Newcastle disease virus activates diverse signaling pathways via Src to facilitate virus entry into host macrophages

Abstract

As an intrinsic cellular mechanism responsible for the internalization of extracellular ligands and membrane components, caveolae-mediated endocytosis (CavME) is also exploited by certain pathogens for endocytic entry [e.g., Newcastle disease virus (NDV) of paramyxovirus]. However, the molecular mechanisms of NDV-induced CavME remain poorly understood. Herein, we demonstrate that sialic acid-containing gangliosides, rather than glycoproteins, were utilized by NDV as receptors to initiate the endocytic entry of NDV into HD11 cells. The binding of NDV to gangliosides induced the activation of a non-receptor tyrosine kinase, Src, leading to the phosphorylation of caveolin-1 (Cav1) and dynamin-2 (Dyn2), which contributed to the endocytic entry of NDV. Moreover, an inoculation of cells with NDV-induced actin cytoskeletal rearrangement through Src to facilitate NDV entry via endocytosis and direct fusion with the plasma membrane. Subsequently, unique members of the Rho GTPases family, RhoA and Cdc42, were activated by NDV in a Src-dependent manner. Further analyses revealed that RhoA and Cdc42 regulated the activities of specific effectors, cofilin and myosin regulatory light chain 2, responsible for actin cytoskeleton rearrangement, through diverse intracellular signaling cascades. Taken together, our results suggest that an inoculation of NDV-induced Src-mediated cellular activation by binding to ganglioside receptors. This process orchestrated NDV endocytic entry by modulating the activities of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPases and downstream effectors.

Importance: In general, it is known that the paramyxovirus gains access to host cells through direct penetration at the plasma membrane; however, emerging evidence suggests more complex entry mechanisms for paramyxoviruses. The endocytic entry of Newcastle disease virus (NDV), a representative member of the paramyxovirus family, into multiple types of cells has been recently reported. Herein, we demonstrate the binding of NDV to induce ganglioside-activated Src signaling, which is responsible for the endocytic entry of NDV through caveolae-mediated endocytosis. This process involved Src-dependent activation of the caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase and downstream effectors, thereby orchestrating the endocytic entry process of NDV. Our findings uncover a novel molecular mechanism of endocytic entry of NDV into host cells and provide novel insight into paramyxovirus mechanisms of entry.

Keywords: Newcastle disease virus; Src; gangliosides; virus entry.

Fig 1

Sialic acid-containing glycoprotein receptors are responsible for NDV entry into HD11 cells via direct fusion with the PM. (A) NDV entered HD11 cells mainly through the endocytic pathway. The internalization assay in HD11 cells was performed with DiOC-labeled NDV. After treatment with trypan blue, confocal microscopy was used to detect the fluorescence of DiOC-labeled NDV (left). Besides, the mean fluorescence intensities of DiOC-labeled NDV that was resistant to and quenched by trypan blue were quantified by confocal microscopy and flow cytometry analysis, respectively (right). (B and C) Treatment with NA reduced the adsorption and internalization of NDV. HD11 cells were treated with NA or PBS and then subjected to adsorption (B) and internalization (C) assays followed by treatment with trypan blue. Flow cytometry was used to determine the MFI of DiOC-labeled NDV. The MFI in reagent-treated cells was calculated relative to that of control cells (×100%). (D) Treatment with MAL and SNA reduced the adsorption and internalization of NDV. HD11 cells were treated with MAL, SNA, or PBS and then subjected to adsorption and internalization assays followed by treatment with trypan blue. Confocal microscopy was used to visualize the cells. (E and F) Treatment with trypsin and chymotrypsin reduced NDV adsorption to cells and the direct fusion of NDV with the PM without affecting the endocytic entry of NDV. HD11 cells were treated with trypsin or PBS and then subjected to adsorption (E) and internalization (F) assays followed by treatment with trypan blue. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. the bars represent means ± standard deviations (SD) of three independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001; NS, no significant difference).

