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Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) interacts with cell surface α3β1 integrin early during in vitro infection of human endothelial cells and fibroblasts and activates the focal adhesion kinase (FAK) that is immediately downstream in the outside-in signaling pathway by integrins, leading to the activation of several downstream signaling molecules. In this study, using real-time DNA and reverse transcription-PCR assays to measure total internalized viral DNA, viral DNA associated with infected nuclei, and viral gene expression, we examined the stage of infection at which FAK plays the most significant role. Early during KSHV infection, FAK was phosphorylated in FAK-positive Du17 mouse embryonic fibroblasts. The absence of FAK in Du3 (FAK−/−) cells resulted in about 70% reduction in the internalization of viral DNA, suggesting that FAK plays a role in KSHV entry. Expression of FAK in Du3 (FAK−/−) cells via an adenovirus vector augmented the internalization of viral DNA. Expression of the FAK dominant-negative mutant FAK-related nonkinase (FRNK) in Du17 cells significantly reduced the entry of virus. Virus entry in Du3 cells, albeit in reduced quantity, delivery of viral DNA to the infected cell nuclei, and expression of KSHV genes suggested that in the absence of FAK, another molecule(s) may be partially compensating for FAK function. Infection of Du3 cells induced the phosphorylation of the FAK-related proline-rich tyrosine kinase (Pyk2) molecule, which has been shown to complement some of the functions of FAK. Expression of an autophosphorylation site mutant of Pyk2 in which Y402 is mutated to F (F402 Pyk2) reduced viral entry in Du3 cells, suggesting that Pyk2 facilitates viral entry moderately in the absence of FAK. These results suggest a critical role for KSHV infection-induced FAK in the internalization of viral DNA into target cells.
The gamma-2 herpesvirus Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8), is etiologically associated with Kaposi's sarcoma (KS) (11) and two lymphoproliferative disorders, namely, body cavity-based B-cell lymphoma (BCBL), or primary effusion lymphoma (10), and some forms of multicentric Castleman's disease (46). KS is a tumor of polyclonal origin with multiple foci of lesions, and the tumor tissue consists of dendritic- and monocytic-origin spindle-shaped endothelial cells mixed with fibroblasts (35, 38). Cell lines with B-cell characteristics established from the lymphomas carry KSHV in a latent form, and BCBL cells carry about 40 to 80 copies of the KSHV genome. About 1 to 3% of these cells spontaneously enter the lytic cycle, and about 20 to 30% of the cells express lytic cycle proteins after stimulation with 12-0-tetradecanoylphorbol-13-acetate (TPA) (20, 36).
KSHV DNA and transcripts have been identified in vivo in KS spindle and endothelial cells, keratinocytes, epithelial cells, B cells, and macrophages (15, 18, 31, 47, 51). In vitro, KSHV has been shown to infect human B, epithelial, and endothelial cells, foreskin fibroblasts (HFF), and keratinocytes, as well as a variety of nonhuman cells, such as owl monkey kidney cells, baby hamster kidney fibroblast cells, Chinese hamster ovary cells, and primary embryonic mouse fibroblasts (2, 4, 6, 15, 23, 32). Unlike infection with alpha- or betaherpesviruses, in vitro infection of target cells with KSHV does not lead to a productive replicative lytic cycle. KSHV establishes latency soon after infection, and the virus genome is lost during successive passages of the infected cells (6, 20). Our recent studies showed that a subset of the lytic transcripts were expressed in the primary human microvascular endothelial cells and fibroblasts soon after infection, and many of these transcripts could not be detected at later time points (23).
Our studies show that KSHV utilizes the ubiquitous cell surface heparan sulfate (HS)-like molecules to bind the target cells (3, 5, 49). We have demonstrated the interaction of virion envelope-associated KSHV glycoprotein gB and gpK8.1A with HS molecules (3, 5, 49). KSHV-gB possesses the integrin-interacting RGD motif, and our studies have demonstrated the interaction of KSHV gB with the host cell surface α3β1 integrin (4). 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, forming focal adhesions (FAs) (19). KSHV-integrin interactions led to the phosphorylation of FAK, which subsequently led to the activation of Src, phosphatidylinositol 3-kinase (PI-3K), protein kinase C-ζ (PKC-ζ), RhoGTPase, mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase 1/2 (ERK1/2) (32). KSHV infection also led to cytoskeletal rearrangements and the formation of structures such as filopodia, lamellipodia, and stress fibers (32). Soluble gB induced extensive cytoskeletal rearrangement in target cells via the induction of a FAK-Src-PI-3K-RhoGTPase signal pathway (42). Inhibition of cellular tyrosine kinases and inhibition of PI-3K blocked the entry of KSHV into target cells (42). Our studies further demonstrated that KSHV induced RhoAGTPases are critical for microtubular acetylation, leading into the modulation of microtubule dynamics, for the movement of KSHV in the cytoplasm, and for the delivery of viral DNA into the infected cell nuclei (33). Soluble KSHV gpK8.1A, but not gB, induced MEK-mediated ERK1/2 phosphorylation as early as 5 min posttreatment, and ERK1/2 phosphorylation facilitated the establishment of KSHV infection (41). These studies demonstrated that besides providing the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with the host cell receptor(s) create an appropriate intracellular environment facilitating infection.
KSHV-infected FAK−/− Du3 mouse embryonic fibroblasts (MEF) (21) with much lower efficiency than FAK+/+ Du17 cells, as detected by the green fluorescent protein (GFP) expression from GFP-KSHV (32). ERK phosphorylation was also significantly reduced in Du3 cells during KSHV infection (32). Src was phosphorylated (42) in FAK+/+ Du17 cells by gB but not in FAK−/− Du3 cells. These experiments indicated an unambiguous role of FAK in KSHV-mediated signal transduction. [3H]thymidine labeled KSHV bound to both Du17 and Du3 cells with equal efficiency, and binding was inhibited by soluble heparin (32). However, KSHV infectivity was reduced by more than 80% in Du3 cells, suggesting a key role for FAK in KSHV infection (32). However, the stage at which KSHV-induced FAK plays a role in infection is not clearly understood.
