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Journal of Virology, August 2007, p. 7941-7959, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.02848-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Hari Raghu, Neelam Sharma-Walia, Mohanan Valiya Veettil, Sathish Sadagopan, Adriana Caballero, Ramu Sivakumar, Laszlo Varga, Virginie Bottero, and Bala Chandran*

Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064

Received 22 December 2006/ Accepted 3 May 2007

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-{zeta}, extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-{kappa}B 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 ß-cyclo dextrin (MßCD) 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 MßCD-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-{kappa}B 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 MßCD 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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL 60064. Phone: (847) 578-8822. Fax: (847) 578-3349. E-mail: bala.chandran{at}rosalindfranklin.edu

{triangledown} Published ahead of print on 16 May 2007.

Journal of Virology, August 2007, p. 7941-7959, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.02848-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Raghu, H., Sharma-Walia, N., Veettil, M. V., Sadagopan, S., Chandran, B. (2009). Kaposi's Sarcoma-Associated Herpesvirus Utilizes an Actin Polymerization-Dependent Macropinocytic Pathway To Enter Human Dermal Microvascular Endothelial and Human Umbilical Vein Endothelial Cells. J. Virol. 83: 4895-4911 [Abstract] [Full Text]  
  • Veettil, M. V., Sadagopan, S., Sharma-Walia, N., Wang, F.-Z., Raghu, H., Varga, L., Chandran, B. (2008). Kaposi's Sarcoma-Associated Herpesvirus Forms a Multimolecular Complex of Integrins ({alpha}V{beta}5, {alpha}V{beta}3, and {alpha}3{beta}1) and CD98-xCT during Infection of Human Dermal Microvascular Endothelial Cells, and CD98-xCT Is Essential for the Postentry Stage of Infection. J. Virol. 82: 12126-12144 [Abstract] [Full Text]  


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