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. 2012 Mar 23;287(13):9804-16.
doi: 10.1074/jbc.M111.312959. Epub 2012 Feb 8.

Transport of Influenza Virus Neuraminidase (NA) to Host Cell Surface Is Regulated by ARHGAP21 and Cdc42 Proteins

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Free PMC article

Transport of Influenza Virus Neuraminidase (NA) to Host Cell Surface Is Regulated by ARHGAP21 and Cdc42 Proteins

Song Wang et al. J Biol Chem. .
Free PMC article

Abstract

Influenza virus neuraminidase (NA) is transported to the virus assembly site at the plasma membrane and is a major viral envelope component that plays a critical role in the release of progeny virions and in determination of host range restriction. However, little is known about the host factors that are involved in regulating the intracellular and cell surface transport of NA. Here we identified the Cdc42-specific GAP, ARHGAP21 differentially expressed in host cells infected with influenza A virus using cDNA microarray analysis. Furthermore, we have investigated the involvement of Rho family GTPases in NA transport to the cell surface. We found that expression of constitutively active or inactive mutants of RhoA or Rac1 did not significantly affect the amount of NA that reached the cell surface. However, expression of constitutively active Cdc42 or depletion of ARHGAP21 promoted the transport of NA to the plasma membranes. By contrast, cells expressing shRNA targeting Cdc42 or overexpressing ARHGAP21 exhibited a significant decrease in the amount of cell surface-localized NA. Importantly, silencing Cdc42 reduced influenza A virus replication, whereas silencing ARHGAP21 increased the virus replication. Together, our results reveal that ARHGAP21- and Cdc42-based signaling regulates the NA transport and thereby impacts virus replication.

