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Previous Article | Next Article 
Journal of Virology, August 2005, p. 10308-10329, Vol. 79, No. 16
0022-538X/05/$08.00+0 doi:10.1128/JVI.79.16.10308-10329.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, Kansas
Received 18 November 2004/ Accepted 13 May 2005
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ABSTRACT |
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-MEK pathway. In addition, KSHV modulated the transcription of several host genes of primary human dermal microvascular endothelial cells (HMVEC-d) and fibroblast (HFF) cells by 2 h and 4 h p.i. Neutralization of virus entry and infection by PI-3K and other cellular tyrosine kinase inhibitors suggested a critical role for signaling molecules in KSHV infection of target cells. Here we investigated the induction of ERK1/2 by KSHV and KSHV envelope glycoproteins gB and gpK8.1A and the role of induced ERK in viral and host gene expression. Early during infection, significant ERK1/2 induction was observed even with low multiplicity of infection of live and UV-inactivated KSHV in serum-starved cells as well as in the presence of serum. Entry of UV-inactivated virus and the absence of viral gene expression suggested that ERK1/2 induction is mediated by the initial signal cascade induced by KSHV binding and entry. Purified soluble gpK8.1A induced the MEK1/2 dependent ERK1/2 but not ERK5 and p38 mitogen-activated protein kinase (MAPK) in HMVEC-d and HFF. Moderate ERK induction with soluble gB was seen only in HMVEC-d. Preincubation of gpK8.1A with heparin or anti-gpK8.1A antibodies inhibited the ERK induction. U0126, a selective inhibitor for MEK/ERK blocked the gpK8.1A- and KSHV-induced ERK activation. ERK1/2 inhibition did not block viral DNA internalization and had no significant effect on nuclear delivery of KSHV DNA during de novo infection. Analyses of viral gene expression by quantitative real-time reverse transciptase PCR revealed that pretreatment of cells with U0126 for 1 h and during the 2-h infection with KSHV significantly inhibited the expression of ORF 73, ORF 50 (RTA), and the IE-K8 and v-IRF2 genes. However, the expression of lytic IE-K5 gene was not affected significantly. Expression of ORF 73 in BCBL-1 cells was also significantly inhibited by preincubation with U0126. Inhibition of ERK1/2 also inhibited the transcription of some of the vital host genes such as DUSP5 (dual specificity phosphatase 5), ICAM-1 (intercellular adhesion molecule 1), heparin binding epidermal growth factor, and vascular endothelial growth factor that were up-regulated early during KSHV infection. Several MAPK-regulated host transcription factors such as c-Jun, STAT1
, MEF2, c-Myc, ATF-2 and c-Fos were induced early during infection, and ERK inhibition significantly blocked the c-Fos, c-Jun, c-Myc, and STAT1 activation in the infected cells. AP1 transcription factors binding to the RTA promoter in electrophoretic mobility shift assays were readily detected in the infected cell nuclear extracts which were significantly reduced by ERK inhibition. Together, these results suggest that very early during de novo infection, KSHV induces the ERK1/2 to modulate the initiation of viral gene expression and host cell genes, which further supports our hypothesis that beside the conduit for viral DNA delivery into the cytoplasm, KSHV interactions with host cell receptor(s) create an appropriate intracellular environment facilitating infection. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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KSHV DNA sequence analyses demonstrate that a large region of the KSHV genome is conserved among herpesviruses and is colinear with gamma-1 Epstein-Barr virus and gamma-2 herpesvirus saimiri (46, 50). KSHV encodes more than 90 open reading frames (ORFs), which are designated as ORFs 4 to 75 by their homology to herpesvirus saimiri ORFs (46, 50). Divergent regions in between the conserved gene blocks contain more than 20 KSHV unique genes, which are designated with the prefix K. Several KSHV-encoded proteins are homologs of host proteins. These genes include K2 (v-interleukin-6), K4 (v-macrophage inhibitory protein II), K3 and K5 (MIR-1 and MIR-2; immunomodulatory proteins), K6 (v-macrophage inhibitory protein I), K7 (antiapoptotic protein), K9 (v-interferon regulatory factor [vIRF]), vIRF2 (K11.1), ORF 16 (vBcl-2), K13 (v-FLICE-inhibitory protein), K14 (vOX-2), ORF 72 (v-cyclin D), and ORF 74 (v-G protein-coupled receptor) (17, 21, 22, 40, 46, 50, 52, 53).
KSHV DNA and transcripts have been identified in vivo in KS spindle cells, human B cells, endothelial cells, keratinocytes, prostate epithelial cells, B cells, and macrophages (8, 11, 16, 53, 69). KSHV infects a variety of cell types in vitro, which include human B, endothelial, epithelial, fibroblast cells, keratinocytes, owl monkey kidney cells, baby hamster kidney (BHK-21) cells, Chinese hamster ovary (CHO) cells, and primary embryonic mouse fibroblast cells (4, 9, 44, 49, 52, 62). Infection is characterized by the expression of latency-associated ORF 73, ORF 72, and K13 genes and the transient expression of a very limited number of lytic genes such as ORF 50, K5, K8, and v-IRF2 (17, 32).
Like other herpesviruses, KSHV encodes a number of envelope-associated glycoproteins, and KSHV glycoproteins gB (ORF 8), gH (ORF 22), gM (ORF 39), gL (ORF 47), and gN (ORF 53) are counterparts to other herpesvirus glycoproteins (45, 50). In addition to these conserved glycoproteins, KSHV also encodes K1, gpK8.1A, gpK8.1B, and K14, which are unique to KSHV (13, 45, 50). Our previous studies showed that KSHV uses cell surface heparan sulfate (HS)-like molecules to bind target cells. We along with others have also demonstrated the interaction of virion envelope-associated KSHV glycoprotein gB and gpK8.1A with HS molecules (2, 3, 65). KSHV gB possesses the integrin binding RGD motif, and we have demonstrated the inhibition of KSHV infectivity by antibodies against 3 and ß1 integrins and soluble 3ß1 integrin (4). Anti-KSHV-gB antibodies immunoprecipitated the virus-3ß1 complex. Radiolabeled virus binding studies suggest that KSHV uses the 3ß1 integrin as one of the cellular receptors for entry into target cells. Using an RTA-dependent reporter 293 T-cell line, Inoue et al. (23) reported the inability of soluble 3ß1 integrin and RGD peptides to block the infectivity of KSHV. However, in this study virus was centrifuged with cells in the presence of polybrene, which may account for the apparent discrepancy. Polybrene is a positively charged cation which complexes with the virus envelope and bypasses the need for receptors (33). This property of polybrene is the basis for its use to increase the infectivity of many viruses and to deliver nucleic acid for gene therapy (33). The nature of other receptor(s) recognized by KSHV and the glycoproteins involved need to be evaluated further.
