![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-
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-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. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
- and ß-herpesviruses, KSHV in vitro infection of target cells does not lead to the typical cascade of its immediate early, early, and late gene expression and progeny virus formation. Instead, KSHV establishes latency, and the viral genome is lost during successive passages of the infected cells (10, 32). Another novel feature of this in vitro latency in human microvascular dermal endothelial (HMVEC-d) and human foreskin fibroblast (HFF) cells is our demonstration of concurrent expression of a limited set of lytic cycle genes with antiapoptotic and immunomodulation functions, including the lytic cycle switch open reading frame 50 (ORF50) or RTA gene (39). While the expression of latent ORF72, ORF73, and K13 genes continues, nearly all lytic genes decline (10, 39). This characteristic expression of limited lytic cycle genes could be allowing KSHV to evade the immune system and to provide the necessary factors and time to establish and/or maintain latency during the initial phases of infection.
KSHV enters HFF cells, BJAB (KSHV- and Epstein-Barr virus [EBV]-negative B lymphoma) cells, and human embryonic kidney epithelial cells (293) by endocytosis (2, 5, 36), and this uptake is severely attenuated in cells pretreated with inhibitors affecting endosomal functions (2, 36). KSHV utilizes ubiquitous cell surface heparan sulfate (HS) to bind to several in vitro target cells such as BJAB, BCBL-1, 293, HFF, and HMVEC-d cells (5), and this interaction is mediated by the virion envelope glycoprotein gB and gpK8.1A (3, 78). Treatment of virus with heparin blocked the binding of radiolabeled virus to both adherent and nonadherent target cells, suggesting that HS probably serves as an initial contact receptor for the target cells. KSHV interacts with 3ß1 integrin molecule of HMVEC-d and HFF cells via its envelope gB (4). Anti-3 or -ß1 antibodies or soluble 3ß1 integrin blocked KSHV infection of HMVEC-d and HFF cells with ca. 50 to 70% reduction in infection as measured by green fluorescent protein (GFP) expression (4). A role of integrin in KSHV infection of B and other cells has not been demonstrated, and integrin could be one of the KSHV receptors in some adherent target cells and not in all cells.
KSHV also utilizes the transporter protein xCT for entry into adherent cells but not into the B cells (37). The xCT molecule is part of the 125-kDa disulfide-linked heterodimeric membrane glycoprotein CD98 (4F2 antigen) complex containing a common glycosylated heavy chain (80 kDa) and a group of 45-kDa light chains (19, 59). It has also been shown that CD98 constitutively associates with ß1 integrin and is involved in membrane clustering and ß1 integrin-mediated signal cascades (26, 27). KSHV also utilizes the dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN; CD209) as a receptor for the infection of myeloid dendritic cells and macrophages (53). These studies suggest that KSHV utilizes multiple receptors for infection of target cells, and these receptors differ according to the cell type. This is not surprising since the related 
-1 EBV enters primary B cells via endocytosis and human epithelial cells via fusion at the cell membrane (12). EBV binds to the B cells via its gp350/220 interaction with the CR2 receptor and subsequently binds to HLA class II via its gp42 in the gH/gL complex (79). In contrast, infection of epithelial cells occurs via gH/gL complex interaction with an unknown receptor and/or via the interaction of the 5ß1 integrin with the EBV-BMRF2 glycoprotein (72).
Integrin interactions with extracellular matrix proteins lead to the assembly of integrins, numerous signaling molecules including focal adhesion kinase (FAK), Src, and p130cas, and cytoskeletal proteins such as talin, paxillin, and vinculin into aggregates on each side of the membrane, creating the formation of focal adhesions (22, 30, 55, 81). KSHV-integrin interactions lead to the phosphorylation of FAK, which subsequently leads to the activation of Src, phosphatidylinositol 3-kinase (PI3-K), protein kinase C- (PKC-), Rho-GTPases, mitogen-activated protein kinase kinase (MEK), and extracellular signal regulated kinase 1/2 (ERK1/2) (46, 61, 62). Soluble gB induced extensive cytoskeletal rearrangement in the target cells via the induction of a FAK/Src/PI3-K/RhoGTPase signal pathway (62). KSHV-induced phosphorylation of FAK is essential for the entry of KSHV into adherent target cells, since entry of KSHV is greatly reduced in FAK-null fibroblasts (40). Inhibitors of cellular tyrosine kinases, Src kinases, PI3-K, and RhoA-GTPases blocked the entry of KSHV into target cells, suggesting a crucial role for Src, PI3-K, RhoA-GTPase, and other tyrosine kinases in KSHV entry (62). RhoA is also involved in the activation of microtubules (MTs) in HFF cells and is essential for the nuclear delivery of KSHV DNA (47).
In cell membranes, glycosphingolipids, cholesterol, and proteins show specific interactions to form microdomains, known as lipid rafts, which are resistant to solubilization in cold, nonionic detergents (63). Lipid rafts have been known to function as sorting centers for the delivery of lipids and proteins to the apical surface (13, 63, 65). Lipid rafts have also been attributed to a number of cellular functions such as signal transduction (64) and kinase activity modulation (24, 48, 82). Recent studies have suggested that lipid rafts play roles in a wide range of cellular events such as signal transduction, apoptosis, cell adhesion, migration, synaptic transmission, cytoskeletal organization, and protein sorting during endocytosis and exocytosis (13, 33, 64, 71). Since many of the host cell preexisting signaling cascade molecules induced by KSHV during its binding and entry into HFF and HMVEC-d cells are known to be associated with lipid rafts, we examined the role of lipid rafts during de novo KSHV infection of HMVEC-d cells.