Here we have examined in detail the stage(s) at which FAK plays important roles in the infectious process. These studies suggest that the reduced infectivity observed in Du3 cells is due to reduced viral entry. In the absence of FAK, we observed the induction of Pyk2 molecules by infection, which partially compensated for the function of FAK and aided in the moderate entry of KSHV into Du3 cells. These studies suggest that FAK plays key roles in the internalization of viral DNA into target cells.
Primary embryonic fibroblasts negative (−/−) for FAK (Du3 cells) and control FAK+/+ wild-type Du17 cells (gifts from D. Ilic, University of California at San Francisco) were grown in Dulbecco's modified Eagle medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and antibiotics (21, 32). 293 cells and human B cells carrying KSHV (BCBL-1) were cultured as described previously (21, 32).
KSHV was purified from BCBL-1 cells according to previously described methods (32). KSHV DNA was extracted from the virions, and copy numbers were quantitated by real-time DNA PCR using primers amplifying the KSHV ORF73 gene (23).
Construction and characterization of adenovirus (Ad) constructs expressing FAK-Wt, FRNK, Pyk2-Wt, and F402 Pyk2 with hemagglutinin (HA) or c-Myc tags were described previously (48). Stock adenovirus preparations and titrations were carried out in 293 cells.
Mouse anti-pY397 FAK and mouse anti-total FAK antibodies were obtained from BD Biosciences, Palo Alto, CA. The mouse anti-pY402 Pyk2 antibody was from Biosource International, Camarillo, CA, and the rabbit polyclonal antibody against total Pyk2 was obtained from Cell Signaling Technologies, Beverly, MA. Mouse monoclonal anti-c-Myc antibodies were obtained from Santa Cruz Biotechnology, Inc., Santa Cruz, CA. The rat monoclonal antibody against HA was from Roche Diagnostics, Indianapolis, IN.
The cytotoxicity of various inhibitors on different cells was determined using a lactate dehydrogenase cytotoxicity assay kit (Promega, Madison, WI) as described previously (42).
Nuclei of the infected mouse fibroblasts were prepared using Nuclei EZ isolation kit (Sigma, St. Louis, MO) by procedures described before (33) with some modifications. Infected or uninfected cells were washed, treated with trypsin-EDTA (0.25% trypsin and 5 mM EDTA) for 5 min to remove the bound and noninternalized virus, washed, and collected. The cells were incubated in lysis buffer on ice for 30 min, and nuclei were pelleted at 500 × g for 5 min, incubated in lysis buffer on ice for 30 min, and concentrated at 500 × g for 5 min. The nuclei separated from the cytoplasmic components were resuspended in nuclear homogenization buffer (33), and 60% iodixanol (Optiprep; Axis-Shield, Oslo, Norway) was added to a final concentration of 25% iodixanol. This was layered over a gradient of equal volumes of 30% and 35% iodixanol solution and centrifuged at 10,000 × g for 15 min at 4°C. The pure nuclear band at the 30-to-35% iodixanol interface was collected, and total DNA was prepared using a DNeasy tissue kit (QIAGEN, Valencia, CA). The purity of the nuclear preparation was confirmed by immunoblotting using anti-lamin B antibodies, and cytoskeletal contamination was assessed by the use of anti-β-tubulin antibodies.
Total DNAs from the viral stocks and cells were prepared using the DNeasy tissue kit (QIAGEN) as described previously (23). Briefly, uninfected and infected cells were trypsinized for 5 min at 37°C and collected with 10 ml of ice-cold DMEM. Cells were pelleted at 1,000 rpm for 10 min, washed, and resuspended in 200 μl of 1× phosphate-buffered saline (PBS), and total DNA was prepared according to the manufacturer's instructions. Total RNA was isolated from infected or uninfected cells using the RNeasy kit (QIAGEN) (23). Target cells were infected with virus for different times and washed to remove unbound virus, and the cells were lysed to prepare RNA samples.
Real-time DNA and reverse transcription-PCR (RT-PCR) were performed to quantify the internalized viral DNA and to monitor KSHV ORF73 mRNA expression, respectively (23).
Detection of expressed proteins in Du17 or Du3 cells was performed according to previously described protocols (32, 48). Du17 or Du3 cells grown in 8-well chamber slides (Nalge Nunc International, Naperville, IL) to about 80% confluence were infected with adenoviruses carrying the following inserts: HA-wt FAK, c-Myc-FRNK, HA-wt Pyk2, and HA-F402 Pyk2. After a 48-h infection, cells were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS, and dried. The cells were blocked with 10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% Tween 20 (TBST) containing 2% bovine serum albumin, followed by incubation with either rat anti-HA or mouse anti-c-Myc antibodies at 1:200 dilutions for 1 h at room temperature. This was followed by a 1-h incubation at room temperature with goat anti-rat or goat anti-mouse antibodies labeled with Alexa 488 (Molecular Probes-Invitrogen Corp., Carlsbad, CA). The fluorescence-positive cells were observed under a fluorescent microscope (Leica Microsystems Inc., Bannockburn, IL) equipped with an Optronics Magnifier digital camera (Goleta, CA).
Twenty micrograms of the whole-cell lysates or immunoprecipitated proteins was separated by electrophoresis in sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose membranes, and the membranes were blocked with 5% milk solution in TBST buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl) containing 0.1% Tween 20 at room temperature for 2 h. The blots were reacted with optimum concentrations of primary antibodies at 4°C overnight and washed five times with washing buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.3% Tween 20). The blots were reacted with alkaline phosphatase-conjugated secondary antibodies (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) for 1 h at room temperature. The antibody reactions were performed, and the bands were detected by chemiluminescent reaction with CDP-Star reagent (Roche Diagnostics, Indianapolis, IN).