Fig 2

Sialic acid-containing gangliosides as receptors for NDV endocytic entry into HD11 cells. (A and B) Treatment with PDMP reduced the adsorption and internalization of CTB. HD11 cells were treated with PDMP or ethanol and then subjected to adsorption and internalization assays of CTB. Flow cytometry was used to analyze the fluorescence of CTB. (C and D) Treatment with PDMP reduced adsorption, the direct fusion of NDV with the PM, and endocytosis of NDV. HD11 cells were treated with PDMP or ethanol and then subjected to adsorption (C) and internalization (D) assays followed by treatment with trypan blue. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (E) The presence of gangliosides in HD11 cells and colocalization of gangliosides with NDV. HD11 cells were inoculated with NDV, and then fixed and incubated with anti-GM1 antibody, anti-GD1a antibody, anti-GT1b antibody, and chicken anti-NDV antibody, respectively. The cells were visualized by confocal microscopy. (F and G) Exogenous GM1 and GD1a gangliosides blocked the adsorption and internalization of NDV. DiOC labelled NDV were pre-incubated with GM1, GD1a or DMSO. Adsorption (F) and internalization (G) assays of NDV followed by treatment with trypan blue were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent means ± SD of three independent experiments (*P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 3

NDV induces tyrosine phosphorylation of Src for entry into HD11 cells. (A and B) Treatment with Genistein inhibited internalization of NDV. HD11 cells were treated with Genistein or DMSO and then subjected to adsorption (A) and internalization (B) assays. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (C) Y416 phosphorylation of Src was induced by NDV during the entry process. HD11 cells were either inoculated with NDV at an MOI of 10 or mock inoculated. At each of the indicated time point, cell lysates were analyzed by Western blotting using antibodies against p-Src (Tyr416) and Src, respectively. GAPDH was used as a control. The relative intensity of p-Src (Tyr416) and Src were normalized to GAPDH. (D) Y416 phosphorylation of Src was induced by NDV in HD11 cells and primary chicken macrophage, rather than DF-1 cells. HD11 cells, primary chicken macrophage and DF-1 cells were either inoculated with NDV at an MOI of 5 and 10 or mock inoculated. At 30 mpi with NDV, the levels of p-Src (Tyr416) and Src by Western blotting as described in Fig. 3C. (E–H) Treatment with Src inhibitors inhibited the internalization of NDV. HD11 cells were pretreated with Saracatinib (E and F) and Dasatinib (G and H), or mock-treated with DMSO, after which adsorption and internalization assays were performed. (I) Treatment with Saracatinib inhibited NDV-induced phosphorylation of Src. HD11 cells were pretreated with either DMSO or Saracatinib. The cells were inoculated or mock inoculated with NDV. At 30 mpi, the levels of p-Src and Src were determined by Western blotting. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 4

NDV-activated Y416 phosphorylation of Src through binding to gangliosides is involved in the entry of NDV into HD11 cells. (A) Overexpression of a Src phospho-defective (PD) mutant reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT Src or PD mutant Src Y416F were inoculated with DiOC-labelled NDV. Then the cells were collected, fixed and treated with trypan blue. Flow cytometry was used to determine the MFI of DiOC-labelled NDV in BFP-positive cells. (B) Knockdown of Src by siRNA. HD11 cells were transfected with siRNA targeting Src (siSrc) or a control siRNA (siNC). Western blotting was used to determine the effect of siRNA knockdown on Src expression. (C and D) Knockdown of Src inhibited the entry of NDV. After transfection with siRNA, the adsorption (C) and internalization (D) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (E) Treatment with PDMP inhibited the NDV-induced phosphorylation of Src. HD11 cells were pretreated with either ethanol or PDMP. The cells were inoculated or mock inoculated with NDV. Western blotting was used to determine the levels of p-Src and Src at 30 mpi with NDV. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 5

Phosphorylation of Cav1 and Dyn2 induced by NDV via Src contributes to endocytic entry. (A) Phosphorylation of Cav1 was induced by NDV during the viral entry process. HD11 cells were inoculated or mock inoculated with NDV. At each of the indicated time point, Western blotting using antibody against Cav1 was used to analyze the cell lysates. In addition, the cell lysates were precipitated with mouse anti-phospho-tyrosine antibody or normal mouse IgG as the isotype control antibody. Western blotting using an anti-Cav1 antibody was used to detect the precipitated samples. GAPDH was used as a control. (B) Overexpression of a Cav1 PD mutant reduced the level of NDV internalization. HD11 cells transfected with the BFP-tagged WT Cav1 or PD mutant Cav1 Y14F were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. (C) Treatment with the Src inhibitor, Saracatinib, inhibited NDV-induced Cav1 phosphorylation. HD11 cells were pretreated with either DMSO or Saracatinib and then inoculated or mock inoculated with NDV. Western blotting was used to determine the levels of p-Cav1 and Cav1 at 30 mpi with NDV as described in Fig. 5A. (D) Dyn2 phosphorylation was induced by NDV during the entry process. Detection of Dyn2 and phosphorylated Dyn2 in NDV-inoculated cells was performed with an anti-Dyn2 antibody and an anti-phospho-tyrosine antibody as described in Fig. 5A. (E) Overexpression of a Dyn2 PD mutant reduced NDV internalization. The internalization assay in HD11 cells transfected with the BFP-tagged WT Dyn2 or PD mutant Dyn2 Y231/597F was performed as described in Fig. 4A. (F) Treatment with the Src inhibitor, Saracatinib, inhibited NDV-induced phosphorylation of Dyn2. HD11 cells were treated with Saracatinib and inoculated with NDV as described in Fig. 5C. Western blotting was used to determine the levels of p-Dyn2 and Dyn2 at 30 mpi with NDV as described in Fig. 5A. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 6

