CIB1 synergizes with EphrinA2 to regulate Kaposi's sarcoma-associated herpesvirus macropinocytic entry in human microvascular dermal endothelial cells

Abstract

KSHV envelope glycoproteins interact with cell surface heparan sulfate and integrins, and activate FAK, Src, PI3-K, c-Cbl, and Rho-GTPase signal molecules in human microvascular dermal endothelial (HMVEC-d) cells. c-Cbl mediates the translocation of virus bound α3β1 and αVβ3 integrins into lipid rafts (LRs), where KSHV interacts and activates EphrinA2 (EphA2). EphA2 associates with c-Cbl-myosin IIA and augmented KSHV-induced Src and PI3-K signals in LRs, leading to bleb formation and macropinocytosis of KSHV. To identify the factor(s) coordinating the EphA2-signal complex, the role of CIB1 (calcium and integrin binding protein-1) associated with integrin signaling was analyzed. CIB1 knockdown did not affect KSHV binding to HMVEC-d cells but significantly reduced its entry and gene expression. In contrast, CIB1 overexpression increased KSHV entry in 293 cells. Single virus particle infection and trafficking during HMVEC-d cell entry was examined by utilizing DiI (envelope) and BrdU (viral DNA) labeled virus. CIB1 was associated with KSHV in membrane blebs and in Rab5 positive macropinocytic vesicles. CIB1 knockdown abrogated virus induced blebs, macropinocytosis and virus association with the Rab5 macropinosome. Infection increased the association of CIB1 with LRs, and CIB1 was associated with EphA2 and KSHV entry associated signal molecules such as Src, PI3-K, and c-Cbl. CIB1 knockdown significantly reduced the infection induced EphA2, Src and Erk1/2 activation. Mass spectrometry revealed the simultaneous association of CIB1 and EphA2 with the actin cytoskeleton modulating myosin IIA and alpha-actinin 4 molecules, and CIB1 knockdown reduced EphA2's association with myosin IIA and alpha-actinin 4. Collectively, these studies revealed for the first time that CIB1 plays a role in virus entry and macropinocytosis, and suggested that KSHV utilizes CIB1 as one of the key molecule(s) to coordinate and sustain the EphA2 mediated signaling involved in its entry, and CIB1 is an attractive therapeutic target to block KSHV infection.

Figure 1. Effect of CIB1 knockdown on de novo KSHV entry.

(A) HMVEC-d cells were either untransduced or transduced with sh-control or sh-CIB1 expressing lentivirus particles and selected with puromycin. Knockdown of CIB1 protein expression was examined by Western blotting for CIB1 (specific shRNA target), tubulin, and integrin β3 (off-target molecules). (B and C) Untransduced, control-shRNA and CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell) for 1 h at 4°C (B) or 2 h at 37°C (C) for binding and entry experiments, respectively. Post-washing, total DNA was isolated and KSHV binding and entry were determined by real-time DNA-PCR for the KSHV ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. For binding studies, results are presented as KSHV DNA copies bound to CIB1-shRNA-transduced and control-shRNA-transduced cells. For entry studies, results are presented as percentage of inhibition of KSHV DNA internalization by sh-CIB1 or sh-control compared with infected untransduced cells. **P = 0.002. (D) Control-shRNA and CIB1-shRNA transduced HMVEC-d cells were infected with DiI-(viral envelope) labeled KSHV (40 DNA copies/cell) for 30 min at 37°C. After washing, cells were processed for immunofluorescence microscopy and co-stained with DAPI. DiI-KSHV was observed with the Alexa-594 filter. White arrows indicate DiI-KSHV staining. (E) (i) Control-shRNA and CIB1-shRNA transduced HMVEC-d cells were infected with BrdU (viral DNA) labeled KSHV (20 DNA copies/cell) for 30 min at 37°C. After washing, cells were fixed, permeabilized, treated with 4N HCl for 10 min to expose BrdU residues, stained with anti-BrdU antibodies followed by Alexa 488-anti-mouse antibodies, co-stained for DAPI, and examined by immunofluorescence. White arrows indicate BrdU positivity. Representative images are shown. (E) (ii) The percentage of cells observed positive for BrdU staining in IFA is presented graphically. A minimum of five independent fields, each with at least 6 cells were chosen. *P = 0.05.

