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-kappaB 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 beta-cyclo dextrin (MbetaCD) 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 MbetaCD-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-kappaB 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 MbetaCD 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.