Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction
Fig 5
CMV productively infects human intestinal epithelial cells.
Nonpolarized HCoEpiC infected with CMV VR1814 at a MOI of 1.0 expressed cytokeratin, an epithelial cell marker (green) and CMV IE (A, red) at 1 day and gB (B, red) at 3 days after inoculation. When plated on collagen-coated transwells for 6 days, HCoEpiC differentiated into a polarized monolayer expressing ZO-1 (D, green). (C) Nonpolarized (A) and polarized HCoEpiC (E) were inoculated from the apical surface with CMV VR1814 at a MOI of 1.0. At day 1 after inoculation, cells were fixed and immunostained for CMV IE, and nuclei were counterstained with DAPI. Images of the infected cells were acquired at magnification 400x, and a percentage of CMV IE-positive nuclei were calculated relative to the total number of DAPI-stained nuclei per image. (F, I) Polarized HCoEpiC were inoculated with CMV VR1814 at a MOI of 1.0. After 1 h virus adsorption, some infected cell cultures were treated with an inhibitory concentration (50 nM) of letermovir, whereas other cultures were treated with an inhibitory concentration (20 μM) of GCV. Infectious viral progeny in the medium at 1, 3, 6 and 9 days as well as in the inoculum (day 0) were quantified in human fibroblasts as the indicator monolayer (F). (G, I) CMV slowly replicated in polarized HCoEpiC during 9 days as indicated by the low number of CMV IE-positive cells (6.7%), which was nevertheless higher than CMV-IE-positive cells treated with GCV or letermovir (****P<0.0001). (H) Cytomegalic cells expressing CMV gB (red) were detected on top of the polarized HCoEpiC monolayer 9 days after inoculation. Nuclei were counterstained with DAPI. Scale bars: 10 μm (A, B, D, E), 50 μm (G), 20 μm (H). Data are shown as the mean ± SEM with n = 10 (C, I) and n = 4 (F). Statistical comparison was performed by the Mann Whitney U-test and was considered significant at ****P<0.0001 (C, I); **P<0.01 (F, CMV infectious units (IU)/ml at day 3 versus day 1).