Figure 1.
Effect of EphA2 knockdown on KSHV entry and infection in HFF cells.
(A) Control or different EphA2 shRNA-transduced HFF cells were infected with KSHV (30 DNA copies/cell) for 10 min at 37°C. After washing, total DNA was isolated and virus entry was determined by real-time DNA PCR for KSHV ORF73 gene. Results are presented as percentage of inhibition of KSHV DNA internalization by sh-EphA2 transduced compared to control shRNA transduced HFF cells incubated with virus. Data shown are means ± SD (n = 3; ** statistical significance, P<0.01). (B) Control or sh-EphA2 transduced (no 4) HFF cells were infected with KSHV for 2 h. At 48 h.p.i., cells were harvested for RNA isolation and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Data represented are means ± SD (n = 3; P<0.01). (C1 and C2) Control or sh-EphA2 transduced HFF cells were either mock infected or infected with KSHV (30 DNA copies/cell) for 2 h at 37°C, washed and cultured in complete media for another 46 h. After washing, cells were fixed and processed for immunofluorescence using rabbit anti-LANA-1 antibody. Representative images are shown. The percentage of cells showing punctate LANA -1 dots is represented in the graphical plot. A minimum of 3 fields having at least 20 cells were chosen. Error bars show means ± SD.
Figure 2.
EphA2 is activated and associated with entry receptors early during KSHV infection.
(A) Serum-starved (8 h) HFF cells were either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for the indicated time periods and subjected to Western blot analysis for phospho-EphA2 (Y594) (pEphA2). The blot was stripped and reprobed for total EphA2 and tubulin was used as loading control. (B) Serum starved HFF cells were either mock infected or infected with KSHV (30 DNA copies/cell) for 5 and 10 min and processed for immunofluorescence analysis using rabbit anti-EphA2 and mouse monoclonal anti-KSHV gpK8.1A antibodies for overnight at 4°C followed by staining with anti-rabbit Alexa 488 and anti-mouse Alexa 594 secondary antibodies. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules. (C) Serum-starved HFF cells were left uninfected or infected with KSHV for the indicated periods of time and immunoprecipitated with anti-α3β1, αVβ3 or αVβ5 antibodies and analyzed for EphA2 by Western blot (first, third and fifth panels). These blots were stripped and reprobed for total β1, β3 and β5 integrin subunits, respectively (second, fourth and sixth panels). For negative control, cell lysates from uninfected or KSHV infected HFF cells were immunoprecipitated with anti-β6 antibody and analyzed for EphA2 by western blotting (seventh panel). The blot was stripped and reprobed with total β6 antibodies (eighth panel). Whole cell lysates were subjected to western blot analysis for input expression of EphA2 (ninth panel) and β6 (tenth panel) and β-actin was used as loading control (eleventh panel). (D) Serum starved HFF cells were either left uninfected or infected with KSHV (30 DNA copies/cell) for 5, 10 and 30 min, washed and processed for immunofluorescence analysis. Cells were permeabilized, blocked with blocking reagents and incubated with (D1) rabbit anti-EphA2 and mouse anti-αVβ3 antibodies or (D2) rabbit anti-EphA2 and mouse anti-αVβ5 antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 or anti-mouse Alexa fluor 594. Representative 2D deconvoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules.
Figure 3.
Endocytic effectors critical for clathrin mediated entry are activated and associated with EphA2 early during KSHV infection.
(A) Serum-starved HFF cells were either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for the indicated time periods and subjected to Western blot analysis for phospho-c-Cbl (p-c-Cbl) (Y731) and phospho-myosin IIA (S-19). For clathrin activation, cell lysates were subjected to immunoprecipitation with anti-clathrin heavy chain antibody followed by Western blot analysis with anti-phosphotyrosine antibody (4G10). Blots were stripped and reprobed for the respective total c-Cbl, myosin and clathrin. (B,C and D) Serum-starved HFF cells were left uninfected (UI) or infected with KSHV for the indicated periods of time and immunoprecipitated with anti-EphA2 antibodies and analyzed for c-Cbl, clathrin or clathrin adaptors AP2 and Epsin15 by Western blots which were stripped and reprobed for EphA2. For heparin treatment of KSHV, virus was pre-incubated with heparin (100 µg/ml) for 1 h at 37°C and used for infecting cells for 5 min. (E) Serum-starved HFF cells either uninfected or infected with KSHV for the indicated time points were subjected to immunofluorescence assay using mouse anti-c-Cbl and rabbit anti-myosin IIA antibodies for 2 h at room temperature followed by staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 594, respectively. Representative 2D convoluted images are shown (E1). The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules. Quantitation of cells showing c-Cbl and myosin IIA colocalization (E2). At least three different microscopic fields having at least 10 cells each were chosen to analyze the colocalization efficiency. Error bars represent mean ± SD, * represents p<0.05 and ** represents p<0.01.