NDV induces rearrangement of actin cytoskeleton via Src. (A) Treatment with the Src inhibitor, Saracatinib, inhibited NDV-induced actin cytoskeletal rearrangement. HD11 cells were pretreated with either DMSO or the Src inhibitor, Saracatinib. Then the cells were inoculated or mock inoculated with NDV. At the indicated time points, the cells were fixed and permeabilized, and F-actin was stained with Phalloidin-iFluor 594 reagent (red). After staining with DAPI (blue), the cells were observed by confocal microscopy. (B–D) Src directly involved in the rearrangement of actin cytoskeleton induced by NDV. (B) Phosphorylation of Src was induced by inoculation of a Src activator, YEEI peptide. HD11 cells were inoculated with YEEI peptide at 37℃ for the indicated time points, the cell lysates were analyzed by Western blotting using antibody against p-Src and Src. (C) Inoculation of YEEI peptide induced rearrangement of actin cytoskeleton. HD11 cells were inoculated with YEEI peptide at 37°C for the indicated time points, then the cells were fixed, stained with Phalloidin-iFluor 594 and observed by confocal microscopy. (D) Knockdown of Src inhibited the NDV-induced rearrangement of actin cytoskeleton. After transfection with siRNA, HD11 cells were inoculated with NDV and the dynamic of F-actin was observed by confocal microscopy as described above. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 7

Rearrangement of actin cytoskeleton is involved in the NDV entry process via both endocytosis and direct fusion with the PM. Treatment with the actin dynamics inhibitors, Cytochalasin D (A and B) and Jasplakinolide (C and D), reduced NDV internalization. HD11 cells were pretreated with Cytochalasin D and Jasplakinolide or mock-treated with DMSO. Next, adsorption and internalization assays were performed. The MFI of DiOC-labelled NDV was analyzed by flow cytometry. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 8

Phosphorylation of LIMK1/CFN is induced by NDV via Src in a PI3K/AKT independent manner. (A) Inoculation of NDV induced LIMK1 and CFN phosphorylation during the entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were analyzed by Western blotting using antibodies against p-CFN (S3), CFN, p-LIMK1 (T508) and LIMK1, respectively. GAPDH was used as a control. (B) Overexpression of a PD mutant CFN reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT CFN or PD mutant CFN S3A were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. (C) Treatment with the Src inhibitor, Saracatinib, inhibited NDV-induced phosphorylation of LIMK1 and CFN. HD11 cells were pretreated with either DMSO or Saracatinib. Then the cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN were determined by Western blotting. GAPDH was used as a control. (D) HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were analyzed by Western blotting using antibodies against p-Akt (S473) and Akt, respectively. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 9

PI3K/AKT downstream Rac1-PAK1 signaling axis is not activated and involved in NDV entry into HD11 cells. (A) GTPase activity of Rac1 was analyzed by using the Rac1 activation assay kit, followed by Western blotting using an anti-Rac1 antibody. The level of GTP-Rac1 was normalized to the total Rac1. (B) The levels of p-PAK1 (T423) and PAK1 were analyzed by Western blotting using corresponding antibodies. GAPDH was used as a control. (C–G) Treatment with the Rac1 and PAK1 inhibitor, NSC23766 and IPA-3, showed no effect on NDV-induced LIMK1/CFN phosphorylation and entry of NDV. (C) HD11 cells were pretreated with NSC23766, IPA-3, or DMSO. Then the cells were inoculated or mock inoculated with NDV. Western blotting was used to determine the levels of p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN at 30 mpi with NDV. GAPDH was used as a control. (D–G) Treatment with the NSC23766 and IPA-3 had no effect on the adsorption and internalization of NDV. HD11 cells were pretreated with NSC23766 (D and E), IPA-3 (F and G), after which NDV adsorption and internalization assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 10