Using soluble gB protein, we have demonstrated the ability of gB to mediate extensive cytoskeletal rearrangement in the target cells via a FAK-Src-PI3-kinase-Rho-GTPases signaling pathway (54). As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) superfamily via PI3K-PKC-MEK pathway (44). Inhibitors specific for phosphatidylinositol (PI) 3-kinase, protein kinase C (PKC), MEK, and ERK1/2 significantly reduced the infectivity of KSHV without affecting its binding to the target cells. Examination of viral DNA entry suggests a role for PI 3-kinase in KSHV entry into the target cells and a role for PKC, MEK, and ERK at a postviral entry stage of infection (44, 54). The role of KSHV envelope glycoproteins in initiating the ERK1/2 induction was unknown.
Here we present several lines of evidences to show that ERK1/2 is induced by KSHV gpK8.1A and gB and that this induction plays a major role in KSHV gene expression. These findings implicate a critical role for KSHV-induced signaling in its infection of target cells and suggest that, by orchestrating the signal cascade, KSHV may create an appropriate intracellular environment to facilitate the infection.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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KSHV.152) carrying BCBL-1 cells (62) were cultured in RPMI 1640 (Gibco BRL) medium (44). All cells were cultured in lipopolysaccharide (LPS)-free medium.
Antibodies, substrates, and chemicals.
Polyclonal rabbit antibodies immunoprecipitating ERK2 (sc-154) and MEK (sc-219) as active kinases were from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif. Rabbit antibodies detecting the phosphorylated forms of ERK1/2 (Thr 202/Tyr 204 phospho-p44/42 MAP kinase), MEK1/2 (Ser 217/221 phospho-MEK1/2), p38 MAP kinase (Thr180/Tyr182 phospho-p38 MAPK) antibodies, and PD98059 (MEK1 inhibitor) were from Cell Signaling Technology, Beverly, Mass. Monoclonal antibodies detecting phosphotyrosine (PY20) and mouse anti-phospho-FAK antibody (Y397) were from Transduction Laboratories, Los Angeles, Calif. Antirabbit and anti-mouse horseradish peroxidase-, fluorescein isothiocyanate-, or tetramethyl rhodamine isothiocyanate-linked antibodies were from KPL, Inc., Gaithersburg, Md. Rabbit (polyclonal) anti-ERK5/BMK1 (pTpY218/220) phospho-specific antibody was from Biosource International, Calif. Rabbit (polyclonal) anti-ERK5/BMK1 antibody was from StressGen Biotechnologies Corp., British Columbia Canada. Monoclonal (MAb) antibodies against MAP kinase (anti-diphosphorylated ERK1/2 MAPK-YT), -tubulin, anti-ß tubulin 1, and ß-actin (clone AC-40) were from Sigma, St. Louis, Mo. NuSieve GTG agarose was from Cambrex Bio Science Rockland, Inc., Maine. Lysophosphatidic acid, LY294002 [20(4-morphodinyl)-8-phenyl-1(4H)-benzopyran-4-one], sorbitol, heparin, sodium orthovanadate, benzamidine, leupeptin, aprotinin, and epidermal growth factor (EGF) were obtained from Sigma. The ERK substrate, myelin basic protein (MBP), was from Upstate Biotechnology, Lake Placid, N.Y. U0126 (1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene), U0124(1,4-diamino-2,3-dicyano-1,4-bis [methylthio]butadiene), Clostridium difficile toxin A (CdTxA),and MAb against lamin B were from Calbiochem, La Jolla, Calif. Protein A Sepharose CL-4B beads were from Amersham Pharmacia Biotech, Piscataway, N.J. Polyclonal rabbit antibody for total p38 was from Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.
Recombinant KSHV proteins.
The generation and purification of recombinant baculovirus-expressed 
TMgB, TMgB-RGA, TMgpK8.1A, and ORF 73 proteins have been previously described (64, 65, 74). Recombinant protein purifications were carried out in buffers prepared with LPS-free water, and stock preparations of proteins were monitored for endotoxin contaminations by standard Limulus assay (Limulus Amebocyte Lysate Endochrome; Charles River Endosafe, Charleston, S.C.) as recommended by the manufacturer.
Virus. Induction of the KSHV lytic cycle in GFP-BCBL-1 cells, supernatant collection, and virus purification procedures were described previously (44), and purity was assessed by general guidelines established in our laboratory (4, 32, 44). KSHV DNA was extracted from the virus, and the copy numbers were quantitated by real-time DNA PCR using primers amplifying the KSHV ORF 73 gene as described previously (32). To prepare replication-defective virus, KSHV was inactivated with UV light (365 nm) for 20 min at a 10-cm distance.
KSHV antibodies.
The generation of rabbit polyclonal antibodies against KSHV TMgB and MAbs against gpK8.1A has been described previously (64, 74).
Cytotoxicity assay. Target cells were tested for their viability in the presence of various concentrations of soluble recombinant proteins or various inhibitors at 37°C using a lactate dehydrogenase cytotoxicity assay kit (Promega, Madison, Wis.) as described previously (54, 64).
Western blotting.
