Plectin regulates the signaling and trafficking of the HIV-1 co-receptor CXCR4 and plays a role in HIV-1 infection

Abstract

The CXC chemokine CXCL12 and its cognate receptor CXCR4 play an important role in inflammation, human immunodeficiency virus (HIV) infection and cancer metastasis. The signal transduction and intracellular trafficking of CXCR4 are involved in these functions, but the underlying mechanisms remain incompletely understood. In the present study, we demonstrated that the CXCR4 formed a complex with the cytolinker protein plectin in a ligand-dependent manner in HEK293 cells stably expressing CXCR4. The glutathione-S-transferase (GST)-CXCR4 C-terminal fusion proteins co-precipitated with the full-length and the N-terminal fragments of plectin isoform 1 but not with the N-terminal deletion mutants of plectin isoform 1, thereby suggesting an interaction between the N-terminus of plectin and the C-terminus of CXCR4. This interaction was confirmed by confocal microscopic reconstructions showing co-distribution of these two proteins in the internal vesicles after ligand-induced internalization of CXCR4 in HEK293 cells stably expressing CXCR4. Knockdown of plectin with RNA interference (RNAi) significantly inhibited ligand-dependent CXCR4 internalization and attenuated CXCR4-mediated intracellular calcium mobilization and activation of extracellular signal regulated kinase 1/2 (ERK1/2). CXCL12-induced chemotaxis of HEK293 cells stably expressing CXCR4 and of Jurkat T cells was inhibited by the plectin RNAi. Moreover, CXCR4 tropic HIV-1 infection in MAGI (HeLa-CD4-LTR-Gal) cells was inhibited by the RNAi of plectin. Thus, plectin appears to interact with CXCR4 and plays an important role in CXCR4 signaling and trafficking and HIV-1 infection.

Fig. 1

Plectin was co-immunoprecipitated with CXCR4. (A) HEK293 cells stably expressing Myc-CXCR4 were exposed to CXCL12 (10 nM) for the indicated time intervals and CXCR4 was immunoprecipitated from the cell lysate using a specific anti-Myc antibody. In a parallel experiment, parental HEK293 cells transiently transfected with vector containing Myc alone were treated with CXCL12 for 10 min, and immunoprecipitation was performed as described above (mock). Proteins were separated by 6% SDS-PAGE and transferred to a nitrocellulose membrane, and coprecipitated proteins were detected by Western blotting. The membrane was stripped and reblotted with a specific anti-Myc antibody to confirm equal loading of protein samples. (B) Quantification of the density of bands representing plectin, which was time-dependently associated with CXCR4, was determined by densitometric scanning. Data are the means ± S.E. from three independent experiments.

Fig. 2

The N-terminal domain of plectin interacted with the C-terminal domain of CXCR4. (A) Diagrams of wild-type and truncation mutant forms of plectin, highlighting structurally distinct regions. (B) HEK293 cells transiently transfected with plasmids encoding HA-tagged full-length plectin or its truncation mutants (1−339, 1−606, 1−1154, 284−1154, and 284−2532) were lysed and proteins were separated by 10% SDS-PAGE. The expression of HA-tagged plectin forms was determined by Western blotting using a specific anti-HA antibody. (C) The GST-CXCR4 C-terminus fusion proteins were incubated with the cell lysate of HEK293 cells transiently expressing the above HA-tagged plectin forms (lanes 2−6). Equal amounts of GST proteins were incubated with the lysate of HEK293 cells overexpressing HA-tagged full-length plectin as control (lane 1). Co-precipitated plectin forms were analyzed by Western blotting using anti-HA antibody as described above.

Fig. 3

Plectin is co-distributed with CXCR4. HEK293 cells stably expressing CXCR4 were treated with or without CXCL12 (10 nM) for 60 min before being fixed in methanol. Cells were incubated with an antibody mixture of a goat polyclonal plectin antibody and a mouse monoclonal CXCR4 antibody for 30 min followed by incubation with a secondary antibody mixture containing a Cy3-conjugated anti-goat IgG and a FITC-conjugated anti-mouse IgG for 30 min. Images are look-through confocal microscopic reconstructions with Z axis projections below and right. Data are representatives of three independent experiments with similar results.