ROCK1-LIMK1/CFN signaling is activated by NDV to modulate viral entry. (A) ROCK1 was activated by NDV during the entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cells lysate were analyzed by Western blotting using antibody against ROCK1. GAPDH was used as a control. (B and C) Treatment with the ROCK1 inhibitor Y-27632 inhibited NDV-induced the activation of ROCK1, LIMK1 and CFN. HD11 cells were pretreated with either DMSO or Y-27632. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of ROCK1 (B), p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN (C) were determined by Western blotting. GAPDH was used as a control. (D and E) Treatment with the ROCK1 inhibitor, Y-27632, reduced NDV internalization. HD11 cells were pretreated with Y-27632 or mock-treated with DMSO, after which adsorption (D) and internalization (E) assays of NDV were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 11

RhoA activated by NDV via Src is responsible for the entry of NDV into HD11 cells through ROCK1-LIMK1/CFN signaling. (A) RhoA was activated by NDV during the entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were harvested and the GTPase activity of RhoA was analyzed using a RhoA activation assay kit, followed by a Western blotting using anti-RhoA antibody. GAPDH was used as a control. (B and C) Treatment with the RhoA inhibitor, Rhosin, inhibited NDV-induced activation of RhoA, ROCK1, LIMK1, and CFN. HD11 cells were pretreated with either DMSO or Rhosin. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-RhoA (B), ROCK1, p-LIMK1 (T508), LIMK1, p-CFN (S3), and CFN (C) were determined by a RhoA activation assay kit and Western blotting. GAPDH was used as a control. (D and E) The RhoA inhibitor, Rhosin, reduced NDV internalization. HD11 cells were pretreated with Rhosin or mock-treated with DMSO, after which NDV adsorption (D) and internalization (E) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (F) Overexpression of a dominant-negative (DN) mutant RhoA reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT RhoA or DN mutant RhoA T19N were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. (G) Treatment with the Src inhibitor, Saracatinib, inhibited NDV-induced activation of RhoA and ROCK1. HD11 cells were pretreated with either DMSO or Saracatinib. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-RhoA and ROCK1 were determined by a RhoA activation assay kit and Western blotting. GAPDH was used as a control. (H) Schematic of NDV-activated RhoA-ROCK1-LIMK1-CFN signaling axis during entry into HD11 cells. The bars represent the means ± SD from three independent experiments. (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 12

MLC is activated by NDV and contributes to the entry of NDV into HD11 cells. (A) MLC phosphorylation was induced by NDV during the entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were analyzed by Western blotting using antibodies against p-MLC (S19) and MLC, respectively. GAPDH was used as a control. (B) Treatment with the MLCK inhibitor, ML-9, inhibited NDV-induced phosphorylation of MLC. HD11 cells were pretreated with either DMSO or ML-9. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of p-MLC (S19) and MLC were determined by Western blotting. GAPDH was used as a control. (C and D) Treatment with the MLCK inhibitor, ML-9, reduced NDV internalization. HD11 cells were pretreated with ML-9 or mock-treated with DMSO, after which NDV adsorption (C) and internalization (D) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. The bars represent the means ± SD from three independent experiments. (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 13

MLC activation is mediated by NDV via Src-RhoA-ROCK1 signaling and facilitates virus entry through regulating dynamics of actin cytoskeleton. (A) Overexpression of a PD mutant MLC reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT MLC or PD mutant MLC S19A were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. (B) Treatment with Src inhibitor, Saracatinib, RhoA inhibitor, Rhosin, and ROCK1 inhibitor, Y-27632, inhibited NDV-induced activation of MLC. HD11 cells were pretreated with either DMSO or Saracatinib, Rhosin, and Y-27632. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of p-MLC (S19) and MLC were determined by Western blotting. GAPDH was used as a control. (C) Treatment with ML-9 inhibited NDV-induced rearrangement of actin cytoskeleton. HD11 cells were pretreated with either DMSO or ML-9. Then the cells were inoculated with NDV. At 30 mpi, F-actin was stained and observed by confocal microscopy as described in Fig. 6A. (D) Schematic of NDV-activated RhoA-ROCK1-MLC signaling axis during entry into HD11 cells. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 14