Target cells grown to confluence in 25-cm2 flasks were serum starved for 24 h, cooled to 4°C, and induced with KSHV proteins or ligands at 37°C. Cells were exposed to kinase inhibitors (U0126 and LY294002) for 1 h at 37°C prior to KSHV infection or protein induction. As a control, cells were treated with U0124 (Calbiochem), a chemical analogue of U0126 without MEK-inhibitory activity (19). After treatment, cells were washed twice with phosphate-buffered saline (PBS), and total protein was extracted. In some experiments, TMgB and TMgpK8.1A were incubated with anti-TMgB and anti-TMgpK8.1A antibodies for 1 h at 37°C before being added to the cells. Total cell lysates (15 µg) were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose membranes, and immunoblotted with antibodies. Immunoreactive bands were developed by enhanced chemiluminescence reaction (NEN Life Sciences Products, Boston, Mass.) and quantified following standard protocols (54).
Immunoprecipitation. For immunoprecipitation reactions, 500 µg of whole-cell extract was mixed with respective antibodies and rocked overnight at 4°C. A total of 100 µl of protein A-Sepharose was then added, and rocking was allowed to continue for another 90 min. Beads were then washed four times with RIPA buffer. Fifty microliters of 2x SDS sample buffer was added to the beads and boiled for 4 min. The insoluble material was removed by centrifugation, and the supernatant was then subjected to Western blot analysis. The membranes were soaked in blocking solution (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5% bovine serum albumin, 0.02% NaN3) at 4°C overnight and then reacted with phospho-specific antibodies overnight at 4°C. The membranes were washed five times with washing buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.3% Tween 20) and probed with secondary antibodies conjugated with horseradish peroxidase or alkaline phosphatase for 1 h at room temperature. These were detected by using either enhanced chemiluminescence as specified by manufacturer (NEN) or CDP-Star (Roche Diagnostics Corp., Indianapolis, Ind.). The bands were scanned and the band intensities were assessed using the ImageQuaNT software program (Molecular Dynamics, Sunnyvale, Calif.).
Immune complex kinase assays.
Immune complexes were washed five times with lysis buffer, twice with kinase buffer (10 mM MgCl2, 25 mM ß-glycerophosphate, 25 mM HEPES, pH 7.5, 5 mM benzamidine, 0.5 mM dithiothreitol, 1 mM Na3VO4). Kinase reactions were initiated by the addition of 2 µCi of [-32P]ATP (Perkin Elmer, Boston, MA) and incubated with 5 µg of substrate (MBP for ERK) in the kinase buffer for 20 min at 30°C. Reactions were terminated by adding 2x SDS sample buffer, boiled for 5 min, and analyzed by SDS-12% PAGE. Gels were dried, and phosphorylated substrate was detected by phosphorimager analyses and by autoradiography. Bands were scanned and the band intensities were assessed.
Immunofluorescence assay. Confluent HFF cells in eight-well chamber slides (Nalge Nunc International, Naperville, Ill.) were incubated with either KSHV or 10% FBS for 30 min at 37°C. For phosphorylated p42/p44 MAPKs staining, cells were fixed with 10% paraformaldehyde and permeabilized with methanol. For total p42/p44 MAPKs staining, methanol-acetone (70:30, vol/vol) at –20°C for fixation and permeabilization was used. Alexa 594-coupled anti-mouse antibodies (Molecular Probes, Eugene, OR) and Alexa 488-coupled anti-rabbit antibodies (Molecular Probes) were used at a 1:250 dilution to detect the phosphorylated p42/p44 MAPK and total p42/p44 MAPK, respectively. Stained cells were washed and viewed with appropriate filters under a fluorescence microscope with the Nikon Magna Firewire digital imaging system.
Preparation of DNA and RNA. Total DNA from the viral stocks and cells were prepared using a DNeasy Tissue kit (QIAGEN, Inc., Valencia, Calif.). Monolayers of 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 1x PBS, and total DNA was prepared according to the manufacturer's instructions. For the preparation of DNA from intact virions, 200 µl of virus stocks was pretreated with 12 µg of DNase I for 15 min at room temperature; the reaction was stopped by EDTA followed by heat inactivation at 70°C, and DNA was prepared. Total RNA was isolated from infected or uninfected cells using an RNeasy kit as described previously (32).
Measurement of KSHV internalization by real-time DNA PCR. Untreated HFF cells or HFF cells incubated with inhibitors were infected with KSHV at 10 DNA copies/cell. After a 2-h incubation, cells were washed twice with PBS to remove the unbound virus, treated with trypsin-EDTA for 5 min at 37°C to remove the bound but noninternalized virus, and washed, and total DNA was isolated using a DNeasy kit. A total of 100 ng of DNA samples, KSHV ORF 73 gene TaqMan probe (32), and Quantitect PCR mix were used. The KSHV ORF 73 gene cloned in the pGEM-T vector (Promega) was used for the external standard. Known amounts of ORF 73 plasmid were used in the amplification reactions along with the test samples. The lower limit of ORF 73 gene detection was 10 to 100 copies, and the most accurate detection was from 100 to 106 copies. Special care was taken to keep the slope of the standard curve close to 3.3 so that the amplification efficiency of the cycles was 2. The cycle threshold (CT) values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples.
DNA nuclear delivery assay.
Pure nuclear fractions were prepared using a Nuclei EZ isolation kit (Sigma) following the manufacturer's instructions. Cells were pretreated with either 300 ng/ml of CdTxA or 10 µM U0126, U0124, or 50 µM LY294002 for 1 h at 37°C. Cells were infected with KSHV for 2 h, washed, treated with trypsin-EDTA to remove noninternalized virus, and lysed on ice for 5 min with a mild lysis buffer (Sigma), and nuclei were concentrated by centrifugation at 500 x g for 5 min. Cytoskeletal components loosely bound to the nuclei were removed from the nuclear pellet by a repeat of lysis and centrifugation procedures as described previously (42). The nuclei were purified and assessed for their purity by immunoblotting with anti-lamin B antibodies, and cytoskeletal contamination was ruled out by immunoblotting with anti-ß-actin and anti--tubulin antibodies.