Figure 4.
Activated EphA2 colocalizes with activated c-Cbl and clathrin.
Serum-starved HFF cells either uninfected or infected with KSHV (30 DNA copies/cell) for the indicated time points were subjected to immunofluorescence assay using rabbit anti-phospho-EphA2 (p-EphA2) and (A) mouse anti-phospho-c-Cbl or (B) clathrin heavy chain antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 and anti-mouse Alexa fluor 594, respectively. Representative 2D convoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules.
Figure 5.
EphA2 associated with integrins and clathrin are localized to non-lipid rafts in KSHV infected HFF cells.
(A) Serum-starved HFF cells were either uninfected or infected with KSHV (30 DNA copies/cell) for the indicated time points. LR and non-LR fractions were isolated and analyzed by Western blotting for the presence of EphA2. Caveolin-1 and CD71 characterize the purity of LR and non-LR fractions, respectively. (B) Western immunoblot analysis showing phosphorylation of EphA2 at the indicated time post-KSHV infection in non-NLR fractions from HFF cells. (C) 150 µg of protein from non-LR fractions of uninfected and KSHV-infected cells was immunoprecipitated with anti- αVβ5 or anti-EphA2 antibodies at 4°C overnight followed by Western blotting with EphA2 or clathrin heavy chain, respectively. Blots were stripped and reprobed for β5 and β3, respectively. CD71 characterizes the purity of non-LR fractions.
Figure 6.
EphA2 recruits KSHV-induced signal molecules necessary for virus entry.
(A) Serum-starved HFF cells were either mock infected or KSHV infected for the indicated time points and immunoprecipitated with EphA2 antibody followed by Western blot analysis for FAK, Src and PI3-K. For heparin treatment of KSHV, KSHV was pre-incubated with heparin (100 µg/ml) for 1 h at 37°C and used for infecting cells for 5 min followed by cell lysate preparation to confirm specificity of virus induced signaling. The blots were stripped and reprobed for total EphA2 as indicated. (B) Serum-starved HFF cells either uninfected or infected with KSHV for the indicated time points were subjected to immunofluorescence assay using rabbit p-EphA2 and either mouse anti-p-FAK (B1), anti-p-Src (B2) or anti-p-PI3-K (B3) antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 and anti-mouse Alexa fluor 594, respectively. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules.
Figure 7.
Effect of EphA2 knockdown on the activation of KSHV induced signalling molecules as well as endocytic effectors.
(A) Control or EphA2 shRNA-transduced HFF cells were either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for the indicated time period and subjected to Western blot analysis for phospho-FAK (p-FAK) (Y397), phospho-Src (Y-416) and phospho-PI3-K (Y458). Blots were stripped and reprobed for the respective total FAK, Src and PI3-K. Efficiency of EphA2 knockdown was analyzed by Western blot with EphA2 antibody and β-actin was used as loading control. (B) Control or EphA2 shRNA transduced HFF cells either mock infected or infected with KSHV (30 DNA copies/cell) were subjected to Western blot analysis for activation (phosphorylation) of the indicated endocytic effector molecules (c-Cbl, myosin IIA and clathrin). For clathrin activation, cell lysates were subjected to immunoprecipitation with clathrin heavy chain antibody followed by Western blot analysis with anti-phosphotyrosine antibody (4G10). Blots were reprobed for the respective total c-Cbl, myosin IIA and clathrin for equal expression. The levels of fold activation are indicated.
Figure 8.
