Background
Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent of the AIDS-defining cancer Kaposi sarcoma (KS). Despite widespread use of antiretroviral therapy, KSHV seroprevalence remains at 40% in HIV+ individuals in the US. HIV infection promotes KS development via angiogenic and pro-inflammatory factors and KSHV infection drives angiogenesis as observed in highly vascularized KS lesions and in vitro infection. In our previously published RNA-Seq data from cancerous skin KS versus matched normal tissue (n=10 patients) we found the pro-angiogenic vascular endothelial growth factor receptor-3 (VEGFR3/FLT4) significantly upregulated 7-fold ± 1.2. This induction was more pronounced in cell culture with a 50-fold increase after KSHV lytic reactivation. FLT4 signaling pathways promote cell proliferation, migration, and angiogenesis, and dysregulated expression is linked to various cancers, but it’s role in KSHV infection remains unknown.
Methods
To probe the role of FLT4 in infection, we performed siRNA depletion of FLT4 in primary lymphatic endothelial cells (LEC) prior to KSHV infection (MOI ~1). We measured viral replication by transcript and protein expression, genome replication, and virion production. We determined significance by paired T-tests between a non-targeting control and the siRNA targeting FLT4. We also performed RNA-sequencing of knockdown samples to interrogate the host transcriptional landscape upon VEGFR3 depletion.
Results
We observed significant increases in lytic replication upon FLT4 depletion with increases in viral genome replication (2-fold), viral transcripts and protein (1.5-fold), infectious progeny (4-fold), and nascent particles (10-fold) with no change in viral entry. RNA-sequencing of FLT4-depleted samples revealed enrichment for host genes involving cell contact and communication. Pathway analysis showed inhibition of angiogenic and tumor progression signaling, including FAK signaling and Molecular Mechanisms of Cancer.
Conclusions
These data suggest that FLT4 may be suppressing lytic infection yet promoting tumorigenesis. We propose a model in which FLT4 is impeding lytic replication such that KSHV preferentially enters latency where it can more favorably drive pro-angiogenic processes. As such, we are investigating the use of FDA-approved VEGFR inhibitors on the viral life cycle and plan to assess effects on host proliferation and pro-angiogenic processes as a new potential strategy to inhibit KSHV-driven disease.