CMV productively infects human intestinal epithelial cells.

<p>Nonpolarized HCoEpiC infected with CMV VR1814 at a MOI of 1.0 expressed cytokeratin, an epithelial cell marker (green) and CMV IE (<b>A,</b> red) at 1 day and gB (<b>B,</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 (<b>D</b>, green). (<b>C</b>) Nonpolarized (<b>A</b>) and polarized HCoEpiC (<b>E</b>) 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. (<b>F, I</b>) 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 (<b>F</b>). (<b>G</b>, <b>I</b>) 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 <b>(</b>****P<0.0001). (<b>H</b>) 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 (<b>A, B, D, E</b>), 50 μm (<b>G</b>), 20 μm (<b>H</b>). Data are shown as the mean ± SEM with n = 10 (<b>C, I</b>) and n = 4 (<b>F</b>). Statistical comparison was performed by the Mann Whitney U-test and was considered significant at ****P<0.0001 (<b>C, I</b>); **P<0.01 (<b>F</b>, CMV infectious units (IU)/ml at day 3 versus day 1).</p

Detection of CMV in rectosigmoid biopsies from untreated HIV/CMV-coinfected individuals.

<p>(<b>A</b>–<b>C</b>) Formalin-fixed paraffin-embedded (FFPE) rectosigmoid biopsies were probed for HIV RNA (fuchsia) by RNAscope ISH. CMV proteins were detected in adjacent tissue sections by chromogenic (<b>D,</b> brown) and fluorescent (<b>E</b>−<b>G</b>, green) IHC using a blend of anti-CMV monoclonal antibodies (Millipore) comprising clone 8B1.2 reacting with an IE protein of 68–72 kDa, clone 1G5.2 reacting with unspecified late CMV protein, and clone 2D4.2 reacting with a late structural protein of 47–55 kDa. The adjacent section of <b>D</b> was immunostained with isotype control IgG (<b>H</b>). Regions of interest outlined by black (<b>A</b>, <b>D</b>) or red (<b>B</b>, <b>C</b>) are shown in the insets. HIV-infected T cells were identified using CD3 detection (<b>B,</b> dark brown), and HIV-infected macrophages were identified using CD163 and CD68 markers (<b>C,</b> dark brown). Putative leukocytes containing CMV structural proteins (<b>D</b>, brown and <b>E</b>, <b>F</b>, green) appeared to be trafficking across the intestinal epithelium identified by cytokeratin (CK, red) immunostaining. Leukocyte phenotype was verified by coimmunostaining for CMV proteins (<b>G</b>, green) and CD45 (<b>G</b>, red). Nuclei were counterstained with hematoxylin (<b>A</b>−<b>D</b>, <b>H</b>) or DAPI (<b>E</b>−<b>G</b>). A HIV-infected CD163⁺CD68⁺ macrophage (arrow) and CD163⁻CD68⁻ putative T cell (arrowhead) are shown in panel <b>C</b>. Scale bars: 100 μm (<b>A</b>–<b>C</b>), 50 μm (<b>D</b>–<b>H</b>). These results are representative of those observed in rectosigmoid biopsies from 3 untreated HIV-infected individuals with asymptomatic CMV infection.</p

Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction

<div><p>Although invasive cytomegalovirus (CMV) disease is uncommon in the era of antiretroviral therapy (ART), asymptomatic CMV coinfection is nearly ubiquitous in HIV infected individuals. While microbial translocation and gut epithelial barrier dysfunction may promote persistent immune activation in treated HIV infection, potentially contributing to morbidity and mortality, it has been unclear whether CMV replication in individuals with no symptoms of CMV disease might play a role in this process. We hypothesized that persistent CMV replication in the intestinal epithelium of HIV/CMV-coinfected individuals impairs gut epithelial barrier function. Using a combination of state-of-the-art <i>in situ</i> hybridization technology (RNAscope) and immunohistochemistry, we detected CMV DNA and proteins and evidence of intestinal damage in rectosigmoid samples from CMV-positive individuals with both untreated and ART-suppressed HIV infection. Two different model systems, primary human intestinal cells differentiated <i>in vitro</i> to form polarized monolayers and a humanized mouse model of human gut, together demonstrated that intestinal epithelial cells are fully permissive to CMV replication. Independent of HIV, CMV disrupted tight junctions of polarized intestinal cells, significantly reducing transepithelial electrical resistance, a measure of monolayer integrity, and enhancing transepithelial permeability. The effect of CMV infection on the intestinal epithelium is mediated, at least in part, by the CMV-induced proinflammatory cytokine IL-6. Furthermore, letermovir, a novel anti-CMV drug, dampened the effects of CMV on the epithelium. Together, our data strongly suggest that CMV can disrupt epithelial junctions, leading to bacterial translocation and chronic inflammation in the gut and that CMV could serve as a target for therapeutic intervention to prevent or treat gut epithelial barrier dysfunction during HIV infection.</p></div

Detection of CMV in rectosigmoid biopsies from ART-suppressed HIV/CMV-coinfected individuals.

<p>IHC for CMV IE, early and late structural proteins using the anti-CMV antibody blend in FFPE rectosigmoid biopsies from ART-suppressed HIV/CMV-coinfected individuals (<b>A</b>) and a HIV-negative, CMV-negative individual <b>(B</b>). The presence of CMV was verified by DNAscope ISH using a probe targeting the noncoding region (120742–122152) of CMV strain Merlin, which corresponds to the UL83 (pp65) sequence (fuchsia) (<b>C, D</b>). Yellow stars indicate similar patterns of IE protein expression (<b>A</b>) and the presence of viral DNA (<b>C</b>) in the nuclei of CMV-infected intestinal cells. (<b>E</b>) CMV early and late proteins (brown) were detected in the cytoplasm of intestinal epithelial cells costained for cytokeratin (fuchsia). (<b>F</b>) HIV RNA was randomly detected by RNAscope ISH in CD68/CD163-positive macrophages (insets) in an adjacent section of tissue shown in (<b>C</b>). Nuclei were counterstained with hematoxylin. Scale bars: 50 μm (<b>A</b>–<b>C</b>, <b>E</b>, <b>F</b>), 100 μm (<b>D</b>). These results are representative of those observed in rectosigmoid biopsies from 9 asymptomatic ART-suppressed HIV/CMV-coinfected individuals.</p

Disruption of intestinal tight junctions was associated with the presence of CMV in rectosigmoid biopsies.

<p>Immunolocalization of ZO-1, a marker of tight junctions (tight junctions, green) and CMV IE, early, and late proteins (red) in FFPE rectosigmoid biopsies by fluorescent IHC. ZO-1 labeling appeared in the epithelial cells of the intestinal crypts and endothelial cells of blood vessels. Results are representative of those observed in rectosigmoid biopsies from 5 HIV-negative CMV-negative (HIV⁻CMV⁻) individuals (<b>A</b>), 3 untreated HIV/CMV-coinfected (HIV⁺CMV⁺) individuals (<b>B</b>), 9 ART-suppressed HIV/CMV-coinfected (HIV⁺CMV⁺) individuals (<b>C</b>), 3 HIV-negative CMV-positive (HIV⁻CMV⁺) individuals (<b>D</b>) and an individual with active CMV infection before (<b>E</b>) and after (<b>F</b>) treatment with valganciclovir. The arrowheads show areas of discontinuous tight junctions (absence of ZO-1 staining). Nuclei were counterstained with DAPI. Scale bars: 20 μm</p

CMV infection decreases TER of polarized HCoEpiC.

<p>At 6 days after plating, polarized HCoEpiC were inoculated with CMV VR1814 from the apical surface at a MOI of 1.0 (<b>A</b>), from the basolateral surface at a MOI of 10 (<b>B</b>), or mock-infected. TER was then measured by Millicell-ERS every 3 days. The shaded area (gray) indicates TER of nonpolarized cells (<100 Ohm*cm² as confirmed by FITC-dextran leakage). Data are shown as the mean ± SEM with n = 5 for each experimental condition. Statistical comparison was performed by the Mann Whitney U-test and was considered significant at (<b>A</b>) **P<0.01 and (<b>B</b>) *P<0.05.</p

CMV-associated disruption of polarized HCoEpiC monolayer integrity is partially attributed to CMV-induced IL-6.