Semiquantitative RT-PCR. Expression kinetics of host genes were analyzed by reverse transcriptase PCR (RT-PCR) using procedures described previously (43). DNase-treated total RNAs were subjected to RT-PCR using specific primers. Successive samples were removed from every three cycles (14 to 41), resolved on agarose gel, and increases in expression (n-fold) were calculated after normalizing to the ß-actin gene. For all genes, integrated density values (IDV) corresponding to the sum of pixel intensities after background corrections were recorded for both the KSHV-infected and U0126-pretreated samples at linear points on the amplification curve and changes in expression were calculated after normalizing to ß-actin gene.
Real-time RT-PCR. The ORF 50 and ORF 73 transcripts were detected by real-time RT-PCR using specific real-time primers and specific TaqMan probes as described previously (32). K5, K8, and v-IRF2 transcripts were detected using the gene-specific real-time PCR primers and specific TaqMan probes (Applied Biosystems, Foster City, Calif.) described in Table 1. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the internal control. The reaction conditions used for the ORF 50 and 73 gene amplifications consisted of four stages: 50°C for 2 min, 60°C for 30 min, and 95°C for 10 min, followed by 44 cycles of 95°C for 15 s and 60°C for 30 s. K5, K8, and v-IRF2 gene amplifications consisted of four stages: 50°C for 2 min, 60°C for 30 min, and 95°C for 10 min, followed by 44 cycles of 95°C for 20 s and 55°C for 30 sec. GAPDH conditions varied slightly in the amplification stage, with denaturation of the strands at 95°C for 20 s and 62°C for 1 min, and were repeated for 40 cycles.
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Transcription factor activation assay.
A total of 5 µg of each nuclear extract was used for this assay. Transcription factors ATF-2, c-Jun, c-Myc, c-Fos, MEF2, and STAT1 in the nuclear extracts were measured using an enzyme-linked immunosorbent assay (ELISA)-based TransAM MAPK Family kit (Active Motif Corp.) per the manufacturer's instructions. In this assay, transcription factors bind to the immobilized oligonucleotide containing the consensus sequences specific for the particular transcription factor, which is detected by a sandwich ELISA. The active form of the transcription factor contained in the nuclear extract specifically binds to this oligonucleotide mixture. The primary antibodies used to detect each of the MAPK-regulated transcription factors recognize an epitope in the phosphorylated ATF-2, c-Jun, c-Myc, MEF2, c-Fos, and STAT1 that is accessible only when these transcription factors are activated and bound to their target DNA. The detection limit for the TransAM MAPK Family kit is <0.5 µg of nuclear extract/well. A competition assay was done by premixing nuclear extracts for 30 min at 4°C with wild-type (WT) consensus and mutated consensus oligonucleotides provided in the kit before addition to the probe immobilized on the plate.
EMSA.
The activator protein 1 (AP1) consensus oligonucleotide binding site for c-Jun homodimer and Jun/Fos heterodimeric complexes and AP1 mutant (AP1m) oligonucleotide (identical to AP1 consensus oligonucleotides with the exception of a CA to TG substitution in the AP1 binding motif) were obtained from Santa Cruz Biotechnology, Inc. The double-stranded oligonucleotides were labeled at the 5' end with [-32P]ATP (Perkin Elmer) using T4 polynucleotide kinase (Gibco BRL). To prepare 32P-labeled probe for RTA-AP1 and RTA-AP1m, single-strand oligonucleotides (66) were purchased from Invitrogen, annealed, and labeled at the 5' end with [-32P]ATP (Perkin Elmer) using T4 polynucleotide kinase (Gibco BRL). Binding reactions were incubated on ice for 20 min and were performed in a 20-µl reaction volume containing 50 mmol/liter NaCl, 10 mmol/liter Tris-HCl, pH 7.5, 1 mmol/liter MgCl2, 0.5 mmol/liter EDTA, 0.5 mmol/liter dithiothreitol, 9% (vol/vol) glycerol, 1 µg of poly(dI-dC), 6 µg of nuclear extract, and labeled probe (10,000 cpm). The resulting DNA-protein complexes were then size fractionated from the free DNA probe by electrophoresis at 200 V on a 6% native polyacrylamide gel (acryl/bisacrylamide ratio of 29/1) containing 2.5% glycerol, 50 mmol/liter Tris-HCl, pH 8.5, 0.4 mol/liter glycine, and 2.7 mmol/liter EDTA. These gels were dried at 80°C for 30 min, exposed to Kodak X-ray film, and visualized with a PhosphorImager (Molecular Dynamics), and the band intensities were calculated. A competition electrophoretic mobility shift assay (EMSA) was performed by adding a 100x molar excess of unlabeled double-stranded oligonucleotide probes, AP1 oligonucleotide (WT), and RTA-AP1 oligonucleotide, for respective treatments.
The nucleotide sequences of the annealed DNA probes used for AP1 consensus, AP1 mutated, RTA-AP1, and RTA-AP1m are shown in Fig. 1.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MEK-ERK signal pathway in target cells infected with KSHV at an MOI of 5 (44). In this study, we examined the ability of KSHV at different MOIs (viral DNA copy/cell) to induce ERK1/2 phosphorylation. When serum-starved HFF cells infected for 30 min were analyzed with anti-phospho-ERK1/2 antibodies for ERK1/2 phosphorylation, compared to uninfected cells (Fig. 2A, top band, lane 1), a dose-dependent increase in ERK1/2 phosphorylation was observed in infected cells (Fig. 2A, top band, lanes 2 to 8). About 1.2- to 1.4-fold induction was observed at a low MOI, which increased to 2- to 5-fold at an MOI of 2 to 10. Equal loading of lysate was confirmed by Western blot reactions with antibodies against the total ERK2 protein (Fig. 2A, lower band). These results demonstrated the steady-state level of endogenous ERK2, thus suggesting that KSHV infection was activating the endogenous or preexisting ERK. The maximum activation by KSHV infection at an MOI of 5 probably reflects target cell saturation. Induction of ERK1/2 by a low MOI of KSHV suggested that this phosphorylation event is probably physiologically relevant.