Figures

FIGURE 1.
FIGURE 1.
Infection of influenza A virus causes a decreased expression of ARHGAP21 revealed by microarray analyses and a increase in GTP-bound Cdc42. A, a gene expression microarray analysis was used to determine the differentially expressed genes in A549 cells infected with or without A/WSN/33 influenza virus (H1N1). All the genes whose expressions were changed by at least 2-fold were clustered and displayed. Shown are results from three independent experiments. B, RT-PCR was performed to examine the expression of ARHGAP21 in A549 cells infected with or without WSN viruses. C, quantitative real-time PCR was performed to examine the expression of Cdc42 and ARHGAP21 in A549 cells infected with or without WSN viruses. Plotted are the average expression levels from three independent experiments. The error bars represent the S.E. *, p < 0.01. D, Cdc42-transfected cells were infected with or without WSN viruses, and the cell lysates were used for pulldown Cdc42 activation assays with PAK-GST protein beads. The lysates and the bound material were fractionated by SDS-PAGE and probed with antibodies as indicated. These results are representative of three identical experiments. E, the relative levels of active Cdc42 in D were quantified by densitometric analyses of the immunoblots. Shown are the average values for control cells and cells infected with WSN (n = 3; *, p < 0.01). The error bars represent the S.E.
FIGURE 2.
FIGURE 2.
Rho-family GTPases are required for efficient transport of NA to the cell surface. A, B, and C, a studying system for examination of the cell surface NA was established and verified. A, immunofluorescence staining was performed using an anti-HA antibody to detect HA-tagged NA protein. Shown are confocal micrographs of HeLa cells transfected with NA wild-type (NA(WT)) or NA(H274Y) mutant. The nuclei were stained with DAPI. Bar, 10 μm. B, 293T cells transfected with NA(WT) or NA(H274Y) were examined for the cell surface NA enzymatic activity. The NA(WT) activity is 100, and the result from NA(H274Y) is shown as the percentage difference in the NA activity with respect to NA(WT). Plotted is the average NA activity from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.01). C, lysates derived from cells in B were analyzed by Western blotting using indicated antibodies. D, shown are confocal micrographs of HeLa cells transfected with NA(WT). The cells were mock-treated or treated with toxin B (100 ng/ml) for 15 min. Bar, 10 μm. E, 293 T cells expressing NA were treated as described in D, and cell surface NA activity was analyzed. The average NA activity from three experiments is plotted such that the NA activity in mock treatment is 100. The error bars represent the S.E. (n = 3; *, p < 0.01). F, HeLa cells expressing NA were treated as described in D. For surface labeling of NA, cells were biotinylated with sulfo-NHS-SS-biotin. The labeled surface proteins were then retrieved from the whole cell lysate (WCL) by streptavidin precipitation and examined by immunoblotting with indicated antibodies. G, the relative levels of NA in F were quantified by densitometry and normalized to input (WCL). Plotted are the results from three experiments. The error bars represent the S.E. (n = 3; *, p < 0.01).
FIGURE 3.
FIGURE 3.
Alteration of RhoA function has no significant effect on the transport of NA to the cell surface. A, HeLa cells transfected with NA cDNA were incubated with or without C3 transferase (1 μg/ml) for 15–30 min and followed by immunofluorescence staining. Shown are confocal micrographs. Bar, 10 μm. B, 293T cells expressing NA were treated with C3 transferase as described in A. The cell surface NA activity was measured as described in Fig. 1. Plotted is the average NA activity from three independent experiments. The error bars represent the S.E. C, HeLa cells were co-transfected with plasmid encoding NA and either pEGFP empty vector, GFP-RhoA(WT), GFP-RhoA(G14V), or GFP-RhoA(T19N). Shown are confocal micrographs. Bar, 10 μm. D, cell transfection was performed as described in C. The cell surface NA activity was quantified, and the average NA activity from three independent experiments is plotted. The error bars represent the S.E. E, lysates from cells in D were analyzed by Western blotting using indicated antibodies.
FIGURE 4.
FIGURE 4.
Rac1 function is not absolutely required for the transport of NA to the cell surface. A, HeLa cells co-transfected with NA cDNA, and either pEGFP vector, GFP-Rac1(WT), GFP-Rac1(G12V), or GFP-Rac1(T17N) were stained with an anti-HA-tagged NA antibody. Shown are confocal micrographs. Bar, 10 μm. B, cell transfection was performed as described in A. The cell surface NA activity was measured. Plotted are the results from three independent experiments. The error bars represent the S.E. C, lysates from cells in B were analyzed by Western blotting using the indicated antibodies. D, shRNA-based knockdown of RhoA was analyzed by immunoblot probed with antibodies as indicated (upper panel). The relative levels of RhoA protein in the upper panel were quantified by densitometry and normalized to the actin bands (lower panel). Plotted are the results from three experiments. The error bars represent the S.E. (n = 3; *, p < 0.001). E, shRNA-based knockdown of Rac1 was analyzed as described in D. Plotted are the average Rac1 levels from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.001). F, cells expressing NA and shRNAs targeting either RhoA, Rac1, or luciferase were measured for the cell surface NA activity. Plotted are the results from three independent experiments. The error bars represent the S.E.