Fig. 4

Effect of plectin RNAi on CXCR4-mediated ERK1/2 activation. (A) HEK293 cells stably expressing CXCR4 were transiently transfected with a scramble siRNA as a control or two plectin specific siRNAs (siRNA-1 and siRNA-2). Cells were treated with CXCL12 (10 nM) for indicated time intervals (0, 5, 30 min) and phosphorylation of ERK1/2 was assessed by Western blot analysis as described above. In a parallel experiment, equal amount of samples were subjected to 6% SDS-PAGE, and Western blot analysis was performed with an anti-plectin antibody to confirm the knockdown of plectin in the specific siRNAs transfected cells. (B) Quantification of the density of bands representing phosphorylated ERK1/2, which was normalized with the density of the total ERK2 bands, was performed using NIH Image software (http://rsb.info.nih.gov/nih-image). Data are the mean ± S.E. of three independent experiments. **P<0.01, compared to the control with the same treatment. (C) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells.

Fig. 5

Effect of plectin RNAi on CXCL12–induced Ca²⁺ mobilization. (A) HEK293 cells stably expressing CXCR4 were transiently transfected with a scramble siRNA or a plectin specific siRNA (siRNA-1). Cells were loaded with Fura-2, stimulated with CXCL12 (10 nM), and analyzed for ability to mobilize intracellular free Ca²⁺. The data shown are representative of at least three independent experiments with similar results. (B) The potency of Ca²⁺ mobilization was quantified from three independent experiments. *P<0.05, compared to the control. (C) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells.

Fig. 6

Effect of plectin RNAi on CXCR4–mediated chemotaxis. (A) HEK293 cells stably expressing CXCR4 were transiently transfected with a scramble siRNA or a plectin specific siRNA (siRNA-1). After transfection, chemotactic migration of the cells toward CXCL12 gradients was assessed. Values represent the mean ± S.E. of three independent experiments. *P<0.05, compared to the control. (B) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells. (C) Jurkat T cells endogenously expressing CXCR4 were transiently transfected with a scramble siRNA or a plectin specific siRNA (siRNA-1). After transfection, chemotactic migration of the cells toward CXCL12 gradients was assessed. Values represent the mean ± S.E. of three independent experiments. *P<0.05, compared to the control. (D) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells. (E) Equal numbers of transfected HeLa cells were plated onto ICAM-1-coated coverslips for 30 min, followed by four washes with adhesion medium to remove nonadhered cells. The number of adhered cells was determined using CyQUANT Cell Proliferation Kit (Molecular Probes, Eugene, OR, USA). Fifteen randomly chosen optical fields were counted in two independent experiments. Bars represent mean ± S.E.M. The data were analyzed using Student's paired t test. *P < 0.05 compared to the control. (F) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells.

Fig. 7

Effect of plectin RNAi on ligand-induced CXCR4 internalization. (A) HEK293 cells stably expressing CXCR4 were transiently transfected with a scramble siRNA or a plectin specific siRNA (siRNA-1). Cells were incubated in HEPES (20 mM)-buffered DMEM at 37°C for 30 min in the presence (dotted lines) or absence of CXCL12 (10 nM) (thick solid lines) before being fixed in 2% formaldehyde in PBS. Cells were incubated with a PE-conjugated monoclonal anti-CXCR4 antibody at 4°C for 60 min. As a control, the parental HEK293 cells without expressing CXCR4 were incubated with the PE-conjugated CXCR4 antibody (thin solid lines). The cell surface CXCR4 receptors were analyzed by flow cytometry equipped with CellQuest software (BD Biosciences). (B) The mean fluorescence intensity (MFI) was quantified and CXCL12-induced reduction of MFI was calculated. *P<0.05, compared to the control. (C) Western blot analysis revealed a remarkable knockdown of the plectin expression in the plectin specific siRNA but not the scramble siRNA transfected cells.

Fig. 8

Effect of plectin RNAi on HIV-1 infectiuon in MAGI-CXCR4 cells. MAGI-CXCR4 cells CXCR4 were transiently transfected with a scramble siRNA or a plectin specific siRNA (siRNA-1). The cells were incubated with 20 ng of p24 antigen of a luciferase reporter virus, NL-Luc-HXB (X4-tropic), for 60 min. Luciferase activities in cell lysates were determined 48 h post-infection. The data are represented as percent infection efficiency, which is the total relative light units measured in control or untreated cells. Data are the means ± S.E. of three different experiments. *P<0.05, compared to the control. (B) Western blot analysis revealed a remarkable knockdown of the plectin expression and reduced gp120 expression in the plectin specific siRNA but not the scramble siRNA transfected cells.