FAK is a cytoplasmic nonreceptor tyrosine kinase, and FAK activation is the first step necessary for the outside-in-signaling by integrins (19, 29, 40). Following integrin-ligand interaction, FAK is autophosphorylated at the Tyr397 residue, which is critical for the subsequent phosphorylation of other FA proteins (19, 29, 40). Tyr397 is the primary site of FAK phosphorylation as well as the initiation of further FAK activation, since it is the binding site for the Src homology domain 2 (SH2 domain) of Src family kinases. These interactions link FAK to the signaling pathways that modify the cytoskeletal rearrangement and activate the mitogen-activated protein kinase cascades (19).
We have previously demonstrated that KSHV induces the phosphorylation of FAK in human endothelial and fibroblast cells and that FAK induction is critical for the subsequent activation of Src, PI-3K, and RhoGTPases (32, 33, 42). For the better understanding of the role of FAK, it was necessary to have a system in which FAK was not expressed. However, such FAK-negative cells of human origin are not available. Since there are no known specific chemical inhibitors of FAK, we have used the FAK-positive and -negative MEF (21). By monitoring KSHV infection for the expression of GFP from the KSHV genome, we have previously shown that FAK+/+ Du17 and FAK−/− Du3 MEF can be infected with KSHV (32). KSHV infection in Du17 cells was comparable to that of human fibroblasts and endothelial cells (32). [3H]thymidine-labeled KSHV bound to both Du17 and Du3 cells with equal efficiency, and binding was inhibited by soluble heparin (32). However, KSHV infectivity was reduced by more than 80% in Du3 cells, suggesting a key role for FAK in KSHV infection (32).
To determine the role of FAK in infection, here we systematically analyzed the stage(s) of KSHV infection at which FAK plays the key role. Initially we determined the ability of KSHV to induce the phosphorylation of FAK in Du17 and Du3 cells. Unless specified otherwise, in all the infection procedures described here, cells were infected with 20 DNA copies/cell of KSHV, because this dose was found to be optimal for infecting Du17 and Du3 cells. Lysates from cells infected with KSHV for different times were tested for the tyrosine phosphorylation of FAK at Tyr397 by Western blot reactions. Compared to that of uninfected Du17 cells (Fig. (Fig.1,1, top panel, lane 1), FAK phosphorylation increased about fivefold when cells were treated with 20% FBS for 5 min (Fig. (Fig.1,1, top panel, lane 8). We have previously shown that as early as 5 min after infection of HFF, KSHV induced the phosphorylation of FAK (4, 32). Similarly, phosphorylation of FAK was observed in Du17 cells as early as 5 min postinfection (p.i.), was sustained up to 30 min p.i., and returned to the basal level by 1 h p.i. (Fig. (Fig.1,1, top panel, lanes 2 to 5). To confirm equal protein loading, lysates were reacted with antibodies against total FAK. Equal amounts of total FAK were detected in all the samples (Fig. (Fig.1,1, lower panel), demonstrating the steady-state level of endogenous FAK. These results suggested that KSHV infection was activating the preexisting FAK in Du17 cells. In contrast, no FAK activation was detected in KSHV-infected Du3 cells (Fig. (Fig.1,1, top panel, lane 9). This is not unexpected, since it was reported previously (44) that these cells were negative for FAK expression (Fig. (Fig.1,1, lower panel, lane 9).
The specificity of FAK phosphorylation by KSHV was confirmed by the significant reduction in FAK activation in cells infected for 15 min with virus pretreated with either 10 μg/ml of heparin or 5 μg/ml of α3β1 integrin (Fig. (Fig.1,1, top panel, lanes 6 and 7, respectively). These results suggested that the phosphorylation of FAK at Tyr397 was indeed due to KSHV infection and that interaction with α3β1 integrin in Du17 cells is critical for the observed induction of FAK phosphorylation.
[3H]thymidine-labeled KSHV bound to Du17 and Du3 cells with equal efficiency (32). Hence, our earlier observation of reduced expression of GFP in Du3 cells after GFP-KSHV infection (32) could be due either to a block in virus entry into these cells, to a block in the movement of virus in the cytoplasm or the delivery of viral DNA into the infected cell nucleus, or to a block in the expression of viral genes in the nucleus. To determine the stage of KSHV infection at which FAK plays the key role, FAK+/+ Du17 and FAK−/− Du3 cells were infected with KSHV at a multiplicity of infection (MOI) of 20 for different lengths of time, washed, treated with trypsin-EDTA for 5 min to remove bound noninternalized virus, washed, and collected, and DNA was extracted. Real-time DNA-PCR was carried out with 100 ng of total DNA from each test reaction to amplify the ORF73 genes, and external copy standards were used to obtain the copy numbers of the amplified ORF73 gene. Since every viral genome contains a single copy of the ORF73 gene, the number of internalized viral DNA molecules could be calculated from the corresponding copy numbers of the ORF73 gene.
Internalized viral DNA could be detected in Du17 cells as early as 30 min p.i., and internalized viral copy numbers increased rapidly during the first 60 to 90 min of infection, reaching a plateau around 120 to 240 min p.i. (Fig. (Fig.2A).2A). In Du3 cells, even though there was about 70% reduction in viral entry, the kinetics of virus entry was similar to that in Du17 cells (Fig. (Fig.2B).2B). These results were reproducible and consistent, with only minor variations between experiments and batches of viruses. Preincubation of virus with 100 μg/ml of heparin blocked more than 90% of KSHV DNA entry (data not shown). Together with the steady increase in the internalized viral DNA, these results demonstrated the specificity of the real-time DNA PCR assay (23). Since we have demonstrated previously that [3H]thymidine labeled KSHV bound to both Du17 and Du3 cells with equal efficiency (32), these results suggested the following: (i) the presence of FAK plays a key role in the postbinding stage of infection; (ii) internalization of significantly more viral DNA in Du17 cells than in Du3 cells suggested that FAK plays a role in KSHV entry into target cells; (iii) the overall reduction in virus entry observed in Du3 cells is not due to delayed entry into these cells. These results differ considerably from those for adenovirus infection (25), since Du3 cells are infected with adenovirus 2 and 5 with the same efficiency as that of FAK-positive Du17 cells (25), thus suggesting a key role for FAK in KSHV infection. However, since about 30% of KSHV was entering Du3 cells, this suggested that in the absence of FAK, there must be other signal mediator molecules that are partially compensating for the FAK function and thus aiding in KSHV entry.
