Effective human herpesvirus 8 infection of human umbilical vein endothelial cells by cell-mediated transmission - PubMed (original) (raw)

Effective human herpesvirus 8 infection of human umbilical vein endothelial cells by cell-mediated transmission

S Sakurada et al. J Virol. 2001 Aug.

Abstract

Cell-free transmission of human herpesvirus 8 (HHV-8) to human cells in vitro has been reported to be difficult, if not impossible. The present experiments were conducted with the idea that cell-cell contact may produce much more effective transmission, so-called cell-mediated transmission. Primary human umbilical vein endothelial cells (HUVECs) were cocultured with an HHV-8-infected lymphoma cell line, BCBL-1 cells. When a ratio of 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated BCBL-1 cells to HUVECs of 10:1 was used, more than 20% of HUVECs were found to express the HHV-8 latency-associated nuclear antigen (LANA) 48 h after the start of coculturing; this value increased to more than 30% after 72 h. HHV-8-encoded ORF26, K8, K8.1, K10, K11, ORF59, and ORF65 proteins were not detected in these HHV-8-infected HUVECs until 72 h. The HHV-8 antigens were not observed in HUVECs cocultured with TPA-treated BCBL-1 cells separated by a membrane. Thirty days after removal of the BCBL-1 cells from the cell-mediated transmission experiment, the HUVECs still expressed LANA and the HHV-8 genome was detected by PCR in these cells. Moreover, the ORF59 protein, a DNA replication-associated protein of HHV-8, was expressed in such HUVECs in the presence of TPA stimulation. These results indicated a far more effective transmission mechanism, cell-cell contact, suggesting the possibility that such a mechanism works in vivo.

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Figures

FIG. 1

FIG. 1

Immunodetection of HHV-8-encoded proteins. (A and B) Adherent cells on chamber slides on which HUVECs were cocultured with TPA-treated BCBL-1 cells. All adherent cells express VWF (in panel A, Alexa 488, green), and some of them express LANA (in panels A and B, Alexa 568, red). These adherent cells do not express LCA (in panel B, Alexa 488, green). (C and D) Centrifuged BCBL-1 cells. BCBL-1 cells express LANA (in panels C and D, Alexa 568, red) and LCA (in panel D, Alexa 488, green) but not VWF (in panel C, Alexa 468, green). (E to I) Expression of LANA (in panels E to G, Alexa 488, green) and vIL-6 (in panels H and I, Alexa 488, green) in HUVECs cocultured with BCBL-1 cells. Propidium iodide (red) was used as a counterstaining agent, and yellow indicates overlap (E to I). (J) No staining of LANA (Alexa-488, green) in HEp-2 cells cocultured with TPA-treated BCBL-1 cells. (K) LANA expression in HUVECs cocultured with BCBL-1 cells 30 days after infection. Green indicates Alexa-488 staining of LANA, and red indicates Alexa-568 staining of VWF. (L) ORF59 protein expression in TPA-treated HUVECs cocultured with BCBL-1 cells 30 days after infection. Green indicates Alexa-488 staining of ORF59 protein, and red indicates Alexa-568 staining of VWF.

FIG. 2

FIG. 2

Percentages of LANA-positive HUVECs cocultured with BCBL-1 cells. Vertical bars indicate standard deviations from three experiments. (A) Time course experiment. Solid, broken, and gray lines indicate the LANA positivity rates in HUVECs with cell-mediated transmission using TPA-stimulated BCBL-1 cells and nonstimulated BCBL-1 cells and cell-free transmission using TPA-stimulated BCBL-1 cells, respectively. BCBL-1 cell/HUVEC ratio, 10/1. (B) Titration of BCBL-1 cells and HUVECs. The ratio of BCBL-1 cells to HUVECs varied from 10−4 to 101. Solid, broken, and gray lines indicate culturing times of 48, 24, and 0 h, respectively.

FIG. 3

FIG. 3

PCR analysis of HHV-8 infected HUVECs. HHV-8 DNA was detected in HUVECs infected with HHV-8 by cell-mediated transmission but not in those infected by cell-free transmission. HHV-8 DNA was also detected in HUVECs 30 days after infection. Lanes: M, 100-bp ladder marker; 1, HUVECs (not cocultured); 2, HUVECs infected by cell-free transmission for 48 h; 3, HUVECs infected by cell-mediated transmission for 48 h; 4, HUVECs 30 days after cell-mediated transmission; 5, HUVECs 30 days after cell-mediated transmission plus TPA; 6, BCBL-1 cells; 7, THP-1 cells; 8, no DNA

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