Non-classical human astrovirus VA1 induces cell line-specific increases in intestinal epithelial permeability associated with claudin remodeling

Astroviruses are non-enveloped viruses with a positive-sense, single-stranded RNA genome. Human astroviruses (HAstVs) are classified into classical strains (HAstV-1 to HAstV-8) and non-classical strains (VA1-6 and MLB1-3). These viruses typically cause self-resolving acute gastroenteritis, but symptoms can be more severe, including extraintestinal infections and viral encephalitis, especially in vulnerable groups, such as children and the immunocompromised. Despite being one of the leading causes of pediatric gastroenteritis worldwide, little is known about the pathogenic mechanisms, and no vaccines or antivirals currently exist. Tight junctions are formed by multiprotein complexes, including claudins and occludin, that separate the intestinal lumen from the host interior and play an important role in intestinal barrier maintenance. HAstV-1 disrupts these tight junctions in polarized Caco-2 monolayers by degrading occludin and E-cadherin, thereby decreasing intestinal barrier permeability. In contrast, this study demonstrated that VA1 decreases transepithelial electrical resistance in polarized T84, but not Caco-2, cells by altering protein levels and localization of select claudins. Specifically, western blot analysis of T84, but not Caco-2, protein lysates showed that protein expression of the pore-forming claudin-2 increased in infected cells, while protein levels of sealing claudins-4 and -18 decreased. In addition, immunofluorescence imaging indicated a relocalization of claudin-3 into the cytosol. Butyrate, a short-chain fatty acid bacterial metabolite found in the intestine, attenuated these barrier changes. These data demonstrate that VA1 induces cell type-specific claudin remodeling associated with decreased epithelial barrier permeability in T84 cells and highlight the differences between classical and non-classical HAstVs in the mechanisms underlying intestinal barrier remodeling.IMPORTANCEHuman astroviruses are prevalent enteric viruses that are a leading cause of pediatric gastroenteritis worldwide. However, little is known about their pathogenic mechanisms, and no vaccines or antivirals are available to treat or prevent infections. Herein, we demonstrate that VA1 infection induces changes in the electrical conductance of polarized T84 cell monolayers and alters claudin protein levels or their cellular localization. This phenotype is attenuated by pre-treatment with the bacterial short-chain fatty acid butyrate and is not observed in infected polarized Caco-2 monolayers. Collectively, these data suggest that VA1 induces cell type-specific barrier alterations through claudin remodeling that differ from the HAstV-1-induced alterations in tight junction complex proteins occludin and E-cadherin, highlighting virus-specific differences in pathogenic mechanisms of classical and non-classical HAstVs.