Even though KSHV entry into Du17 and Du3 cells increased steadily over time and reached a plateau around 120 to 240 min p.i., we also observed about 24% reduction in virus entry after 8 h p.i. compared to 4 h p.i. (Fig. (Fig.2A).2A). Since KSHV reprograms the host cell transcription machinery (34), it is possible that modulation of several host cell genes involved in endocytosis and signal pathways may potentially be responsible for the observed reduction at 8 h p.i.
FAK activation leads to the activation of several downstream signaling molecules such as Src, PI-3K, PKC-ζ, RhoGTPase, MEK, and ERK1/2. Recently, we have demonstrated the role of RhoGTPases in the movement of KSHV in the cytoplasm and in the delivery of viral DNA into the infected cell nuclei (33). Results in Fig. Fig.22 show that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection (32) is probably due to a block in virus entry into these cells. To further confirm this observation and to rule out the possibility that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection is not due to a block in the movement of virus in the cytoplasm or the delivery of viral DNA into the infected cell nucleus, Du17 and Du3 cells were infected with KSHV for 3 h, a time point of maximum internalized viral load, and nuclei were isolated according to procedures described before (33). The purity of nuclei was confirmed by immunoblotting using anti-lamin B antibodies, and cytoskeletal contamination was assessed by the use of anti-β-tubulin antibodies for the detection of α-tubulin protein (data not shown) (33). DNA was prepared from the isolated nuclei, and 100 ng of DNA from each sample was used to quantitate viral DNA by real-time PCR for KSHV ORF73. Total DNA was also prepared from a replicate sample of the infected cells and was subjected to real-time PCR for the estimation of total internalized viral DNA copy numbers.
In agreement with the results shown in Fig. 2, a significant reduction in KSHV DNA entry was observed in Du3 cells compared to Du17 cells (Fig. 3A and B). Viral delivery into the nuclei was also correspondingly reduced significantly in Du3 cells compared to Du17 cells, with about 71% reduction (Fig. (Fig.3B).3B). However, when the copy numbers of viral DNA associated with infected Du3 cell nuclei were compared to the total amount of viral DNA entering Du3 cells, we observed a ratio of entry similar to that for Du17 cells. In the representative example shown in Fig. Fig.3A,3A, of the 13,630 total viral DNA copies detected in Du17 cells, about 52% (7,102 DNA copies) were detected in the nuclei. Similarly, of the 3,939 total DNA copies detected in Du3 cells, about 54% (2,122 DNA copies) were associated with the nuclei of infected cells. This suggested that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection (32) is not due to a block in the movement of virus in the cytoplasm or in the delivery of viral DNA into the infected cell nucleus but is due to reduced entry into the target cells. These results also reaffirm that in the absence of FAK, there must be other signal mediator molecules that are partially compensating for FAK function and aiding in KSHV entry. In addition, these studies suggest a similarity in the induction of a signal cascade that is critical for the delivery of viral DNA into the nuclei in the FAK-positive and FAK-negative cells.
FAK activation leads to the activation of MEK and ERK1/2. Recently, we have demonstrated the role of ERK1/2 in KSHV gene expression (41). Results shown in Fig. Fig.22 and 3A and B show that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection (32) is probably due to a block in virus entry into these cells and not to a block in the movement of virus in the cytoplasm or the delivery of viral DNA into the infected cell nucleus. To further confirm this observation and to rule out the possibility that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection is not due to a block in the expression of viral genes in the nucleus, Du17 and Du3 cells were infected with KSHV for different times; then virus was removed and total RNA was prepared by procedures described before (23). Two hundred fifty nanograms of DNase I-treated RNA from each experiment was used in real-time RT-PCRs to quantitate the viral transcripts expressed in the infected cells. As shown in Fig. 3C and D, KSHV ORF50 and ORF73 messages were detected in both Du17 and Du3 cells soon after infection. In agreement with KSHV gene expression in human endothelial cells shown previously (23), the maximum expression of ORF50 was detected at 2 h p.i. in Du17 and Du3 cells, was reduced drastically thereafter, and was almost undetectable at 24 h p.i. in both cells (Fig. (Fig.3C).3C). In agreement with the reduction in virus entry and the reduction in DNA associated with infected cell nuclei, compared to Du17 cells, there was about 60% and 70% reduction in the ORF50 transcripts in Du3 cells at 1 and 2 h p.i., respectively (Fig. (Fig.3C3C).
ORF73 transcript levels in both cell types were very low during the early hours of infection compared to ORF50 levels (Fig. (Fig.3D).3D). The levels increased gradually in the observed 24 h postinfection. These results are in agreement with our earlier observations with HMVEC-d and HFF (23). Similar to the lower ORF50 levels, there was a reduction of about 70 to 80% in ORF73 transcripts in Du3 cells compared to Du17 cells. However, when the copy numbers of viral transcripts in Du3 cells was compared to the total amount of viral DNA entering Du3 cells, we observed a ratio of viral gene expression similar to that for Du17 cells. This suggested that the reduced expression of GFP observed in Du3 cells after GFP-KSHV infection (32) is not due to a block in the movement of virus in the cytoplasm, the delivery of viral DNA into the infected cell nucleus, or viral gene expression but is due to reduced entry into the target cells.