PAK2 phosphorylation induced by NDV is involved in MLC activation and viral entry. (A) PAK2 phosphorylation was induced by NDV during the viral entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were analyzed by Western blotting using antibodies against p-PAK2 (T402) and PAK2. GAPDH was used as a control. (B and C) Treatment with the PAK2 inhibitor, Staurosporine, inhibited NDV-induced phosphorylation of PAK2 and MLC. HD11 cells were pretreated with either DMSO or Staurosporine. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of p-PAK2 (T402), PAK2 (B), p-MLC (S19) and MLC (C) were determined by Western blotting. GAPDH was used as a control. (D and E) Treatment with the PAK2 inhibitor, Staurosporine, reduced the level of NDV internalization. HD11 cells were pretreated with Staurosporine or mock-treated with DMSO, after which NDV adsorption (D) and internalization (E) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (F) Overexpression of a PD mutant PAK2 reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT PAK2 or PD mutant PAK2 T402A were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 15

Cdc42 is responsible for the activation of PAK2-MLC signaling and viral entry. (A) Cdc42 was activated by NDV during the viral entry process. HD11 cells were inoculated or mock inoculated with NDV. At the indicated time points, the cell lysates were harvested and the GTPase activity of Cdc42 was analyzed using a Cdc42 activation assay kit followed by Western blotting using anti-Cdc42 antibody. GAPDH was used as a control. (B and C) Treatment with Cdc42 inhibitor ML141 inhibited NDV-induced activation of Cdc42, PAK2, and MLC. HD11 cells were pretreated with either DMSO or ML141. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-Cdc42 (B), p-PAK2 (T402), PAK2, p-MLC (S19) and MLC (C) were determined by a Cdc42 activation assay kit and Western blotting. GAPDH was used as a control. (D and E) Treatment with the Cdc42 inhibitor, ML141, reduced the level of NDV internalization. HD11 cells were pretreated with ML141 or mock-treated with DMSO, after which NDV adsorption (D) and internalization (E) assays were performed. Flow cytometry was used to analyze the MFI of DiOC-labelled NDV. (F) Overexpression of a DN mutant Cdc42 reduced the internalization of NDV. HD11 cells transfected with the BFP-tagged WT Cdc42 or DN mutant Cdc42 T17N were inoculated with DiOC-labelled NDV. The internalization assay was performed as described in Fig. 4A. The bars represent the means ± SD from three independent experiments (* P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 16

NDV activates Cdc42-PAK2-MLC signaling axis via Src, rather than Cdc42-N-WASP-Arp2/3, to promote viral entry. (A and B) Treatment with Src inhibitor, Saracatinib, inhibited NDV-induced activation of Cdc42, PAK2, and MLC. HD11 cells were pretreated with either DMSO or Saracatinib. The cells were inoculated or mock inoculated with NDV. At 30 mpi with NDV, the levels of GTP-Cdc42 (A), p-PAK2 (T402), PAK2, p-MLC (S19), and MLC (B) were determined with a Cdc42 activation assay kit and Western blotting. GAPDH was used as a control. (C and D) Treatment with the N-WASP inhibitor, Wiskostatin, and (E and F) Arp2/3 complex inhibitor, CK636, had no effect on the adsorption and internalization of NDV. HD11 cells were pretreated with Wiskostatin, CK-636 or mock-treated with DMSO, and NDV adsorption and internalization assays were performed as described above. (G) Schematic of NDV-activated Cdc42-PAK2-MLC signaling axis during entry into HD11 cells. The bars represent the means ± SD from three independent experiments (*P < 0.05; ** P < 0.01; *** P < 0.001; NS, no significant difference).

Fig 17

Schematic of NDV entry pathways into HD11 cells. (A) NDV entry through CavME. By binding to gangliosides located in caveolae, Src tyrosine kinase was activated by NDV, leading to activation of caveolae-associated Cav1 and Dyn2, as well as specific Rho GTPase, RhoA and Cdc42, and downstream effectors, CFN and MLC, orchestrating the endocytic entry of NDV. The endocytosed virus-containing caveolar vesicles were subsequently trafficked to the early endosome, where viral-cell membrane fusion occurs [12]. (B) By binding to glycoproteins, viral-cell membrane fusion occurs at the PM. Src-mediated actin cytoskeletal rearrangement also contributes to the direct fusion of the NDV with the cell PM.