ERK1/2 and MEK1/2 induced by Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) early during infection of target cells are essential for expression of viral genes and for establishment of infection

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) in vitro target cell infection is characterized by the expression of the latency-associated genes ORF 73 (LANA-1), ORF 72, and K13 and by the transient expression of a very limited number of lytic genes such as lytic cycle switch gene ORF 50 (RTA) and the immediate early (IE) lytic K5, K8, and v-IRF2 genes. During the early stages of infection, several overlapping multistep complex events precede the initiation of viral gene expression. KSHV envelope glycoprotein gB induces the FAK-Src-PI3K-RhoGTPase (where FAK is focal adhesion kinase) signaling pathway. As early as 5 min postinfection (p.i.), KSHV induced the extracellular signal-regulated kinase 1 and 2 (ERK1/2) via the PI3K-PKCzeta-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 transcriptase 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, STAT1alpha, 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 STAT1alpha 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.

Figures

FIG. 1.

Nucleotide sequences of the annealed DNA probes for AP1 consensus, AP1 mutants, RTA-AP1, and RTA-AP1m.

FIG. 2.

Induction of ERK1/2 by KSHV. (A) ERK1/2 phosphorylation by different MOIs of KSHV. (Top band) Serum-starved HFF cells were either uninfected (lane 1) or infected with at different MOIs (DNA copies/cell) of KSHV for 30 min at 37°C (lanes 2 to 8). Ten micrograms of cell lysates was resolved by SDS-10% PAGE, subjected to Western blotting, and reacted with anti-phospho ERK1/2 antibodies. (B) Kinetics of ERK1/2 induction by KSHV. (Top band) Serum-starved HFF cells were either uninfected (lane 1) or infected at an MOI of 5 with KSHV for indicated time points (lanes 2 to 6), or treated with 20% FBS for 15 min (lane 7). Cell lysates were resolved by SDS-10% PAGE and subjected to Western blotting with anti-phospho ERK1/2 antibodies. (C) ERK1/2 phosphorylation in serum-fed HFF cells. (Top band) HFF cells grown in the presence of serum were either uninfected (lane 5) or infected with KSHV at an MOI of 5 for indicated time points (lanes 4 to 1). Cell lysates were resolved by SDS-10% PAGE and subjected to Western blotting with anti-phospho ERK1/2 antibodies. The bottom bands of panels A, B, and C show membranes that were stripped and reprobed with anti-ERK2 antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence reactions, and the band intensities were assessed. The ERK1/2 phosphorylation in the uninfected cells was considered 1 for comparison and expressed as an increase in phosphorylation (n-fold) of ERK1/2. Each point represents the average ± the standard deviation of three experiments. (D) KSHV infection triggers nuclear translocation of phosphorylated ERK1/2. Serum-starved HFF cells in eight-well chamber slides were either uninfected (frames 1 and 4) or infected with KSHV (MOI, 10) for 30 min (frames 2 and 5), or incubated with 20% FBS for 30 min (lanes 3 and 6), and then collected, permeabilized, and stained with anti-phospho p42/p44 MAPK monoclonal antibodies and anti-p42/p44 MAPK polyclonal antibodies recognizing phosphorylated (activated) and total p42/p44 MAPKs, respectively. Magnification, ×100.

FIG. 2.