Here, we show that KSHV viral gene expression is significantly reduced by lipid raft disruption in HMVEC-d cells. Lipid raft disruption did not affect the binding of KSHV to HMVEC-d cells. Although lipid raft disruption resulted in an increase of internalized viral DNA, nuclear delivery of viral DNA was significantly reduced in these cells. Methyl ß-cyclo dextrin (MßCD) treatment resulted in the reduced association of KSHV capsids with MTs. An increase in KSHV induced Src phosphorylation and no alteration in FAK and ERK activation were observed in these cells. In contrast, significant reduction in KSHV-induced activation of PI3-K, RhoA-GTPase, and NF-B; lipid raft association of PI3-K, RhoA-GTPase, and Diaphanous-2 (Dia-2; a RhoA-GTPase-activated adaptor molecule involved in MT activation); and reduction in acetylation of MTs were observed in lipid raft-disrupted cells. These studies suggest that lipid rafts of microvascular dermal endothelial cells are essential for KSHV infection and gene expression, and this could be due to their potential roles in the modulation of KSHV-induced PI3-K, RhoA-GTPase, and Dia-2 signal molecules playing roles in postbinding and entry stages of infection.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Antibodies and reagents.
MßCD, nystatin, heparin, LY294002 [20(4-morphodinyl)-8-phenyl-1(4H)-benzopyran-4-one], cytochalasin D, wortmannin, chlorpromazine, NH4Cl, tetradeconyl-phorbol acetate, lysophosphatidic acid, Triton X-100, and monoclonal antibodies to acetylated tubulin, total tubulin, and ß-actin were obtained from Sigma, St. Louis, MO. Antibody to pERK, pNFB was from Cell Signaling, Denver, MA. U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenlythio butadiene) and PP2 were obtained from Calbiochem, La Jolla, CA. Mouse anti-phospho FAK (Y397), Mouse anti-caveolin and anti-FAK (total) antibodies were from BD Biosciences, San Jose, CA. Anti-phospho Src (PY418) and anti-Src (total) antibodies were obtained from Upstate Biotechnology, Lake Placid, NY. Anti-PI3-K, RhoA, mouse antihuman transferrin receptor, and Dia-2 antibodies were from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. 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, and Molecular Probes, Eugene, OR. The Amplex Red Cholesterol assay kit and Vybrant lipid raft labeling kit were purchased from Molecular Probes. The FACE PI3-Kinase ELISA kit was purchased from Active Motif, Carlsbad, CA. The G-LISA-RhoA activation assay kit was purchased from Cytoskeleton, Denver, CO.
Cytotoxicity assays. Target cells were incubated with Dulbecco modified Eagle medium containing different concentrations of various inhibitors for 4 h. Supernatants were collected and assessed for cellular toxicity by using an LDH cytotoxicity assay kit (Promega, Madison, WI) in accordance with the manufacturer's recommendations.
Preparation of DNA and RNA. Isolation of total DNAs from the viral stocks and HMVEC-d cells by DNeasy tissue kit (QIAGEN, Inc., Valencia, CA) and isolation of total RNA from infected or uninfected cells using an RNeasy kit were carried out as described previously (39, 61).
Measurement of KSHV DNA internalization and nuclear delivery. Untreated HMVEC-d cells or HMVEC-d cells incubated with nontoxic concentrations of different inhibitors for 1 h at 37°C were infected with GFP-KSHV at an MOI of 10 viral DNA copies per cell. After different time points of incubation with virus, cells were washed twice with HBSS to remove unbound virus. Cells were treated with 0.25% trypsin-EDTA for 5 min at 37°C to remove the bound, noninternalized virus. Detached cells were washed twice to remove the virus and trypsin-EDTA. Total DNA isolated from cells or from nuclear fractions was tested by real-time PCR for ORF73 as described previously (47).
Real-time RT-PCR. The ORF50 and ORF73 transcripts were detected by real-time reverse transcription-PCR (RT-PCR) using gene-specific real-time primers and specific TaqMan probes as described previously (39).
Amplex Red Cholesterol assay.
HMVEC-d cells were washed twice with phosphate-buffered saline (PBS) and then incubated at 37°C for 1 h with different concentrations of MßCD in Dulbecco modified Eagle medium. After two washes with PBS, cells were harvested for cholesterol measurement assays as described by the manufacturer (Molecular Probes). In brief, 106 cells in 80 µl of PBS were lysed by three cycles of freeze-thawing, followed by ultrasonication (three bursts of 20 s each at room temperature). Cholesterol was extracted from the cell lysate by the addition of chloroform (200 µl) and methanol (200 µl) to the sonicated lysate (50 µl). The bottom chloroform layer was collected and evaporated under a vacuum. The residual cholesterol was dissolved in ethanol (50 µl) and assayed using the Amplex Red reagent, and the fluorescence intensity was measured in the microplate reader (Synergy HT Multi-Detection Microplate Reader) using excitation in the range of 530 to 560 nm and emission detection at 
590 nm. For each point, the background fluorescence from the no-cholesterol control was subtracted, and the percentage of remaining cholesterol after MßCD treatment was calculated by dividing the fluorescence value of treated cells by the total fluorescence of untreated cells.