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KSHV induces the p42/p44 ERK1/2 MAPK in the presence of serum. To mimic the effect of KSHV during the initial stage of infection in quiescent cells and to determine the induction of preexisting signal pathways, we routinely use serum-starved confluent target cells (44, 54). To determine whether KSHV-induced ERK phosphorylation is a specific response to the infectious process or a compensatory mechanism induced by serum starvation, HFF cells in the presence of serum were infected with KSHV at an MOI of 5. Compared to the uninfected serum-starved cells (Fig. 2B, lane 1), the background ERK1/2 activity in the uninfected cells with serum was much higher (Fig. 2C, top band, lane 5). Despite this background level, similar to serum-starved cells, KSHV induced a twofold increase in ERK phosphorylation as early as 5 min p.i. (Fig. 2C, top band, lane 4), which was maintained at 1.8-fold over background at 15, 30, and 60 min p.i. (Fig. 2C, top band, lanes 3, 2, and 1, respectively). Compared to serum-starved infected cells (Fig. 2A and B), levels of ERK phosphorylation did not decrease to the basal level at 60 min in serum-fed KSHV-infected cells. Equal loading of total lysate between the treatments and the steady-state level of endogenous ERK2 were demonstrated by Western blot reactions with antibodies against the total ERK2 protein (Fig. 2C, lower band). These results confirmed the activation of endogenous or preexisting ERK by KSHV in the target cells and suggested that ERK induction is a consequence of infection and not a response to serum starvation.
KSHV triggers the rapid nuclear translocation of p42/p44 MAPK. Phosphorylation and dephosphorylation play significant roles in the signaling cascades, and the subcellular location of a phosphorylated protein is important for its activity and inactivity. The p44 and p42 MAPK isoforms (ERK1 and ERK2) are serine/threonine kinases, and their activity is positively regulated by phosphorylation mediated by MEK1 and MEK2, which phosphorylate ERK1T202 and Y204 and ERK2 T185 and Y187 to activate their kinase activities (71). Among all the members of the MAPK signaling cascade, ERK1 and ERK2 have been shown to be the only key mediators of signal transduction transmitting signals from the cell surface to the nucleus. Upon signal induction, the MEK remains cytoplasmic, whereas ERKs anchored to MEK in the cytoplasm of resting cells translocate to the nucleus, a process which is rapid, reversible, and controlled by the strict activation of the MAPK cascade (34, 63).
Since KSHV induced ERK1/2 early during infection, we examined the infected cells using monoclonal antibody against the MAP kinase synthetic diphosphopeptide. This antibody specifically recognized the active, doubly phosphorylated forms but not the inactive mono- and nonphosphorylated forms of ERKs (73). Infection with KSHV (MOI of 10) for 30 min induced the rapid nuclear translocation of phosphorylated ERK1/2 in >90% of infected cells; an example is shown in Fig. 2D, frame 2. This is similar to the translocation observed in cells treated with 20% FBS (Fig. 2D, frame 3). KSHV-induced ERK1/2 translocation correlated well with the rapid phosphorylation of ERK1/2, as demonstrated in Fig. 2B. In contrast, appreciable amounts of phosphorylated p42/p44 MAPKs were not detected in the nuclei of uninfected cells (Fig. 2D, frame 1). When the subcellular localization of total p42/p44 MAPKs was observed in these cells, the inactive p42/p44 MAPKs pool was primarily located in the cytoplasm (Fig. 2D, frames 4, 5, and 6). These results demonstrated that KSHV triggers the rapid nuclear entry of phosphorylated p42/p44 MAPKs in the infected cells.
UV-inactivated KSHV induces the p42/p44 ERK1/2 MAPK pathway. To determine whether KSHV gene expression is essential for ERK1/2 activation, replication-incompetent KSHV was prepared by UV irradiation. After 20 min of UV exposure, binding of [3H]thymidine-labeled KSHV to HFF cells was not affected (data not shown). When KSHV entry into the target cells was measured by quantitative real-time DNA PCR (32), UV-inactivated virus entered the target cells as efficiently as the untreated virus (Fig. 3A). As demonstrated previously (32), immediately following infection with live KSHV (10 DNA copies/cell), a quantitative increase in expression of the latency-associated ORF 73 gene was seen, as well as expression of a limited number of the lytic cycle-associated ORF 50, K8, and K5 genes (Fig. 3B, left). In contrast, no such increase in KSHV gene expression was observed in cells infected with equal MOIs of UV-inactivated KSHV (Fig. 3B, right). However, similar to live KSHV (Fig. 3C, upper band, lane 3), a robust ERK1/2 activation was observed in target cells incubated with UV-inactivated KSHV, and about 2.3-, 3.7-, 4.8-, and 2.7-fold ERK1/2 activations were detected at 5, 15, 30, and 60 min p.i., respectively (Fig. 3C, upper band, lanes 4 to 7). These results suggested that the early kinetics of ERK activation of KSHV was not dependent on KSHV gene expression but probably depended upon signal induction mediated by KSHV envelope glycoprotein interactions with host cell receptor molecule(s) during the binding and entry stages of infection.
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3ß1 integrin molecule (4). Soluble gB but not soluble gpK8.1A induced the integrin-dependent preexisting FAK-Src-PI-3K-RhoGTPase signal pathways and induced the substrate adhesion of target cells (54, 64). To determine the role of gB and gpK8.1A in ERK1/2 activation, lysates from HFF cells induced for 30 min with different concentrations of TMgB, TMgB-RGA, TMgpK8.1A, and ORF 73 proteins were analyzed for ERK1/2 phosphorylation (Fig. 4A). No induction was seen in cells incubated with DMEM, and incubation for 15 min with DMEM containing 20% FBS induced a rapid sixfold phosphorylation of ERK (Fig. 4A, top band, lanes 1 and 2). Phosphorylation of ERK1/2 was not observed in cells incubated with various concentrations of TMgB, TMgB-RGA, and ORF 73 proteins (Fig. 4A, top band, lanes 3 to 6, 11 to 14, and 15, respectively). In contrast, TMgpK8.1A induced ERK phosphorylation in a dose-dependent manner (Fig. 4A, top band, lanes 7 to 10), and maximum induction was observed with 2 µg/ml of TMgpK8.1A (Fig. 4A, lane 9). Incubation with even low (0.5 µg/ml) concentrations of TMgpK8.1A could induce a twofold ERK phosphorylation. These results demonstrated that like KSHV infection, gpK8.1A was activating the endogenous or preexisting ERK.
