Host range of Kaposi's sarcoma-associated herpesvirus in cultured cells

Abstract

Difficulties in efficiently propagating Kaposi's sarcoma-associated herpesvirus (KSHV) in culture have generated the impression that the virus displays a narrow host range. Here we show that, contrary to expectation, KSHV can establish latent infection in many adherent cell lines, including human and nonhuman cells of epithelial, endothelial, and mesenchymal origin. (Paradoxically, the only lines in which we have not observed successful latent infection are cultured lymphoma cell lines.) In most latently infected lines, spontaneous lytic replication is rare and (with only two exceptions) is not efficiently induced by phorbol ester treatment-a result that explains the failure of most earlier studies to observe efficient serial transfer of infection. However, ectopic expression of the KSHV lytic switch protein RTA from an adenoviral vector leads to the prompt induction of lytic replication in all latently infected lines, with the production of infectious KSHV virions. These results indicate (i) that the host cell receptor(s) and entry machinery for KSHV are widely distributed on cultured adherent cells, (ii) that latency is the default pathway of infection, and (iii) that blocks to lytic induction are frequent and largely reside at or upstream of the expression of KSHV RTA.

FIG. 1.

KSHV infects a wide array of cell lines. (A) HeLa, HFF, CV-1, and 293 cells were infected with concentrated stocks of KSHV and then examined 36 hpi for expression of LANA (green), the KSHV latent antigen, and ORF59 protein (red), an early lytic protein, by IFA. The nuclei are stained with DAPI (blue). (B) TIME, CV-1, SLK, HFF, 3T3, and CHO cells were infected with threefold serial dilutions of a KSHV stock for 2 h. At 48 hpi, the percentage of LANA-positive nuclei was determined by IFA and plotted for each dilution and cell type.

FIG. 2.

KSHV DNA in latent and induced cell lines. Cells were latently infected with KSHV for 2 h and then either washed and maintained in complete media or infected with AdGFP or AdRTA. Cells were harvested 36 hpi and lysed, and the DNA was separated on a Gardella gel. Shown are the Southern blots of the Gardella gels probed for the viral K8.1 gene and ORF73. Linear, linear KSHV DNA; circular, circular episomal KSHV DNA.

FIG. 3.

Lytic markers of KSHV in infected cells. Cells latently infected with KSHV were induced by RTA overexpression. At 36 h postinduction, cells were stained for LANA (green) and the nuclei were stained with DAPI (blue). Cells were also stained for ORF59 protein (left panels) and K8.1 (right panels), shown in red.

FIG. 4.

Production of infectious virus in induced cell lines. (A) Virus in the supernatants of induced KSHV-infected TIME, 293, CV-1, HFF, and SLK cells was concentrated by ultracentrifugation. Cells were induced by overexpression of RTA. Serial dilutions of the concentrated virus were then used to infect TIME cells. The infected TIME cells were stained for LANA at 48 hpi. The percentage of LANA-positive cells at each dilution for each cell type is shown. (B) Virus was harvested from the supernatants of induced TIME, 293, CV-1, HFF, and SLK cells infected in the presence or absence of 0.5 mM PFA. The percentage of LANA-positive cells for each cell type in the presence or absence of PFA is shown.