Evidence of inability of human cytomegalovirus to reactivate Kaposi's sarcoma-associated herpesvirus from latency in body cavity-based lymphocytes

Abstract

Background: Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 (HHV-8)) has been determined to be the most frequent cause of malignancies in AIDS patients. It is associated primarily with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), as well as with multicentric Castleman's disease (MCD).(2) The switch from the latent to the lytic stage is important in the maintenance of malignancy and viral infection. So far, the mechanism of its reactivation has not been fully understood.

Objectives: Human cytomegalovirus (HCMV) and KSHV might infect the same cells, and it was found by other groups that several viruses could reactivate KSHV from latency. We investigate whether HCMV infection could reactivate KSHV from latency in body cavity-based lymphocyte (BCBL-1) cells.

Study design and results: Laboratory strains of HCMV cannot infect B cells. In this article, we demonstrate that the UL131-repaired HCMV (vDW215-BADrUL131) derived from AD169 strain is able to infect B lymphocytes. We directly infected KSHV latent cells including BCBL-1 with vDW215-BADrUL131 to evaluate the ability of HCMV to reactivate KSHV. Inconsistent with previous reports in human fibroblast cells, our results provide direct evidence that HCMV is unable to reactivate KSHV from latency-to-lytic infection in BCBL-1 cell lines. As a control, herpes simplex virus type 1 (HSV-1) was shown to be able to reactivate KSHV.

Conclusions: Our observations, different from others, suggest that reactivation mechanisms for KSHV might vary in different cells.

Fig. 1

Infectivity of laboratory strain and UL131-repaired HCMV in B cells. A. after infection of laboratory strains of HMCV in cells for the time as indicated, cells were collected and lysed in Lamile buffer and applied in 7.5% PAGE; the viral proteins were detected with Western blot assay. B. UL131-repaired HCMV, vDW215-BADrUL131 infection in B cells were detected by Western blot assay. C. BCBL-1 cells in 12-well plates were infected with HCMV AD169 or vDW215-BADrUL131 at MOI of 1 and the medium was changed at 12 hours after infection. The cells and medium was collected as the days post infection as indicated and the viral particles were released by thaw-and freeze for 3 cycles. After centrifugation, the supernatant were applied for PFU assay. Data from triplicates were obtained and statistically analyzed; the error bars stand for the standard error from the three independent experiments.

Fig. 2

KSHV reactivation in BCBL-1 cells by HCMV, HSV-1, and TPA. BCBL-1 cells were infected with vDW215-BADrUL131 (left) or HSV-1 (right) for the time indicated, and the cells were treated with TPA for 48 hours and were collected and lysed in Lamile buffer; cellular proteins and viral proteins were detected with Western blot assay.

Fig. 3

Immunofluorescence to detect HCMV infection in BCBL-1 cells. BCBL-1 cells were infected with vDW215-BADrUL131, untreated (A and B) or treated (C and D) with TPA, and were washed with PBS and fixed with 1% paraformaldehyde and cytospun to slides for immunofluorescence assay using different antibioses, as indicated, to detect viral proteins: IE1/2 (A1 and B1) and MCP (C1 and D1) of HCMV in green; RTA (A2 and C2) and K8 (B2 and D2) of KSHV in red. Total cells were shown with DAPI blue (A3 to D3). Three different colors were merged and shown in A4 through D4.