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TMgpK8.1A.
Lysates from HFF cells induced with 2 µg/ml of TMgpK8.1A for different time points were analyzed for ERK activity in an in vitro immune-complex kinase assay. Quantitation of 32P-MBP (Fig. 4B, top band) as a measure of ERK activity showed that compared to uninduced cells (Fig. 4B, lane 1, and 4C), TMgpK8.1A induced a 2.5- and 3-fold increase in ERK phosphorylation at 5 and 15 min of incubation, respectively (Fig. 4B, lanes 2 and 3, and C). This activation reached a maximum of fivefold by 30 min (Fig. 4B, lane 4, and C) and decreased close to the basal levels by 60 to 120 min (Fig. 4A, lanes 5 to 6, and C). FBS used as a positive control induced a rapid activation of ERK in 15 min (Fig. 4A, top panel, lane 7, and C). In contrast, incubation with either TMgB (30 min), or with TMgB-RGA (30 min) had no effect on ERK activity (Fig. 4A, top band, lanes 8 and 9, respectively). Western blot analysis of the same lysates with anti-phospho ERK1/2 antibodies demonstrated the increase in the phosphorylated forms of ERK1/2 with kinetics similar to the kinase assay (Fig. 4B, middle band), thus confirming the induction of ERK activity. Equal loading of total lysate between different treatments was confirmed by Western blot reactions with antibodies against the total ERK1/2 protein (Fig. 4B, bottom band).
Cell type specificity of ERK1/2 activation.
To determine whether the ERK activation is a cell type-specific phenomenon, ERK1/2 phosphorylation was tested in HMVEC-d (Fig. 5A). Compared to HFF cells, the endogenous level of phospho-ERK1/2 in HMVEC-d was much lower (Fig. 5A, top band, lane 14). Similar to HFF cells and in comparison to uninduced cells (Fig. 5A, lane 14), treatment of serum-starved HMVEC-d with 2 µg/ml of TMgpK8.1A for 5 and 15 min resulted in about 1.5- and 5-fold increases in ERK phosphorylation (Fig. 5A, lanes 12 and 11, respectively). However, unlike HFFs, the maximum induction of ERK1/2 was observed at 15 min in HMVEC-d (Fig. 5A, lane 11), which decreased to twofold by about 30 min (Fig. 5A, lane 10). In contrast, incubation with either 2 µg/ml of TMgB-RGA or 2 µg/ml ORF 73 for 5, 15, and 30 min had no effect on ERK phosphorylation in HMVEC-d (Fig. 5A, top band, lanes 6 to 1, respectively). Even though no ERK1/2 induction was observed in HFF cells induced with TMgB, incubation of HMVEC-d for 15 min with TMgB induced a twofold ERK1/2 phosphorylation (Fig. 5A, top band, lane 8). These results suggested that interaction of KSHV glycoproteins with cell surface receptors and the onset of signaling cascades differ from one cell type to another and that KSHV gpK8.1A is the major inducer of ERK1/2 phosphorylation in HMVEC-d and HFF cells.
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TMgpK8.1A.
To confirm that the ERKl/2 phosphorylation was induced by purified soluble TMgpK8.1A and not by contaminating protein(s), TMgpK8.1A was preincubated with increasing concentrations of heparin or chondroitin sulfate for 90 min at 4°C before incubation with HFF cells. Incubation of TMgpK8.1A with 1, 10, and 100 µg/ml of heparin reduced the ERKl/2 induction in dose-dependent manner by 45%, 70%, and 79%, respectively (Fig. 5B, top band, lanes 4 to 2, respectively). Only 16% inhibition of ERK phosphorylation was seen with 100 µg/ml of chondroitin sulfate (Fig. 5B, lane 1). The total ERK-2 levels remained same during different treatments (Fig. 5B, bottom band).
To confirm that we were detecting ERK1/2 induction driven by TMgpK8.1A, TMgpK8.1A was preincubated with anti-gpK8.1A MAb 4D6 antibodies or with anti-ORF 59 MAb 11D1 for 1 h at 37°C before incubation with HFFs. Incubation with anti-gpK8.1A antibodies reduced the ERKl/2 induction at 30 min in a dose-dependent manner (Fig. 5C, top band, lanes 2 to 6). Quantitation demonstrated a 21%, 33%, 64%, and 82% reduction in ERK1/2 induction at 40, 60, 80, and 100 µg/ml concentrations, respectively (Fig. 5D). No inhibition was seen with anti-ORF 59 MAb even at a concentration of 100 µg/ml (Fig. 5D and C, lane 7). Western blot analyses of immune complexes detected equal amounts of total ERK1/2 protein, thus demonstrating that equal ERKs were immunoprecipitated (Fig. 5C, bottom band). Preincubation of KSHV with 100 µg/ml of anti-gB and -gpK8.1A antibodies reduced the ERK1/2 induction 59% and 67%, respectively (data not shown). These results demonstrated that ERK was specifically induced by soluble gpK8.1A or by KSHV infection and not by the contaminating host cell factors and/or LPS or any component from the medium.
MEK1/2 is an upstream inducer of TMgpK8.1A-activated ERK1/2.
Since MEK1/2 is an upstream kinase that induces ERK1/2, we used the phosphorylation-specific antibodies to examine MEK1/2 induction by TMgpK8.1A. Compared to the uninduced HFF cells (Fig. 6A, top band, lane 2), phosphorylation of MEKl/2 by TMgpK8.1A increased in a time-dependent manner, from twofold at 5 min to a peak activation of fivefold at 30 min, decreasing at 60 min (Fig. 6A, top band, lanes 3 to 6) and to background level at 90 min (data not shown). KSHV infection for 30 min also induced the MEK1/2 phosphorylation by sixfold (Fig. 6A, top band, lane 1). In contrast, the total MEK or ß-actin levels remained unaffected (Fig. 6A, middle and bottom bands, respectively). The observed kinetics of MEK1/2 induction was similar to the kinetics of ERK1/2 induction (Fig. 4B and 6A).
