Latent KSHV infection increases the vascular permeability of human endothelial cells.

Kaposi sarcoma-associated herpesvirus (KSHV) is associated with 3 different human malignancies: Kaposi sarcoma (KS), primary effusion lymphoma, and multicentric Castleman disease. The KS lesion is driven by KSHV-infected endothelial cells and is highly dependent on autocrine and paracrine factors for survival and growth. We report that latent KSHV infection increases the vascular permeability of endothelial cells. Endothelial cells with latent KSHV infection display increased Rac1 activation and activation of its downstream modulator, p21-activated kinase 1 (PAK1). The KSHV-infected cells also exhibit increases in tyrosine phosphorylation of vascular endothelial (VE)-cadherin and ß-catenin, whereas total levels of these proteins remained unchanged, suggesting that latent infection disrupted endothelial cell junctions. Consistent with these findings, we found that KSHV-infected endothelial cells displayed increased permeability compared with uninfected endothelial cells. Knockdown of Rac1 and inhibition of reactive oxygen species (ROS) resulted in decreased permeability in the KSHV-infected endothelial cells. We further demonstrate that the KSHV K1 protein can activate Rac1. Rac1 was also highly activated in KSHV-infected endothelial cells and KS tumors. In conclusion, KSHV latent infection increases Rac1 and PAK1 activity in endothelial cells, resulting in the phosphorylation of VE-cadherin and ß-catenin and leading to the disassembly of cell junctions and to increased vascular permeability of the infected endothelial cells.

Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas.

Primary effusion lymphoma (PEL) constitutes a subset of non-Hodgkin lymphoma whose incidence is highly increased in the context of HIV infection. Kaposi sarcoma-associated herpesvirus is the causative agent of PEL. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays a critical role in cell proliferation and survival, and this pathway is dysregulated in many different cancers, including PEL, which display activated PI3K, Akt, and mammalian target of rapamycin (mTOR) kinases. PELs rely heavily on PI3K/Akt/mTOR signaling, are dependent on autocrine and paracrine growth factors, and also have a poor prognosis with reported median survival times of less than 6 months. We compared different compounds that inhibit the PI3K/Akt/mTOR pathway in PEL. Although compounds that modulated activity of only a single pathway member inhibited PEL proliferation, the use of a novel compound, NVP-BEZ235, that dually inhibits both PI3K and mTOR kinases was significantly more efficacious in culture and in a PEL xenograft tumor model. NVP-BEZ235 was effective at low nanomolar concentrations and has oral bioavailability. We also report a novel mechanism for NVP-BEZ235 involving the suppression of multiple autocrine and paracrine growth factors required for lymphoma survival. Our data have broad applicability for the treatment of cytokine-dependent tumors with PI3K/mTOR dual inhibitors.

X-box-binding protein 1 activates lytic Epstein-Barr virus gene expression in combination with protein kinase D.

Epstein-Barr virus (EBV) establishes a latent form of infection in memory B cells, while antibody-secreting plasma cells often harbor the lytic form of infection. The switch between latent and lytic EBV infection is mediated by the two viral immediate-early proteins BZLF1 (Z) and BRLF1 (R), which are not expressed in latently infected B cells. Here we demonstrate that a cellular transcription factor that plays an essential role in plasma cell differentiation, X-box-binding protein 1 (XBP-1), also activates the transcription of the two EBV immediate-early gene promoters. In reporter gene assays, XBP-1 alone was sufficient to activate the R promoter, whereas the combination of XBP-1 and protein kinase D (PKD) was required for efficient activation of the Z promoter. Most importantly, the expression of XBP-1 and activated PKD was sufficient to induce lytic viral gene expression in EBV-positive nasopharyngeal carcinoma cells and lymphoblastoid cells, while an XBP-1 small interfering RNA inhibited constitutive lytic EBV gene expression in lymphoblastoid cells. These results suggest that the plasma cell differentiation factor XBP-1, in combination with activated PKD, can mediate the reactivation of EBV, thereby allowing the viral life cycle to be intimately linked to plasma cell differentiation.

BZLF1 activation of the methylated form of the BRLF1 immediate-early promoter is regulated by BZLF1 residue 186.

The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome.

DNA methylation promotes gene silencing, yet the Epstein-Barr virus immediate-early protein, BZLF1 (Z), converts the virus from the latent to the lytic form of infection even when the viral genome is highly methylated. Here we show that methylation of CpG motifs in Z-responsive elements of the viral BRLF1 immediate-early promoter enhances Z binding to, and activation of, this promoter. Demethylation of the viral genome impairs Z activation of lytic viral genes. Z is the first transcription factor that preferentially binds to, and activates, a methylated promoter. These results identify an unexpected mechanism by which Epstein-Barr virus circumvents the inhibitory effects of viral genome methylation.