Radiolabeled KSHV binding assay. HMVEC-d cells were preincubated with nontoxic doses of various inhibitors before the addition of [3H]thymidine-labeled, density gradient-purified KSHV (5,000 cpm) (3, 5). As a control, labeled KSHV was incubated with 100 µg of heparin/ml for 90 min at 4°C, added to HMVEC-d cells, and incubated for 90 min at 4°C. After incubation, the cells were washed five times and lysed with 1% sodium dodecyl sulfate (SDS) and 1% Triton X-100, and the radioactivity was precipitated with trichloroacetic acid and counted in a scintillation counter.
Western blot analysis.
Total cell lysates prepared from HMVEC-d cells were resolved on SDS-polyacrylamide gels, transferred onto nitrocellulose paper, and immunoblotted with primary antibodies (anti-pSrc, pFAK p-ERK, and pNF-B). To confirm equal protein loading, blots were reacted with antibodies against human ß-actin. Horseradish peroxidase- or alkaline phosphatase-conjugated secondary antibodies were used for detection. Immunoreactive bands were developed by either an enhanced chemiluminescence reaction (NEN Life Sciences Products, Boston, MA) or CDP-Star (Roche Diagnostics Corp., Indianapolis, IN) and quantified by following standard protocols (62).
Immunofluorescence assay (IFA) for ORF73 protein detection. Confluent HMVEC-d cells grown in eight-well chamber slides were either treated with 5 mM MßCD or 50 µg of nystatin for 1 h or left untreated, washed, and infected with 10 multiplicities of infection (MOI) (DNA copies per cell) of KSHV for 2 h; complete medium was added, followed by incubation for 72 h. The cells were fixed in 3.7% paraformaldehyde, permeabilized in 0.5% Triton X-100, and incubated overnight with rabbit anti-ORF73 antibodies (1:80) in 5% bovine serum albumin (BSA). Cells were washed with PBS, incubated with anti-rabbit Alexa Fluor 488 antibodies for 1 h at room temperature, washed, mounted with antifade reagent containing DAPI (4',6'-diamidino-2-phenylindole), and visualized under a Nikon fluorescent eclipse 80i microscope. Images were processed by using Metamorph imaging software.
IFA for acetylated tubulin detection. HMVEC-d cells (90% confluence) in eight-well chamber slides were serum starved for 6 to 8 h. These were untreated or treated with 5 mM MßCD alone for 1 h or treated with 5 mM MßCD for 1 h and then infected with 10 MOI KSHV for various time points. Cells were washed, fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, labeled with a 1:100 dilution of primary monoclonal antibody for acetylated tubulin for 1 h, washed, incubated with 1:500 anti-mouse secondary Alexa Fluor 488 for 1 h at room temperature, washed again, mounted with antifade reagent containing DAPI, and visualized under a Nikon fluorescent eclipse 80i microscope. Images were processed by using Metamorph imaging software.
Colocalization studies with lipid raft marker GM1. Confluent HMVEC-d cells in eight-well chamber slides were uninfected or infected with KSHV or treated with various inhibitors at 37°C for 1 h and then infected with KSHV for the required time points. These cells were washed and labeled for lipid raft marker GM1 (lipid raft labeling kit) according to the manufacturer's instructions. Briefly, a 1:1,000 dilution of CT-B conjugate at 4°C was added for 10 min and washed thrice with PBS, and a 1:200 dilution of anti-CT-B antibody was added for 15 min at 4°C. These were washed, fixed with 3.7% paraformaldehyde, permeabilized in 0.5% Triton X-100, and stained with primary phospho-Src, anti-P-85, and anti-RhoA antibodies overnight at 4°C. The cells were washed, incubated with secondary anti-mouse Alexa Fluor 488 for 1 h at room temperature, washed, mounted with antifading agent containing DAPI, and examined under a fluorescence microscope.
Cytoplasmic trafficking of KSHV capsid. Serum-starved HMVEC-d cells in chamber slides were preincubated with 10 µg of nocodazole or 5 mM MßCD or LY294002 (PI3-K inhibitor) for 1 h and infected with a high MOI of KSHV (100 viral DNA copies/cell) to facilitate visualization of viral capsids. These cells were washed, fixed for 15 min with 3.7% formaldehyde in PBS at room temperature, permeabilized for 3 min with 0.5% Triton X-100, and blocked for 30 min with 10% normal goat serum (Sigma) in PBS. The cells were incubated for 1 h at room temperature with a 1:100 dilution of rabbit polyclonal immunoglobulin G (IgG) against KSHV capsid ORF65 protein (42) and 1:500 dilution of a mouse anti-tubulin monoclonal antibody. After being washed with PBS, the cells were incubated with goat anti-mouse IgG-Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594 (10 µg/ml) for tubulin and ORF65, respectively, for 1 h at room temperature, washed, and mounted with antifade reagent containing DAPI before examination.