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TMgpK8.1A treatment, HFF cells were preincubated with different concentrations of U0126 for 1 h at 37°C and then incubated with TMgpK8.1A (2 µg/ml) for 30 min. U0126 is a potent and specific covalent binding inhibitor of MEKl/2, and shows little, if any, effect on the kinase activities of PKC, Abl, cRaf-1, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4 (17). U0126 pretreatment inhibited the TMgpK8.1A-induced ERK phosphorylation in a dose-dependent manner without affecting the total ERK levels (Fig. 6B, bottom band, lanes 2 to 5). Control cells treated either with dimethyl sulfoxide (data not shown) or U0124, an analogue of U0126 with no inhibitory effect on MEK1/2, failed to block ERK1/2 phosphorylation (Fig. 6B, top band, lane 1). These results demonstrated that ERK1/2 activation by TMgpK8.1A is dependent upon MEK1/2 kinase. This is similar to our earlier results where we have demonstrated that MEK1/2 is an upstream inducer of KSHV-stimulated ERK1/2 pathway (44).
KSHV and TMgpK8.1A activate ERK1/2 but not ERK5 and p38-MAPK.
To determine whether gpK8.1A induces other MAPK pathways, phosphorylation-specific antibodies were used to examine the induction of big MAPK (ERK5) and LPS- or stress-activated p38-MAPK. Unlike the other MAPK cascades that seem to serve one set of extracellular stimuli such as stress or mitogens, the ERK5 cascade is activated by both stress responses and mitogens (1, 29). The ERK5 cascade mediates its function mainly through the regulation of transcription by directly phosphorylating and activating several transcription factors including c-Myc (18), MEF2 family members (30), and c-Fos (28). Treatment of HFF cells with 0.5 M sorbitol used as positive control induced the phosphorylation of ERK5 (Fig. 6C, top band, lanes 2 to 5), and the total level of ERK5 was maintained constant in these cell lysates (Fig. 6C, bottom band, lanes 1 to 7). In contrast, no phosphorylation of ERK5 was seen in cells infected with KSHV or treated with TMgpK8.1A (Fig. 6C, top band, lanes 6 and 7). Similarly, phosphorylation of p38-MAPK was not observed with KSHV infection or with TMgpK8.1A (Fig. 6D, bottom band, lanes 6 and 7). Treatment with 0.5 M sorbitol induced an appreciable level of p38-MAPK phosphorylation (Fig. 6D, top band, lanes 2 to 4). In contrast, the total p38 or ß-actin levels remained unaffected (Fig. 6D, middle and bottom bands, respectively). These results suggested that KSHV and TMgpK8.1A preferentially induced the MEK1/2-ERK1/2 pathway.
KSHV-induced MEK1/2 and ERK1/2 do not play a role in the internalization of virus. In our earlier studies, in cells preincubated with U0126 and ERK-antisense oligonucleotides, we observed the neutralization of GFP-KSHV infectivity as measured by the immunofluorescence assay measurement of GFP expression (44). Since this inhibition was observed without affecting KSHV binding to the target cells (44), to determine whether this is due to a role of ERK1/2 in viral internalization, we carried out a quantitative real-time DNA PCR assay (32). By this method, we have previously shown that internalized viral DNA could be detected in HFF cells as early as 5 min p.i., increasing rapidly during the first 60 to 90 min of infection and reaching a plateau at around 90 to 120 min p.i. (32). Preincubation of KSHV with 100 µg of heparin per ml blocked the viral DNA entry by more than 90% (Fig. 7A). Treatment of HFF cells with the U0126 did not show any significant reduction of KSHV DNA internalization (Fig. 7A). In contrast, preincubation of cells with the PI-3K inhibitor LY294002 at 50 and 100 µM concentrations reduced the internalization by about 64 to 79% (Fig. 7A), which reaffirmed our earlier conclusion that PI-3K plays a role in the entry stage of KSHV infection (44, 54). These results, together with inhibition of infectivity by the inhibitors of MEK (44, 54), suggest a role for activation of the MEK pathway subsequent to the virus internalization stage of infection.
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Inhibition of ERK1/2 blocks the expression of KSHV latent ORF 73 gene expression. To analyze the potential role of ERK1/2 activation in KSHV gene expression, HFF cells and HMVEC-d were preincubated with U0126 at 37°C for 1 h and infected with KSHV for 2 h, and viral messages collected at different times p.i. were quantitated by real-time RT-PCR. We have previously shown that immediately after infection of HMVEC-d and HFF cells, KSHV expressed the latent ORF 73 and 72 and K13 genes as well as the lytic cycle switch ORF 50 gene (32). In this present study also we observed a steady increase in ORF 73 gene expression in HMVEC-d and HFF cells (Fig. 8A and B). This expression was significantly reduced in U0126-pretreated HMVEC-d at 1, 2, 4, and 8 h p.i. with about 83%, 87%, 94%, and 95% reduction, respectively (Fig. 8C). Similarly, ORF 73 expression was significantly reduced in U0126-pretreated HFF cells at 2, 4, and 8 h p.i. with about 81%, 86%, and 93% reduction, respectively (Fig. 8D). The effect of ERK inhibitor on ORF 73 gene expression was maintained throughout the observed period with about 77% reduction even at 24 h p.i. (data not shown). The specificity of these reactions was demonstrated by the absence of contaminating DNA in any of the DNase-treated RNA samples. Even though we observed slight differences in the copy numbers of transcripts in different experiments and batches of viruses, the patterns of gene expression were closely similar and highly reproducible.