Figure 2. Effect of CIB1 knockdown on de novo KSHV infection.

(A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

Figure 3. CIB1 association with KSHV in macropinocytic blebs early during de novo KSHV infection in endothelial cells.

(A) Uninfected HMVEC-d cells or cells infected with KSHV (20 DNA copies/cell) for 5 min or 30 min were reacted with anti-CIB1 antibodies and anti-KSHV glycoprotein gB antibodies, and co-stained with DAPI. Boxed areas from the merged images are enlarged in the rightmost panel and arrows indicate colocalization of CIB1 with virus particles at the indicated time points. (B) Infected cells at 10 min p.i. were stained for KSHVgB, CIB1, and DAPI, which were merged with the deconvoluted DIC images. Arrows indicate the association of viral particles with CIB1 in KSHV induced blebs. (C) DIC images show KSHV induced membrane blebs in HUVEC cells at 5 and 10 min post-infection.

Figure 4. CIB1 colocalization with purified recombinant KSHV glycoproteins.

HMVEC-d cells were left untreated or treated with 3 µg of purified recombinant gBΔTM (A) or ΔTMgpK8.1A (B) proteins at 4°C for 1 h for binding and transferred to 37°C for 10 min for subsequent internalization. Cells were fixed, permeabilized, blocked, stained for respective glycoproteins and co-stained for CIB1 to examine colocalization using immunofluorescence microscopy. Representative deconvoluted images are shown. White arrows indicate colocalization. Boxed areas are enlarged in the right most panels.

Figure 5. Transmission electron microscopic observation of HMVEC-d cells early during KSHV infection.

HMVEC-d cells (10⁶) were infected with purified KSHV (20 DNA copies/cell) at 37°C for 5 and 10 min. Post-infection, cells were washed, fixed, processed for transmission EM, and embedded in 812 resin. Thin sections were made and visualized under a JEOL 100CXII transmission electron microscope. (A–I) Red arrows indicate HMVEC-d cell membrane protrusions induced by enveloped KSHV virion particles at various stages of wrapping and engulfment of KSHV during the process of endocytosis. Magnification 87,000 X (A–F, H and I); G: Enlarged.

Figure 6. CIB1 association with actin protrusions during de novo KSHV infection in endothelial cells.

HMVEC-d (A) and HUVEC (B) cells were either uninfected or infected with KSHV (20 DNA copies/cell) for the indicated time points. Cells were then processed for double immunofluorescence analysis using AF488 phalloidin for actin staining and with anti-CIB1 antibodies. Representative deconvoluted images at 5 min post-infection are shown. Boxed areas from merged panels are enlarged in the rightmost panel below. Arrows indicate the association of CIB1 with actin protrusions (blebs) in KSHV infected cells.

Figure 7. CIB1 association with KSHV productive trafficking.

(A) HMVEC-d cells were incubated with medium containing Alexa 488 conjugated LysoTracker alone (no virus, uninfected) or with DiI-labeled KSHV (40 DNA copies/cell) and Alexa 488 conjugated LysoTracker at 37°C for 10 min. Cells were fixed and processed for confocal immunofluorescence analysis using mouse anti-CIB1 antibodies and co-stained with DAPI. (B) HMVEC-d cells were left uninfected or DiI-labeled KSHV infected (40 DNA copies/cell) at 37°C for 10 min and were processed for confocal immunofluorescence using Rab5 and CIB1 antibodies. (A & B) Boxed areas are enlarged in the right most panels for uninfected cells, and left most panels for infected cells. Representation of line-scan signal intensity plots for triple staining was performed on the enlarged infected cell area (bottom panel).

