HHV-8-target cell interaction induces the actin cytoskeleton reorganization. Serum-starved HFF cells incubated with different ligands at 37°C were collected at various time points, fixed, permeabilized, and stained for polymerized actin by rhodamine-labeled phalloidin for 20 min at room temperature. (A) Unstimulated control cells; (B) cells incubated with 20 ng of LPA/ml for 5 min; (C and D) cells incubated with DMEM with 10% FBS for 15 and 30 min, respectively; (E) cells mock infected for 5 min; (F, G, and H) cells infected with GFP-HHV-8 for 5, 15, and 30 min, respectively. Open arrowheads indicate accumulated actin stress fibers. Closed arrowheads indicate hair-like membrane filopodia extensions. Arrows indicate sites of membrane ruffling.

Activation of ERK by HHV-8 is via the recruitment of PKC-ζ in a PI 3-kinase-dependent manner. (A) Kinetics of PKC-ζ activation by HHV-8. Lysates from mock-infected, HHV-8 infected, or known PKC-ζ inducer (LPS and dPP)-treated HFF cells were immunoprecipitated with anti-PKC-ζ antibodies and subjected to in vitro kinase assays with [γ-³²P]ATP and MBP as described for Fig. 3A (top panel). Western blotted immune complexes were tested with anti-PKC-ζ antibody (bottom panel). (B and C) PI 3-kinase inhibitors inhibit PKC-ζ activation by HHV-8. HFF cells were preincubated with different concentrations of the PI 3-kinase inhibitors wortmannin and LY294002 for 1 h at 37°C and infected with HHV-8 for 30 min. Lysates were tested in Western blot analysis with either phospho-PKC-ζ antibodies (top panel) or PKC-ζ antibody (lower panel). (D and E) PI 3-kinase and PKC-ζ inhibitors inhibit ERK1/2 activation. HFF cells were preincubated with PI 3-kinase or PKC-ζ inhibitors for 1 h at 37°C and then infected with HHV-8 for 30 min, and lysates tested in Western blots with phospho-ERK1/2 or total ERK2 (top and bottom panels, respectively).

Integrin α3β1 is one of the critical elements required for the signal induction by HHV-8. (A) Surface expression of human integrin α3 in CHO cells. CHO-B2 cells transfected with pCDNA3-human α3 integrin cDNA (CHO-B2-B3) or with control pCDNA3 plasmid (CHO-B2-pCDNA3) were grown in eight-well chamber slides, fixed with 0.1% paraformaldehyde, and tested by surface IFA staining with anti-human integrin α3 antibodies. (B) Induction of ERK1/2 by HHV-8 in human integrin α3 expressing CHO cells. Serum-starved CHO-B2-B3 and CHO-B2-pCDNA cells were either mock infected or infected with HHV-8 for 30 min. In parallel experiments, HHV-8 was incubated with 5 μg of soluble α3β1 or α5β1 integrin/ml for 1 h at 37°C before infection of the cells. ERK1/2 was immunoprecipitated and subjected to in vitro kinase assays (top panel) and immunoblot (bottom panel) as described for Fig. 3A. In the middle panel, ³²P-labeled MBP bands (top panel) were quantitated, and the ERK activity in mock-infected HFF cells was considered as “1” for comparison with the fold induction in the infected cells.

HHV-8 activates PI 3-kinase in the target cells via its interactions with α3β1 integrin. (A) Induction of PI 3-kinase by HHV-8. Serum-starved HFF cells were mock infected (lane 1) or infected with HHV-8 (lane 2) for 5 min. Cells were also preincubated with the PI 3-kinase inhibitors wortmannin or LY294002 for 1 h at 37°C and then infected with virus in the presence of inhibitors for 5 min (lanes 3 and 4). The phosphotyrosine residue in PI 3-kinase was dephosphorylated by incubating the immunoprecipitates with 0.5 U of PTPase 1B for 2 h at 30°C before an assay for PI 3-kinase activity (lane 5) was carried out. Virus was also preincubated with 5 μg of soluble α3β1 or α5β1/ml for 1 h at 37°C before infection of the cells (lanes 6 and 7). Equal protein concentrations of cell lysates were immunoprecipitated with PY20 antibodies, assayed for PI 3-kinase activity by using sonicated PIP₂ as the substrate, and resolved with radiolabeled PIP₃ by ascending thin-layer chromatography, and PIP₃ spots were detected by autoradiography. (B) Kinetics and quantitation of PI 3-kinase activity induced by HHV-8. Serum-starved HFF cells were mock infected for 15 min or infected with HHV-8 for the indicated time points or treated with FBS for 15 min and assayed for PI 3-kinase activity as described in Fig. 2A. Radioactive PIP₃ spots were quantitated and expressed as fold change of PI 3-kinase activity over mock-infected cells.

