RhoA-GTPase Facilitates Entry of Kaposi's Sarcoma-Associated Herpesvirus into Adherent Target Cells in a Src-Dependent Manner

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) (human herpesvirus 8) binds to adherent target cell surface heparan sulfate molecules via its envelope glycoproteins gB and gpK8.1A, to integrins via gB, to the transporter CD98/xCT complex, and possibly to another molecule(s). This is followed by virus entry overlapping with the induction of preexisting host cell signal pathways, such as focal adhesion kinase, Src, phosphatidylinositol 3-kinase (PI3-K), Rho-GTPases, protein kinase C-ζ, and extracellular signal-regulated kinase 1/2. Here, using hemagglutinin-tagged plasmids expressing wild-type, dominant-positive, and dominant-negative forms of RhoA in HEK (human embryonic kidney) 293 cells, we investigated the role of RhoA-GTPase in virus entry. The dominant-negative form of RhoA GTPase and treatment of target cells with Clostridium difficile toxin B (CdTxB), a specific inactivator of Rho-GTPases, significantly blocked KSHV entry. KSHV infection induced closely similar levels of FAK and PI3-K in all three cell types. In contrast, very strong Src activation was observed in KSHV-infected dominant-positive RhoA cells compared to wild-type cells, and only moderate Src activation was seen in dominant-negative cells. Inhibition of Src activation by CdTxB and reduction of RhoA activation by Src inhibitors suggest that KSHV-induced Src is involved in RhoA activation, which in turn is involved in a feedback-sustained activation of Src. Since the decreased entry in RhoA dominant-negative cells may be due to inefficient signaling downstream of RhoA, we examined the induction of RhoA-activated Dia-2, which is also known to induce Src. Dia-2 coimmunoprecipitated with activated Src, which was inhibited by Src inhibitors, in the infected cells. Together with the reduced virus entry in RhoA dominant-negative cells, these results suggest that activated RhoA-dependent Dia-2 probably functions as a link between RhoA and Src in KSHV-infected cells, mediating the sustained Src activation, and that KSHV-induced Src and RhoA play roles in facilitating entry into adherent target cells.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), or human herpes virus 8, is a γ-2 herpesvirus associated with the pathogenesis of KS, the most common AIDS-related malignancy (10). KSHV is also associated with two lymphoproliferative disorders, namely body cavity-based B-cell lymphoma (BCBL) or primary effusion lymphoma (9) and multicentric Castleman's disease (57). In vivo, KSHV infects several cell types, including human B cells, macrophages, keratinocytes, endothelial cells, and epithelial cells (14, 24, 44, 71). Like other herpesviruses, KSHV establishes latent infection, and KSHV DNA is present in a latent form in the vascular endothelial and spindle cells of KS lesions. KSHV lytic cycle proteins are also detected in a low percentage of infiltrating inflammatory monocytes of KS lesions (14, 24, 58, 64). Viral latent proteins and lytic proteins are believed to contribute to the pathogenesis of KS lesions (14, 24, 57, 58, 64).

In vitro, KSHV infects a variety of cell types, including lymphoytes, endothelial cells, epithelial cells, fibroblasts, keratinocytes, owl monkey kidney cells, baby hamster kidney (BHK-21) cells, and Chinese hamster ovary (CHO) cells (4, 23, 45, 51, 58, 67). Unlike α- and β-herpesviruses, in vitro KSHV infection of target cells does not lead to a productive replicative lytic cycle. KSHV establishes latency soon after infection, and virus genome is lost during successive passages of the infected cells (7, 10, 27). Our recent studies showed that a subset of the lytic transcripts were expressed in the primary endothelial and fibroblast cells soon after infection, and many of these transcripts could not be detected at later time points (35).

KSHV enters human fibroblast cells (2), B cells (5), and epithelial cells (30) via endocytosis. Our previous studies have shown that KSHV interacts with the ubiquitous cell surface heparan sulfate (HS)-like molecules of adherent epithelial, endothelial, and fibroblast cells as well as B cells (3, 5, 68). KSHV envelope glycoproteins gB and gpK8.1A mediate the interaction with HS molecules (3, 68). KSHV-gB, through its integrin-binding RGD motif, interacts with the host cell surface α3β1 integrin and utilizes α3β1 integrin as one of the cellular receptors for entry into the human endothelial and fibroblast target cells (4). A recent study showed that KSHV also utilizes the transporter protein xCT for entry into adherent cells but not into B cells (34). The xCT molecule involved in glutamate/cystine exchange is part of the cell surface 125-kDa disulfide-linked heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex containing a common glycosylated heavy chain (80 kDa) and one of a group of 45-kDa light chains (11, 55). It is interesting that the CD98 complex usually associates with β1 integrin and has been shown to be involved in membrane clustering and β1 integrin-mediated signal cascades (19, 20).

Integrin interactions with extracellular matrix proteins lead to the assembly of integrins; numerous signaling molecules, including focal adhesion (FA) kinase (FAK), Src, and p130cas; and cytoskeletal proteins like talin, paxillin, and vinculin into aggregates on each side of the membrane, leading to the formation of FAs (12, 25, 52, 70). KSHV-integrin interactions led to the phosphorylation of FAK, which subsequently led to the activation of Src, phosphatidylinositol 3-kinase (PI3-K), protein kinase C-ζ (PKC-ζ), Rho-GTPases, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase 1/2 (ERK1/2) (45, 60, 61). KSHV infection also led to cytoskeletal rearrangements and the formation of structures such as filopodia, lamellipodia, and stress fibers (45). Soluble gB induced extensive cytoskeletal rearrangement in the target cells via the induction of a FAK-Src-PI3K-Rho-GTPase signal pathway (61).

We are examining the mechanism by which the KSHV-induced signal pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA. KSHV-induced phosphorylation of FAK is essential for the entry of KSHV into adherent target cells, since entry of KSHV is greatly reduced in the FAK null fibroblasts (36). PI3-K and cellular tyrosine kinase inhibitors blocked the entry of KSHV into target cells, suggesting a crucial role for PI3-K and tyrosine kinases in KSHV entry (61). Rho-GTPases belong to the Ras superfamily of proteins and function as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state. RhoA, Rac1, and Cdc42 are the best-studied members of this family (28). Besides playing a role in cytoskeleton reorganization, Rho-GTPases participate in a wide variety of cellular processes, including proliferation, differentiation, microtubule stabilization, endocytosis, vesicle trafficking, cytoplasmic transport, and gene expression (17, 54). In KSHV-infected human foreskin fibroblast (HFF) cells, RhoA promoted the actin stress fibers, whereas Rac1 and Cdc42 mediated the lamellipodia and filopodial extensions, respectively (61).

When we examined the role of KSHV-induced cytoskeleton dynamics in the infectious process and its interlinkage with signal pathways, we observed that depolymerization of microtubules (MT) did not affect KSHV binding and internalization but inhibited the nuclear delivery of viral DNA and infection (46). In contrast, depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, and infection. Early during infection, KSHV induced the acetylation of microtubules and the activation of RhoA and Rac-1-GTPases. Inactivation of Rho-GTPases by Clostridium difficile toxin B (CdTxB) significantly reduced the microtubular acetylation and delivery of viral DNA to the nucleus. In contrast, activation of Rho-GTPases by Escherichia coli cytotoxic necrotizing factor 1 significantly augmented the nuclear delivery of viral DNA. Among the Rho-GTPase-induced downstream effector molecules known to stabilize the microtubules, activation of RhoA-GTP dependent Dia-2 was observed with no significant activation in the Rac and Cdc42-dependent PAK1/2 and stathmin molecules. Nuclear delivery of viral DNA increased in cells expressing a dominant-positive RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA (46). Confocal microscopic examination showed colocalization of KSHV capsid with the microtubules, which was abolished by the destabilization of microtubules with nocodazole and by the PI3-K inhibitor affecting the Rho-GTPases. These results suggested that KSHV induces the RhoA-GTPases and in doing so modulates the microtubules and promotes the trafficking of viral capsid and the establishment of infection. This was a first demonstration of involvement of virus-induced host cell signal pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus.