Detergent-free method of raft microdomain isolation. Raft microdomains were purified from HMVEC-d cells by a previously described method (67) with modifications. Briefly, HMVEC-d cells grown to 80 to 90% confluence in a 150-cm2 flask were washed twice with Hanks balanced salt solution. HMVEC-d cells were left uninfected, infected with 10 MOI KSHV for 30 min, or treated with 5 mM MßCD for 1 h; washed thoroughly; and then infected with 10 MOI of KSHV for 30 min. After infection, cells were washed with ice-cold PBS. Cells were then scraped into 2 to 4 ml of PBS and collected into cold 5-ml tubes. The cells were spun at 250 x g for 5 min. The supernatant was removed, and the cell pellet was resuspended with 1 ml of 500 mM Na2CO3 per tube. The content of tube was transferred to the Dounce homogenizer (kept on ice), and cells were lysed with 20 strokes using the tight-fitting pestle. The cell lysate was collected into a 5-ml tube, and further disruption of the membranes was achieved by passing the lysate 10 times through a 23-gauge needle. These cell lysates were sonicated three times for 15 s each time, and 1 ml of cell lysate was mixed with 1 ml of cold 90% sucrose-MBS {25 mM MES [2-(N-morpholinoethanesulfonic acid) plus 0.15 M NaCl [pH 6.5]}. This was overlaid onto 2 ml of cold 35% sucrose-MBS-Na2CO3 and 1.5 to 2 ml of cold 35% sucrose-MBS-Na2CO3 and centrifuged for 16 to 18 h at 45,000 rpm in SW-55 rotor at 4°C. Then, 500-µl fractions from the top to the bottom of the gradient were collected and stored at –80°C until processed. The pH of the fractions was adjusted with 1 N HCl, and protein levels in the isolated fractions were estimated by the microBCA method. A total of 20 µg of the lysate from the fractions was immunoblotted for caveolin as a raft marker and for transferrin as a nonraft marker.
Immunoprecipitation of raft fractions for PI3-K phosphorylation.
Portions (150 µg) of raft fraction lysates were immunoprecipitated by incubation with PI3-K p85 (Z-8) antibody and probed with anti-phosphotyrosine monoclonal antibody PY20. The total p85 (PI3-K) level was measured by testing the whole-cell lysate in Western blot assays with PI3-K p85 (Z-8) antibody. Immunoreactive bands were visualized scanned and quantitated by using Alphaease FC (Fluor Chem) software from Alpha Innotech, San Leandro, CA.
Measurement of PI3-K induction by FACE PI3-K ELISA. The FACE (for fast activated cell-based) ELISA kit is designed to detect activated proteins in mammalian cells. The phospho-PI3-K p85 antibody raised against a synthetic phospho-(Tyr) p85 motif binding peptide (preferentially recognizes peptides and proteins containing phosphotyrosine at the consensus YXXM motif). The total PI3-K p85 antibody recognizes the total level of PI3-K p85 proteins regardless of the phosphorylation state. The FACE PI3-kinase kits can be used to study phosphorylated PI3-K relative to the cell number and also to determine PI3-K phosphorylation relative to the total PI3-K protein found in the cells. After the phospho-PI3-K and total PI3-K signals have been normalized for cell number, a comparison of the ratio of phosphorylated PI3-K to total PI3-K for each of the cell growth conditions is made. Briefly, HMVEC-d cells (90% confluence) serum starved for 6 to 8 h were left untreated or treated with 5 mM MßCD for 1 h, infected with 10 MOI KSHV for various time points, fixed in 4% formaldehyde, and assayed for PI3-K according to the manufacturer's instructions.
Determination of RhoA activity by G-LISA RhoA activation assay. This assay is based on the principle that a Rho-GTP-binding protein is linked to the 96-well plates. The active GTP-bound Rho in the cell lysates binds to the wells, while the inactive GDP-bound Rho is removed during the washing steps. The bound active RhoA is detected with a RhoA specific antibody and quantitated by absorbance. The degree of RhoA activation is determined by comparing readings from the activated cell lysates versus the nonactivated cell lysates. The inactivation of RhoA is achieved by serum starvation of cells prior to the experiment for 6 to 8 h. HMVEC-d cells (90% confluence) serum starved for 6 to 8 h were left untreated or were treated with 5 mM MßCD for 1 h and infected with KSHV (10 DNA copies/cell) for various time points, and lysates were collected and tested for RhoA activation according to the manufacturer's instructions. To determine RhoA activity after PI3-K inhibition, HMVEC-d cells were untreated or pretreated with PI3-K inhibitors LY294002 (100 µM) and wortmannin (500 nM) and then infected with 10 MOI of KSHV for various time points. The RhoA-GTPase inhibitor, Clostridium difficile toxin B, was used as a control.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
To confirm these observations, we next examined the infected cells by immunofluorescence techniques. In HMVEC-d cells infected with 10 MOI of KSHV/cell, ca. 50% of cells exhibited the characteristic nuclear staining of ORF73 protein at 72 h p.i. (Fig. 1Ea and b). In contrast, in cells pretreated with lipid raft inhibitors, a dramatic reduction in the number of ORF73-expressing cells were observed (Fig. 1Ec to f), with ca. 86% reduction in 5 mM MßCD-treated cells (Fig. 1Ec and d) and ca. 80% reduction in 50 µg of nystatin-treated cells (Fig. 1Ee and f). The expression of ORF50 protein was also inhibited by ca. 80 to 90% with 5 mM MßCD or 50 µg of nystatin treatments (data not shown). These results further validated our observation that lipid rafts play an important role in KSHV infection.