Since the absence of FAK resulted in a significant reduction in virus entry, we next determined whether introducing FAK exogenously could enhance KSHV entry in Du3 cells. About 80% confluent Du3 cells were infected for 2 h at an MOI of 10 with adenovirus carrying HA-tagged wild-type FAK (Ad-FAKWt) or with adenovirus expressing GFP (Ad-GFP). These cells were first washed and then incubated for 48 h at 37°C, and FAK expression was determined by immunofluorescence using anti-HA tag antibodies. Bright cytoplasmic fluorescence was detected in all cells after 48 h p.i. (Fig. (Fig.4A,4A, panels 1 and 2). The specificity of this reaction was seen by the absence of reactivity with anti-HA tag antibodies in cells infected with control Ad-GFP (Fig. (Fig.4A,4A, panel 3). GFP was detected in all cells infected with Ad-GFP (data not shown). These results demonstrated the expression and proper localization of FAK.
Cells infected with adenovirus constructs were washed after 48 h, serum starved for 24 h, and infected with KSHV for 15 min. The induction of FAK phosphorylation by KSHV in Du3 cells infected with Ad-FAKWt and Ad-GFP was next determined. Du3 cells infected with Ad-GFP did not express FAK, and hence no phospho-FAK could be detected after KSHV infection (Fig. (Fig.4B,4B, top panel, lanes 1 and 2). Du3 cells infected with Ad-FAK expressed HA-FAK, as evidenced by the detection of total FAK (Fig. (Fig.4B,4B, middle panel, lanes 3, 4, and 5). Phosphorylation of FAK was not observed in uninfected serum-starved Du3 cells expressing exogenous FAK (Fig. (Fig.4B,4B, top panel, lane 3). In contrast, KSHV infection readily induced the phosphorylation of FAK at Tyr397 in these cells (Fig. (Fig.4B,4B, top panel, lane 4), and a ~4- to 5-fold increase in FAK phosphorylation was seen. The specificity of these reactions was confirmed by the induction of FAK by 20% FBS, which was comparable with phosphorylation induced by virus infection (Fig. (Fig.4B,4B, top panel, lane 5). To confirm equal protein loading, membranes were stripped and reprobed with anti-β-actin antibodies. Equal amounts of actin were detected in all of the samples (Fig. (Fig.4B,4B, lower panel).
To determine whether Du3 cells expressing FAK will support enhanced virus entry, cells infected with adenovirus constructs were washed after 48 h and infected with KSHV for 2 h. KSHV entry into Du3 cells infected with Ad-GFP (Fig. (Fig.4C)4C) was comparable to entry into non-adenovirus-infected Du3 cells (data not shown). In contrast, a significant increase in viral entry was observed in Du3 cells expressing HA-FAK from Ad-FAKWt (Fig. (Fig.4C).4C). Phosphorylation of exogenously expressed HA-FAK by KSHV infection together with increased entry into these cells clearly demonstrated that FAK plays an important role in KSHV entry into target cells.
Since exogenously expressed FAK augmented KSHV entry into Du3 cells, we next determined whether overexpression of FRNK could inhibit viral entry into Du17 cells. FRNK is used as a dominant inhibitor of FAK, since it is a naturally expressed protein deficient in the kinase domain of FAK and lacks the catalytic activity and the downstream signaling properties of FAK (40). Expression of FRNK has been shown to promote the dephosphorylation of FAK, probably due to the competitive displacement of FAK from FAs (40).
Du17 cells were infected by adenoviruses expressing c-Myc-FRNK molecules (Ad-FRNK) or with Ad-GFP for 2 h, washed, and incubated for 48 h at 37°C. When FRNK expression was determined by anti-c-Myc antibodies, cytoplasmic fluorescence was detected in all cells after 48 h p.i., thus demonstrating the expression and proper localization of FRNK (Fig. (Fig.5A,5A, panels 1 and 2). The specificity of this reaction was seen by the absence of reactivity with anti-c-Myc antibodies in control Ad-GFP-infected cells (Fig. (Fig.5A,5A, panel 3).
The extent of induction of FAK phosphorylation by KSHV in Du17 cells previously infected with Ad-FRNK and Ad-GFP was then determined. FAK was phosphorylated in Du17-Ad-GFP cells after KSHV infection (Fig. (Fig.5B,5B, top panel, lane 2), while no phosphorylation was detected in mock-infected cells (Fig. (Fig.5B,5B, top panel, lane 1). In Du17 cells overexpressing FRNK, phosphorylation of FAK by FBS was considerably reduced (Fig. (Fig.5B,5B, top panel, lane 5), with about 2.5-fold reduction compared to KSHV-infected Du17 cells. A similar reduction was also observed after KSHV infection of Du17-Ad-FRNK cells (Fig. (Fig.5B,5B, top panel, lane 4). Equal amounts of total FAK were detected in all these cells, demonstrating that the expression of FAK is not affected by the expression of FRNK (Fig. (Fig.5B.5B. middle panel). To confirm equal protein loading, membranes were stripped and reprobed with anti-β-actin antibodies. Equal amounts of actin were detected in all the samples (Fig. (Fig.5B,5B, lower panel). These results demonstrated the reduction of phosphorylation of FAK at the Tyr397 position following the overexpression of FRNK.
When the effect of FRNK expression on KSHV entry was examined, a significant reduction in the internalization of viral DNA was observed in Du17 cells expressing FRNK compared to internalization in Du17-Ad-GFP cells, (Fig. (Fig.5C).5C). This, together with the reduced FAK phosphorylation in FRNK-expressing Du17 cells, further confirmed the role of FAK in KSHV entry into Du17 cells.
Even though KSHV entry into FAK−/− Du3 cells was reduced significantly compared to internalization in Du17 cells, about 30% of input virus still entered the Du3 cells. Moreover, viral DNA was transported into the nucleus, and viral genes were expressed, in Du3 cells (Fig. (Fig.22 and and3).3). This suggested that either FAK is not the only molecule involved in KSHV entry or some other molecule compensates for its function in its absence in Du3 cells. Pyk2 is a nonreceptor protein-tyrosine kinase with ~45% overall amino acid sequence identity with FAK. Pyk2 has been shown to be expressed and phosphorylated in Du3 cells in the absence of FAK (44). Pyk2 complements some of the functions of FAK in its absence through interaction with downstream signaling molecules (44). Hence, we next examined the expression and phosphorylation of Pyk2 during KSHV infection.