Since RhoA-GTPase is critical for the formation of various types of endocytic vesicles and their movement, we investigated the role of RhoA-GTPase in KSHV entry and the upstream events leading to RhoA activation in HEK (human embryonic kidney) 293 cells stably expressing wild-type (WT), dominant-positive (G14V^+/+), and dominant-negative (T19N^−/−) forms of RhoA. Here we demonstrate that KSHV entry is decreased in cells expressing the dominant-negative form of RhoA. Increased Src activation was observed in dominant-positive RhoA cells, and Src is essential for KSHV internalization. We also demonstrate the interactions between RhoA, Src, and Dia-2. These results suggest that RhoA signaling and the associated Src activation are essential for KSHV entry into adherent target cells.

MATERIALS AND METHODS

Cells.

HMVEC-d (human dermal microvascular endothelial cells) (CC-2543; Clonetics, Walkersville, Md.), HFF cells (Clonetics), HEK 293 cells, and BCBL-1 cells (KSHV-carrying human B cells) used in this study were propagated and maintained as per procedures described previously (2, 4, 5, 45).

Generation of stable 293 cells expressing RhoA-WT, RhoA-G14V, and RhoA-T19N.

Establishment and characterization of 293 cells transfected with the HA-tagged WT, G14V^+/+, and T19N^−/− mutants of RhoA plasmids have been described previously (46). Essentially, 2.5 μg of plasmid DNA obtained from the Guthrie cDNA resource center was used for transfections, and each cell line carrying either a WT or mutant RhoA plasmid was first characterized by a GTP-loading assay to confirm its phenotype. Cell lines were further characterized by immunofluorescence microscopy.

For immunofluorescence detection, cells which stably express HA-tagged WT, RhoA-G14V, and RhoA-T19N were seeded into an 8-well chamber slide (Nalge Nunc International, Naperville, IL), grown at 37°C for 48 h, and rinsed twice with 1× phosphate-buffered saline (PBS [pH 7.4]), fixed with 2% paraformaldehyde for 15 min, and permeabilized with 0.2% Triton X-100 in 1× PBS for 5 min at room temperature. PBS containing 5% bovine serum albumin (BSA) was used for blocking. After blocking for 45 min, cells were incubated with primary rat anti-HA antibody and fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 1 h. Following washing with PBS, slides were mounted with mounting medium and examined under a Nikon fluorescent microscope equipped with a Metamorph digital imaging system.

Virus.

Induction of the KSHV lytic cycle in BCBL-1 cells, supernatant collection, and virus purification procedures were performed as described previously (45), and virus purity was assessed according to general guidelines established in our laboratory (4, 35, 45). KSHV DNA was extracted from the virus, and the copy numbers were quantitated by real-time DNA PCR using primers amplifying the KSHV open reading frame (ORF) 73 gene as described previously (35).

Antibodies and reagents.

Polyclonal rabbit immunoglobulin G antibodies against PI3-K p85α (Z-8), RhoA (110), and goat polyclonal Dia-2 (C-15) were from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Mouse antiphosphotyrosine (PY20) antibodies, CdTxB, U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2 aminophenylthio)butadiene], SU6656, and PP2 were obtained from Calbiochem, La Jolla, Calif. Mouse anti-phospho-FAK (Y397) and anti-FAK (total) antibodies were from BD Biosciences, San Jose, Calif. Anti-phospho-Src (PY418) and anti-Src (total) antibodies were obtained from Upstate Biotechnology, Lake Placid, N.Y. LY294002 [20(4-morphodinyl)-8-phenyl-1(4H)-benzopyran-4-one], lysophosphatidic acid (LPA), tetradecanoyl phorbol acetate, leupeptin, aprotinin, and Triton X-100 were from Sigma, St. Louis, Mo. The rat monoclonal antibody against HA was from Roche Diagnostics, Indianapolis, Ind. Anti-goat, anti-rabbit, and anti-mouse antibodies linked to horseradish peroxidase, alkaline phosphatase, fluorescein isothiocyanate, Alexa 488, and Alexa 594 were purchased from KPL Inc., Gaithersburg, Md., or Molecular Probes, Eugene, Oreg. Protein A-Sepharose CL-4B beads were from Amersham Pharmacia Biotech, Piscataway, N.J.

Cytotoxicity assays.

Target cells were incubated with Dulbecco's minimal essential medium (DMEM) containing different concentrations of various inhibitors for 4 h. Supernatants were collected and assessed for cellular toxicity by using an lactate dehydrogenase cytotoxicity assay kit (Promega, Madison, Wis.) in accordance with the manufacturer's recommendations.

Measurement of KSHV internalization by real-time DNA PCR.

Target cells treated with inhibitors or untreated cells were infected with KSHV at 10 DNA copies per cell (multiplicity of infection [MOI], 10). After 2 h of incubation, cells were washed twice with PBS to remove the unbound virus, treated with trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus, and washed. Total DNA was isolated from infected or uninfected cells using a DNeasy kit (QIAGEN, Inc., Valencia, Calif.) as described previously (35). A total of 100 ng of DNA samples, KSHV ORF 73 gene specific primers, TaqMan probe (35), and Quantitect PCR mix were used. The KSHV ORF 73 gene cloned in the pGEM-T vector (Promega) was used for the external standard. Known amounts of ORF 73 plasmid were used in the amplification reactions along with the test samples. The cycle threshold values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples.

Determination of RhoA activity.

Affinity precipitation reactions using the GST-Rho-binding domain (GST-RBD) of rhotekin (Cytoskeleton Inc., Denver, Colo.), which precipitated GTP-bound RhoA, were done per the manufacturer's recommendations to determine the amounts of activated cellular RhoA after KSHV infection. Briefly, target cells were infected with KSHV at an MOI of 10 for different time points, washed with PBS, and lysed in radioimmunoprecipitation assay (RIPA) buffer (45). The lysates were clarified, normalized to equal amounts of total proteins, and incubated with glutathione beads containing bound GST-RBD for 90 min at 4°C. Bound RhoA was resolved by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) and immunoblotted with rabbit polyclonal antibodies against RhoA. Immunoreactive bands were visualized, and band intensities were assessed (see Fig. 5). The bands were scanned, and their intensities were assessed and quantified, with GTP-bound RhoA in mock-infected cells being considered onefold activation for comparison to infected cells.

FIG. 5.

KSHV induces phosphorylation of FAK (Tyr397), Src (Tyr418), and PI3-K (p85α). WT, RhoA-G14V, and RhoA-T19N cells that had been serum starved for 48 h were left uninfected or infected with KSHV at an MOI of 10 for 5 min, 10 min, 15 min, and 30 min; induced with 20 ng/ml of LPA for 10 min; or stimulated with 20% of FBS for 10 min. Cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail. Cellular debris was removed by centrifugation at 13,000 × g for 20 min at 4°C. (A) FAK (Tyr397). Equal amounts of protein samples [WT (a), RhoA-G14V (b), and RhoA-T19N (c) cells] were resolved by SDS-7.5% PAGE, subjected to Western blotting, and reacted with mouse anti-phospho-FAK antibodies (Tyr397) (p-FAK; top panels) or anti-FAK antibodies (t-FAK; bottom panels). (B) Src (Tyr418). Equal amounts of protein samples [WT (a), RhoA-G14V (b), and RhoA-T19N (c) cells] were resolved by SDS-7.5% PAGE, transferred onto nitrocellulose membranes, and probed with anti-phospho-Src (PY418) (p-Src; top panels) antibodies for 3 h. The lysates were also analyzed by Western blotting with anti-Src antibodies (t-Src; bottom panels). (C) PI3-K (p85α). Equal protein concentrations (300 μg) of WT (a), RhoA-G14V (b), and RhoA-T19N (c) cell lysates were immunoprecipitated with PY20 antibodies for 2 h at 4°C. Immune complexes were washed four times with ice-chilled RIPA buffer containing protease inhibitors, bound proteins were eluted by boiling in 50 μl of 2× Laemmli buffer for 3 min and centrifuged, and the supernatants were subjected to Western blot analysis. The nitrocellulose membrane was probed with PI3-K p85α (Z-8) antibody for 2 h (top panels; p-P85α). Bottom panels show t-P13-K (total P13-K). Immunoreactive bands for each blot were visualized, and band intensities (middle panels) were assessed as described in the legend to Fig. 3 and expressed as increase in fold phosphorylation of Src over that of uninduced cells. Each blot represents data from a minimum of three separate experiments.

