| Summary: | Adenoviruses are non-enveloped, dsDNA tumor viruses responsible for a breadth of pathogenesis including acute respiratory disease and viral myocarditis. Gap junctions, which are formed by connexin proteins, directly couple the cytoplasms of apposed cells enabling immunological, metabolic, and electrical intercellular communication. The gap junction protein connexin43 (Cx43; gene name – GJA1) is the most widely expressed human connexin protein and is the predominant connexin in the working myocardium. Given the immunological role for Cx43 gap junctions, we hypothesized that gap junctions would be targeted during adenoviral infection. We find reduced Cx43 protein due to suppression of GJA1 transcription dependent upon β-catenin during adenoviral infection, with viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation at residues previously demonstrated to alter gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation, with complementary studies in human induced pluripotent stem cell derived-cardiomyocytes revealing Cx43 gap junction remodeling concomitant with reduced ZO-1 complexing. These findings demonstrate specific targeting of gap junction function by Ad5 leading to disruptions in intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts.
Intercellular junction proteins belonging to classically defined unique junctions exhibit extensive cross-talk and interdependency for expression and localization. We find reduced connexin43 (Cx43) phosphorylation at a known internalization motif, leading us to hypothesize that gap junctions are maintained during adenoviral infection in order to stabilize intercellular junctions and adenoviral receptors therein. Utilizing immunofluorescence confocal microscopy, we demonstrate that Cx43 reductions are primarily cytosolic with Cx43 preservation at the plasma membrane. Click-IT chemistry, a non-radioactive pulse-chase technique, reveals that Cx43 ½ life is extended during adenoviral infection. In order to test if remaining Cx43 exists in de facto gap junctions (i.e. not undocked or cytosolic connexons) we utilized 1 % Triton X-100 solubility fractionation and find Cx43 is indeed primarily junctional during adenoviral infection. Having demonstrated increases in junctional Cx43, we next asked how tightly coupled cells were during adenoviral infection and by ECIS measurements of electrical resistance we demonstrate a transient increase in mechanical coupling during infection. Our future aims are to uncover changes in Coxsackievirus and adenovirus receptor (CAR) protein localization to determine if adenoviral-induced changes to subcellular architecture predisposes neighboring cells to infection and enhances viral spread. These findings will add to the existing model of adenoviral infection and more broadly, contribute to the therapeutic design of adenoviral vectors for cancer and gene therapy. === Doctor of Philosophy === The human heart will beat more than 3 billion times during the average lifetime. This is accomplished by billions of individual heart muscle cells, called cardiomyocytes, contracting in synchrony. Cardiomyocytes require direct cell to cell communication in order to receive the proper cues and work in concert. Outside of the heart, including the lining of the lungs which acts as a first line of defense against invading pathogens, direct cell to cell communication is important for mounting proper immune responses. A primary means by which cells communicate directly with neighboring cells is through gap junctions which are formed of proteins called connexins. Six connexin proteins form a channel in the cell surface that binds to a similar channel on an apposing cell to create a continuous gap junction channel, coupling the cell interiors directly. The most widely expressed human connexin, and the most abundant connexin in the heart, is connexin43 (Cx43; gene name – GJA1). Adenoviruses are pathogens commonly associated with respiratory illnesses in addition to more serious diseases including viral myocarditis, or infection of the heart. Given that Cx43 gap junctions enable direct intercellular communication important in initiating immune responses, we hypothesized that adenovirus would target Cx43 and gap junctions during infection.
We find reduced Cx43 protein in cells infected with human adenovirus, and revel that the expression of the GJA1 gene is suppressed. We next focused on potential signaling pathways that are changed during adenoviral infection. β-catenin is a factor with several cellular roles including regulating expression of specific genes including GJA1 (Cx43). We demonstrate β-catenin is activated during adenoviral infection and that this is necessary for reducing Cx43 transcripts. A pathway that activates β-catenin in this manner is the PI3K/Akt signaling axis, which has previously been shown to be turned on during adenovirus infection by a viral protein called E4orf1. We find the adenoviral protein E4orf1 is sufficient to induce β-catenin activation revealing a potential therapeutic target for future studies. We next determined that direct cell to cell communication through gap junctions is reduced before loss of the gap junction protein Cx43 during infection. Gap junctions are modified by the cell to change their ability to couple cells independently of protein levels alone and we find gap junction modifications consistent with altered communication ability. Furthermore, the gap junction protein Cx43 interacts with the cellular skeleton protein Zonula Occludens-1 (ZO-1) during movement into and out of gap junction clusters. We determined alterations in Cx43/ZO-1 interactions consistent with gap junction remodeling. In complimentary studies we find the same gap junction remodeling in cardiomyocytes revealing arrhythmogenic potential during acute adenoviral infection in human heart cells.
Localized with gap junctions are several other junction proteins including the Coxsackievirus and adenovirus receptor (CAR) which is critical in cardiac development and also the primary receptor for species C adenoviruses (used in our studies). CAR expression has been demonstrated to alter Cx43 levels and indeed, many junctional proteins influence the expression and/or localization of other junctional proteins. Interestingly, despite reduced Cx43 levels and reduced gap junction function (cell to cell communication), we detected decreases in a gap junction modification that is associated with gap junction degradation, suggesting that new gap junction protein Cx43 is not being made but already synthesized Cx43 is degraded more slowly. We hypothesized Cx43 is maintained during adenoviral infection in order to recruit other junctional components, principally CAR, on uninfected neighbor cells to predispose them to infection. We observed using microscopy that Cx43 reductions are primarily inside the cell but Cx43 is preserved on the cell surface and at junctions between cells. We next asked if the protein is being degraded more slowly and find Cx43 exists for longer in infected cells signifying that it is being degraded more slowly. Utilizing a fractionation technique to separate gap junction connexin from connexon that is non-junctional or inside the cell, we detect an increase in junctional Cx43, revealing maintenance of Cx43 gap junction structures. Having now identified adenoviral-mediated maintenance of Cx43 gap junction structures, we next wanted to test for changing in mechanical coupling (i.e. how tightly are the cells connected to one another) where we demonstrate an increase in mechanical coupling during adenoviral infection. Our future directions are to determine if this increase in Cx43 gap junction maintenance and mechanical coupling is concomitant with changes in CAR expression/localization on uninfected neighboring cells and if altered, does this predispose uninfected neighbors of infected cells to infection.
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