Lipid Rafts of Primary Endothelial Cells Are Essential for Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8-Induced Phosphatidylinositol 3-Kinase and RhoA-GTPases Critical for Microtubule Dynamics and Nuclear Delivery of Viral DNA but Dispensable for Binding and Entry

Abstract

Early during de novo infection of human microvascular dermal endothelial (HMVEC-d) cells, Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8 [HHV-8]) induces the host cell's preexisting FAK, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, Diaphanous-2 (Dia-2), Ezrin, protein kinase C-ζ, extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB signal pathways that are critical for virus entry, nuclear delivery of viral DNA, and initiation of viral gene expression. Since several of these signal molecules are known to be associated with lipid raft (LR) domains, we investigated the role of LR during KSHV infection of HMVEC-d cells. Pretreatment of cells with LR-disrupting agents methyl β-cyclo dextrin (MβCD) or nystatin significantly inhibited the expression of viral latent (ORF73) and lytic (ORF50) genes. LR disruption did not affect KSHV binding but increased viral DNA internalization. In contrast, association of internalized viral capsids with microtubules (MTs) and the quantity of infected nucleus-associated viral DNA were significantly reduced. Disorganized and disrupted MTs and thick rounded plasma membranes were observed in MβCD-treated cells. LR disruption did not affect KSHV-induced FAK and ERK1/2 phosphorylation; in contrast, it increased the phosphorylation of Src, significantly reduced the KSHV-induced PI3-K and RhoA-GTPase and NF-κB activation, and reduced the colocalizations of PI3-K and RhoA-GTPase with LRs. Biochemical characterization demonstrated the association of activated PI3-K with LR fractions which was inhibited by MβCD treatment. RhoA-GTPase activation was inhibited by PI3-K inhibitors, demonstrating that PI3-K is upstream to RhoA-GTPase. In addition, colocalization of Dia-2, a RhoA-GTPase activated molecule involved in MT activation, with LR was reduced. KSHV-RhoA-GTPase mediated acetylation and aggregation of MTs were also reduced. Taken together, these studies suggest that LRs of endothelial cells play critical roles in KSHV infection and gene expression, probably due to their roles in modulating KSHV-induced PI3-K, RhoA-GTPase, and Dia-2 molecules essential for postbinding and entry stages of infection such as modulation of microtubular dynamics, movement of virus in the cytoplasm, and nuclear delivery of viral DNA.

Kaposi’s sarcoma-associated herpesvirus interacts with EphrinA2 receptor to amplify signaling essential for productive infection

“…Constitutive macropinocytosis and membrane ruffling induced in oncogene-transformed fibroblasts have been shown to be due to constant activation of Src, PI3-K, and PLC-C (45). Our earlier studies have shown that disruption of LRs can increase global pSrc activity and viral entry, but lead into nonproductive infection (9). Hence, localized and regulated amplification of Src-PI3-K signaling in LRs by EphA2 appears to be crucial for productive KSHV entry.…”

“…Disruption of HMVEC-d cell lipid rafts (LRs) by MβCD results in increased Src activation and virus entry. However, MβCD inhibits PI3K, RhoA, Dia-2, and NFκB activation; microtubule acetylation; nuclear delivery of viral DNA; and viral gene expression (3)(4)(5)(6)(7)(8)(9). Nuclear delivery of KSHV genomic DNA does not lead to lytic gene expression and progeny virus formation; instead, KSHV expresses its latent genes and establishes latency (3).…”

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production

“…It is of interest that apart from lytic infection, the interactions between gamma-herpeviruses and Rho GTPase signaling remain active even during non-productive infection, facilitating either the establishment of latency 41 or affecting cell motility and invasion and promoting the oncogenic properties of these viruses. 36,42 Less information is available for b-herpesviruses. During the initial phase of HCMV infection, total and activated-RhoA levels are decreased and this contributes to the disruption of actin stress fibers, 43 a phenomenon that facilitates HCMV nuclear translocation.…”

Role of Membrane Rafts in Viral Infection

“…In addition to its role in entry of non enveloped the role of membrane rafts in entry of enveloped viruses has been investigated in studies using influenza virus (Orthomyxoviridae) [57][58][59][60][61], human immunodeficiency virus type 1 (HIV-1; Retroviridae) [62][63][64][65][66][67], human T lymphotropic virus 1 (HTLV-1; Retroviridae) [68,69], Ebola virus (Filoviridae) [70], Marburg virus (Filoviridae) [70,71], Epstein-Barr virus (EBV; herpesviridae) [72,73], herpes simplex virus-1 (HSV-1; herpesviridae) [74] including porcine herpesvirus-1 pseudorabies virus [75], human herpesvirus-6 (HHV-6; Herpesviridae) [76], human herpesvirus-8 (HHV-8; Herpesviridae) [77], vaccinia virus (Poxviridae) [78], coronavirus including severe acute respiratory syndrome coronavirus (SARS-CoV; Coronaviridae) [79][80][81][82][83][84][85], West Nile virus (WNV; Flaviviridae) [86], dengue virus (DEN; Flaviviridae) [87][88][89], Japanese encephalitis virus (JEV; Flaviviridae) [89], human hepatitis C virus (HCV; Flaviviridae) [90,91], Semliki Forest virus (Togaviridae) [92][93][94][95], Sindbis virus (Togaviridae) …”

“…Phosphatidylinositol 3-kinase (PI3K), RhoAGTPase, and Diaphanous-2 (a RhoA-GTPase-activated adaptor molecule involved in microtubule activation) act as signal molecules in the entry process of the virus [77]. Reduction of HHV-8 infection and decrease in the cellular signals associated with viral infection in raft-disrupted cells suggest that membrane rafts in microvascular dermal endothelial cells are essential for HHV-8 infection and gene expression.…”