De novo infection and serial transmission of Kaposi's sarcoma-associated herpesvirus in cultured endothelial cells

Abstract

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is central to the pathogenesis of the endothelial neoplasm Kaposi's sarcoma (KS) and is also linked to the rare B-cell tumor known as primary effusion lymphoma (PEL). Latently infected PEL cell lines can be induced to enter the lytic cycle and produce KSHV virions. However, such cells do not support de novo infection or serial propagation of KSHV. These limitations have prevented the development of systems for the genetic analysis of KSHV and have impeded a deeper understanding of KS pathogenesis. Here we show that human dermal microvascular endothelial cells immortalized by expression of telomerase can be readily infected by KSHV virions produced by PEL cells. Infection is predominantly latent, but a small subpopulation enters the lytic cycle spontaneously. Phorbol ester (tetradecanoyl phorbol acetate [TPA]) treatment of latently infected cells leads to enhanced induction of lytic KSHV replication, resulting in foci of cytopathic effect. There is no cytopathic effect or viral DNA expansion when infected TIME cells (telomerase-immortalized microvascular endothelial cells) are TPA induced in the presence of phosphonoacetic acid (PAA), an inhibitor of herpesvirus replication. Supernatants from phorbol-induced cultures transfer latent KSHV infection to uninfected cells, which can likewise be induced to undergo lytic replication by TPA treatment, and the virus can be further serially transmitted. Serial passage of the virus in TIME cells is completely inhibited when TPA treatment is done in the presence of PAA. Latently infected endothelial cells do not undergo major morphological changes or growth transformation, and infection is lost from the culture upon serial passage. This behavior faithfully recapitulates the behavior of spindle cells explanted from primary KS biopsies, strongly supporting the biological relevance of this culture system. These findings suggest that either the stability or the growth-deregulatory potential of the KSHV latency program in endothelial cells is more limited than might be predicted by analogy with other oncogenic viruses.

FIG. 1.

Immunofluoresence of KSHV-infected and uninfected TIME cells. (A) KSHV-infected TIME cells stained with anti-LANA antibody 48 h after infection. (B) DAPI nuclear stain of panel A. (C) Higher magnification of two TIME cells, stained with anti-LANA antibody, 48 h after infection. (D) DAPI stain of panel C. (E) Uninfected TIME cells stained with anti-LANA. (F) DAPI stain of panel E. (G and I) Infected TIME cells stained with anti-ORF 59 and anti-K8.1, respectively. (H and J) DAPI stains of panels G and I, respectively.

FIG. 2.

Enhancement of KSHV infection of TIME cells by different factors. TIME cells were infected for 2 h with serial dilutions of KSHV in the absence or presence of serum, Polybrene (8 μg/ml), dextran sulfate (Dex. Sul.), PEG, or Polybrene and PEG as indicated. At 48 h after infection the number of LANA-positive cells for each dilution was assessed, and the fold increase in the number of cells expressing LANA was plotted. SFM, serum-free medium.

FIG. 3.

Tubule formation by TIME cells on Matrigel medium. Uninfected and KSHV-infected TIME cells (25,000 cells) were plated on Matrigel matrix plugs in each well of a 24-well plate for 6 to 8 h, and tubule formation was examined by light microscopy.

FIG. 4.

KSHV lytic induction and plaque formation in TIME cells treated with TPA. (A) Anti-ORF 59 immunofluorescence of KSHV-infected TIME cells induced for 3 days with TPA. (B) DAPI stain of panel A. (C and D) Light microscope pictures of infected TIME cells (1% LANA positive) 5 days after TPA induction. (E) Anti-ORF 59 immunofluorescence of infected TIME cells 3 days after TPA induction. (F) DAPI staining of panel E.

FIG. 5.

TIME cell DNA digested and hybridized to a radiolabeled fragment of KSHV. Total DNA was isolated from TIME cells at the indicated cell passage after infection. Cells were left untreated throughout the passages or were treated with TPA for 3 days (passage [P] 1), with TPA for 6 days (passages 3 and 5), or with TPA and PAA for 3 to 6 days after the indicated passage. Uninfected cells were passaged and treated identically to the infected cells. After isolation, DNA was digested with BamHI, separated, and probed with a 1-kb BamHI fragment from the GPCR region of KSHV.

FIG. 6.

Serial passage of KSHV through TIME cells. Virions from BCBL-1 cells were used to infect TIME cells. The cells were subsequently split 1:2, and then one flask was induced with TPA for 4 days. Virions were harvested from the supernatant and used to infect fresh TIME cells. At 48 h after infection, a subset of cells were stained with anti-LANA (tetramethyl rhodamine isocyanate), DAPI, and anti-ORF 59 (fluorescein isothiocyanate) (serial passage 2). Virions from the second serial passage after one split and 5 days of TPA induction were used to infect fresh TIME cells, and 72 h later these cells were stained with anti-LANA (tetramethyl rhodamine isocyanate), DAPI, and anti-ORF 59 (fluorescein isothiocyanate) (serial passage 3).