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. 2008 Dec;82(23):11902-12.
doi: 10.1128/JVI.01042-08. Epub 2008 Sep 24.

Kaposi's sarcoma-associated herpesvirus disrupts adherens junctions and increases endothelial permeability by inducing degradation of VE-cadherin

Li-Wu Qian  1 , Whitney Greene, Fengchun Ye, Shou-Jiang Gao
Affiliations

Kaposi's sarcoma-associated herpesvirus disrupts adherens junctions and increases endothelial permeability by inducing degradation of VE-cadherin

Li-Wu Qian et al. J Virol. 2008 Dec.

Abstract

Kaposi's sarcoma (KS) is a vascular tumor of proliferative endothelial cells caused by KS-associated herpesvirus (KSHV) infection. Aberrant vascular permeability is a hallmark of KS manifested as multifocal edematous skin and visceral lesions with dysregulated angiogenesis and vast inflammatory infiltrations. In this study, we showed that KSHV infection increased the permeability of confluent endothelial monolayers to serum albumin, blood-derived cells, KSHV-infected cells, and KSHV virions. KSHV-induced permeability was associated with the disruption of adherens junctions and the degradation of vascular endothelial cadherin (VE-cadherin) protein. Both the inactivation of KSHV virions by UV irradiation and the blockage of de novo protein synthesis with cycloheximide failed to reverse the KSHV-induced disruption of adherens junctions. However, soluble heparin that blocked KSHV entry into cells completely inhibited KSHV-induced permeability. Furthermore, the KSHV-induced degradation of VE-cadherin was dose dependent on the internalized virus particles. Together, these results indicate that KSHV infection induces vascular permeability by inducing VE-cadherin degradation during virus entry into cells. KSHV-induced aberrant vascular permeability could facilitate virus spread, promote inflammation and angiogenesis, and contribute to the pathogenesis of KSHV-induced malignancies.

