Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface heparan sulfate (HS) and ␣3␤1 integrin during the early stages of infection of human dermal microvascular endothelial cells (HMVEC-d) and human foreskin fibroblasts (HFF), and these interactions are followed by virus entry overlapping with the induction of preexisting host cell signal pathways. KSHV also utilizes the amino acid transporter protein xCT for infection of adherent cells, and the xCT molecule is part of the cell surface heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex known to interact with ␣3␤1 and ␣V␤3 integrins. KSHV gB mediates adhesion of HMVEC-d, CV-1, and HT-1080 cells and HFF via its RGD sequence. Anti-␣V and -␤1 integrin antibodies inhibited the cell adhesion mediated by KSHV-gB. Variable levels of neutralization of HMVEC-d and HFF infection were observed with antibodies against ␣V␤3 and ␣V␤5 integrins. Similarly, variable levels of inhibition of virus entry into adherent HMVEC-d, 293 and Vero cells, and HFF was observed by preincubating virus with soluble ␣3␤1, ␣V␤3, and ␣V␤5 integrins, and cumulative inhibition was observed with a combination of integrins. We were unable to infect HT1080 cells. Virus binding and DNA internalization studies suggest that ␣V␤3 and ␣V␤5 integrins also play roles in KSHV entry. We observed time-dependent temporal KSHV interactions with HMVEC-d integrins and CD98/xCT with three different patterns of association and dissociation. Integrin ␣V␤5 interaction with CD98/xCT predominantly occurred by 1 min postinfection (p.i.) and dissociated at 10 min p.i., whereas ␣3␤1-CD98/xCT interaction was maximal at 10 min p.i. and dissociated at 30 min p.i., and ␣V␤3-CD98/xCT interaction was maximal at 10 min p.i. and remained at the observed 30 min p.i. Fluorescence microscopy also showed a similar time-dependent interaction of ␣V␤5-CD98. Confocal-microscopy studies confirmed the association of CD98/xCT with ␣3␤1 and KSHV. Preincubation of KSHV with soluble heparin and ␣3␤1 significantly inhibited this association, suggesting that the first contact with HS and integrin is an essential element in subsequent CD98-xCT interactions. Anti-CD98 and xCT antibodies did not block virus binding and entry and nuclear delivery of viral DNA; however, viral-gene expression was significantly inhibited, suggesting that CD98-xCT play roles in the post-entry stage of infection, possibly in mediating signal cascades essential for viral-gene expression. Together, these studies suggest that KSHV interacts with functionally related integrins (␣V␤3, ␣3␤1, and ␣V␤5) and CD98/xCT molecules in a temporal fashion to form a multimolecular complex during the early stages of endothelial cell infection, probably mediating multiple roles in entry, signal transduction, and viral-gene expression.The ␥-2 herpesvirus Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpesvirus 8, is etiologically associated with KS; primary effusion lymphoma, or body cavitybased B-cell lymphoma (BCBL); and multicentric Castleman...

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.

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