Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.
Highlights
Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), a c-2 lymphotropic herpesvirus with a double-stranded DNA genome of,160 kb in length, is the etiological agent of hyper-proliferative disorders such as Kaposi’s sarcoma (KS), primary effusion B-cell lymphoma (PEL), and plasmablastic multicentric Castleman’s disease (MCD) [1,2,3]
We show that reactive oxygen species (ROS) activate nuclear factor E2-related factor 2 (Nrf2), a master transcriptional regulator of genes involved in ROS homeostasis, apoptosis, glucose metabolism and angiogenesis
We observed that infection of endothelial cells deficient in Nrf2 resulted in downregulation of multiple genes important in KSHV pathogenesis, such as COX-2 and Vascular endothelial growth factor (VEGF), and affected proper expression of two hallmark KSHV genes, lytic ORF50 and latent ORF73
Summary
Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV-8), a c-2 lymphotropic herpesvirus with a double-stranded DNA genome of ,160 kb in length, is the etiological agent of hyper-proliferative disorders such as Kaposi’s sarcoma (KS), primary effusion B-cell lymphoma (PEL), and plasmablastic multicentric Castleman’s disease (MCD) [1,2,3]. KSHV genome and transcripts are detected in the KS lesion fibroblasts, monocytes, and cells of epithelial origin and the expression of multiple latent and lytic genes in the infected cells, aided by the concomitant action of pro-inflammatory cytokines released by these cells, drives the excessive proliferation and hyperplasia of endothelial cells that lead to their spindle-shaped morphology [5]. HMVEC-d cells provide an excellent in vitro model for studying the early events that follow de novo infection of endothelial cells because i) they are naıve, primary cells permissive to KSHV infection, ii) they are derived from the same cells that eventually transform into the characteristic spindle-shaped morphology in KS lesions, and iii) are not transformed, exhibit
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