HHV-8 induces MEK1/2 but not cRaf-1 in the target cells. (A) Kinetics of MEK1/2 induction. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with 20% FBS for the indicated times. Cell lysates were resolved by SDS-10% PAGE and probed with anti-phospho-MEK1/2 antibodies. Membranes were stripped and reprobed with anti-MEK1/2 antibodies (middle panel) or with anti-β actin antibodies (bottom panel). (B) Inhibition of MEK1/2 inhibits the ERK1/2 activity induced by HHV-8. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 for 15 min (lane 2) or 30 min (lane 4), or incubated with 20% FBS for 15 min (lane 6). Cells were also preincubated with 10 μM MEK1/2 inhibitor U0126 for 1 h at 37°C and infected with virus in the presence of inhibitors for 15 and 30 min (lanes 3 and 5, respectively). Cell lysates were Western blotted and reacted with anti-phospho-ERK1/2 antibodies (top panel) or with anti-ERK2 antibodies (bottom panel). (C) HHV-8 does not induce cRaf-1 in the target cells. Serum-starved HFF cells were mock infected (lane 1), infected with HHV-8 (lanes 2 to 6), or treated with LPS (1 μg/ml; lane 7) or TPA (10 nM; lane 8) for the indicated times. Lysates were either immunoprecipitated with total cRaf-1 antibody and subjected to a kinase assay with MEK as substrate (top panel) or used for Western analysis with either phospho-cRaf-1 or total cRaf-1 antibody (middle and lower panels, respectively).

Activation of MAPK-ERK1/2 by HHV-8 during early times postinfection. (A) Kinetics of MAPK-ERK1/2 induction in HFF cells by HHV-8. Serum-starved HFF cells were either mock infected (lane 1), infected with HHV-8 for different time points (lanes 2 to 6), treated with DMEM containing 20% FBS for 15 min (lane 7), or infected with HSV-2 at an MOI of 5 for 60 min (lane 8). Equal protein concentrations of cell lysates were immunoprecipitated with anti-ERK antibodies, and immune complexes were incubated with [γ-³²P]ATP and MBP for 20 min at 30°C, boiled in sample buffer, and analyzed by SDS-12% PAGE. A portion of the lysate was resolved by SDS-10% PAGE and subjected to Western analysis with anti-phospho-ERK1/2 antibodies (middle panel). Equal total lysate being resolved is shown by probing with anti-ERK2 antibodies (bottom panel). The bands were scanned, and intensities were assessed. (B) Quantitation of ERK activity induced by HHV-8. ³²P-labeled MBP bands were quantitated, and the ERK activity in mock-infected HFF cells was considered as “1” for comparison to infected cells. (C) Kinetics of ERK1/2 induction in HMVEC-d cells. In the top panel, serum-starved HMVEC-d cells were either mock infected (lane 1) or infected with HHV-8 for different time points (lanes 2 to 6). Cell lysates were resolved by SDS-PAGE and Western blotted with anti-phospho-ERK1/2 antibodies. Membranes were then stripped and reprobed with anti-ERK2 antibodies (bottom panel). (D) Inhibition of ERK induction by HHV-8 neutralizing antibodies. Serum-starved HFF cells were mock infected (lane 1) or infected with HHV-8 (lane 2), with virus preincubated with different concentrations of rabbit anti-HHV-8 gB IgG antibodies (lanes 3 to 6)or preimmune IgG antibodies (lane 7) for 30 min. Lysates were subjected to a kinase assay (top panel) or immunoprecipitate was Western blotted with ERK2 antibodies (bottom panel) as described in the Fig. 3A legend. (E) HHV-8 activates MAPK-ERK1/2 but not p38-MAPK. Serum-starved HFF cells were mock infected, infected with HHV-8, or treated 0.5 M sorbitol for the indicated times. Cell lysates were tested with anti-phospho p38-MAPK antibodies (top panel) or with anti-β actin antibodies (lower panel).