Induction of ERK1/2 by KSHV. (A) ERK1/2 phosphorylation by different MOIs of KSHV. (Top band) Serum-starved HFF cells were either uninfected (lane 1) or infected with at different MOIs (DNA copies/cell) of KSHV for 30 min at 37°C (lanes 2 to 8). Ten micrograms of cell lysates was resolved by SDS-10% PAGE, subjected to Western blotting, and reacted with anti-phospho ERK1/2 antibodies. (B) Kinetics of ERK1/2 induction by KSHV. (Top band) Serum-starved HFF cells were either uninfected (lane 1) or infected at an MOI of 5 with KSHV for indicated time points (lanes 2 to 6), or treated with 20% FBS for 15 min (lane 7). Cell lysates were resolved by SDS-10% PAGE and subjected to Western blotting with anti-phospho ERK1/2 antibodies. (C) ERK1/2 phosphorylation in serum-fed HFF cells. (Top band) HFF cells grown in the presence of serum were either uninfected (lane 5) or infected with KSHV at an MOI of 5 for indicated time points (lanes 4 to 1). Cell lysates were resolved by SDS-10% PAGE and subjected to Western blotting with anti-phospho ERK1/2 antibodies. The bottom bands of panels A, B, and C show membranes that were stripped and reprobed with anti-ERK2 antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence reactions, and the band intensities were assessed. The ERK1/2 phosphorylation in the uninfected cells was considered 1 for comparison and expressed as an increase in phosphorylation (n-fold) of ERK1/2. Each point represents the average ± the standard deviation of three experiments. (D) KSHV infection triggers nuclear translocation of phosphorylated ERK1/2. Serum-starved HFF cells in eight-well chamber slides were either uninfected (frames 1 and 4) or infected with KSHV (MOI, 10) for 30 min (frames 2 and 5), or incubated with 20% FBS for 30 min (lanes 3 and 6), and then collected, permeabilized, and stained with anti-phospho p42/p44 MAPK monoclonal antibodies and anti-p42/p44 MAPK polyclonal antibodies recognizing phosphorylated (activated) and total p42/p44 MAPKs, respectively. Magnification, ×100.

FIG. 3.

Effect of UV inactivation on ERK1/2 induction. (A) Entry of UV-inactivated KSHV. HFF cells were infected with UV-inactivated KSHV or live KSHV (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. This was normalized, and internalized KSHV genomes per 100 ng of DNA samples, as measured by ORF 73 copies, were estimated by real-time DNA PCR. The CT values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. Data are presented as percentages of internalized viral DNA. Each reaction was done in duplicate, and each point represents the average ± standard deviation of three experiments. (B) Effect of UV inactivation on the kinetics of KSHV gene expression. HFF cells were infected with UV-inactivated KSHV or KSHV (10 DNA copies/cell). At indicated time points p.i., RNA was isolated and treated with DNase I, and 250 ng of DNase-treated RNA was subjected to real-time RT-PCR with ORF 73, ORF 50, K5, and K8 gene-specific primers and TaqMan probes (32). The relative copy numbers of viral transcripts were calculated using a standard graph generated by using known concentrations of DNase-treated in vitro transcribed ORF 73, ORF 50, K5, and K8 transcripts in real-time RT-PCR and normalized with GAPDH. Each reaction was done in duplicate and each point represents the average ± standard deviation of three independent experiments. (C) Kinetics of ERK1/2 induction by UV-inactivated KSHV. (Top band) Serum-starved HFF cells were either uninfected (lane 1) or infected with KSHV at an MOI of 10 (lane 3) or with UV-inactivated KSHV for indicated time points (lanes 4 to 7), or treated with 20% FBS for 15 min (lane 2). Membranes were stripped and reprobed with anti-ERK2 antibodies (bottom band). P-ERK, phosphorylated ERK.

FIG. 4.

Induction of MAPK-ERK1/2 by KSHV gpK8.1A and gB. (A, top band) Serum-starved HFF cells were either uninduced (lane 1) or induced with 20% FBS for 15 min (lane 2) or treated with different concentrations of ΔTMgB (lanes 3 to 6), ΔTMgpK8.1A (lanes 7 to 10), or ΔTMgB-RGA (lanes 11 to 14), or 4.0 μg/ml of ORF 73 (lane 15) proteins for 30 min. The increase in phosphorylation (n-fold) was quantitated as described in the legend Fig. 2A and C. Bottom band, total ERK2 (B) Kinetics of ERK1/2 induction by ΔTMgpK8.1A. (Top band) Serum-starved cells were uninduced (lane 1) or induced with 2.0 μg/ml of ΔTM gpK8.1A for 5, 15, 30, 60, and 120 min (lanes 2 to 6, respectively), or treated with FBS for 15 min (lane 7), or treated with 2.0 μg/ml ΔTMgB for 30 min (lane 8), or treated with 2.0 μg/ml ΔTMgB-RGA for 30 min (lane 9). Equal protein concentrations of cell lysates were immunoprecipitated with anti-ERK2 antibodies, and immune complexes were incubated with [γ-32P]ATP and myelin basic protein for 20 min at 30°C, boiled in sample buffer, and analyzed by SDS-12% PAGE. A portion of these immunoprecipitated samples were reacted in Western blot reactions with anti-total ERK2 antibodies (bottom band). Equal protein concentrations of cell lysates were reacted in Western blot reactions with anti-phospho ERK1/2 antibodies (middle panel). (C) Quantitation of ERK activity induced by ΔTMgpK8.1A. 32P-MBP bands were scanned and the increase in phosphorylation (n-fold) was quantitated as described in the legends of Fig. 2A and C. Each point represents the average ± the standard deviation of three experiments. p-ERK1/2, phosphorylated ERK1/2.

