Patrick W. Ford

Raf promotes human herpesvirus-8 (HHV-8/KSHV) infection.

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposis sarcoma (KS) and other tumors. Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway has been associated with a variety of tumors, including AIDS-related KS. The oncoprotein Raf is situated at a pivotal position in regulating the MAPK pathway. Hence, we analysed the effect of oncoprotein Raf on HHV-8 infectious entry into target cells. Here we report Raf expression to significantly enhance HHV-8 infection of target cells. These findings implicate a role for Raf not only in the infectious entry of HHV-8 but also in modulating KS pathogenesis.

Raf-Induced Vascular Endothelial Growth Factor Augments Kaposi's Sarcoma-Associated Herpesvirus Infection

ABSTRACT Recombinant green fluorescent protein encoding Kaposis sarcoma-associated herpesvirus (rKSHV.152) infection of β-estradiol stimulated human foreskin fibroblasts (HFF) or HFF/ΔB-Raf[FF]:ER (expressing a weaker form of B-Raf) could be enhanced to levels comparable to that of HFF/ΔB-Raf[DD]:ER cells by pretreating cells with soluble vascular endothelial growth factor (VEGF). Conversely, VEGF expression and infection efficiency typically observed in β-estradiol stimulated HFF/ΔB-Raf[DD]:ER cells could be lowered significantly by treating with VEGF small interfering RNA. In addition, we observed enhancement of the KSHV infection in HFF cells transfected with human VEGF121. These results confirm the ability of Raf-induced VEGF to augment KSHV infection of cells.

Vascular endothelial growth factor augments human herpesvirus-8 (HHV-8/KSHV) infection.

Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposi’s sarcoma (KS) and other tumors. The Raf oncoprotein enhances HHV-8 infection of target cells. In addition, we have previously demonstrated that Raf induces vascular endothelial growth factor (VEGF) expression. VEGF is a growth factor that has autocrine growth activity and has been implicated in the formation of the spindle shape cell morphology characteristic of Kaposi’s sarcoma (KS). The aim of this study was to test the hypothesis that VEGF enhances infection of HHV-8. Herein, we demonstrate that the soluble VEGF enhanced green florescence protein encoding (GFP)-HHV-8 (rKSHV.152) infection of human foreskin fibroblasts (HFF) and not of 293 cells. We found this to be in part, due to the fact that HFF inherently produces significantly lower concentrations of VEGF when compared to 293 cells. Treating 293 cells (but not HFF) with a VEGF receptor (VEGFR) inhibitor significantly lowered infection. Furthermore, transfecting 293 cells with VEGF specific si-RNA did not alter the binding of HHV-8 to cells; but significantly lowered VEGF expression and thus GFP-HHV-8 infection. Interestingly, lowering VEGF expression in 293 cells wtih VEGF specific si-RNA did not completely inhibit GFP-HHV-8 infection. We conclude that VEGF is not a requirement for HHV-8 infection; but VEGF plays a major role in augmenting infection at a post binding stage of entry. These findings suggest that targeting VEGF/VEGFR may prove efficacious in controlling HHV-8 associated pathogenesis.

Differential regulation of the attachment of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected human B cells to extracellular matrix by KSHV-encoded gB and cellular αV integrins

Kaposis sarcoma‐associated herpesvirus (KSHV) has two modes of replications: latent and lytic replications. Reactivation from latency is dictated, in part, by the cell cycle. Herein, we have attempted to delineate the importance of cell cycle in KSHV pathogenesis by exploring the expression pattern of cell‐surface receptors during different phases of the cell cycle. αV integrin expression is augmented during S phase in fibroblasts, epithelial and KSHV‐infected cells. Using a Matrigel system, we pioneer the concept that KSHV‐infected primary effusion lymphoma cells can attach to extracellular matrix proteins. This attachment is mediated primarily via αV integrins or virally encoded gB, and occurs preferentially in cells from S phase or cells from S phase actively supporting a lytic infection respectively. Such an ability of infected B cells to attach to endothelial cells may also aid in the dissemination of infection. The keystone of this work is that for the first time, we describe the ability of KSHV‐infected B cells to preferentially use cellular (αV) or viral (gB) receptors to specifically bind cells, depending upon the stage of the cell cycle and infection.

β1 Integrins Mediate Tubule Formation Induced by Supernatants Derived from KSHV-Infected Cells

Objective: Angiogenesis is defined as the formation of new blood vessels. In a recently concluded study, we identified Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected cells derived from primary effusion lymphoma (PEL) to overexpress vascular endothelial growth factor (VEGF) that had the propensity to mediate tubule formation on a Matrigel, an indicator of angiogenesis. The objective of this study was to determine the receptor molecules that mediate the tubule formation induced by the supernatant derived from KSHV-infected PEL cells. Methods: The identity of receptor(s) that play a role in mediating tubule formation driven by PEL supernatant was determined by the classical in vitro angiogenesis assay conducted on a Matrigel. Results: RGD peptides, antibodies, and siRNA specific to β1 integrins significantly lowered the ability of the PEL supernatants to induce tubule formation by endothelial cells. β1 Integrins mediated tubule formation to comparable levels in endothelial cells that were incubated with supernatants derived from uninduced or TPA-induced PEL cells. Interestingly, the β1 integrins did not seem to have a major role in cellular attachment. Conclusion: We report for the first time a critical role for β1 integrins in angiogenesis supported by the supernatant from KSHV-infected PEL cells.

Raman tweezers provide the fingerprint of cells supporting the late stages of KSHV reactivation

Kaposi’s sarcoma‐associated herpesvirus (KSHV) has both latent and lytic phases of replication. The molecular switch that triggers a reactivation is still unclear. Cells from the S phase of the cell cycle provide apt conditions for an active reactivation. In order to specifically delineate the Raman spectra of cells supporting KSHV reactivation, we followed a novel approach where cells were sorted based on the state of infection (latent versus lytic) by a flow cytometer and then analysed by the Raman tweezers. The Raman bands at 785, 813, 830, 1095 and 1128 cm−1 are specifically altered in cells supporting KSHV reactivation. These five peaks make up the Raman fingerprint of cells supporting KSHV reactivation. The physiological relevance of the changes in these peaks with respect to KSHV reactivation is discussed in the following report.