FAK is essential for signal induction by HHV-8. (A) FAK and ERK expression in Du cells. Primary mouse embryonic fibroblasts dominant negative for FAK (Du3^−/−) and control FAK positive (Du17^+/+) were grown in chamber slides, washed, fixed with acetone, and reacted with anti-FAK or anti-ERK antibodies and fluorescein isothiocyanate- and rhodamine-labeled second antibodies. (B) FAK expression is essential for HHV-8 infectivity in the target cells. Du17/Du3 cells grown in eight-well chamber slides were infected with 100 μl of GFP-HHV-8, incubated at 37°C for 2 h, washed, and incubated with growth medium for 3 days. GFP-expressing cells were enumerated under a fluorescence microscope. Several areas of Du3 cells in a single chamber need to be searched to identify a single GFP-expressing cell. An average of eight GFP-expressing cells per well was seen in the Du3 cells infected with 100 μl of GFP-HHV-8 compared to 300 green fluorescent cells in the Du17 cells. (C) FAK expression is not essential for HHV-8 binding to the target cells. [³H]thymidine labeled, purifiedHHV-8 (5,000 cpm) was mixed with 10 μg of heparin or chondrotin sulfate A/ml for 90 min at 4°C and then added to Du3 and Du17 cells. After incubation for 90 min at 4°C with the virus, cells were washed, lysed, and precipitated with trichloroacetic acid, and the cell-associated virus counts per minute (cpm) were counted. In the absence of heparin, ca. 21% of the input HHV-8 radioactivity became associated with the cells. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments. (D) FAK expression is essential for ERK induction by HHV-8. Serum-starved Du17 and Du3 cells were either mock infected or infected with HHV-8 for 30 min. In another experiments, cells were incubated with U0126 for 1 h, and infected with HHV-8 for 30 min in the presence of inhibitor. ERK1/2 was immunoprecipitated and subjected to in vitro kinase assays (top panel), or total lysate was immunoblotted with anti-phospho-ERK1/2 antibodies (middle panel) or with total anti-ERK-2 antibodies (bottom panel) as described for Fig. 3A. ³²P-labeled MBP bands were quantitated, and the ERK activity in mock-infected cells was considered as “1” for comparison with infected cells.

Induction of cellular kinases by HHV-8 is essential for infectivity. (A) Neutralization of GFP-HHV-8 infectivity by inhibitors of PI 3-kinase, PKC-ζ, and MEK. HFF cell monolayers in eight-well chamber slides were incubated with DMEM containing different concentrations of nontoxic doses of LY294002 (PI 3-kinase inhibitor), myr-ζ (PKC-ζ specific inhibitory peptide), and U0126 (MEK inhibitor) at 37°C for 1 h before infection with GFP-HHV-8. After incubation for 2 h at 37°C with virus in the presence of inhibitors, cells were washed and further incubated with growth medium for 3 days at 37°C. Green fluorescent cells indicative of GFP-HHV-8 entry and infection were counted. In control wells, ca. 300 green fluorescent HFF cells per well were detected. Each reaction was done in triplicate and each point represents the average ± the standard deviation of three experiments. (B) ERK1/2 antisense oligonucleotide decreases the expression of ERK. HFF cells were transfected with AS-1/2 or control SC-1/2 oligonuclotides with Lipofectin at the indicated concentrations for 48 h. Cell lysates were analyzed by Western blot analysis with ERK2 antibodies (top panel) or β-actin (bottom panel) (C) ERK1/2 antisense oligonucleotide treatment reduces the infectivity of HHV-8. HFF cells were transfected with AS-1/2 or control SC-1/2 oligonucleotides for 48 h, infected with GFP-HHV-8 for 2 h at 37°C in the presence of oligonucleotides, washed, and incubated for 3 days at 37°C with growth medium containing the same concentration of oligonucleotides. Green fluorescent cells indicative of GFP-HHV-8 entry and infection were counted. In control wells, ca. 300 green fluorescent HFF cells per well were detected. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments. (D) Effect of PI 3-kinase, PKC-ζ, MEK, and ERK inhibitors on HHV-8 internalization into the target cells. HFF cells were incubated at 37°C for 1 h with neutralizing concentrations of wortmannin (300 nM), LY294002 (100 μM), myr-ζ (10 μM), U0126 (100 μM), and controls (DMSO; myr-α/β) or transfected with AS-1/2 (0.5 μM), and SC-1/2 for 48 h at 37°C. Untreated HFF cells or HFF cells pretreated with various inhibitors were infected with GFP-HHV-8 at an MOI of ∼5 73-IU for 2 h in the presence of the inhibitors. After infection, unbound HHV-8 was removed by washing the cells. Bound, noninternalized virus was removed by treating the cells with trypsin-EDTA for 5 min. Cells were washed, and the internalized viral DNA was isolated. To quantitate the HHV-8 PCR assays, a standard was prepared by cloning the ORF 25 PCR product into the pGEM-T vector and quantitated by UV absorption. Log₁₀ dilutions of the standard DNA (10⁶ to 10¹ copies) were amplified along with the virus stock and internalized DNA. The products were resolved on a 1.2% agarose gel stained with ethidium bromide, and the intensity was measured. The composite shows the PCR results from the copy number standard (top panel) and samples from HHV-8-infected untreated HFF cells and cells treated with the various kinase inhibitors. (E) Quantitation of the effect of kinase inhibitors on HHV-8 DNA internalization. The intensities of the bands in the agarose gels were scanned, and the copy numbers of internalized HHV-8 DNA in the absence and in the presence of kinase inhibitors were calculated by extrapolating the intensity of the band against the standard curve. Approximately 29% of the input vial DNA was internalized. Each reaction was done in triplicate, and each point represents the average ± the standard deviation of three experiments.