FIG. 5.

Characterization of ERK1/2 induction. (A) Kinetics of ERK1/2 induction in HMVEC-d. Serum-starved HMVEC-d were either uninduced (lane 14) or induced with DMEM containing 20% FBS for 15 min (lane 13) or treated with 2.0 μg/ml of ΔTMgpK8.1A (lanes 12 to 10), 2.0 μg/ml of ΔTMgB (lanes 9 to 7), ΔTMgB-RGA (lanes 6 to 4), or 2.0 μg/ml of ΔTM gpK8.1A (lanes 3 to 1) for indicated time points. Equal protein concentrations of cell lysates were resolved by SDS-PAGE and subjected to Western blotting with anti-phospho ERK1/2 antibodies (top band). Membranes were stripped and reprobed with anti-ERK2 antibodies (bottom band). The increase in phosphorylation (n-fold) was quantitated as described in the legends of Fig. 2A and C. Each blot is representative of at least three independent experiments. (B) Inhibition of ERK induction by heparin. Serum-starved HFF were either uninduced (lane 6) or induced with ΔTMgpK8.1A preincubated with 1, 10, and 100 μg/ml of heparin (lanes 4 to 2, respectively) or 100 μg/ml of chondroitin sulfate C (lane 1). Cell lysates were reacted in Western blots with anti-phospho ERK1/2 antibodies. Membranes were stripped and reprobed with anti-ERK2 antibodies (bottom band). ERK activity in cells incubated with ΔTMgpK8.1A protein alone was considered 100%, and data are presented as the percentage of inhibition of ERK phosphorylation. (C) Inhibition of ERK induction by ΔTMgpK8.1A neutralizing antibodies. Serum-starved HFF were induced with 2 μg/ml of ΔTMgpK8.1A for 30 min (lane 1), with gpK8.1A preincubated with different concentrations of anti-gpK8.1A MAb 4D6 (lanes 2 to 6), or with 100 μg/ml of anti-KSHV ORF 59 MAb 11D1 (lane 7) before being added to the cells. Lysates were subjected to a kinase assay (top band) as described in the legend of Fig. 4B or immunoprecipitates were Western blotted with anti-phospho ERK1/2 antibodies (bottom band). Equal protein concentrations of cell lysates were probed with anti-total ERK2 antibodies (bottom band). (D) Quantitation of ERK activity induced by ΔTMgpK8.1A. 32P-MBP bands were scanned and quantitated. ERK activity in HFF cells incubated with 2 μg/ml of ΔTMgpK8.1A protein was considered 100%, and data are presented as percentage of inhibition of ERK phosphorylation. Each point represents the average ± the standard deviation of three experiments. p-ERK1/2, phosphorylated ERK1/2.

FIG. 6.