Serum-starved Du3 and Du17 cells were infected with KSHV at an MOI of 20 DNA copies/cell for different time intervals, and the lysates were tested for the tyrosine phosphorylation of Pyk2. As shown in Fig. Fig.6,6, Pyk2 was phosphorylated at Tyr402 in Du3 cells as early as 5 min p.i., and no phosphorylation was observed in the uninfected cells (Fig. (Fig.6,6, top panel, lanes 1 and 2). Pyk2 phosphorylation by KSHV was more persistent than FAK phosphorylation in Du17 cells. About six- to sevenfold phosphorylation was observed in Du3 cells as late as 1 h p.i., a level that decreased to about twofold by 2 h p.i. (Fig. (Fig.6,6, top panel, lanes 2 to 6). Pyk2 was also phosphorylated by preincubating cells with 20% FBS for 5 min (Fig. (Fig.6,6, top panel, lane 9). The specificity of the reaction was confirmed by the absence of phosphorylated Pyk2 when cells were infected with virus preincubated with heparin or α3β1 integrin (Fig. (Fig.6,6, top panel, lanes 7 and 8). As in previous reports (44), since Pyk2 is not expressed in Du17 cells, no phosphorylation was detected in KSHV-infected Du17 cells (Fig. (Fig.6,6, top and lower panels, lane 10). Total Pyk2 levels remained similar in all Du3 cell samples (Fig. (Fig.6,6, lower panel, lanes 1 to 9), indicating that the expression of Pyk2 was not altered during KSHV infection and suggesting that KSHV infection was activating the preexisting Pyk2 in FAK-negative Du3 cells.
Since Pyk2 was phosphorylated in Du3 cells during KSHV infection but not in Du17 cells, its role in viral entry was next examined. Tyr402 is the autophosphorylation site of Pyk2 interacting with signal molecules such as Src, and this phosphorylation site corresponds to Tyr397 of FAK. HA-F402 Pyk2 has the tyrosine residue replaced at amino acid position 402 with phenylalanine (Y402 to F) and has been shown to inhibit downstream signaling events of Pyk2 mediated through Src (44).
An adenovirus vector carrying the mutated form of Pyk2 (Ad-F402 Pyk2) was introduced into Du3 cells prior to KSHV infection. Du3 cells cultured in 8-well chamber slides were infected with Ad-F402 Pyk2 or Ad-GFP, and the expression of F402 Pyk2 was tested at 48 h p.i. by immunofluorescence reactions using anti-HA antibodies. HA-F402 Pyk2 expression was observed in the cytoplasm of the infected Du3 cells (Fig. (Fig.7A,7A, panels 1 and 2), confirming the expression of these proteins. In contrast, the antibody did not recognize the GFP in control cells infected with Ad-GFP, indicating the specificity of the reaction (Fig. (Fig.7A,7A, panel 3). Du3 cells were infected with Ad-GFP or Ad-F402 Pyk2 for 48 h, serum starved for 24 h, infected with KSHV at an MOI of 20 for 30 min, and examined for Pyk2 phosphorylation. In Du3 cells expressing F402 Pyk2, phosphorylation of Pyk2 by FBS was considerably reduced (Fig. (Fig.7B,7B, top panel, lane 5). Compared to that in control Ad-GFP cells, KSHV infection induced about sixfold Pyk2 phosphorylation in Du3-Ad-GFP cells (Fig. (Fig.7B,7B, top panel, lanes 1 and 2). In contrast, only twofold Pyk2 phosphorylation was observed in Du3-Ad-F402 Pyk2 cells (Fig. (Fig.7B,7B, lane 5). To confirm equal protein loading, membranes were stripped and reprobed with anti-β-actin antibodies. The total Pyk2 levels in the Du3-Ad-F402 Pyk2 cells were slightly elevated, probably due to the expression of Ad-F402 Pyk2 in these cells (Fig. (Fig.7B,7B, lower panel, lanes 3 to 5). Total Pyk2 levels remained similar in Ad-GFP cells (Fig. (Fig.7B,7B, lower panel, lanes 1 to 2), indicating that the expression of Pyk2 was not altered during KSHV infection. Equal amounts of actin were detected in all the samples (Fig. (Fig.7B,7B, lower panel).
Next, we tested whether the reduction in phosphorylation of wild-type Pyk2 due to the expression of F402 Pyk2 will lead to reduced entry of KSHV. Du3 cells infected with either Ad-GFP or Ad-F402 Pyk2 for 48 h were infected with KSHV for 2 h, cells were collected, and total DNA was prepared and subjected to real-time DNA PCR for detection of the KSHV ORF73 gene. KSHV entry was significantly reduced, by about 50%, in Du3-Ad-F402 Pyk2 cells compared to Du3 cells infected with Ad-GFP. These results demonstrated the role of Pyk2 in KSHV entry in the absence of FAK and confirmed that phosphorylation of Pyk2 is important in the process of entry into Du3 cells.
Pyk2 is activated by Ca2+ ions and plays a major role in calcium signaling. Calcium chelators have been shown to inhibit Pyk2 phosphorylation, leading to the successive blocking of many downstream signaling events (27). The effect of EGTA, an extracellular Ca2+ chelator, on the phosphorylation of Pyk2 was next tested. Du3 cells were first treated with 5 mM EGTA in 10 μM NaOH for 5 s and then infected with KSHV for 30 min, and lysates were tested for the tyrosine phosphorylation of Pyk2. Infection of Du3 cells in the presence of 10 μM NaOH used for dissolving EGTA did not affect KSHV-induced Pyk2 phosphorylation, and about fourfold induction over phosphorylation in serum-starved uninfected cells was observed (Fig. (Fig.8A,8A, top panel, lanes 1 and 2). In contrast, infection of Du3 cells pretreated with 5 mM of EGTA in 10 μM NaOH reduced Pyk2 phosphorylation almost to the basal level (Fig. (Fig.8A,8A, top panel, lane 4). The amounts of total Pyk2 remained similar in all the samples, indicating that EGTA treatment did not affect the expression of Pyk2 (Fig. (Fig.8A,8A, top panel). These experiments clearly demonstrated that the phosphorylation of Pyk2 induced by KSHV was inhibited by EGTA.