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Inhibition of ERK1/2 blocks the expression of KSHV lytic ORF 50 gene expression. The 110-kDa KSHV immediate early ORF 50 (RTA) protein is a transcriptional activator and acts as a molecular switch for KSHV reactivation (17, 21, 40, 53, 59, 68). As demonstrated previously (32), we observed a steady increase in ORF 50 gene expression in HMVEC-d and HFF cells with slightly varying kinetics (Fig. 8A and B). Copy numbers of ORF 50 transcripts reached a peak at 2 h p.i. in HMVEC-d and declined rapidly at subsequent time points (Fig. 8A). In contrast, in HFF cells, the peak level of ORF 50 gene expression was observed at 8 h p.i., followed by a sharp decline by 24 h p.i. (Fig. 8B). The observed ORF 50 expression was significantly reduced in U0126-pretreated cells at all time points (Fig. 8C and D). About 20%, 84%, 80%, and 60% reduction was observed at 1, 2, 4, and 8 h p.i. in HMVEC-d, respectively (Fig. 8C). In HFF cells, about 66%, 60%, and 58% reduction was observed at 2, 4, and 8 h p.i., respectively (Fig. 8D). After 24 h p.i., about 40% inhibition in ORF 50 expression was observed in HFF cells pretreated with MEK inhibitor (data not shown). These results suggested that the ERK1/2 pathway also plays a role in the transcription initiation of KSHV ORF 50 transcripts.
Inhibition of ERK1/2 blocks the expression of KSHV immediate early lytic K8 and v-IRF2 genes but not K5 gene expression. Our studies have shown that in addition to the latent and lytic ORF 50 genes, KSHV also expressed a limited number of lytic cycle genes early during infection (32). Though the detection of viral transcripts by gene array in the earlier study could also represent messages carried in the virion particles, further examination revealed a steady quantitative increase in early lytic K5, K8, and v-IRF2 gene expression in the infected HMVEC-d and HFF cells with various kinetics (Fig. 9A and B). KSHV ORF 50 gene activates the expression of K8 and v-IRF2 genes (35, 66, 67, 59), and, as expected, the expression kinetics of K8 and v-IRF2 genes (Fig. 9A and B) followed the ORF 50 expression kinetics (Fig. 8A and B). Expression of K8 and v-IRF2 reached a peak around 2 h and 8 h p.i. in HMVEC-d and HFF cells, respectively, and declined sharply thereafter (Fig. 9A and B). Pretreatment of HMVEC-d with U0126 inhibited the expression of K8 by about 37%, 73%, 44% and 7% at 1, 2, 4, and 8 h p.i., respectively (Fig. 9C), and by about 83%, 97%, 71%, and 33% at 2, 4, 8, and 24 h, respectively, in HFF cells (Fig. 9D). U0126 pretreatment also affected the v-IRF2 expression by about 32%, 68%, 59%, and 12% at 1, 2,4, and 8 h p.i., respectively, in HMVEC-d (Fig. 9C) and by about 50%, 45%, 95%, and 24% at 2, 4, 8, and 24 h p.i., respectively, in HFF cells (Fig. 9D). Maximum inhibition correlated with the time point at which maximum expression of K8 and v-IRF 2 genes was observed. Unlike K8 and v-IRF2, ERK1/2 inhibition had minimal impact on K5 gene expression in HMVEC-d and HFF cells (Fig. 9C and D). K5 was inhibited by about 23%, 16%, 5%, and 4% at 1, 2, 4, and 8 h, respectively, in HMVEC-d (Fig. 9C) and by about 12% and 37% at 4 h and 8 h, respectively, in HFF cells (Fig. 9D). Since expression of K8 and v-IRF2 depends upon the expression of ORF 50 (35, 59, 66, 67), these results suggested that the ERK1/2 pathway plays a pivotal role in KSHV transcription initiation. The limited impact by ERK1/2 inhibition over K5 gene expression may probably be due to the ORF 50-dependent and -independent expression of the K5 gene (46). These differential effects over KSHV gene expression by U0126 also suggested the specificity of the observed reduction in viral RNA copy numbers.
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Pretreatment of HFF cells with U0126 had no effect on the expression of the MAP3K8 gene, which was shown to be induced by KSHV about threefold at about 4 h p.i. (43). Similarly, the IE-3 gene, whose expression was induced by KSHV by about seven- and eightfold at 2 and 4 h p.i., was not affected (Fig. 10C). The expression of HB-EGF was highest at 2 h p.i. (18-fold), and U0126 treatment did not affect this expression. However, when the induction was about fourfold at 4 h p.i., about 25% inhibition was seen with U0126. In contrast, about 17- and 14-fold induction of ICAM-1 expression was observed at 2 and 4 h p.i.; about 77% inhibition was observed after 2 h in U0126-pretreated HFF cells (Fig. 10C). These results suggested that activation of ERK is not only involved in the maintenance of viral gene expression but also in the activation of some of the host genes induced by KSHV early during infection. These differential effects over host cell gene expression by U0126 also suggested that the observed reductions in viral RNA copy numbers were not due to a nonspecific shutdown of host transcriptional apparatus.
KSHV-induced ERK plays a role in the activation of MAPK family transcription factors.
Since our studies suggested that the ERK1/2 pathway plays a role in the transcription initiation of KSHV ORF 50 and ORF 73 transcripts and some cellular genes, to determine whether this is due to the ability of ERK1/2 to modulate a variety of host transcription factors, we next examined whether KSHV infection induces the activity of various transcription factors. Using an ELISA-based technique, nuclear extracts from the uninfected and infected HFF cells were assessed for the ability of host transcription factors to bind to their respective wild-type DNA sequences. Infection of HFF cells with KSHV increased the activity of MAPK-regulated transcription factors, which was dependent upon the time of infection; the data in Fig. 11A show a representative example of the activity of MAPK-regulated c-Jun, STAT1, MEF2, c-Myc, ATF-2, and c-Fos in HFF cells infected with KSHV for 120 min. The levels of these transcription factors were barely detectable in the serum-starved uninfected cells (data not shown). When values of uninfected cells were subtracted from values of infected cells, substantial inductions of c-Jun, STAT1, MEF2, c-Myc, ATF-2, and c-Fos were observed (Fig. 11A). This activation was lowest for MEF-2 and highest for c-Fos and c-Jun, while similar levels of induction were observed for c-Myc, STAT1, and ATF-2 (Fig. 11A).
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