Lipid raft disruption does not affect KSHV binding to HMVEC-d cells but enhances KSHV entry into HMVEC-d cells. To determine whether treatment of HMVEC-d cells with various concentrations of MßCD and nystatin efficiently extracted cholesterol and to check whether the cholesterol levels remain depleted even after 24 h, we next assayed the cellular cholesterol levels. As shown in Fig. 2A and B, the cellular cholesterol levels decreased in a dose-dependent manner at both 1 h and 24 h after treatment with nontoxic doses of drugs. With 25-, 50-, and 100-µg concentrations of nystatin, we observed a reduction of ca. 40, 50, and 70%, respectively (Fig. 2A and B). Similarly, ca. 50, 60, and 70% reduction in cholesterol levels was observed with 2.5, 5, and 10 mM concentrations of MßCD, respectively (Fig. 2A and B). The depleted cholesterol levels remained low even after 24 h (Fig. 2B). In contrast, no significant change in cellular cholesterol levels was observed when cells were treated with 10 µg of nocodazole (MT-disrupting agent) or 1 mM NH4Cl (endocytic vesicle acidification inhibitor) for 1 h at 37°C (Fig. 2A). These results demonstrated that under our testing conditions both MßCD and nystatin treatment were efficient in sequestering or chelating cholesterol from lipid rafts of HMVEC-d cells and that depleted cellular cholesterol levels remained low even after 24 h of drug treatment.
|
To determine whether reduction in KSHV gene expression in lipid raft-disrupted cells was due to interference in viral entry, total viral DNA internalization (cytoplasmic and nuclear) was measured by real-time DNA PCR. Similar to our earlier studies (62), we observed ca. 70 to 80% reduction in viral DNA internalization by preincubating virus with heparin or by pretreating cells with the PI3-K inhibitor LY294002 (Fig. 2D). No significant inhibition in KSHV-DNA internalization was seen when cells were treated with chlorpromazine or NH4Cl or cytochalasin D or U0126 (Fig. 2D). Surprisingly, we observed an increase in KSHV internalization in cells pretreated with 5 mM MßCD and 50 µg of nystatin (Fig. 2E and F). We compared the entry kinetics of KSHV internalization in untreated or MßCD- or nystatin-treated cells at 15, 30, 60, and 120 min p.i. When KSHV entry in untreated cells at each time point was taken as 100%, we observed a significant increase in viral DNA internalization at all time points in lipid raft-disrupted cells (Fig. 2E and F). KSHV-DNA internalization was maximal at the 2-h time point, with ca. 140 and 250% in MßCD- and nystatin-treated cells, respectively (Fig. 2E and F). These results suggested that intact lipid rafts probably play a critical checkpoint role in the orderly entry of KSHV, and their disruption probably affected the equilibrium of KSHV entry into HMVEC-d cells.
Lipid raft disruption decreases the nuclear delivery of KSHV-DNA in HMVEC-d cells. To determine whether the observed reduction in KSHV gene expression despite the increased KSHV entry into the lipid raft disrupted cells was due to interference at the postentry steps such as delivery of KSHV-DNA to the nuclei, we next examined the kinetics of KSHV- DNA delivery into the infected cell nuclei. For this, the nuclear fractions of KSHV-infected HMVEC-d cells at various time points were isolated by cell lysis and gradient purification (47). Nuclear fractions were positive for the nuclear marker, lamin B (Fig. 3A, upper panel, lane 2), while the cytoskeletal marker tubulin was absent in these nuclear fractions (Fig. 3A, lower panel, lane 2). These results demonstrated that our nuclear fractions were mostly free of cytoskeletal contamination.
|
When cells were pretreated with 5 mM MßCD and 50 µg of nystatin, we observed ca. 60 and 80% inhibition of nucleus-associated KSHV-DNA, respectively (Fig. 3C). Similar to our earlier observation in HFF cells (47), incubation of HMVEC-d cells with 10 µg of nocodazole blocked ca. 50% of nuclear viral DNA delivery (Fig. 3C). In contrast, pretreatment with cytochalasin D, NH4Cl, or chlorpromazine did not inhibit the nuclear delivery of KSHV-DNA (Fig. 3C). These studies demonstrated that despite increased KSHV entry in lipid raft-disrupted endothelial cells, there were defects in the delivery of KSHV-DNA to the infected cell nuclei, suggesting that these defects were probably responsible for the observed inhibition of viral gene expression.
Lipid raft disruption reduced the association of KSHV capsids with MTs and cytoplasmic trafficking in HMVEC-d cells.
To further verify these observations, we monitored KSHV capsid movement in the cytoplasm using our well-characterized antibodies against KSHV capsid protein ORF65 (42) and anti--tubulin antibodies to visualize MTs. At 1 h p.i. in the absence of drug treatments, abundant accumulation of KSHV capsids was detected as fine red dots that colocalized with MTs and near the infected cell nuclei (Fig. 4Aa to c). In contrast, in endothelial cells pretreated with 5 mM MßCD for 1 h at 37°C, although KSHV capsids were distributed throughout the cytoplasm, the majority of these did not colocalize with the MTs (Fig. 4Ad to f). Interestingly, KSHV-induced MT aggregation was comparatively less prominent in MßCD-treated cells and, instead, less-organized disrupted MTs and thickened rounded plasma membranes were prominently visible (Fig. 4Ad). Cells treated with 5 mM MßCD alone for 1 h at 37°C similarly exhibited less-organized disrupted MTs and thickened rounded plasma membranes (Fig. 4Bb and see Fig. 9Be). We have shown previously that treatment with the MT-disrupting agent nocodazole inhibited the MT-dependent transport of KSHV via dynein motor toward the nuclei of infected HFF cells (47). Similarly, in endothelial cells treated with nocodazole as a control, we observed the absence of MT aggregation, localization of KSHV capsid near the periphery of infected cell plasma membranes, and significant reduction in perinuclear accumulation of viral capsids (Fig. 4Ag to i). The specificity of these observations was shown by the absence of staining with preimmune IgG antibodies (data not shown).
