Astrovirus increases epithelial barrier permeability independently of viral replication

Abstract

Astrovirus infection in a variety of species results in an age-dependent diarrhea; however, the means by which astroviruses cause diarrhea remain unknown. Studies of astrovirus-infected humans and turkeys have demonstrated few histological changes and little inflammation during infection, suggesting that intestinal damage or an overzealous immune response is not the primary mediator of astrovirus diarrhea. An alternative contributor to diarrhea is increased intestinal barrier permeability. Here, we demonstrate that astrovirus increases barrier permeability in a Caco-2 cell culture model system following apical infection. Increased permeability correlated with disruption of the tight-junction protein occludin and decreased the number of actin stress fibers in the absence of cell death. Additionally, permeability was increased when monolayers were treated with UV-inactivated virus or purified recombinant human astrovirus serotype 1 capsid in the form of virus-like particles. Together, these results demonstrate that astrovirus-induced permeability occurs independently of viral replication and is modulated by the capsid protein, a property apparently unique to astroviruses. Based on these data, we propose that the capsid contributes to diarrhea in vivo.

FIG. 1.

HAstV-1 increases barrier permeability in apical, but not basal, infection. Differentiated Caco-2 monolayers grown on permeable supports were mock infected (diamonds) or were infected with HAstV-1 (MOI 10) (squares) from the apical (A) or the basal (B) surface. TER levels were measured from 0 to 48 hpi. (C and D) The degree of paracellular permeability was measured from 4 to 48 hpi by monitoring the migration of FD-4 to the basal chamber over mock-infected (gray) or HAstV-infected (black) Caco-2 monolayers infected either apically (C) or basally (D). Error bars represent standard deviations, and asterisks represent statistical significance (P < 0.05) compared to values for mock infection.

FIG. 2.

HAstV-1's effects are dose dependent and require virus-cell interactions. (A) Differentiated Caco-2 cells were mock infected (triangles) or were infected with HAstV-1 virus at an MOI of ∼10 (light gray squares), 5 (dark gray squares), or 1 (black squares), and TER levels were monitored from 0 to 48 hpi. Error bars represent standard errors of the means, and asterisks represent statistical significance (P < 0.05) compared to values for mock infection. (B) Differentiated Caco-2 cells were treated with mock-infected cell lysate (dark triangles), mock-infected lysate preincubated with anti-HAstV-1 antibody (open triangles), HAstV-1 alone (MOI 10) (dark squares), HAstV-1 preincubated with irrelevant antibody (open diamonds), or HAstV-1 preincubated with anti-HAstV-1 antibody (open squares), and TER levels were monitored from 0 to 48 hpi. Data are representative of at least two experiments.

FIG. 3.

HAstV-1 does not increase cell death. Cell viability was measured for mock- or HAstV-1-infected (MOI, 10) differentiated Caco-2 cells at 4, 12, 24, and 36 hpi by trypan blue exclusion. No significant difference between mock-infected cells and HAstV-1-infected cells was noted at any time point. Error bars represent standard deviations.

FIG. 4.

HAstV-1 inactivation. Differentiated Caco-2 cells were mock infected (A) or were infected with infectious HAstV-1 (B) or UV-treated HAstV-1 at 50 mJ/cm² (C) or 100 mJ/cm² (D). At 24 hpi, monolayers were screened for HAstV-1 antigen by immunofluorescence. Inserts depict nuclei staining by DAPI. All images are at ×40 magnification.

FIG. 5.

HAstV-1-induced barrier permeability is independent of viral replication. (A) Differentiated Caco-2 cells were mock infected (diamonds), infected with HAstV-1 (MOI, 10) (squares), or infected with UV-inactivated HAstV-1 (circles). TER levels were measured from 0 to 24 hpi. Error bars represent standard errors of the means, and asterisks indicate P < 0.05 compared to values for mock infection. (B) At the indicated times postinfection, TER levels of mock-treated (n ≥ 3) (closed diamonds), HAstV-1-infected (n ≥ 3) (closed squares), PBS-treated (n = 3) (open diamonds), or purified HAstV-1 VLP-treated (35 μg; n ≥ 3) (open squares) monolayers were determined. Results are presented as percentages of the initial TER readings. Error bars represent standard deviations, and asterisks indicate significance between mock- and HAstV-1-infected samples; #, significance between PBS- and capsid-treated samples (P < 0.05).

FIG. 6.

Purification of HAstV-1 VLPs. (A) HAstV-1 ORF2 was expressed in Sf9 cells by baculovirus, and capsid protein was purified by size-exclusion chromatography and examined by Western blot analysis. (B) VLPs then were verified by electron microscopy. Bar, 100 nm.

FIG. 7.

HAstV-1 infection disrupts the actin cytoskeleton. Differentiated Caco-2 cells grown on inserts were treated with mock-infected lysates (A, D, and G), UV-inactivated HAstV-1 (C, F, and I), or HAstV-1 virus (B, E, and H). At 24 hpi, monolayers were fixed and stained for actin (A to F) and HAstV-1 capsid protein (G to I). Protein localization was visualized by epifluorescent microscopy. All images are at ×40 magnification; arrows identify regions of protein disruption.

FIG. 8.

HAstV-1 infection disrupts occludin. Differentiated Caco-2 cells grown on inserts were mock (A), UV-inactivated HAstV-1 (C), or HAstV-1 (B) infected. At 24 hpi, monolayers were fixed and stained for occludin. Protein localization was visualized by confocal microscopy. All images are at ×40 magnification; arrows identify regions of protein disruption between neighboring cells.