Measurement of FAK and Src phosphorylation and PI3-K induction.

Target cells grown to confluence were serum starved by incubation with DMEM at 37°C for 48 h and infected with KSHV at 10 MOI for different time points at 37°C. Cells were lysed in RIPA lysis buffer containing a protease inhibitor cocktail. Cellular debris was removed by centrifugation at 13,000 × g for 20 min at 4°C. The protein concentration of the clarified supernatant was determined with a micro-bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.). Each sample was heated at 95°C for 5 min, resolved by SDS-10% PAGE, and transferred to a nitrocellulose membrane. Phosphorylated FAK and Src contents were assessed by immunoblotting with anti-phospho-FAK (Y397) and anti-phospho-Src (PY418) antibodies, respectively. To confirm equal protein loading, membranes were stripped and reprobed with goat anti-rabbit pp125 FAK and anti-Src antibodies.

To determine PI3-K induction, cell lysates were immunoprecipitated by incubation with antiphosphotyrosine monoclonal antibody PY20 and probed with PI3-K p85α (Z-8) antibody. The total p85 (PI3-K) level was measured by testing the whole-cell lysate in Western blot assays with PI3-K p85α (Z-8) antibody. Immunoreactive bands were visualized by development with the enhanced chemiluminescence reaction kit (NEN Life Sciences Products, Boston, Mass.). Bands were scanned and quantitated with the ImageQuant software program (Molecular Dynamics).

Immunofluorescence colocalization analysis.

WT, RhoA-G14V, RhoA-T19N, and HMVEC-d cells grown in 8-well chamber slides were serum starved and infected with KSHV at an MOI of 10 or 50 for 10 min. After infection, the cells were washed, fixed for 15 min with 2% paraformaldehyde in PBS at room temperature, permeabilized for 5 min with 0.2% Triton X-100, and blocked for 45 min with 5% BSA in PBS. The cells were incubated with a 1:100 dilution of rat anti-HA antibodies for 1 h at room temperature and with a 1:200 dilution of rabbit anti-phospho-Src antibodies, mouse anti-phospho-FAK antibodies, or rabbit anti-RhoA antibodies for 1 h at room temperature and washed. This was followed by 1 h of incubation at room temperature with goat anti-rat, goat anti-mouse, or goat anti-rabbit antibodies labeled with Alexa 488 or Alexa 594. After being washed with PBS, the cells were mounted with antifade reagent containing DAPI (4,6-diamidino-2-phenylindole). The fluorescence-positive cells were observed under a fluorescence microscope equipped with the Nikon Metamorph digital imaging system.

Coimmunoprecipitation.

WT cells that had been serum starved for 48 h were incubated with Src kinase inhibitor PP2 (5 μM) for 1 h at 37°C and then infected with KSHV at an MOI of 10 at 37°C in the presence or absence of inhibitors for 10 min. Cells were lysed in RIPA lysis buffer, and the lysates were immunoprecipitated with Src or Dia-2 antibodies together with protein G-Sepharose beads. The immune complexes were then analyzed by Western blotting with Dia-2 or p-Src to detect their association or with anti-Src or Dia-2 to verify equal amounts of samples in the immunoprecipitates.

RESULTS

RhoA-GTPase is essential for KSHV entry into human endothelial and fibroblast cells.

Our previous studies have demonstrated the activation of host cell RhoA-GTPase by KSHV very early during infection of fibroblast cells and a role for RhoA-GTPase in the nuclear delivery of viral DNA (46). As an initial step to investigate whether RhoA has a role in virus entry, we used CdTxB that specifically inactivates the Rho family of GTPase members such as Rho, Rac, and Cdc42 by monoglucosylation at threonine 37 in Rho and threonine 35 in Rac and Cdc42 (32, 33). Monolayers of HMVEC-d and HFF cells were pretreated with nontoxic concentrations of CdTxB for 90 min to block endogenous Rho-GTPase before infection with KSHV at an MOI of 10 for 2 h, a time point of maximum internalization of viral DNA in these cells (36). Internalization of viral DNA determined by real-time DNA PCR analysis of viral ORF 73 copy numbers demonstrated a dose-dependent inhibition of virus entry into CdTxB-treated cells, with about 15%, 29%, and 54% reduction in HMVEC-d cells and about 22%, 35%, and 56% reduction in infected HFF cells at 100-, 200-, and 300-ng concentrations of CdTxB, respectively (Fig. 1) . Treatment of HMVEC-d and HFF cells with 10 μM U0126, a selective inhibitor for MEK/ERK, did not show any significant inhibition of KSHV entry. These results suggest a role for Rho-GTPases in KSHV entry into adherent target cells.

FIG. 1.

Dose-dependent inhibition of KSHV entry by Rho-GTPase inhibitor CdTxB. HMVEC-d and HFF cells were incubated with serum-free DMEM containing different nontoxic concentrations (100, 200, and 300 ng/ml) of CdTxB for 90 min. As a control, cells were pretreated for 1 h with 10 μM U01216, a selective inhibitor of MEK/ERK. The cells were infected with KSHV at an MOI of 10 in the presence or absence of inhibitors at 37°C and incubated for 2 h, washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) for 5 min to remove bound noninternalized virus, washed, and collected, and total DNA was prepared. The KSHV ORF 73 gene in 100 ng of DNA was amplified by real-time DNA PCR, and copy numbers were calculated from the standard graph generated by real-time DNA PCR using known concentrations of a cloned ORF 73 gene. Each reaction was carried out in duplicate, and each point represents the average standard deviation of three experiments.

To further investigate the role of RhoA-GTPase in virus entry, we established HEK 293 cell lines transfected with the HA-tagged plasmids expressing WT, G14V, and T19N forms of RhoA. In the RhoA-T19N mutant, substitution of threonine 19 for asparagines creates a GDP-bound conformation, rendering the GTPase inactive and unable to interact with effector proteins, thus preventing the activation of wild-type endogenous RhoA. In the RhoA-G14V mutant, substitution of glycine by valine at residue 14 lowers the intrinsic GTPase activity. This causes the GTPase to be locked in the GTP-bound conformation, and thus RhoA is always in the active GTP-bound state and interacting continually with effector proteins (18, 53).

Since inactive cytosolic RhoA has been shown to translocate to the membranes upon activation (26), we examined the expression of HA-tagged RhoA in 293 cell lines grown in the presence of FBS by using anti-HA antibody. A diffuse distribution of RhoA throughout the cytoplasm was detected in RhoA-T19N cells (Fig. 2A, panel c). In addition to the diffused RhoA, an activated form of RhoA at the plasma membrane was also detected in WT RhoA cells (Fig. 2A, panel a), and increased plasma membrane accumulation of RhoA was observed in the RhoA-G14V cells (Fig. 2A, panel b). The entry kinetics of KSHV in WT, RhoA-G14V, and RhoA-T19N cells was next investigated by extracting DNA from cells harvested at different times after KSHV infection followed by real-time DNA PCR. As shown in Fig. 2B, at all time points, entry of KSHV into cells expressing RhoA-T19N was less than that into WT and RhoA-G14V cells. Reductions of about 32%, 28%, 60%, and 51% compared to WT cells were observed at 10 min, 30 min, 60 min, and 120 min postinfection (p.i.), respectively. In contrast, entry of KSHV increased by >20% in cells expressing RhoA-G14V. The extent of inhibition of KSHV internalization seen at 2 h in cells expressing RhoA-T19N (Fig. 2B) was similar to that seen with CdTxB-treated HMVEC-d and HFF cells, as seen in Fig. 1. More than 80% of KSHV entry in all cell types was blocked by preincubating virus with 100 μg/ml of heparin (data not shown), thus demonstrating the specificity of the virus entry assay. These results suggested that RhoA-GTPase plays a role in KSHV entry in addition to the nuclear delivery of viral DNA.