Figure 8. CIB1 association with macropinocytic marker dextran early during de novo KSHV infection in endothelial cells.

HMVEC-d cells were incubated with medium containing Texas Red labeled dextran (A) or Texas Red labeled transferrin (B) alone (no virus, Uninfected) or with KSHV (20 DNA copies/cell) and Texas Red labeled dextran or Texas Red labeled transferrin at 37°C for the indicated time points. Cells were fixed and processed for immunofluorescence analysis using mouse anti-CIB1 antibodies and co-stained with DAPI. Boxed areas in the merged panel are enlarged in the rightmost panel and arrows indicate colocalization of molecules. Representative deconvoluted immunofluorescence images are shown.

Figure 9. Effect of CIB1 knockdown on productive KSHV trafficking in endothelial cells.

(A) Untransduced or CIB1 sh-RNA transduced HMVEC-d cells were incubated with KSHV (20 DNA copies/cell) and FITC labeled dextran for 30 min at 37°C. After washing, cells were harvested, fixed, permeabilized, and examined by FACS. Unstained HMVEC-d cells were used as negative control. The results are shown in a histogram as a percentage of fluorescent cells with a fluorescence intensity higher than the unstained negative control. The mean fluorescence intensity (MFI) as an indication of dextran uptake was measured and the table shows representative MFI values from three independent experiments. (B) Serum starved control or CIB1- shRNA transduced cells were infected with KSHV (20 DNA copies/cell) for 10 min, washed, and processed for double immunofluorescence staining using mouse anti-KSHV glycoprotein gpK8.1A and rabbit anti-Rab5 antibodies, followed by anti-mouse Alexa Fluor 594 and anti-rabbit Alexa Fluor 488 antibodies, respectively. Representative deconvoluted images are shown. Boxed areas from the merged panel are enlarged in the rightmost panel and arrows indicate KSHV particles in proximity to Rab5 positive vesicles (green).

Figure 10. Effect of CIB1 knockdown on macropinocytic uptake of KSHV during de novo infection of endothelial cells.

(A) Control shRNA or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell) for 10 min, washed, and processed for double immunofluorescence staining using rabbit anti-KSHV glycoprotein gB antibodies, followed by anti-rabbit Alexa Fluor 488 antibodies and rhodamine-phalloidin for actin staining. Representative deconvoluted images are shown. Boxed areas from the merged panel are enlarged and shown in the rightmost panel below. Arrows indicate the localization of viral particles in infected cells. (B) The percentage of bleb-containing cells were counted under bright field and presented graphically. At least 10 different microscopic fields were observed and analyzed as a proportion of the total number of DAPI-stained cells. Error bars show ± SD.

Figure 11. CIB1 association with membrane lipid rafts of HMVEC-d cells early during de novo KSHV infection.

(A) Serum-starved (8 h) HMVEC-d cells were either mock or KSHV infected (20 DNA copies/cell) for the indicated time points. Caveolin-1 and CD71 characterize the purity of LR and non-LR fractions by dot blot, respectively. LR and NLR fractions were isolated and analyzed for CIB1 and EphA2 levels by Western blot and (B) total cell lysates from the same experiment were also assessed for CIB1 expression with tubulin as loading control. (C) Serum-starved (8 h) HMVEC-d cells were either left uninfected or infected (20 DNA copies/cell) for the indicated time points with KSHV (20 DNA copies/cell), washed, and processed for double immunofluorescence using mouse anti-CIB1 and goat anti-Flotillin-1 (LR marker) antibodies, followed by Alexa 594 and 488 antibodies, respectively. Representative deconvoluted immunofluorescence images are shown. Arrows indicate CIB1 and Flotillin-1 colocalization at the cell periphery.