FIGURE 5.
FIGURE 5.
Cdc42 regulates the transport of NA to the cell surface. A, shown are confocal micrographs of HeLa cells co-transfected with NA and either pEGFP vector, GFP-Cdc42(WT), GFP-Cdc42(Q61L), or GFP-Cdc42(T17N). Bar, 10 μm. B, cell transfection was performed as described in A. The cell surface NA activity was measured, and the results from three independent experiments are plotted. The error bars represent the S.E. (n = 3; **, p < 0.01; ***, p < 0.05). C, lysates from cells in B were analyzed by immunoblotting using indicated antibodies. D, the interference efficiency of shRNAs targeting Cdc42 was analyzed by real-time PCR. Plotted are the average expression levels from three identical experiments. The error bars represent the S.E. *, p < 0.001. E, shRNA-based knockdown of Cdc42 was analyzed by Western blotting using the antibodies as indicated (left panel). The levels of Cdc42 protein in the left panel were quantified by densitometry and normalized to the actin bands (right panel). Plotted are the average Cdc42 levels from three experiments. The error bars represent the S.E. (n = 3; *, p < 0.001). F, shown are HeLa cells expressing NA and GFP + shRNA vectors targeting Cdc42, ARHGAP21, or luciferase control. Bar, 10 μm. G, cells expressing NA and shRNAs targeting Cdc42, ARHGAP21, or luciferase were measured for the cell surface NA activity. Plotted are the results from three independent experiments. The error bars represent the S.E. (n = 3; **, p < 0.01).
FIGURE 6.
FIGURE 6.
The change in cell surface NA amount is because of altered anterograde transport. A, to determine the endocytosis of NA, HeLa cells transfected with NA cDNA were biotinylated with sulfo-NHS-SS-biotin at 4 °C. Cells were then warmed to 37 °C and mock-treated or treated with toxin B (100 ng/ml) followed by treatment with reduced GSH to cleave surface biotin. Control cells were biotinylated and maintained at 4 °C. The biotinylated proteins that were freshly endocytosed were precipitated with streptavidin-bound agarose and examined by immunoblotting using the indicated antibodies. Shown are representative data from three independent experiments with similar results. B, to examine the anterograde transport of NA, HeLa cells transfected with NA cDNA were first blocked by sulfo-NHS-acetate and then treated with toxin B at 37 °C. Cell surface biotinylation was then performed, and biotinylated proteins were precipitated with streptavidin-bound beads and examined by immunoblotting as described in A. C, the levels of NA protein transported to the plasma membrane in B were quantified by densitometry and normalized to input (whole cell lysate (WCL)). Plotted are the results from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.01). D, HeLa cells were co-transfected with NA and either pEGFP vector or Cdc42(Q61L). The endocytosis of NA was examined as described in A. E, HeLa cells were co-transfected with NA and either pEGFP vector or Cdc42(Q61L). The transport of NA to the cell surface was examined as described in B. F, the levels of NA protein transported to the plasma membrane in E were quantified as described in C. Plotted are the average values from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.01).
FIGURE 7.
FIGURE 7.
ARHGAP21 is involved in regulating transport of NA to the cell surface. A, shown are HeLa cells co-transfected with NA-coding cDNA and either pEGFP vector or plasmid encoding a GFP-fused ARHGAP21 containing the ARF-binding and GAP domains. Bar, 10 μm. B, 293T cells were co-transfected with plasmids as described in A, and the cell surface NA activity was quantified. Plotted is the average NA activity from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.01). C, lysates from cells in B were analyzed by immunoblotting using the indicated antibodies. D, the interference efficiency of shRNAs targeting ARHGAP21 was analyzed by RT-PCR. E, the interference efficiency of shRNAs targeting ARHGAP21 was analyzed by quantitative real-time PCR. Plotted are the average expression levels from three independent experiments. The error bars represent the S.E. (n = 3; *, p < 0.001).
FIGURE 8.
FIGURE 8.
Silencing of Cdc42 or ARHGAP21 has significant effects on influenza A virus replication. A, A549 cell lines stably expressing the shRNAs targeting either Cdc42, ARHGAP21, or control luciferase were infected with A/WSN/33 influenza virus at multiplicity of infection of 0.05. The supernatants of cell culture were harvested and examined for the viral titers by hemagglutinin assay at different time points post-infection as indicated. B and C, Cdc42 knockdown A549 cells, ARHGAP21 knockdown A549 cells, and control cells were infected with influenza A virus as described in A. Viral titers in the supernatants of these cells were examined by plaque assay (post-infection = 24 h) in Madin-Darby canine kidney cells. B, shown are representative of plaques formed by influenza A virus and stained with 0.165 mg/ml neutral red. C, the average viral titers from three independent experiments are plotted. The error bars represent the S.E. (n = 3; *, p < 0.01; **, p < 0.005). PFU, plaque-forming units.

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