|
|
Lipid raft disruption does not influence KSHV-induced FAK activation in HMVEC-d cells. Early during the infection of adherent HMVEC-d and HFF cells, KSHV induced the integrin-dependent host cell preexisting FAK, Src, PI3-K, and Rho-GTPase signal pathways, as well as microtubular acetylation (46, 47). Inactivation of Rho-GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus (47). Nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA (47, 73). KSHV capsid colocalization with MT was also abolished by PI3-K inhibitor affecting the Rho-GTPases (47). These results suggested that KSHV induced the Rho-GTPases and, in doing so, modulates the MTs and promotes the trafficking of viral capsid and the establishment of infection. These observations, coupled with the results presented above demonstrating the effect of lipid raft disruption on MT dynamics and trafficking of KSHV, suggested that KSHV-induced signaling pathways may depend upon intact lipid rafts. Hence, we next examined the activation of signaling molecules by KSHV in lipid raft-disrupted cells.
KSHV-induced FAK has been shown to be essential for virus entry into the adherent target cells (40). Similar to our earlier observations, KSHV induced robust FAK phosphorylation by 5-fold at 5 min p.i. of HMVEC-d cells, which then stabilized at 3-fold at 30, 60, and 120 min p.i (see supplemental Fig. 1A at http://66.99.255.20/cms/micro/Chandran.cfm). MßCD alone did not alter FAK phosphorylation in the uninfected cells. In cells pretreated with MßCD for 1 h at 37°C and then infected with KSHV, no significant change in FAK phosphorylation levels was observed (see supplemental Fig. 1A at http://66.99.255.20/cms/micro/Chandran.cfm). Equal loading of total lysates was confirmed with ß-actin antibodies. These results suggested that the lipid raft does not play an active role in KSHV-induced FAK phosphorylation and that increased virus entry seen in lipid raft-disrupted cells (Fig. 4d-h) was not due to enhanced FAK activation.
Lipid raft disruption does not influence KSHV-induced ERK1/2 activation. KSHV infection induces a rapid transient MEK1/2 and ERK1/2 phosphorylation in HMVEC-d and HFF cells, and ERK1/2 plays a critical role in the initiation of ORF50 and ORF73 gene expression, as well as host gene expression (61). Similar to our earlier observations, KSHV induced a robust ERK1/2 phosphorylation by about 2-, 4-, and 3-fold at 15, 30, and 60 min p.i., respectively, which returned to the basal level at 120 min p.i. (see supplemental Fig. 1 at http://66.99.255.20/cms/micro/Chandran.cfm). Interestingly, when uninfected cells were treated with MßCD alone for 1 h at 37°C, we observed a twofold induction of ERK1/2 phosphorylation. This is similar to other studies (17), demonstrating that the activation of ERK1/2 by MßCD alone in COS-1 and NIH 3T3 cells is due to cross-linking and ligand-independent activation of epidermal growth factor receptor. When cells were pretreated with MßCD for 1 h at 37°C and then infected with KSHV, we observed a moderate augmentation in ERK1/2 activation with about 3-, 4-, 4-, and 3-fold inductions at 15, 30, 60, and 120 min p.i., respectively (see supplemental Fig. 1 at http://66.99.255.20/cms/micro/Chandran.cfm). These results suggested that the observed reduced viral gene expression in lipid raft-disrupted cells (Fig. 1) was probably not due to the absence and/or reduction in KSHV-induced ERK1/2 necessary for viral gene expression.
Lipid raft disruption decreased the KSHV-induced NF-B.
We have recently shown that NF-B also plays a role in influencing viral gene expression (56). When HMVEC-d cells were infected with KSHV (10 MOI/cell) there was a rapid NF-B activation as early as 15 min p.i. (see supplemental Fig. 1 at http://66.99.255.20/cms/micro/Chandran.cfm). As previously reported (56), there was a sustained activation of NF-B until 120 min of our observation (see supplemental Fig. 1 at http://66.99.255.20/cms/micro/Chandran.cfm). When the cells were pretreated with 5 mM MßCD, there was a 50% reduction in NF-B phosphorylation at 15 min p.i. and nearly 25 to 35% reduction at 30, 60, and 120 min p.i., respectively. Equal loading of total lysates was confirmed with ß-actin antibodies (see supplemental Fig. 1 at http://66.99.255.20/cms/micro/Chandran.cfm). These results suggest that the observed reduced viral gene expression in lipid raft disrupted cells could be due to the reduction in the KSHV-induced NF-B which is necessary for viral gene expression.
Lipid raft disruption increased KSHV-induced Src phosphorylation in HMVEC-d cells. To check whether lipid raft disruption affected Src phosphorylation, lysates prepared from HMVEC-d cells before and after treatment with 5 mM MßCD were probed with antibodies against phospho-Src PY418. Src phosphorylation in uninfected cells was taken as onefold (Fig. 5B, upper panel, lane 1). Similar to our earlier observations, KSHV induced a robust Src phosphorylation by about 2-, 3-, and 4-fold at 15, 30, and 60 min p.i., respectively (Fig. 5B, upper panel, lanes 2 to 4). Cholesterol depletion by MßCD has been shown to constitutively phosphorylate Src in NIH 3T3 cells (49). Similarly, as seen for ERK1/2 activation (unpublished data), we also observed a twofold Src phosphorylation in uninfected cells treated with 5 mM MßCD alone (Fig. 5B, upper panel, lane 8). When MßCD-pretreated cells were infected with KSHV, we observed about 5-, 4-, and 4-fold Src activation at 15, 30, and 60 min p.i., respectively (Fig. 5B, upper panel, lanes 5 to 7). These results demonstrated the augmentation of KSHV-induced Src by lipid raft disruptions and suggested that, since Src is critical for KSHV entry (73), this increased Src activation in the presence of MßCD may potentially be responsible for the observed increased KSHV entry in lipid raft-disrupted cells (Fig. 2D).