EphA2 knockdown has a negative effect on KSHV trafficking in the early endosome due to defective endocytosis.
(A) Control sh-RNA or EphA2-sh-RNA-transduced or untransduced HFF cells were serum starved and either mock infected (UI) or infected with KSHV (30 DNA copies/cell) for 5 min at 37°C and were processed for immunofluorescence assay using clathrin adaptor AP2 and mouse-gpK8.1A antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 or mouse Alexa fluor 594. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules. Representative 2D convoluted images are shown (A1). Quantification of KSHV particles colocalized with AP2 molecules was performed by counting three different fields having atleast 10 cells each and error bars represent mean ± SD, ** represents p<0.01 (A2). (B) Control or EphA2 sh-RNA-transduced HFF cells either mock infected (UI) or infected with medium containing Alexa 594-transferrin along with KSHV (30 DNA copies/cell) for 5 min at 37°C were subjected to immunofluorescence assay using rabbit anti-gB antibody for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488. Representative 2D convoluted images are shown (B1). The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules. Quantification of KSHV particles colocalized with Transferrin molecules was performed by counting three fields with atleast 10 cells each and error bars represent mean ± SD, ** represents p<0.01 (B2). (C) (C1) Serum starved control sh-RNA or EphA2 sh-RNA transduced HFF cells, either mock infected or infected with KSHV for 10 min at 37°C, were washed and processed for immunofluorescence using rabbit anti Rab5 and mouse anti-gpK8.1A antibodies for 2 h at room temperature followed by staining with anti-rabbit Alexa fluor 488 or mouse Alexa fluor 594. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules. (C2) Quantitation of KSHV accumulated in Rab5 positive endosomes was performed by counting three different fields having at least 10 cells each and error bars represent mean ± SD, ** represents p<0.01.
Figure 9.
c-Cbl directed polyubiquitination of EphA2 early during KSHV infection.
(A) Control or c-Cbl-siRNA-transfected HFF cells either mock infected (UN) or infected with KSHV (30 DNA copies/cell) for 5 min were lysed and subjected to Western blot analysis for c-Cbl expression (First panel). β-actin was used as loading control (second panel). 200 µg of the lysates prepared under the stringent conditions for ubiquitination were immunoprecipitated with anti-EphA2 antibody followed by Western blotting with either total anti-ubiquitin antibody P4D1 (mouse anti-mono- and anti-polyubiquitin antibody) (third panel) or FK-1 antibody detecting polyubiquitination (Fourth panel). The blot was stripped and reprobed for EphA2 (Fifth panel). For heparin treatment of KSHV, KSHV was pre-incubated with heparin (100 µg/ml) for 1 h at 37°C and used for infecting cells for 5 min. (B) Validation of the differential nature of EphA2 polyubiquitination. 200 µg of the whole-cell lysate prepared under stringent conditions from fig. 9. A experiment was immunoprecipitated with rabbit anti-EphA2 antibody and was subjected to Western blot analysis for either Lys-48 (first panel) or Lys-63 (second panel) specific polyubiquitination. Blots were stripped and reprobed for EphA2. For heparin treatment of KSHV, KSHV was pre-incubated with heparin (100 µg/ml) for 1 h at 37°C and used for infecting cells for 5 min followed by cell lysate preparation to confirm specificity of virus induced events. (C) Schematic representation of Myc-EphA2 deletion mutants and HA-c-Cbl substitution mutants in comparison to the wild type proteins (Wt). For EphA2 constructs, numbers indicate amino acid position within the sequence. TM, Transmembrane; KD, Kinase domain; SAM, Sterile alpha motif. For c-Cbl mutants amino acid substitutions are indicated. Glycine (G) was substituted to Glutamic acid (E). C1–C7 represents conserved Cysteine residues and H represents the Histidine. Substitutions to either Alanine (A) or Asparagine (N) were made at the indicated positions. TKB, Tyrosine kinase binding domain; RF, Ring finger domain; PRO, Proline rich region; LZ, Leucine zipper. (D) HEK 293 cells were transiently cotransfected with Myc-EphA2 (Wt, ΔKD and ΔSAM) and HA-c-Cbl (Wt, Cbl-C3HC4C5 and Cbl-G306E) as indicated or with vector controls. 48 h post-transfection, cells were mock infected or infected with KSHV (30 DNA copies/cell) for 5 min and cell lysates prepared under stringent conditions were used for coimmunoprecipitation with Myc antibody followed by Western blot analysis with polyubiquitin specific FK-1 (First panel, low exposure blot; second panel, high exposure blot). To analyse Myc-EphA2 and HA-cCbl interaction in a separate experiment, lysates from HEK 293 cells overexpressing different combinations of Myc-and HA-tagged Wt and mutant proteins were immunoprecipitated with anti-Myc antibody followed by western blot with HA antibody (Third panel). Blots were stripped and reprobed for Myc (Fourth panel). Whole cell lysates were subjected to Western blot analysis for HA expression (input) (Fifth panel) and β-actin from whole cell lysates was used as loading control (Sixth panel).