<p>Polarized HCoEpiC derived from two donors (#1 and #2) were inoculated with CMV VR1814 at a MOI of 1.0; TER was measured 9 days after inoculation by Millicell-ERS (<b>A</b>), and culture medium was collected for quantification of TNF-<i>α</i> (Β<b>),</b> IL-6 (<b>C</b>), and IL-1β (<b>D</b>) by ELISA. (<b>E</b>) Polarized HCoEpiC were inoculated with CMV at a MOI of 5.0 or mock infected. Some infected cells were treated before and during infection with anti-human IL-6 mouse IgG or isotype control IgG at 0.1 mg/ml (5 times the 50% neutralization dose). The shaded area (gray) indicates TER of nonpolarized cells. Data are shown as the mean ± SEM with n = 4 (<b>A</b>-<b>D</b>) and n = 6 (<b>E</b>) for each experimental condition. Statistical comparison was performed by the Mann Whitney U-test and was considered significant at (<b>A</b>-<b>D</b>) *P<0.05; (<b>E</b>) **P<0.01 (CMV infected and treated with anti-IL-6 IgG versus CMV infected and treated with isotype control IgG at day 1), *P<0.05 (CMV infected and treated with anti-IL-6 IgG versus CMV infected and treated with isotype control IgG at day 3 and 6; mock infected versus CMV infected and treated with anti-IL-6 IgG at day 1). Dpi, days postinfection.</p

CMV infection rapidly depletes epithelial cells of the human fetal intestine differentiated <i>in vivo</i>.

<p>(<b>A</b>, <b>B</b>) Human fetal intestine implanted subcutaneously into SCID mice differentiates <i>in vivo</i> over 4 weeks. Gut implant cross-sections stained with hematoxylin and eosin (H&E) (<b>A</b>) and immunostained for human cytokeratin (<b>B</b>, green). (<b>C</b>) Histologic sections of gut implants 7 days after CMV VR1814 inoculation with 5.7 log₁₀ IU were coimmunostained for CMV IE proteins (red) and human cytokeratin (green). The region of interest outlined by the white oblong is magnified in (<b>D</b>). Data are representative of those obtained in 3 independent experiments using gut tissues from three donors. Nuclei were counterstained with hematoxylin (<b>A</b>) and DAPI (<b>B</b>−<b>D</b>). Scale bars: 200 μm (<b>A</b>), 100 μm (<b>B</b>, <b>C</b>), 50 μm (<b>D</b>).</p

Bevirimat Inhibits p24 Release from the Capsid p25 Precursor in Human Thymocytes.

<p>Thymocytes dispersed from NL4-3- and SP1/A1V-infected SCID-hu Thy/Liv implants were cultured in the presence of the indicated range of concentrations of bevirimat for 2 days. Purified virions was lysed and individual viral proteins were detected with HIV-1 Ig. Note the target-specific and dose-dependent reduction of CAp25 cleavage in the presence of bevirimat. The presence of the drug does not affect the stoichiometry of the precursor Pr55Gag polyprotein nor does it inhibit cleavage of the matrix protein, MAp17 (lower panel, longer exposure).</p

Protease Inhibitor-Resistant HIV-1 has Impaired CA-SP1 Cleavage.

<p>Wild-type NL4-3 and protease mutant NL4-3 PR_I54V+V82A virions were collected from transfected 293T cells grown in the presence (20 µM) or absence of bevirimat and analyzed for particle maturation by Western blot using a p24 monoclonal antibody. The protease mutant is deficient in CA-SP1 cleavage and is comparable to bevirimat-treated wild-type virus, however, the drug has a more dramatic effect on CA-SP1 cleavage in HIV-1 PR_I54V+V82A.</p