infected primary astrocytes, as demonstrated by a reduced number of dye-coupled cells after a scrape-loading Lucifer yellow dye transfer assay. Upon MHV infection, a significant downregulation of Cx43 was observed in the virus-infected mouse brain. This study demonstrates that astrocytic Cx43 expression and function can be modulated due to virus stress and can be an appropriate model to understand the basis of cellular mechanisms involved in the alteration of gap junction intercellular communication (GJIC) in CNS neuroinflammation. IMPORTANCE We found that MHV infection leads to the downregulation of Cx43 in vivo in the CNS. In addition, results show that MHV infection impairs Gap junction communication (GJC) is one of the pivotal requirements in all vertebrates for maintaining cellular homeostasis (1). Gap junctions span the plasma membranes of closely opposed cells to align end to end, forming intercellular channels which allow the exchange of small molecules (less than 1 kDa). They are composed of two hemichannels contributed by two opposing cells. These hemichannels or connexons are formed by six protein subunits, called connexins (Cx). Connexin proteins consist of an intracellular N terminus, four transmembrane domains, two extracellular loops, one cytoplasmic loop, and an intracellular C terminus (2). In the central nervous system (CNS), the major cell type which is coupled by gap junctions is the astrocyte. Astrocytes also form gap junction channels between other neuronal cells to form panglial networks to provide metabolic support and maintain homeostasis in the CNS.Astrocytes, being the most abundant cells in this panglial system, perform essential metabolic functions by maintaining the balance of fluid, ions, pH, and some neurotransmitters in...

Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection

“…These phenomena affect functional channel formation between primary astrocytes. Similar alterations of Cx43 expression and localization are also observed in the mouse brain (19), but the molecular mechanism of Cx43 retention in the ER-ERGIC was not described. For efficient infection and cell-to-cell spread, many viruses have developed mechanisms for utilizing cytoskeletal elements.…”

“…Weakly adherent microglia cells came off into suspension, and these cells suspended in the culture medium were removed. The remaining adherent monolayers of cells were used as enriched astrocyte cultures for further experimentation (19).…”

“…A neurotropic demyelinating strain of coronavirus, MHV-A59, was used to understand the mechanism of retention of Cx43 in the intracellular compartment, similar to previous studies (19). Primary astrocytes were infected with inoculation medium (DMEM containing 1% penicillin-streptomycin and 1% glutamine with 2% FBS) containing MHV-A59 at an MOI of 2.…”

“…Immunofluorescence studies were done according to a protocol described previously (19). For standard immunofluorescence, primary astrocytes were plated on etched glass coverslips.…”

“…The beads were washed three times, and finally the bound material was eluted in SDS-PAGE sample buffer. Samples were boiled and separated by SDS-PAGE, transferred to PVDF, and probed for target protein by Western blotting, as described previously (19). The sources and dilutions of primary antibodies are listed in Table 1.…”