We next tested whether KSHV entry into Du3 cells was inhibited in the presence of EGTA. Du3 cells preincubated with 2 and 5 mM of EGTA in 10 μM NaOH for 5 s were infected with KSHV at an MOI of 20 for 2 h in the presence of the chelator. As shown in Fig. Fig.8B,8B, viral entry into Du3 cells was significantly reduced in the presence of EGTA. Though 5 mM EGTA inhibited viral entry more, a significant reduction was observed even at the 2 mM concentration. However, no significant inhibition of viral entry was observed in Du17 cells in the presence of EGTA (Fig. (Fig.8C),8C), indicating that EGTA probably does not affect the integrity of integrin molecules and FAK activation. Since EGTA treatment resulted in the dephosphorylation of Pyk2 along with the reduction of viral entry in Du3 cells, these experiments further confirmed the role of Pyk2 in KSHV entry in the absence of FAK.
An interaction with a host cell surface receptor(s) is the first essential step in any viral infection of target cells. Though binding to the target cells could occur at low temperatures, entry of a virus or viral genome into the cells and subsequent movement in the cytoplasm are energy-dependent phenomena. Virus interactions with specific target cell receptors have probably evolved to accommodate these needs, and the manipulation of preexisting host cell signal pathways via cell surface receptors is probably one of the best strategies to fulfill these requirements. Even though several host cell surface receptors have been identified for human and animal herpesviruses, how these interactions with cell surfaces facilitate infection is largely unexplored. KSHV is the first herpesvirus shown to interact with integrin molecules (4). Within minutes of its binding to target cells, KSHV induces a variety of preexisting host cell signal pathway molecules, such as FAK, Src, PI-3K, RhoGTPases, PKC-χ, MEK, and ERK1/2 (32, 41, 42). Chief among them is the integrin-dependent protein component FAK, and our studies here demonstrate that FAK plays a critical role in KSHV entry.
Since FAK-null human cells were not available, FAK-null mutant MEF (FAK−/−) and their wild-type counterpart Du17 cells were used (21). The present study demonstrates that KSHV entered the mouse cells with an efficiency similar to that of primary human fibroblasts or endothelial cells, and the kinetics of viral gene expression was remarkably similar to that reported in the human primary cells (23). Our previous study demonstrated reduced infectivity in the absence of FAK (32). Since infection was monitored by counting the number of GFP-expressing cells 48 h after GFP-HHV-8 infection, it was not clear whether the reduced infectivity was due to reduced viral entry, slow movement of virus particles to the nucleus, or downregulation of viral gene expression. The assays carried out here were designed to differentiate the role of FAK in the various steps of KSHV infection. If FAK phosphorylation and the subsequent activation of signal cascades played no role in viral entry but played a role in nuclear delivery of internalized virus particles, we should have observed no reduction in entry into Du3 cells but reduced nuclear entry and gene expression. Similarly, if FAK activation played no role in viral entry and in nuclear delivery of internalized virus particles but played a role in viral gene expression, we should have seen no reduction in entry and nuclear delivery of viral DNA, and we should have observed reduced viral gene expression. Instead, we observed a 70% reduction in KSHV entry into Du3 cells. However, the ratio of delivery of viral DNA into the nuclei and viral gene expression to the internalized viral DNA in Du3 cells was comparable to that in Du17 cells. This demonstrated that the reduced infectivity in Du3 cells was certainly due to reduced viral entry. The role played by FAK at the entry stage of KSHV infection was also confirmed with the introduction of Wt-FAK into Du3 cells and the expression of FRNK in Du17 cells.
Even though integrins are used by several diverse groups of viruses, the mechanism by which the integrin-virus interactions facilitate the infection is not well studied. Our demonstration of the role of FAK in KSHV entry is the first for these viruses. Adenovirus types 2 and 5 (group C) bind to a primary receptor, the coxsackie virus B Ad receptor (CAR). The CAR-docked particles activate integrin coreceptors, and this triggers a variety of cell responses, including endocytosis (25). Ad2/Ad5 endocytosis is clathrin mediated and involves the large GTPase dynamin and the adaptor protein 2. A second endocytic process, macropinocytosis, is induced simultaneously with viral uptake. Macropinocytosis requires integrins, F-actin, PKC, and the Rho family of small G-proteins but not dynamin. A major question arising from studies of adenovirus entry is whether integrins promote virus entry via specific cell signaling events. Adenovirus induces the ERK1/ERK2 mitogen-activated protein kinases via FAK and consequently cell motility. However, FAK activation appears to have little role in integrin-mediated adenovirus endocytosis (25). FAK is phosphorylated and activated upon adenovirus entry, but this kinase is not required for virus uptake into cells or infection, since FAK-null Du3 cells were infected with an efficiency similar to that of parental FAK+/+ DU17 cells (25). Expression of FRNK also failed to inhibit adenovirus entry into the wild-type cells. Adenovirus internalization requires PI-3K activation-dependent p130Cas, and entry was blocked by the pharmacologic inhibitors of PI-3K (25). Our studies show that the presence of FAK is critical for KSHV entry, which is remarkably different from adenovirus infection. FAK has been shown to play a significant role in human cytomegalovirus-induced smooth muscle cell migration (48). FAK was also phosphorylated during the infection of CaSki cells with herpes simplex virus types 1 and 2 (HSV-1 and -2) (12), and a recent study showed the role of FAK in the infection of target cells by HSV-2 (13). Further studies are essential to determine the role of FAK and the associated signal pathways in KSHV entry and infection.