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Figures

FIG. 1.
FIG. 1.
KSHV infection increases the permeability of confluent endothelial monolayers to serum albumin, blood-derived U937 cells, KSHV-infected BCBL-1 cells, and KSHV virions. (A) Kinetics of endothelial permeability to serum albumin following KSHV infection. Confluent endothelial monolayers were mock infected or infected with KSHV and assayed for endothelial permeability to methylene blue-conjugated serum albumin at different hpi. The amount of serum albumin that migrated to the lower chamber was determined by measuring the optical density at 405 nm. (B) Endothelial permeability of KSHV-infected confluent endothelial monolayers to VybrantDiO cell-labeled U937 cells at 24 hpi. The number of U937 cells that migrated to the lower chamber was counted with a fluorescence microscope. (C) Kinetics of endothelial permeability to BCBL-1 cells following KSHV infection. BCBL-1 cells containing BAC36 and expressing GFP were used for the assay. The number of BCBL-1 cells that migrated to the lower chamber was counted with a fluorescence microscope. (D) Endothelial permeability of KSHV-infected confluent endothelial monolayers to KSHV virions at 24 hpi. Virus preparations containing 105 infectious units in 250 μl culture medium were added to the monolayers and incubated for 1 h. The amount of infectious virions that penetrated the endothelial monolayers and reached the lower chamber was titrated for infectious units.
FIG. 2.
FIG. 2.
KSHV infection disrupts the adherens junctions. (A) VE-cadherin staining of KSHV-infected confluent endothelial monolayers at different hpi. (B) Mock- and KSHV-infected confluent endothelial monolayers stained for VE-cadherin protein (green), KSHV LANA (red), and nuclei (blue) at 24 hpi.
FIG. 3.
FIG. 3.
Expression kinetics of VE-cadherin protein and transcripts following KSHV infection. (A) Western-blotting analysis of VE-cadherin protein in mock- or KSHV-infected endothelial cells at different hpi. β-Tubulin was used to calibrate protein loading. (B) Quantification of VE-cadherin protein levels at the different hpi shown in panel A. The VE-cadherin protein amount of mock-infected endothelial cells at 0.25 hpi was set at 100%. (C to D) Expression of VE-cadherin (C) and LANA (ORF73) (D) transcripts in mock- or KSHV-infected endothelial cells at different hpi quantified by reverse transcription real-time quantitative PCR (RT-qPCR). RT-qPCR for GAPDH was used as a loading control. The level of VE-cadherin transcripts of mock-infected endothelial cells at 1 hpi was set at 1. The level of LANA transcripts of KSHV-infected endothelial cells at 1 hpi was set at 1. LANA transcripts were not detected in mock-infected cells at any time point.
FIG. 4.
FIG. 4.
Disruption of adherens junctions in UV-irradiated KSHV (UV-KSHV)-infected confluent endothelial monolayers but not in those treated with conditioned medium from KSHV-infected endothelial cells. (A and B) Adherens junctions revealed by VE-cadherin staining in mock- or KSHV-infected confluent endothelial monolayers at 6 hpi. (C) VE-cadherin staining in confluent endothelial monolayers treated with KSHV-conditioned medium for 6 h. Conditioned medium was obtained from confluent endothelial monolayers that displayed a clear disruption of adherens junctions after infection with KSHV for 6 hpi. The medium was subjected to high-speed centrifugation to eliminate any virions and cellular debris. A new confluent endothelial monolayer was then incubated with the medium for 6 h and stained for VE-cadherin. (D) VE-cadherin staining of a UV-KSHV-infected confluent endothelial monolayer at 6 hpi. (E) Representative pictures showing viral particles revealed by ORF65 staining in endothelial cells infected with KSHV or UV-KSHV at 4 hpi. (F) Infectivity of KSHV and UV-KSHV was examined by infecting cells for 2 days, staining for the expression of KSHV LANA, and calculating the percentages of positive cells. (G) Internalized viral particles in cells infected by KSHV or UV-KSHV for 4 h. Viral particles were detected by staining for the ORF65 protein.
FIG. 5.
FIG. 5.
The disruption of adherens junctions and the degradation of VE-cadherin protein during the KSHV primary infection of confluent endothelial monolayers are mediated by virus entry and do not require de novo synthesis of viral and cellular proteins. (A) Western-blotting analysis of VE-cadherin protein in mock-, KSHV-, and UV-KSHV-infected confluent endothelial monolayers with (+) or without (−) the presence of CHX. The infection was carried out for 4 h. β-Tubulin was used to calibrate protein loading. (B) Quantification of the VE-cadherin protein levels shown in panel A. The VE-cadherin protein level of mock-infected endothelial cells without the presence of CHX was set at 100%. (C) KSHV infection disrupted adherens junctions in the presence of CHX. Confluent endothelial monolayers were either mock-infected or infected with KSHV with or without the presence of CHX for 4 h and stained for VE-cadherin. (D) KSHV infection increased endothelial permeability to serum albumin in the presence of CHX. Confluent endothelial monolayers were either mock-infected or infected with KSHV with or without the presence of CHX. The permeability of the cells to serum albumin was examined as described in Fig. 1A. (E) UV-KSHV infection induced endothelial permeability to serum albumin, which was inhibited by soluble heparin. Confluent endothelial monolayers were either mock-infected or infected with KSHV or UV-KSHV with or without the presence of soluble heparin. The permeability of the cells to serum albumin was examined as described in the legend to Fig. 1A.
FIG. 6.
FIG. 6.
Kinetics of VE-cadherin protein degradation during the KSHV primary infection of confluent endothelial monolayers. (A) Western-blotting analysis for VE-cadherin protein was carried out with confluent endothelial monolayers mock-infected or infected with KSHV in the presence of CHX at different hpi. (B) Quantification of the VE-cadherin protein levels at the different hpi shown in panel A. The VE-cadherin protein level of mock-infected cells at 0 hpi was set at 100%.
FIG. 7.
FIG. 7.
The KSHV-induced degradation of VE-cadherin protein is dose dependent on the internalization of viral particles but not infectivity. (A) Western-blotting analysis for VE-cadherin protein was carried out with confluent endothelial monolayers mock-infected or infected with KSHV with different vpc in the presence of CHX at different hpi. (B) Quantification of the VE-cadherin protein levels shown in panel A. The VE-cadherin protein levels at 0 hpi were set at 100%. (C) Infectivity and internalized viral particles in cells infected with the same doses of vpc as described in panel A. The actual internalized viral particles were revealed by staining for the ORF65 protein at 4 hpi. KSHV infectivity was examined by staining for the expression of KSHV LANA and calculating the percentages of positive cells.
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