FIG. 2.

(A) Fluorescence microscopy examination of RhoA protein distribution. HEK 293 cell lines transfected with HA-tagged plasmids expressing RhoA-WT (a), RhoA-G14V^+/+ (b), and RhoA-T19N^−/− (c) were grown on 8-well chamber slides in DMEM medium containing 5% FBS for 48 h and washed with PBS. The cells were fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 for 5 min, blocked, and tested with rat anti-HA antibodies for determination of RhoA localization. Asterisks indicate nuclei. Arrows indicate membrane localization of activated RhoA. Scale bars, 20 μm. (B) Kinetics of KSHV entry into 293 RhoA-WT, RhoA-G14V, and RhoA-T19N cells. WT, RhoA-G14V, and RhoA-T19N cells were infected with KSHV at an MOI of 10 for different time points (10, 30, 60, and 120 min) and washed. For control, virus was preincubated with 100 μg of heparin per ml for 1 h at 37°C before addition to the cells. Infected cells were washed, treated with trypsin-EDTA for 5 min, washed, and collected, and total DNA was prepared. KSHV ORF 73 gene copy numbers were calculated by real-time DNA PCR as described in the legend to Fig. 1. Data are presented as percentages of KSHV DNA internalization obtained when WT cells were incubated with the virus alone for 2 h. Each experiment was done in duplicate, and each point represents the average standard deviation of three experiments.

KSHV infection increased the RhoA-GTPase activity in WT and RhoA-G14V cells.

To analyze the role of RhoA-GTPase activity in regulating KSHV entry, WT, RhoA-G14V, and RhoA-T19N cells were serum starved and infected with KSHV at an MOI of 10. Lysates from different times p.i. were used in GST-RBD binding followed by Western blot analysis as described in Materials and Methods. Compared to the mock-infected cells (Fig. 3A, top panel, lane 1), KSHV induced an increase in RhoA-GTPase activity as early as 5 min p.i. (1.5-fold) in WT cells, which peaked at 10 min p.i. (3.1-fold) and decreased thereafter, with increases of about 2.8-fold and 1.6-fold at 15 min and 30 min p.i., respectively (Fig. 3A, top panel, lanes 2 to 5). A similar kinetics of induction involving much higher levels of RhoA-GTPase activity, with about 2.5-, 4.8-, 4.5-, and 3.1-fold increases at 5 min, 10 min, 15 min, and 30 min p.i., respectively, was observed in RhoA-G14V cells (Fig. 3B, top panel, lanes 2 to 5). In contrast, infection of RhoA-T19N cells with KSHV did not induce any appreciable level of RhoA-GTPase (Fig. 3C, top panel, lanes 2 to 5), suggesting that interaction with downstream effectors is abrogated in RhoA-T19N cells. KSHV-induced RhoA-GTPase activity in infected cells was comparable to the induction caused by incubation with 20 ng of LPA for 10 min (Fig. 3A to C, lanes 6). The total cellular RhoA level remained unchanged both before and after infection (Fig. 3A to C, bottom panels), indicating the steady-state level of endogenous RhoA in these cells and thus demonstrating that KSHV induced the preexisting endogenous RhoA.

FIG. 3.

KSHV induces RhoA-GTPase activity. WT (A), RhoA-G14V (B), and RhoA-T19N (C) cells were serum starved for 48 h and infected with KSHV at an MOI of 10. Equal amounts of cell lysates from mock-infected cells, cells infected with KSHV at different time points (5 min, 10 min, 15 min, and 30 min), or cells treated with LPA 20 ng/ml (10 min) were used to capture the GTP-bound forms of RhoA-GTPase by affinity precipitation with GST-RBD beads. The proteins captured by beads were analyzed by SDS-12% PAGE and immunoblotted with anti-RhoA (A to C, top panels). The bottom panels show normalized cell lysates analyzed for total RhoA as a loading control.

In our previous study (46), when we used virus at an MOI of 5 in HFF cells, we observed RhoA-GTPase activity similar to virus at an MOI of 10 in 293 cells. The difference in induction of RhoA activity between these studies could be due to cell type specificity of RhoA activation.

KSHV entry is reduced in cells treated with PI3-K and Rho-GTPase inhibitors.

Among the hallmarks of integrin interaction with ligands are reorganization and remodeling of the actin cytoskeleton, which are controlled by the Rho family of small GTPases, such as RhoA, Rac, and Cdc42 (25, 28, 70). Early during infection, KSHV induced the polymerization of actin and morphological changes in target cells (61). Upon FAK and Src phosphorylation, these Rho-GTPases can also be activated by PI3-K via intermediate mediators (65). Our earlier studies demonstrated that KSHV-induced PI3-K signaling occurs upstream of the Rho-GTPase activation site (61). The PI3-K inhibitor, LY294002 inhibited the KSHV-induced PI3-K activity, reduced the entry of virus into the target cells (45), and blocked the KSHV-induced Rho-GTPase activity and the accompanying morphological changes in the infected cells without affecting FAK and Src activities (45, 61).

To determine the specificity of the observed RhoA role in KSHV entry, WT, RhoA-G14V, and RhoA-T19N cells were treated with LY294002 prior to infection. At 20 and 50 μM concentrations, LY294002 reduced the virus internalization by ∼50% and 60% in WT (Fig. 4A), RhoA-G14V (Fig. 4B), and RhoA-T19N (Fig. 4C) cells compared to the entry into the respective untreated control cells. To establish the role of RhoA in KSHV entry into WT, RhoA-G14V, and RhoA-T19N cells, we used CdTxB to inhibit target cell RhoA. As shown in Fig. 4, treatment of WT (Fig. 4A) and RhoA-G14V (Fig. 4B) cells with CdTxB decreased the entry of KSHV in a dose-dependent manner, with a maximum inhibition of 60% at the nontoxic dose of 300 ng, but had very little effect (∼20%) in the RhoA-T19N cells (Fig. 4C) compared to the respective untreated control cells. The specificity of inhibition was demonstrated by the absence of significant reduction in virus entry into cells treated with MEK inhibitor U0126. These results were consistent with our earlier observation that PI3-K lies upstream of RhoA-GTPase and plays a role in KSHV entry, and they further demonstrated a role for RhoA-GTPase in KSHV entry.

FIG. 4.

PI3-K and Rho-GTPase inhibitors block entry of KSHV into target cells. WT (A), RhoA-G14V (B), and RhoA-T19N (C) cells were pretreated with 20 μM and 50 μM LY294002, an inhibitor of PI3-K, for 1 h or with different nontoxic concentrations of CdTxB (100 to 300 ng/ml) for 90 min and then infected with KSHV at an MOI of 10 in the presence or absence of inhibitors at 37°C for 2 h and washed. Cells pretreated with U0126, a specific inhibitor of MEK/ERK, and also infected with KSHV at an MOI of 10 were used as a control. Cells were treated with trypsin-EDTA for 5 min, washed, and collected, and total DNA was prepared. KSHV ORF 73 gene copy numbers were calculated by real-time DNA PCR as described in the legend to Fig. 1.

KSHV-induced activation of upstream signaling molecules of RhoA: phosphorylation of FAK was unaffected in WT, RhoA-G14V, and RhoA-T19N cells.

