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

Abstract

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.

B-Raf–dependent expression of vascular endothelial growth factor–A in Kaposi sarcoma–associated herpesvirus-infected human B cells

“…Northern blotting was performed at 0, 12, 24, and 48 hours after transfection as per the standard protocols to monitor B-Raf mRNA expression ( Figure 3A). 9,10 The level of B-Raf mRNA was significantly suppressed in HFF, BCBL-1, and BJAB cells by siRNA when compared with a (NS)siRNA control ( Figure 3A). The effect of siRNA on B-Raf expression in BC-1, BC-2, and BCP-1 cells was comparable to BCBL-1 cells (data not shown).…”

“…Northern blotting to monitor VEGF and ␤-actin expression was performed using a DIG Luminescent Detection Kit (Roche, Indianapolis, IN) as per the manufacturer's recommendations. 4,9 Analyses of mutation in B-Raf Total RNA was extracted and reverse transcriptase-polymerase chain reaction (RT-PCR) performed as per earlier protocols. 4,9 A 2-L sample of the cDNA was subjected to PCR analysis to determine the expression of B-Raf exons 11 and 15 using specific primers.…”

“…9,10,16 Briefly, 1 ϫ 10 6 cells were washed twice in RPMI and incubated in phenol red-free RPMI supplemented with 5% FBS at 37°C. After 24 hours incubation (considered as 0 h for experiments in Figure 3A), the target cells were transfected with either ds short interfering RNAs (siRNAs) or the nonspecific (NS) controls using lipofectamine 2000 as per manufacturer's recommendations (Invitrogen).…”

“…After 24 hours incubation (considered as 0 h for experiments in Figure 3A), the target cells were transfected with either ds short interfering RNAs (siRNAs) or the nonspecific (NS) controls using lipofectamine 2000 as per manufacturer's recommendations (Invitrogen). 9,10 At 0, 12, 24, and 48 hours after transfection, total RNA was isolated from the cells and subjected to Northern blotting to monitor the expression of B-Raf and ␤-actin mRNA as per protocol mentioned in the "Materials and methods" section describing Northern blotting. In another set of experiments, cell culture supernatants were collected at 0, 12, 24, and 48 hours after siRNA transfection and VEGF-A expression was monitored by enzyme-linked immunosorbent assay (ELISA) as per protocols described in the "Materials and methods" section describing the monitoring of VEGF-A expression.…”

“…4 In separate studies, we found VEGF-A to enhance KSHV infection of fibroblasts and epithelial cells. 9,10 Interestingly, constitutive activation of the components (Ras/Raf) of the mitogen-activated protein kinase (MAPK) pathway of signaling as well as VEGF expression has been a common feature with KSHV pathogenesis. 11,12 Hence, in this study we attempted to analyze the relationship between the expression of B-Raf and VEGF-A in KSHV-infected hematopoietic cells.…”