As has been shown for FAK, Pyk2 has also been shown to participate in various signaling events. However, unlike that of FAK, Pyk2 signaling could also be nonintegrin related. Pyk2 was not expressed in Du17 cells (44), and it was expressed in MEF only in the absence of FAK. The mechanism by which FAK expression regulates the expression of Pyk2 is not known. Our results show that Pyk2 plays a role in the KSHV entry process in Du3 cells. During KSHV infection of Du3 cells, but not Du17 cells, Pyk2 was phosphorylated at Tyr402, which is functionally equivalent to Tyr397 of FAK. The role of Pyk2 was further confirmed by the introduction of F402 Pyk2 into Du3 cells. Expression of F402 Pyk2 has previously been shown to reduce the tyrosine phosphorylation of wild-type Pyk2, resulting in the inhibition of downstream signaling events such as ERK phosphorylation (44). Overexpression of F402 Pyk2 in osteoblast-like cells inhibited bone resorption, like a deletion in Src in these cells, indicating that phosphorylation at Tyr402 was important in the interaction with Src (30). The expression of this autophosphorylation site mutant form of Pyk2 in Du3 cells resulted in reduced phosphorylation of Pyk2 during KSHV infection, indicating that Pyk2 had a role at this stage and that phosphorylation of Pyk2 was directly correlated with viral entry into Du3 cells. Studies of Pyk2 inhibition by the Ca2+ chelator EGTA further confirmed these results. The effect of expression of Pyk2 along with FAK is not known, and it can be speculated that Pyk2 may compete for the target molecules, thus resulting in reduced viral entry.
The present study did not address the mechanism by which KSHV entry is facilitated by FAK and Pyk2. KSHV enters human fibroblast cells (2), B cells (5), and epithelial cells (22) via endocytosis. We hypothesized that KSHV binding- and entry-activated signaling pathways may play roles at the following stages of viral infection: (i) to aid in virus entry through the formation of either clathrin-coated vesicles, caveolin coated vesicles, or macropinocytic vesicles, and/or an ill-defined route of non-clathrin, non-caveola-dependent vesicles, and their movement; (ii) to aid in the release of viral capsids with viral DNA from these vesicles into the cytoplasmic environment; (iii) to aid in the movement of viral capsids to the nuclear membrane boundary; (iv) to aid in the release and delivery of viral DNA into the nucleus; and (v) to aid in viral gene expression.
Based on the endocytic mode of KSHV entry into the target cell, certain reasonable scenarios can be hypothesized (Fig. (Fig.9).9). Numerous recent reports show that endocytosis and signal pathways are highly interlinked (1, 9, 14, 17, 24, 26, 28, 37, 39, 45). Protein components of signal transduction cascades assemble at clathrin-coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane (9, 14, 28). 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 (50). Src-mediated tyrosine phosphorylation of clathrin regulates clathrin translocation to the plasma membrane (1, 50). Clathrin subsequently interacts with a number of other essential proteins, as well as dynamin (1, 26). Src-dependent phosphorylation also regulates dynamin self-assembly and ligand-induced endocytosis by releasing the internalized endocytic vesicles from the plasma membrane (1). Through interactions with growth factor receptor bound protein 2 (Grb2), murine son of sevenless protein (mSOS) is recruited to the plasma membrane to activate Ras. Src-dependent phosphorylation initiates the assembly of a plasma membrane-associated Ras activation complex. Rho and Rab-GTPases activated by PI-3K and Ras are critical for the formation of various types of endocytic vesicles and their movement, and for microtubule and microfilament reorganization (9, 14, 24, 28, 45).
The interplay between virus-induced signaling with endocytosis and the movement of capsids in the cytoplasm has not been thoroughly analyzed. In recent years, it has become increasingly clear that integrins and the associated signaling pathways are the converging point for the signals induced by other receptor tyrosine kinases, including growth factor receptors, and cross talk initiated by integrin-ligand interactions plays vital roles in various cellular processes (43, 50). The first step in any integrin-ligand interaction is the induction of two major overlapping signaling pathways, namely, (i) the FAK/Src signaling pathways and (ii) the caveolin-1/Shc signaling pathways (19). The converging point for both pathways is the activation of Ras via Grb2 and mSOS (19). FAK activation also results in the induction of other signaling pathways via the induction of Src kinase (Fig. (Fig.9).9). FAK interacts with a number of signaling and cytoskeletal proteins, including Src, paxillin, PI3-K, p130Cas, and Grb2. These interactions link FAK to signaling pathways that modify the cytoskeleton, aid in the formation, movement, and recycling of endocytic vesicles, and aid in the activation of other cascades (7, 8, 16, 17, 19, 24). FAK activation has been shown to be essential for invasion by certain bacteria (7), and for endocytosis of integrin-bound photoreceptors (17). Activation of FAK by KSHV thus may aid in virus entry by virtue of its critical role in endocytosis (Fig. (Fig.9).9). The immediate converging point of FAK and Pyk2 induction is the activation of Src (Fig. (Fig.9)9) (7, 19, 30). KSHV infection and incubation with purified soluble KSHV gB induced comparable levels of Src phosphorylation in both Du17 and Du3 cells (42). Since Src activation is the initial critical step that leads to the phosphorylation of clathrin, dynamin, Ras, and other associated molecules necessary for the formation of endocytic vesicles, as well as for the activation of PI-3K and RhoGTPases involved in the movement and recycling of endocytic vesicles, the role of FAK and Pyk2 in KSHV could be due to their ability to activate Src. Further studies are in progress to decipher the role of Src in KSHV infection and in the formation of endocytic vesicles and their movement.
This study was supported in part by Public Health Service grant AI 057349 and the Rosalind Franklin University of Medicine and Science—H. M. Bligh Cancer Research Fund to B.C. and by a University of Kansas Medical Center Biomedical Research Training program postdoctoral fellowship to H.H.K.