Figure 10.
Effect of c-Cbl knockdown on the association of EphA2 with clathrin.
(A) Control or c-Cbl-si-RNA-transfected HFF cells either mock infected (UI) or infected with KSHV (30 DNA copies/cell) or KSHV pre-incubated with heparin (100 µg/ml) for 1 h at 37°C, for 5 min were lysed and subjected to immunoprecipitation with EphA2 antibody followed by western blot analysis with anti-Clathrin antibody. Blot was stripped and reprobed for EphA2. (B) Serum-starved control si-RNA or c-Cbl-si-RNA transfected HFF cells either uninfected or infected with KSHV for 5 min were subjected to immunofluorescence assay using rabbit p-EphA2 and mouse anti-clathrin antibodies overnight at 4°C followed by staining with anti-rabbit Alexa fluor 488 and anti-mouse Alexa fluor 594, respectively. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules.
Figure 11.
c-Cbl regulates clathrin mediated productive entry of KSHV without affecting its association with EphA2.
(A and B) Serum starved, control si-RNA or c-Cbl-si-RNA transfected, HFF cells either uninfected or infected with KSHV for 5 min were subjected to immunofluorescence assay using anti-rabbit KSHV gB and mouse anti-clathrin antibodies (A1) or anti-rabbit EphA2 and anti-mouse KSHV gpK8.1 antibodies (B1), respectively, overnight at 4°C followed by staining with either anti-rabbit Alexa 594 and anti-mouse Alexa 488 (A1) or vice versa (B1). Representative 2D convoluted images are shown. The enlarged pictures represent boxed regions within the merged panels and the white arrows represent colocalization of the indicated molecules. (A2 and B2) Quantification of KSHV colocalized with clathrin or EphA2 was performed by counting three different fields having atleast 10 cells each and error bars represent mean ± SD, ** represents p<0.01. (C) Control or c-Cbl-si-RNA-transfected HFF cells were incubated with medium containing LysoTracker green along with or without KSHV (30 DNA copies/cell) for 30 min at 37°C and were processed for immunofluorescence assay using anti-gB antibody followed by Alexa 594 anti-rabbit secondary antibody. Representative 2D convoluted images are shown. The white boxes within the merged panels are shown as enlarged pictures and the white arrows represent colocalization of the indicated molecules.
Figure 12.
Schematic model depicting the proposed mechanism of EphA2 dependent KSHV entry in human foreskin fibroblast cells via clathrin mediated endocytosis.
(1) Binding and interaction of KSHV with HFF cell surface heparan sulfate and integrins (αVβ3, αVβ5 or α3β1) is followed by their association with EphA2 in the non-LR region. (2) EphA2 coordinates formation of active signaling complex among integrins, c-Cbl and myosin IIA with concomitant induction of FAK, Src and PI3-K signaling necessary for endocytic entry of associated virus. (3) c-Cbl directed polyubiquitination (K63-linked) of EphA2 helps interaction with accessory proteins Epsin15 and adaptor protein AP-2. (4) These interactions promote activation, recruitment and assembly of clathrin to the formation of clathrin coated pits (CCP). (5) Together, such complex signaling and associated events trigger internalization of KSHV into clathrin coated pits, dynamin dependent release of endocytic vesicles [22] that lead to trafficking of KSHV into the Rab5 early endosome followed by a productive KSHV infection and gene expression.