ΔTMgpK8.1A induces MEK1/2. (A) Kinetics of MEK1/2 induction. (Top band) Serum-starved HFF were either infected with KSHV (5 DNA copies/cell) for 30 min (lane 1), uninduced for 60 min (lane 2), induced with ΔTMgpK8.1A for 5, 15, 30, and 60 min (lanes 3 to 6, respectively), or treated with DMEM containing 20% FBS for 15 min (lane 7). Western-blotted equal protein concentrations of cell lysates were reacted with anti-phospho MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle band) or with anti-β-actin antibodies (bottom band). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by ΔTMgpK8.1A. Serum-starved HFF cells were uninduced (lane 7) or induced with ΔTMgpK8.1A for 30 min (lane 6). Cells were also preincubated with either MEK1/2 inhibitor U0126 (lanes 2 to 5) or MEK1/2 inhibitor analogue U0124 (lane 1) for 1 h at 37°C and then induced with ΔTMgpK8.1A for 30 min in the presence of inhibitors. Cell lysates were subjected to Western blotting and reacted with anti-phospho ERK1/2 antibodies (top band) or with anti-ERK2 antibodies (bottom band). ERK activity in cells incubated with ΔTMgpK8.1A protein alone was considered 100%, and data are presented as percentage of inhibition of ERK phosphorylation. (C) KSHV and ΔTMgpK8.1A activate ERK1/2 but not ERK5. (Top band) Serum-starved HFF cells were either uninduced (lane 1), induced with 0.5 M sorbitol for different time points (lanes 2 to 5), or infected with KSHV (5 DNA copies/cell) for 30 min (lane 6) or 2 μg/ml of ΔTMgpK8.1A for 30 min (lane 7). Cell lysates were subjected to Western blotting and reacted with anti-phospho-ERK5 antibodies. Membranes were stripped and reprobed with anti-total ERK5 antibodies (bottom band). Each blot is representative of at least three independent experiments. The ERK5 phosphorylation in the uninduced cells was considered 1 for comparison. (D) KSHV and ΔTMgpK8.1A activate ERK1/2 but not p38 MAPK. (Top band) Serum-starved HFF were either uninduced (lane 1), induced with 0.5 M sorbitol for the indicated times (lanes 2 to 5), infected with KSHV (5 DNA copies/cell) for 30 min (lane 6), or treated with 2 μg/ml of ΔTM gpK8.1A for 30 min (lane 7). Cell lysates were subjected to Western blotting and reacted with anti-phospho p38 antibodies. Membranes were stripped and reprobed with anti-β-actin antibodies (bottom band). Each blot is representative of at least three independent experiments. The p38 phosphorylation in the uninduced cells was considered 1 for comparison.

FIG. 7.

Effect of ERK1/2 inhibition on virus entry and nuclear delivery of viral DNA. (A) Inhibition of ERK1/2 does not block viral DNA internalization. HFF cells or HFF cells incubated with different concentrations of inhibitors for 1 h at 37°C were infected with KSHV at 10 DNA copies/cell. For a control, virus was preincubated with 100 μg/ml of heparin for 1 h at 37°C before being added to the cells. 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. This was normalized, and number of KSHV genome copies were estimated by real-time DNA PCR of ORF73. The CT values were used to plot the standard graph and to calculate the relative copy numbers of viral DNA in the samples. Data are presented as the percentage of inhibition of KSHV DNA internalization obtained when the cells were incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of three experiments. **, statistically significant (P < 0.001). (B) Inhibition of MEK1/2 and ERK 1/2 does not influence the nuclear delivery of KSHV DNA. HFF cells or HFF cells preincubated for 1 h with nontoxic doses of LY294002 (50 μM), CdTxA (300 ng/ml), U0126 (10 μM), or U0124 (10 μM) were infected with 5 DNA copies/cell of KSHV for 3 h in the presence of inhibitors. Nuclear fractions were purified and assessed for purity, and total DNA was normalized to contain 100 ng/5 μl was analyzed by real-time DNA PCR using KSHV ORF73 primers. Copy standards and nontemplate controls were run in parallel. A standard graph generated by real-time PCR of known concentrations of a cloned ORF 73 gene was used to calculate the relative viral DNA copy numbers. Data are presented as percentages inhibition of KSHV DNA associated with the infected cell nuclei relative to cells incubated with virus alone. Each reaction was done in duplicate, and each bar represents the mean ± standard deviation of three experiments. **, statistically significant (P < 0.001).

FIG. 8.

Kinetics of KSHV ORF 73 and ORF 50 gene expression in the presence of U0126. (A and B) HMVEC-d and HFF cells were infected with KSHV (10 DNA copies/cell) for 2, 4, 8, and 24 h; RNA was isolated and treated with DNase I for 1 h. A total of 250 ng of DNase-treated RNA was subjected to real-time RT-PCR with ORF 73 and 50 gene-specific primers and TaqMan probes (32). Standard graphs generated using known concentrations of DNase-treated in vitro transcribed ORF 73 and ORF 50 transcripts were used to calculate the relative copy numbers of viral transcripts and were normalized with GAPDH. Each reaction was done in duplicate, and each point represents the average ± standard deviation of three independent experiments. **, statistically significant (P < 0.001). (C and D) Histogram depicting the percent inhibition of KSHV ORFs 73 and 50 expression in the presence of U0126. Data represent the average ± standard deviation of three experiments. **, statistically significant (P < 0.001).