Figure 12. CIB1 association with KSHV macropinocytosis associated signal molecules and regulation of signal pathways early during de novo infection of HMVEC-d cells.

(A) Serum-starved (8 h) HMVEC-d cells or CIB1-shRNA transduced HMVEC-d cells were either mock or KSHV infected (20 DNA copies/cell) for the indicated time points, immunoprecipitated with anti-CIB1 antibodies and analyzed by Western blot (WB) for CIB1 and other signal molecules (as indicated). Cells treated with 10% FBS for 10 min were used as positive control. As negative control, cells were infected for 10 min with KSHV pre-incubated with Heparin (50 µg/ml) for 1 h at 37°C. 20 µg of whole-cell lysate (WCL) proteins was analyzed by Western blot to check for total protein levels of CIB1 and indicated signal molecules. The measured levels of associations are given in the results sections. (B) Serum starved (8 h) control or CIB1-shRNA transduced HMVEC-d cells were either left uninfected or KSHV infected (20 DNA copies/cell) for the indicated time points and were subjected to Western blot analysis for the indicated phosphorylated (activated) signal molecules. The blots were stripped and reprobed for the respective total molecules and tubulin as a loading control. The band intensities were measured as described in the methods section and expressed as increased fold phosphorylation of each molecule over uninfected cells. These are given in the results sections.

Figure 13. Mass spectrometric analysis of immunoprecipitates of anti-CIB1 and EphA2 antibodies with lysates from KSHV infected HMVEC-d cells.

(A) Serum-starved (8 h) HMVEC-d cells were either mock or KSHV infected (20 DNA copies/cell) for 10 min and immunoprecipitated either with anti-CIB1 or anti-EphA2 antibodies. Immunoprecipitated proteins were separated by SDS-PAGE gel and gel slices (as indicated by the band #) were analyzed by mass spectrometry. (B) Serum starved (8 h) HMVEC-d cells were either left uninfected or KSHV infected (20 DNA copies/cell) for the indicated time points and immunoprecipitated either with anti-CIB1 or anti-EphA2 antibodies. Immunoprecipitates were subjected to Western blot analysis for the indicated molecules identified by mass spectrometry. Blots were then stripped and probed for total EphA2 and CIB1 levels. 20 µg of whole cell lysate (WCL) protein was also analyzed by Western blot to check for all indicated signal molecules at the total protein level. (C) Serum starved (8 h) control or CIB1-shRNA transduced HMVEC-d cells were either left uninfected or KSHV infected (20 DNA copies/cell) for 10 min, immunoprecipitated with anti-EphA2 antibodies and immunoprecipitates were subjected to Western blot analysis for total EphA2 and the indicated molecules identified by mass spectrometry. As negative control, cells were infected for 10 min with KSHV pre-incubated with heparin (50 µg/ml) for 1 h at 37°C. 20 µg of whole cell lysate protein was analyzed by Western blot to check for the indicated molecules at the total protein level with tubulin as a loading control.

Figure 14. Model depicting the impact of CIB1 shRNA on KSHV macropinocytosis and productive de novo infection in HMVEC-d cells.

CIB1-shRNA transduction in HMVEC-d cells resulted in the following consequences which are depicted by the red lines: (i) CIB1 protein level was reduced by >90%; (ii) CIB1 association with EphA2 and the associated KSHV induced Src-PI3-K-c-Cbl signal complex was significantly reduced; (iii) KSHV induced EphA2 activation was not sustained and as a consequence, downstream Src and Erk1/2 signal amplification was significantly abrogated; (iv) EphA2 association with Src-PI3-k-c-Cbl signal complex was substantially reduced; (v) EphA2 association with actin modulating myosin IIA and alpha actinin-4 were almost abolished, (vi) as a consequence, actin cytoskeletal rearrangement and membrane blebbing was inhibited, which (vii) impaired KSHV macropinocytosis, productive trafficking, and establishment of de novo infection.