|
Lipid raft disruption downregulates KSHV-induced PI3-K activation in HMVEC-d cells. PI3-K is an important regulator of cell proliferation, survival, and growth, and the phosphorylation of PI3-K leads to the downstream activation of a variety of cell signaling pathways (76). Early during the infection of target cells, PI3-K was induced by KSHV, which is critical for virus entry and induction of Rho-GTPases (61). Since PI3-K has been shown to be associated with lipid raft microdomains (24), we examined here the association of KSHV-induced PI3-K with lipid rafts and measured the phospho PI3-K and total PI3-K levels.
A rapid induction of PI3-K activity with about 2.5- and 3-fold activations at 5 min and 15 min p.i., respectively, was observed in KSHV-infected HMVEC-d cells, which peaked to 6-fold at 30 min and remained at 3-fold at 60 min p.i. (Fig. 6A). MßCD alone did not alter PI3-K phosphorylation significantly in the uninfected cells (data not shown). In contrast, infection of cells pretreated with MßCD resulted in the drastic reduction of PI3-K activity, with only about 1- to 1.5-fold induction throughout the 60-min period of observation (Fig. 6A). When serum-starved untreated HMVEC-d cells or cells preincubated with MßCD for 1 h were infected with KSHV and examined at 30 min (a time point of maximum PI3-K activation) (Fig. 6B), only very minimal colocalization of PI3-K with lipid rafts was observed in the uninfected cells (Fig. 6Ba to c). In contrast, very prominent PI3-K (P85) colocalization with lipid rafts was observed throughout the untreated infected cells (Fig. 6Bd to f). As observed in Fig. 5A, lipid raft spots were vastly reduced by MßCD treatment in the uninfected cells (Fig. 6Bg to i), with negligible PI3-K and lipid raft colocalization (Fig. 6Bg-i). In lipid raft-disrupted KSHV-infected cells, we observed a substantial reduction in PI3-K and lipid raft colocalizations (Fig. 6Bj to l), which were dispersed and strikingly different from the high concentrations observed in virus-infected untreated cells (compare Fig. 6Bd to f to Fig. 6Bj to l).
|
Taken together, these results suggested that lipid rafts play an active role in the modulation of KSHV-induced PI3-K activation and that the observed decreased PI3-K levels could play a significant role(s) in the postentry stages of KSHV infection in endothelial cells by modulating the downstream host cell signaling effector molecules and the associated cascades.
Lipid raft disruption downregulates KSHV-induced RhoA-GTPase activation in HMVEC-d cells. RhoA, Rac1, and Cdc42, the best-studied members of the Rho-GTPase family, are the major downstream effector molecules of PI3-K and function as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state. Besides their role in cytoskeleton reorganization, Rho-GTPases participate in a wide variety of cellular processes, including proliferation, differentiation, MT stabilization, endocytosis, vesicle trafficking, cytoplasmic transport, and gene expression (47). In KSHV-infected HFF cells, RhoA promoted the actin stress fibers, whereas Rac1 and Cdc42 mediated the lamellipodia and filopodial extensions, respectively (47). We have demonstrated that KSHV-induced RhoA-GTPase is critical for virus entry, microtubular acetylation, and the delivery of viral DNA to the nucleus (47, 73). These observations, together with the present study demonstrating the defective transport of virus to the nucleus by lipid raft disruption, prompted us to examine interlinks between KSHV-induced RhoA-GTPase activation and lipid rafts.
When RhoA-GTPase activation was examined by the G-LISA method, we observed a rapid 3.5-fold RhoA-GTPase activation by KSHV as early as 1 min p.i., peaking at 4-fold at 5 min p.i. and persisting at 2-fold during the 60 min of observation (Fig. 7A). Treatment of cells with MßCD alone did not significantly alter RhoA-GTPase phosphorylation (data not shown). When serum-starved HMVEC-d cells were pretreated with 5 mM MßCD for 1 h and then infected with KSHV for various time points, RhoA-GTPase activity was drastically inhibited between 1 and 30 min p.i. (Fig. 7A). This inhibition appears to be reversible slowly since 1.5-fold activation was seen at 60 min p.i. When uninfected cells were examined by IFA, only very minimal colocalizations of RhoA with lipid rafts were observed (Fig. 7Ba to c). At 5 min p.i., a time point of maximum RhoA-GTPase activation (Fig. 7A), we observed very prominent RhoA colocalization with lipid rafts throughout the untreated infected cells (Fig. 7Bd to f). Only negligible RhoA and lipid raft colocalization was seen in MßCD treated cells (Fig. 7Bg to i). In lipid raft-disrupted KSHV-infected cells, a substantial reduction in RhoA colocalization compared to untreated infected cells was seen (Fig. 7Bj to l).
|
To determine whether inhibition of PI3-K affected trafficking of KSHV capsid, HMVEC-d cells pretreated with 100 µM LY294002 were infected with 100 MOI of KSHV for 60 min. As shown in Fig. 4Aa and b, we observed the microtubular thickening and association of KSHV capsid with the MTs in untreated infected cells (Fig. 7Da to c), In contrast, the treatment with LY294002 resulted in the disruption of the MT network and, consequently, the absence of perinuclear accumulation of the KSHV capsids and lack of their MT association were observed (Fig. 7Dd to f).