The above results demonstrated that KSHV stimulated RhoA-GTPase activity in WT and RhoA-G14V cells. Since the RhoA-T19N cells show reduced affinity for nucleotides and abrogate downstream signaling (53, 59), the decreased entry observed in RhoA-T19N cells may be due to inefficient signaling downstream of RhoA. Our previous findings demonstrated that FAK, Src, and PI3-K function as upstream molecules of Rho-GTPase induction in the KSHV-induced integrin-mediated signaling pathway (61). The activated FAK is autophosphorylated at Tyr397 and provides a recognition site for the SH2 domain of Src proteins (15) and other intracellular signaling molecules, including the p85 subunit of PI3-K. To decipher the stage of the KSHV entry pathway at which RhoA exerts its effect, we studied the upstream events leading to RhoA activation in infected cells.

KSHV interaction with integrin results in FAK phosphorylation early during infection of adherent target cells, such as HMVEC-d, HFF, and FAK^+/+ mouse embryonic fibroblast Du17 cells (4, 36, 61). Our studies in FAK null mouse embryonic fibroblast Du3 cells demonstrated that FAK is crucial for KSHV entry (36). To determine the FAK activity in WT, RhoA-G14V, and RhoA-T19N cells, serum-starved cells were either uninfected or infected for different times with KSHV, and lysates were tested for tyrosine phosphorylation of FAK at Tyr397 by Western blot reactions. As shown in Fig. 5Aa (top panel, lanes 2 to 5), phosphorylation of FAK in infected WT cells increased in a time-dependent manner, with about 1.6-, 2.3-, 2.4-, and 2.1-fold induction at 5 min, 10 min, 15 min, and 30 min p.i., respectively. Similar kinetics of increased phosphorylation of FAK were observed in the RhoA-G14V cells (Fig.5Ab, top panel, lanes 2 to 5) and in the RhoA-T19N cells (Fig.5Ac, top panel, lanes 2 to 5). Compared to uninfected cells (Fig. 5A, top panels, lanes 1), treatment with 20 ng of LPA for 10 min as a control also induced closely similar levels of FAK phosphorylation in all three cell types (Fig. 5A, top panels, lanes 6). Equal amounts of total FAK were detected in all the samples (Fig. 5A, bottom panels), which not only confirmed the equal protein loading but also indicated that KSHV infection was inducing the phosphorylation of the preexisting FAK molecules. The kinetics of the observed FAK activity in 293 cells closely resembled the FAK induction kinetics observed in HMVEC-d, HFF, and Du17 cells (4, 36, 61). These results suggested that KSHV-induced FAK activation is not affected by over- or underexpression of RhoA-GTPase.

KSHV induces a higher level of Src in RhoA-G14V cells and reduced Src activation in RhoA-T19N cells.

The phosphorylation of Src increased by about 2.3-, 3.7-, and 4.5-fold at 5 min, 10 min, and 15 min p.i., respectively, in WT cells (Fig.5Ba, top panel, lanes 2 to 5). Src phosphorylation in RhoA-G14V cells showed a much higher level of Src phosphorylation, with about 8.9-, 12.9-, and 7.5-fold induction at 5 min, 10 min, and 15 min p.i., respectively (Fig.5Bb, top panel, lanes 2 to 5), compared to uninfected cells (Fig.5Bb, top panel, lane 1). In contrast, Src phosphorylation levels in RhoA-T19N cells were increased to a lesser extent, with about 1.8-, 1.9-, and 2.6-fold at 5 min, 10 min, and 15 min p.i., respectively (Fig.5Bc, top panel, lanes 2 to 5). At 30 min p.i., Src activity returned rapidly to baseline levels in all three cell types (Fig. 5B, top panels, lanes 5). Incubation of cells with 20 ng of LPA for 10 min also induced a rapid phosphorylation of Src (Fig. 5B, top panels, lanes 6) which was comparable to that in the infected samples at 10 min p.i. The lysates analyzed by Western blotting with total anti-Src (Fig. 5B, bottom panels) shows that the total level of Src remained unchanged in all the samples, indicating that KSHV infection was inducing the phosphorylation of the preexisting Src molecules.

These studies demonstrated that the level of Src phosphorylation was significantly lower in RhoA-T19N cells (Fig.5Bc, top panel, lanes 2 to 5) than in WT and RhoA-G14V cells. The kinetics of Src tyrosine phosphorylation closely resembled the kinetics of RhoA activation in these cells (compare Fig. 3A and B). Although Src phosphorylation is an upstream event of RhoA activity in integrin-mediated signaling (6, 42), the strong activation of Src observed in infected WT and RhoA-G14V cells was decreased in RhoA-T19N cells. Together with the strong Src activation in RhoA-G14V cells, this suggested that though KSHV infection induces Src activity independent of activation of RhoA, activated RhoA is probably involved in a feedback activation of Src.

Similar levels of PI3-K phosphorylation are induced by KSHV in WT, RhoA-G14V, and RhoA-T19N cells.

PI3-K induction was observed to occur upstream of Rho-GTPase activation in the KSHV- or KSHV gB-induced signaling pathways (61). To determine the kinetics of PI3-K activation, we examined phosphorylation of the p85 subunit of PI3-K in uninfected cells and cells infected with KSHV at different time points. Cell lysates were immunoprecipitated with PY20 and subjected to Western blotting and analyzed for the p85 subunit of PI3-K. PI3-K activity reached a peak at 10 min p.i. and decreased thereafter in WT (Fig.5Ca, top panel, lanes 2 to 5), RhoA-G14V (Fig.5Cb, top panel, lanes 2 to 5), and RhoA-T19N cells (Fig.5Cc, top panel, lanes 2 to 5). The expression levels observed after infection were similar in WT, RhoA-G14V, and RhoA-T19N cells. In cells treated with 20% FBS for 10 min, PI3-K phosphorylation was increased (Fig. 5C, lanes 6) compared to that in uninfected cells (Fig. 5C, top panels, lanes 1). The total PI3-K level remained unchanged in all the samples (Fig. 5C, bottom panels). These results, together with the inhibitor studies shown in Fig. 4 demonstrating PI3-K role in virus entry, indicated that PI3-K signaling occurs upstream or independently of RhoA-GTPase activation.

Sustained Src activation requires the induction of RhoA-GTPase in KSHV-infected cells.

From our observation of the strong activation of Src by KSHV in RhoA-G14V cells and its abolition in RhoA-T19N cells, we hypothesized that increased RhoA activity promotes increased phosphorylation of Src. To investigate this hypothesis, we analyzed Src phosphorylation upon infection in cells preincubated with the Rho-GTPase inhibitor CdTxB. Treatment with CdTxB decreased the Src phosphorylation levels by >90% in WT (Fig. 6A, lanes 2) and RhoA-G14V (Fig. 6B, lanes 2) cells. The specificity of these reactions was shown by the inhibition of Src by SU6656, a Src inhibitor (Fig. 6A and B, lanes 3). These results demonstrated a role for KSHV-induced RhoA in the sustained Src activation. Since CdTxB blocked virus entry (Fig. 4) as well as the activation of Src, taken together, these data suggested that Src and RhoA play important roles in KSHV entry.

FIG. 6.

