Use of the red fluorescent protein as a marker of Kaposi's sarcoma-associated herpesvirus lytic gene expression

Abstract

A hallmark of all herpesvirus is the ability to exist in either a latent, or lytic, state of replication, enabling the lifelong infection of its host. Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) can efficiently establish a latent infection in a variety of cell types in vitro, making it a valuable model for the study of latency and reactivation. To facilitate the identification of KSHV lytic replication, and allow subsequent experiments with live cells, a recombinant virus, rKSHV.219, was constructed using JSC-1 cells that expresses the red fluorescent protein (RFP) from the KSHV lytic PAN promoter, the green fluorescent protein (GFP) from the EF-1alpha promoter, and with the gene for puromycin resistance as a selectable marker. rKSHV.219 from JSC-1 cells was used to infect Vero cells for purification of the recombinant virus. Vero cells were also used for the production of rKSHV.219 at levels of 10(5)-10(6) infectious units (IU) of virus per milliliter using a combination of KSHV/RTA expressed from a baculovirus vector, BacK50, and butyrate. Virus produced from Vero cells was used to infect human fibroblasts (HF), 293, DU145, T24, HaCaT, and HEp-2 cells, and in all cells except 293 cells, only a latent infection was established with GFP expression, but no RFP expression. In 293 cells, 10-15% of cells showed lytic gene expression. Both primary and immortalized microvascular endothelial cells (MVEC) were also infected with rKSHV.219, and reduced spontaneous lytic replication was found in immortalized cells. In all cells used in this study, rKSHV.219 efficiently established latent infections from which the virus could be reactivated to productive lytic replication. This work also demonstrated strong synergy between KSHV/RTA and butyrate for the activation of KSHV lytic replication and the production of infectious virus.

γδ+ T Cells Involvement in Viral Immune Control of Chronic Human Herpesvirus 8 Infection

“…We have previously shown that the fluorescent proteins expressed by rKSHV.219 can be used as indicators of viral entry and infection. As reported before, upon treatment with sodium butyrate the JSC/ rKSHV.219 cell line was able to support viral replication with the release of infectious viral particles (23). The virus yield can easily be determined using a susceptible cell line by evaluating the number of GFP-expressing cells.…”

“…C1R, a human lymphoblastoid cell line, was provided by V. Groh (Fred Hutchinson Cancer Research Center). The rKSHV.219 infected JSC-1 cell line (JSC/rKSHV.219) has been described elsewhere (23).…”

“…Briefly, rKSHV.219 lytic replication was induced in latently infected Vero cells (10 5 cells/well in a 6-well plate) using sodium butyrate (0.1 M, Sigma) and a recombinant baculovirus expressing KSHV ORF50 as described elsewhere (23). After overnight incubation, culture medium was removed and cells were washed with PBS.…”

“…Cell-free supernatants were recovered 48 h later. Virus titrations were performed as described elsewhere (23). Briefly, virus yield was determined using a susceptible cell line by evaluating the number of GFP-expressing cells.…”

“…PBMC from HHV-8-infected patients or uninfected controls were stimulated in the presence of IL-2 and IL-7 with or without infectious viral particles. Infectious viral particles were obtained from HHV-8-infected Vero cells in which lytic viral replication was artificially triggered as previously described (23). As shown in Table II, stimulation with HHV-8 infectious viral particles demonstrated no significant expansion among either ␣␤ ϩ CD4 ϩ CD8 ϩ or ␥␦ ϩ V␦1 ϩ V␦2 ϩ T cell subsets in PBMC from uninfected individuals.…”