FIG. 9.

Kinetics of KSHV K5, K8, and v-IRF-2 in the presence of ERK inhibitor U0126. (A and B) DNase-treated total RNAs isolated from HMVEC-d and HFF cells infected with KSHV (10 DNA copies/cell) with and without U0126 were subjected to real-time RT-PCR as described in the legend of Fig. 8. (C and D) Histogram depicting the percent inhibition of KSHV ORF expression in the presence of U0126. Data represent the average ± standard deviation of three experiments.

FIG. 10.

Effect of ERK inhibitor U0126 on host gene expression. (A) Semiquantitative RT-PCR confirmation of DUSP5 gene expression in U0126-treated and untreated KSHV-infected HFF cells. DNase-treated total RNAs were subjected to RT-PCR using specific primers. Successive samples were removed from every three cycles (14 to 41) and resolved on agarose gel, and the change in DUSP5 expression (n-fold) was calculated after normalizing to the β-actin gene. Ethidium bromide-stained RT-PCR amplified DUSP5 gene products after gel electrophoresis are shown. Representative amplification of the β-actin gene and reactions of DNA-PCR (-RT) are shown. (B) Quantitation of differential amplification of KSHV-infected and U0126-treated HFF cell RNA samples. IDVs 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 expression were created after normalizing to the β-actin gene. Representative graph depicts the change in DUSP5 expression (n-fold) as calculated from the change in the IDV. (C) Histogram representing the percent inhibition of host genes induced by KSHV infection in the presence of U0126. Expression of the specific gene by KSHV at indicated time points was considered 100%.

FIG. 11.

Effect of ERK inhibitor U0126 on the activation of MAPK-dependent transcription factors. (A) Monitoring MAPK-regulated transcription factor activity. Nuclear extracts prepared from uninfected HFF cells and HFF cells infected with KSHV for 120 min were tested for the activity of MAPK-regulated transcription factors by incubating the nuclear extracts to the plate-immobilized oligonucleotides containing various transcription factor-specific sites, followed by ELISA with phosphorylation-specific antibodies to the respective transcription factors. For competition, soluble oligonucleotides containing various transcription factor-specific sites (wild type) or its mutant form were added to the nuclear extracts. Positive controls provided for each transcription factor were used simultaneously. The histogram represents the activation levels of c-Jun, STAT1α, MEF2, c-Myc, ATF-2, and c-Fos in the nuclear extracts from KSHV-infected HFF cells. Data represent the average ± standard deviation of three experiments, and values shown here are after the subtraction of values from uninfected cells. (B) Histogram depicting the percent inhibition of DNA binding of MAPK-dependent transcription factors in nuclear extracts from HFF cells pretreated with U0126 and then infected with KSHV for different time points. Percent inhibition was calculated with respect to the DNA binding activities in KSHV-infected HFF cells without U0126 pretreatment. Data represent the average ± standard deviations of three experiments.

FIG. 12.

Effect of ERK inhibitor U0126 on AP1 binding to the ORF50 promoter. (A) EMSA experiment showing binding of nuclear extracts prepared from virus-infected and uninfected HFF pretreated or untreated with ERK inhibitor U0126 to the wild-type AP1 as well as RTA-AP1. Specificity of the DNA-protein interaction was assessed by competition EMSA using a 100 times the molar excess of unlabeled double-stranded oligonucleotide AP1 probe (lane 5) and unlabeled RTA-AP1 (lane 6) as competitors for respective treatments. Each EMSA is representative of at least three independent experiments. The binding of AP1 in the uninfected cells was considered 1 for comparison and expressed as the increase in DNA binding (n-fold) in nuclear extracts prepared from virus-infected HFF cells. (B) EMSA experiment showing the binding of proteins present in the nuclear extracts prepared from HFF cells infected with KSHV, with and without pretreatment with ERK inhibitor U0126 to the mutated target sites of AP1 (AP1m) and RTA (RTA-AP1m). Each EMSA is representative of at least three independent experiments.