Inhibition of Src kinases and Rho-GTPases inhibits Src kinase phosphorylation of Src at Tyr418. WT (A) and RhoA-G14V (B) cells that had been serum starved for 48 h were incubated with the Src kinase inhibitor SU6656 (10 μM) for 1 h at 37°C or the Rho-GTPase inhibitor CdTxB (300 ng) and then infected with KSHV at an MOI of 10 at 37°C in the presence or absence of inhibitors and incubated for 10 min. Cells were lysed in RIPA lysis buffer containing protease inhibitor cocktail. Samples were resolved by SDS-7.5% PAGE, transferred onto nitrocellulose membranes, and probed with anti-phospho-Src (Tyr418) antibody (p-Src; top panels) for 2 h. Immunoreactive bands were visualized with an alkaline phosphatase-conjugated secondary antibody and quantitated as described in the legend to Fig. 3. Equal protein loading was confirmed by stripping the membranes and reprobing with anti-Src antibody (bottom panels). (C). Src kinase inhibitor decreases RhoA-GTPase activation. Serum-starved RhoA-WT cells were incubated with the Src kinase inhibitor PP2 (5 μM) for 1 h at 37°C or the Rho-GTPase inhibitor CdTxB (300 ng) and then infected with KSHV at an MOI of 10 at 37°C in the presence or absence of inhibitors and incubated for 10 min. Equal amounts of cell lysates from uninfected, cells infected with KSHV, or cells treated with 20 ng of LPA/ml (10 min) were used to capture the GTP-bound forms of RhoA-GTPase by affinity precipitation with GST-RBD beads. The proteins captured by beads were analyzed by SDS-12% PAGE and immunoblotted with anti-RhoA. Fold ind., fold induction. (D and E) Inhibition of Src kinases blocks KSHV entry. WT and RhoA-G14V cells were incubated with DMEM or DMEM containing PP2 (10 μM), SU6656 (10 μM), or U0126 (10 μM) for 1 h at 37°C and infected with KSHV at an MOI of 10 in the presence or absence of inhibitors at 37°C. After 2 h of incubation, cells were washed with PBS to remove the unbound virus, treated with trypsin-EDTA for 5 min at 37°C, and washed, and then total DNA was isolated. Total DNA was normalized, and KSHV ORF 73 copy numbers were estimated by real-time DNA PCR. The cycle threshold values were used to plot the standard graph and calculate relative copy numbers of viral DNA in the samples. Data are presented as percentages of the inhibition of KSHV DNA internalization obtained when cells were incubated with the virus alone. Cells pretreated for 1 h with U0126, a selective inhibitor for MEK/ERK, and infected with KSHV at an MOI of 10 were used as a control. Each reaction was done in duplicate, and each point represents the average standard deviation of three experiments.

Sustained RhoA-GTPase activation requires the induction of Src in KSHV-infected cells.

Since Src activity also occurs upstream of RhoA activity in the integrin-mediated signaling pathway (6, 42), to determine whether Src activity is required for the activation of RhoA-GTPase, we measured RhoA-GTPase activity in Src inhibitor PP2-treated infected cells. As shown above, KSHV induced a rapid RhoA-GTPase response (Fig. 6C, lane 4). In cells pretreated with PP2 and exposed to KSHV for 10 min, about a 58% reduction in RhoA-GTPase activity (Fig. 6C, lane 2) and a 92% reduction after CdTxB treatment (Fig. 6C, lane 3) were observed. These experiments further demonstrated that KSHV-induced Src is involved in the activation of RhoA, which in turn is involved in feedback activation of Src.

Src is essential for KSHV entry.

To further ascertain the role of Src in KSHV infection, cells were preincubated with the Src kinase inhibitor SU6656 prior to infection. SU6656 inhibited the KSHV-induced activation of Src in WT (Fig. 6A, lanes 3) and RhoA-G14V (Fig. 6B, lanes 3) cells. When we examined the entry of KSHV into WT and RhoA-G14V cells treated with nontoxic doses of the Src inhibitors PP2 and SU6656, KSHV entry was greatly diminished in both WT (Fig. 6D) and RhoA-G14V (Fig. 6E) cells. More than 75% reduction was observed with the PP2 in both cell types, and no significant inhibition was observed with U0126, a MAP kinase inhibitor (Fig. 6D and E). Since previous studies from our laboratory have shown that MAP kinases are not involved in the entry of KSHV (45, 60), we used the MAP kinase inhibitors as controls to ensure the specificity of other inhibitory drugs used to study the entry of KSHV. These results demonstrated the KSHV-induced Src is critical for KSHV entry.

Activated RhoA colocalizes with Src in KSHV-infected 293 cells.

Results presented above suggested that increased RhoA activity promotes increased phosphorylation of Src in WT and RhoA-G14V cells. To further support this observation, immunofluorescence microscopy was used to analyze the locations of RhoA and phospho-Src in infected cells. Serum-starved cells were incubated with KSHV for 10 min at 37°C, washed, permeabilized, fixed, and stained with anti-HA antibodies (to detect RhoA) and anti-phospho-Src antibodies. Colocalization of activated RhoA and Src was detected in the membranes of WT cells (Fig. 7A), and stronger colocalizations were observed in the periphery of RhoA-G14V cells (Fig. 7B). Though activated Src was observed in RhoA-T19N cells, membrane translocation was not easily observed (Fig. 7C), a fact that is consistent with the absence of activated RhoA. No colocalization was seen in the uninfected control cells (Fig. 7E). RhoA and Src colocalization observed in RhoA-G14V cells induced with 20 ng of LPA (Fig. 7D) was comparable to that of the infected cells. The increased colocalization of Src with RhoA further strengthens our conclusion that enhanced RhoA-GTPase activity results in an increase in Src activation in RhoA-G14V cells and WT cells.

FIG. 7.

Visualization of colocalization of RhoA with Src in 293 cells by immunofluorescence assay. WT (A), RhoA-G14V (B), and RhoA-T19N (C) cells that had been serum starved for 48 h were infected with KSHV at an MOI of 10 for 10 min, RhoA-G14V cells were left uninfected (E), and RhoA-G14V cells were induced with 20 ng/ml of LPA for 10 min (D), and all were washed with PBS. Cells were fixed and permeabilized with 0.2% Triton X-100 for 5 min, blocked with 5% BSA for 45 min at room temperature, washed, and incubated with anti-HA antibody for RhoA and anti-phospho-Src antibody (p-Src) for 1 h at room temperature. These cells were washed with PBS and stained with FITC-conjugated (green) secondary antibody for RhoA and Alexa 594-labeled (red) secondary antibody for Src and incubated for 1 h at room temperature. In the merged images, white arrows indicate colocalization of RhoA with Src. Stained cells were viewed with appropriate filters under a fluorescence microscope with the Nikon Magna Firewire digital imaging system. All magnifications, ×40. Scale bars, 20 μm.

Activated RhoA colocalizes with Src in KSHV infected HMVEC-d cells.

Since the studies shown above were performed in stably transfected 293 cell lines, to further examine the RhoA role in increased Src activity we used the HMVEC-d primary cells, an in vivo target cell of KSHV. Similar to 293 cells and in contrast to uninfected cells, increased phospho-Src colocalization with RhoA was observed in infected cells (Fig. 8A). These results demonstrated that the signaling pathways described here are not cell type specific and that KSHV induced these signal cascades in the biologically relevant cells.

FIG. 8.

(A) Colocalization of RhoA with Src in HMVEC-d cells. HMVEC-d cells that had been serum starved for 8 h were uninfected or infected with KSHV at an MOI of 10 and incubated for 10 min, washed, fixed in 4% paraformaldehyde for 15 min, and then permeabilized with 0.2% Triton X-100 for 5 min. Cells incubated with anti-RhoA and anti-phospho-Src primary antibodies were stained with Alexa 488- and 594-labeled secondary antibodies for RhoA and p-Src, respectively. Nuclei were stained with DAPI (blue). In the merged image, white arrows indicate colocalization of RhoA with Src. All magnifications, ×40. Scale bars, 20 μm. (B) Colocalization of Src with FAK in HMVEC-d cells. Cells infected with KSHV at an MOI of 10 and uninfected cells were fixed, permeabilized, and stained for anti-phospho-FAK monoclonal antibody (Alexa 488 labeled; green) and anti-phospho-Src monoclonal antibody (Alexa 594 labeled; red). Nuclei were stained with DAPI (blue). In the merged image, white arrows indicate colocalization of Src with FAK. All magnifications, ×40. Scale bars, 20 μm.

Activated FAK colocalizes with Src in KSHV-infected HMVEC-d cells.