These results demonstrated that lipid raft disruption significantly affected KSHV-induced activation of PI3-K and RhoA-GTPase. Since these molecules are critical for KSHV movement toward the infected cell nuclei (47), the data suggested that the observed reduced viral gene expression in lipid raft-disrupted cells (Fig. 1) could be attributed to reduced activation of PI3-K and RhoA-GTPase by KSHV in lipid raft-disrupted cells.
Lipid raft disruption downregulates KSHV-induced RhoA activation of Dia-2 molecule. Dia-2, a diaphanous-related formin family of molecules activated by RhoA, is involved in mediating the interaction between RhoA and Src and forms part of the signal-transduction cascade leading to rearrangement of the cytoskeleton. Activation of Dia-2 by RhoA has been shown to stimulate the formation of stable MTs that are capped, and Dia-2 promotes this capping by directly binding to MTs (48). More importantly, we have demonstrated that KSHV-induced RhoA-GTPase is essential for the nuclear trafficking of viral DNA and in the regulation of MT dynamics (47, 73). KSHV infection of HFF and 293 cells increased the RhoA-dependent activation of Dia-2 early during infection (47, 73). Our studies suggested that KSHV-induced RhoA-GTPases probably control the MT dynamics via the activation of Dia-2 and thus facilitate the efficient trafficking of viral DNA to the infected cell nuclei.
To determine whether the reduced PI3-K and RhoA activation by KSHV in lipid raft-disrupted cells also results in the modulation of Dia-2 activity, we examined the association of Dia-2 with lipid rafts. When serum-starved untreated HMVEC-d cells or cells preincubated with MßCD for 1 h were infected with KSHV and examined at 10 min, a time point of maximum Dia-2 activation (47), only very minimal colocalization of Dia-2 with lipid rafts was observed in the uninfected cells (Fig. 8a to c). In contrast, very prominent Dia-2 colocalization with lipid rafts was seen in the infected cells (Fig. 8d to f). The specificity of this reaction was shown by the complete absence of Dia-2/lipid raft colocalization in cells pretreated with PP2 (Fig. 8g to i). In cells treated with MßCD alone, we observed a minimal amount of Dia-2 and lipid raft colocalization (Fig. 8j to l). In lipid raft-disrupted KSHV-infected cells, we observed a significant reduction in Dia-2 and lipid raft colocalizations (Fig. 8m to o) compared to untreated virus-infected cells (Fig. 8d to f).
|
Lipid raft disruption downregulates KSHV-induced acetylation of MTs in endothelial cells.
Acetylation and deactylation are powerful and dynamic methods of controlling MT dynamics (51), and KSHV induces the acetylation of MTs in the infected HFF cells via the induction of PI3-K, RhoA-GTPase, and Dia-2 (47). Since hyperacetylation is a quantitative indication of changes in MT stabilization, we next quantified the levels of MT acetylation by Western blot analysis. Moderate levels of acetylated MTs were observed in uninfected cells (Fig. 9, upper panel, lane 13). In contrast, MTs were heavily acetylated in infected cells, with 8-fold induction as early as 5 min p.i. (Fig. 9, upper panel, lane 1); this increased to 10-fold between 15 and 60 min p.i. and persisted at about 7- to 8-fold during the 120 min of observation (Fig. 9, lanes 2 to 5). The levels of total tubulin remained unaltered, thus demonstrating the specificity of acetylation (Fig. 9, lower panel). KSHV-induced hyperacetylation was comparable to the induction by lysophosphatidic acid, a known inducer (Fig. 9A, upper panel, lane 12). Treatment with 5 mM MßCD for 1 h alone moderately induced MT acetylation by 2-fold (Fig. 9A, upper panel, lane 11). Infection of cells treated with MßCD inhibited the virus-induced MT acetylation by ca. 50% at 15 min p.i. (Fig. 9A, upper panel, lane 7).
The observed acetylation was also confirmed by determining the relative amounts of acetylated tubulin in KSHV-infected cells by IFA. Serum-starved uninfected HMVEC-d cells exhibited the classical pattern of finely spread out loose bundles of MTs radiating from the perinuclear nucleation center of MTs, the microtubular organizing center, toward the edges of the cell (Fig. 9Ba). As early as 5 min p.i., KSHV induced the aggregation of MTs, leading to an increased thickening of MT bundles (Fig. 9Bb), which were sustained for the 30 min of observation (Fig. 9Bb to d and inset panel c). These results demonstrated the modulation of MT dynamics early during infection by KSHV, resulting in transient reorganization of the MT network into bundles, replacing the normal MT cytoskeletal morphology. Cells pretreated with 5 mM MßCD alone exhibited less-organized disrupted MTs and thickened rounded plasma membranes (Fig. 9Be, inset). When lipid raft-disrupted cells were infected, the robust bundling of acetylated tubulin observed during KSHV infection alone was not observed. Instead, as seen in Fig. 4D, the bundles were disorganized, MT thickening was absent, and the less-organized disrupted MTs and thickened rounded plasma membranes were prominently visible in these cells (Fig. 9Bf to h and Fig. 9Bg, inset). Taken together, these results suggested that KSHV profoundly influences MT dynamics during the early stages of endothelial cell infection and that lipid rafts play important roles in KSHV-induced MT acetylation and aggregation that are essential for the effective delivery of viral DNA to the infected cell nuclei.
