Productive Lytic Replication of a Recombinant Kaposi's Sarcoma-Associated Herpesvirus in Efficient Primary Infection of Primary Human Endothelial Cells

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to the development of Kaposi's sarcoma (KS), a vascular spindle cell tumor primarily consisting of proliferating endothelial cells. Although KSHV has been shown to infect primary human endothelial cells and convert them into spindle shapes, KSHV infection is largely latent, and efforts to establish a highly efficient and sustainable infection system have been unsuccessful. A recombinant KSHV, BAC36, that has high primary-infection efficiency in 293 cells has been obtained

Kaposi's Sarcoma-Associated Herpesvirus Glycoprotein K8.1 Is Dispensable for Virus Entry

“…Transfection experiments with 293 cells revealed that both BAC36 and BAC36⌬K8.1 genomes expressed a low level of ORF59, indicating that under transient transfection conditions, some viral genomes may be activated for lytic replication in the absence of exogenously provided TPA or RTA; this notion is consistent with previous observations (61). These results are also in agreement with recent observations that infection of primary human umbilical vein endothelial cell cultures occurs in two phases: (i) a permissive phase, in which the cultures undergo active viral lytic replication and infectious virus production; and (ii) a latent phase, in which the surviving cells from the lytic phase switch to latent infection, with a small number of cells undergoing spontaneous viral lytic replication (19). However, in most experiments, trans-activation by RTA produced at least two-to threefold more cells in which KSHV replicated in a lytic phase, as evidenced by the expression of the ORF59 and K8.1 genes.…”

“…On the next day, the viral inoculum was removed and fresh medium was added. Infectivity was determined 2 days postinfection by counting the number of GFP-expressing cells by fluorescence microscopy as described previously (19).…”

“…Recently, it was suggested that KSHV may have a role in the development of primary pulmonary hypertension (14). KSHV can infect a variety of human cell types, including B, T, endothelial, epithelial, fibroblast, and keratinocyte cells, and nonhuman cell types, including owl monkey kidney and baby hamster kidney fibroblast cells (9,12,16,17,19,26,29,32,33,43,45,56,61).…”

“…KSHV lytic replication was further enhanced through the addition of TPA and alpha interferon for 5 days, at which point supernatants of transfected cells were clarified of cellular debris and tested for the presence of infectious KSHV. The relative infectivities of supernatant viruses were determined by monitoring the number of GFP-expressing cells (see Materials and Methods) after infection of 293 cells (19) (Fig. 5A, panels A, B, and C).…”

Cell proliferation and survival induced by Toll-like receptors is antagonized by type I IFNs

“…These processes, in addition to effective immunity against pathogens, may be involved in maintenance of self-tolerance and prevention of autoimmunity (45). The ability of the induced type I IFN to prevent the proliferative effect of signaling through these TLRs, and to contribute to the induction of apoptosis may cripple the ability of viruses or other pathogens to replicate more efficiently in proliferating cells (46)(47)(48).…”