The activation of FAK, as well as that of the downstream signaling molecule Src and the p85 subunit of PI3-K, is an early integrin focal-adhesion-mediated signaling event following KSHV infection (61). To determine whether the activated Src associates with FAK, we examined tyrosine phosphorylation of FAK at 397 and Src at 418 in KSHV-infected HMVEC-d cells. Infection of cells with KSHV led to the appearance of active phosphorylated Src at the edge of the plasma membranes. We also detected the increased staining of tyrosine-phosphorylated FAK 397 in infected cells. As shown in Fig. 8B, the activated FAK colocalized with Src at the plasma membranes of infected cells. This further demonstrated the association between activated FAK and Src early during KSHV infection.

Src is associated with RhoA-GTPase activated Diaphanous-2 molecule.

The RhoA-T19N cells show reduced affinity for nucleotides and abrogate downstream signaling (53, 59). Since the decreased entry observed in RhoA-T19N cells may be due to inefficient signaling downstream of RhoA, we next examined the RhoA downstream effector molecules that may also play roles in the feedback activation of Src. Among the effector molecules, we selected to examine diaphanous-related formin family molecules, including Dia-2, for several reasons. First, recent studies indicate the involvement of Dia-2 in mediating the interaction between RhoA and Src (29, 56). Secondly, we have demonstrated that KSHV-induced RhoA-GTPase is essential for the nuclear trafficking of viral DNA and in the regulation of MT dynamics (46). RhoA-activated Dia-2 forms part of the signal transduction cascade that leads to cytoskeleton rearrangement. Activation of Dia-2 by RhoA has been shown to stimulate the formation of stable microtubules that are capped, and Dia promotes this capping by directly binding to microtubules (48, 49). We have shown recently that KSHV infection increased the RhoA-dependent activation of Dia-2 as early as 1 min p.i. and that this continued to increase throughout the initial 10 min, reaching a plateau between 20 and 30 min and being sustained for about 60 min before reaching the baseline (46). Our studies suggested that KSHV-induced RhoA-GTPases probably control the MT dynamics via the activation of Dia-2, thus facilitating efficient trafficking of viral DNA to the infected cell nuclei.

Since KSHV-induced RhoA activates Dia-2, which is known to stimulate Src, we reasoned that RhoA-activated Dia-2 may associate with Src in the infected 293 cells. To determine whether Src associates with Dia-2, infected and uninfected cell lysates were immunoprecipitated with anti-Dia-2 antibody and then subjected to Western blotting with anti-p-Src antibody to detect the presence of associated Src in the immune complexes. In KSHV-infected cells, Src was detected in association with Dia-2 (Fig. 9A, lane 2), which decreased by about 70% in the presence of the Src inhibitor PP2 (Fig. 9A, lane 3). Dia-2 interaction with Src was also observed when the lysates were immunoprecipitated with an antibody against Src and blotted with Dia-2 antibody (Fig. 9B, lane 2), which was also inhibited significantly by PP2 (Fig. 9B, lane 3).

FIG. 9.

Src associates with Dia-2 in KSHV-infected cells. (A and B) Serum-starved RhoA WT cells and cells incubated with PP2 were infected with KSHV at an MOI of 10 and incubated for 10 min. Uninfected and infected cell lysates were immunoprecipitated (IP) using Dia-2 antibody followed by Western blotting (WB) with p-Src antibody (A) or immunoprecipitated using Src antibody followed by immunoblotting with Dia-2 antibody (B). Anti-Src or Dia-2 antibodies were used to verify equal amounts of samples in the immunoprecipitates. (C to F) Colocalization of Src with Dia-2. Serum-starved WT cells (C) or WT cells incubated with PP2 (5 μM; E) for 1 h at 37°C or RhoA-T19N (F) cells were infected with KSHV at an MOI of 50 and incubated at 37°C for 10 min. Uninfected (D) and infected cells were fixed with 2% paraformaldehyde in PBS for 30 min. The cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min and then incubated with p-Src and Dia-2 primary antibodies. These were visualized by incubation with the Alexa 594-labeled anti-rabbit antibody (red) for p-Src and anti-goat FITC-conjugated secondary antibody (green) for Dia-2. Arrows indicate colocalization of Dia-2 with Src. The inset at the bottom of panel C is an enlarged view of colocalization. All magnifications, ×40. Scale bars, 20 μm.

When we examined the colocalization of Dia-2 with active Src in KSHV-infected WT cells, Src colocalization with Dia-2 in the cell periphery was observed at 10 min p.i. (Fig. 9C). In contrast, no significant colocalization was observed in the uninfected WT cells (Fig. 9D). In PP2-treated infected WT cells, a diffuse staining pattern of p-Src was observed, and Dia-2 colocalization was not observed along the periphery of these cells (Fig. 9E). PP2 disrupted the functional interaction between phospho-Src and Dia-2 proteins and their membrane localization in the infected cells (Fig. 9E). Interestingly, in cells expressing RhoA-T19N, Src activation and membrane localization were observed (Fig. 9F); however, activated Src did not colocalize with Dia-2. These results suggested that in the absence of RhoA activation, Dia-2 is not activated, resulting in the absence of association of Dia-2 with Src and the feedback activation of Src. Similar results were also found for CdTxB-treated WT cells infected with KSHV (data not shown). These findings indicate that interaction between Src and Dia-2 is induced by RhoA-GTPase activation and that Dia-2 could function as a link between RhoA and Src in KSHV-infected cells and in the feedback activation of Src, thus facilitating entry and target cell infection.

DISCUSSION

Entry into target cells by herpesviruses is a multistep process starting with the attachment of virus to the cell surface receptors and subsequent penetration into the cytosol either by direct fusion of the viral envelope at the host cell surface or by internalization via endocytosis where fusion occurs with the endosome membrane, followed by transport to the nuclear periphery and release of the viral genome into the nucleus. Herpesviruses interact with several host cell surface receptor(s) of the target cells, which could occur at low temperature. However, entry of virus or viral genome into the cells and subsequent movement in the cytoplasm are energy-dependent phenomena. Herpesviruses, like other viruses, have probably evolved to trigger a cascade of preexisting signaling events involving the activation and phosphorylation of several signaling molecules and thus facilitating virus entry and infection of target cells. Even though several host cell surface receptors have been identified for human and animal herpesviruses, how these interactions with cell surfaces facilitate the infection is not fully understood.

KSHV is the first herpesvirus shown to interact with integrin molecules in endothelial and fibroblast cells (4). Within minutes of its binding to adherent target cells, KSHV induces a variety of host cell preexisting signal pathway molecules, such as FAK, Src, PI3-K, Rho-GTPases, PKC-ζ, MEK, and ERK1/2 (45, 60, 61). Our previous studies have demonstrated the role of FAK and PI3-K in virus entry, the role of RhoA-GTPase in microtubule modulation and transport of capsid in cytoplasm and nuclear delivery via the induction of Dia-2 molecule (36, 45, 46), and a role for ERK1/2 in the initiation of lytic (ORF 50) and latent (ORF 73) gene expression (60). These studies support our hypothesis that KSHV manipulates the host cell signal cascades to create an appropriate intracellular environment facilitating infection.

Rho-GTPases are believed to play active roles in pinocytosis, clathrin- and caveolae-dependent endocytosis, and macropinocytosis (16, 22, 37, 47, 52). Adenovirus types 3 and 5 interact with integrin molecules and stimulate host cell signal cascades such as those of PI3-K and Rho-GTPases. Adenovirus endocytosis requires actin cytoskeleton reorganization which is stimulated by RhoA-GTPase, and RhoA-GTPase has been shown to play roles in adenovirus endocytosis. Similar to our findings for KSHV, CdTxB decreased the entry of adenovirus (39). The role of RhoA-GTPase in regulating the KSHV infection was clearly demonstrated by the inhibition of virus entry by CdTxB in the HMVEC-d cells, a natural in vivo target cell of KSHV. This observation is further supported by the increased entry observed in RhoA-G14V cells and significant reduction in RhoA-T19N cells. The inhibition observed in RhoA-T19N cells was similar to the inhibition by CdTxB observed in HFF and HMVEC-d cells. Since the RhoA-T19N cells show a reduced affinity for nucleotides and abrogate downstream signaling (53, 59), the decreased entry observed in RhoA-T19N cells may be due to inefficient signaling downstream of RhoA.

RhoA-GTPase activity increased during the initial stages of infection in WT, and a higher level of activation was observed in RhoA-G14V mutant cells. However, in contrast to infection in WT and the RhoA-G14V mutant cells, RhoA-GTPase activation did not occur in RhoA-T19N cells. This demonstrated that the RhoA-T19N mutant cell is unable to form RhoA-GTP upon stimulation by virus, whereas both WT and RhoA-G14V cells are able to form active RhoA-GTPases. Integrins and growth factor receptors act as upstream mediators of Rho-GTPase activation (12, 29, 43). This suggests that in WT and RhoA-G14V cells RhoA-GTPases are stimulated by interaction of KSHV with integrins. Integrins are known to activate RhoA-GTPase in a Src-dependent manner (6), and the GTP-bound form of RhoA interacts with its effector molecules (28). Our previous studies with KSHV-gB suggested that FAK, Src, and PI3-K activities are the upstream events in the integrin-mediated activation of RhoA-GTPase (61). Increased phosphorylation of FAK correlated with enhanced entry of KSHV (36). The activation of Rho-GTPase in KSHV-infected fibroblast cells is dependent on PI3-K, suggesting that RhoA lies downstream of PI3-K (61). Importantly, similar kinetics and enhanced phosphorylation of FAK and PI3-K observed in different forms of RhoA cell lines, including WT, RhoA-G14V, and RhoA-T19N, show that these events are linked to KSHV entry independent of RhoA activation. Along with FAK, KSHV gB interaction with integrin leads to Src kinase phosphorylation (61). The overall Src kinase activation in KSHV-infected RhoA-T19N cells was modest, which is in contrast to the very high level of phosphorylation of Src in RhoA-G14V cells and WT cells, indicating that an increased level of RhoA-GTP promotes feedback phosphorylation of Src.

Roles for Src kinase in the internalization and trafficking of coxsackievirus and poxvirus have been reported recently (13, 50). Similarly, the inhibition of KSHV entry by Src-specific inhibitors suggests that Src plays a major role in the entry stage of KSHV infection. The RhoA-GTPase activation and phosphorylation of Src kinase was time dependent, and the kinetics of Src phosphorylation closely resembled the kinetics of RhoA activation in WT and RhoA-G14V cells. The increased phosphorylation of Src in WT and RhoA-G14V cells compared to that in RhoA-T19N cells suggests that the integrin-mediated activation of RhoA-GTP is involved in the reverse activation of Src, thereby leading to elevated cellular levels of phospho-Src. RhoA colocalized with Src when overexpressed and associated with plasma membrane in infected cells. It has been demonstrated that functional Rho is required for subcellular localization of proteins such as Src (21). The activation of Src by RhoA may be due to the interaction of formin homology domain 1 of Rho with Src homology domain 3 (56, 66).

The link between Src and RhoA in the activation and signaling during viral infection has not yet been fully understood. Since the RhoA-T19N cells show reduced affinity for nucleotides and abrogate downstream signaling (53, 59), we reasoned that the decreased entry observed in RhoA-T19N cells may be due to inefficient signaling downstream of RhoA, possibly having to do with a molecule involved in the feedback activation of Src that is critical for virus entry. Dia-2 fits the criteria since it is activated by RhoA (31, 48): Src homology domain 3 binds to Dia-2 formin homology 1 domains and leads to the activation of Src (56, 66), and Dia-2 is activated via RhoA-GTPase early during KSHV infection (46). The role of Dia-2 in the feedback activation of Src during KSHV infection is supported by the association of Src with Dia-2 in the infected cell plasma membranes. The absence of colocalization with Dia-2 despite the membrane localization of Src in RhoA-T19N cells suggests that Src is activated prior to association with the RhoA-GTPase and Dia-2 in infected cells. Together with reduced entry into the RhoA-T19N cells, these observations clearly show that Dia-2 links signals from RhoA-GTPases and Src in KSHV-infected cells and may be coordinating KSHV internalization.

KSHV enters adherent target cells by endocytosis, and several reports show that endocytosis and signal pathways are highly interlinked (1, 38, 40, 52, 62, 63). Protein components of signal transduction cascades assemble at endocytic vesicles and remain associated with endocytic vesicles following their dynamin-dependent or independent release from the plasma membrane (8, 38). Following ligand interaction with receptors, associated tyrosine kinases are activated by autophosphorylation on tyrosine residues and recruit signaling complexes to the plasma membrane, which then rapidly translocate to clathrin, caveolae, and other vesicles (69). Src-mediated tyrosine phosphorylation of clathrin regulates clathrin translocation to the plasma membrane (1, 69). Clathrin subsequently interacts with a number of other essential proteins, such as AP2, Eps15, and dynamin (1, 40). Src-dependent phosphorylation also regulates dynamin self assembly and ligand-induced endocytosis by releasing the internalized endocytic vesicles from the plasma membrane (1). Src-dependent phosphorylation initiates the assembly of a plasma membrane-associated Ras activation complex. Rho- and Rab-GTPases activated by PI3-K and Ras are critical for the formation of various types of endocytic vesicles and their movement as well as for microtubule and microfilament reorganization (8, 38, 41, 62).

KSHV interaction with integrin; induction of FAK, Src, and PI3-K, the upstream mediators of Rho-GTPases; activation of Src required for the formation of endocytic vesicles and their movement; and activation of RhoA and the associated Dia-2 required for microtubular stabilization suggest that KSHV has evolved to manipulate signal pathways to aid in its entry and in the movement of its capsid/tegument in the cytoplasm (Fig. 10). The association of Dia-2-Src complex formation mediated by RhoA may contribute to increase the rate of virus internalization by promoting interactions between the various molecules involved in endosome formation, thereby increasing the rate of vesicle formation and subsequent trafficking. Studies to decipher the role of KSHV-induced signal pathways in the formation of endocytic vesicles and their movement are in progress. Further understanding of the various components of signal cascades manipulated by KSHV would lead to a better understanding of KSHV interactions with host target cells and their outcomes, all of which would eventually lead to the development of better control measures against KSHV infection.

FIG. 10.

Schematic diagram depicting the overlapping dynamic phases of early stages of KSHV infection of adherent target cells, KSHV-induced signal pathways, including RhoA, and their role in infection. KSHV binds to adherent target cell surface HS molecules via its envelope glycoproteins gpK8.1A and gB (3, 5, 68) followed by interaction with α3β1 integrin via gB (4) binding to CD98-xCT molecules (34) and possibly to other yet to be identified molecule(s). Interactions with cell surface receptors trigger host cell preexisting signal cascades, and KSHV enters the target cells via endocytosis (2, 5, 30). FAK activated via interaction with integrin is essential for virus entry (4, 36). Src is activated by FAK, leading to the activation of PI3-K and Rho-GTPases (61). RhoA activates Dia-2, which in turn augments Src activation, all of which are probably essential for the formation of endocytic vesicles and their movement in the cytoplasm. Endosome moves in the cytoplasm, and capsid is released, probably facilitated by induced signal pathways such as PKC-ζ (45). RhoA-GTPase facilitates the transport of capsid towards the nucleus by inducing MT stabilization and regulating MT dynamics via Dia-2 (46). The endocytic vesicles with virus or released capsid/tegument complexes bind to dynein motor components, transported along the MT to reach the nuclear vicinity, and deliver the viral DNA into the nucleus (46). Arrows depict the potential stages of virus entry and infection at which the induced signaling events play roles.