Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication

Abstract

During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process.

Importance: Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes.

FIG 1

The host cellular protein A5 is upregulated at the cell surface after IAV infection. A549 (A) or MDCK (B) cells were either left uninfected or infected with A/PR/8/34, A/Udorn/72, or A/WSN/33 viruses (MOI of 10). At 24 hpi, flow cytometry analysis was performed with an anti-A5 antibody (closed histograms) or an isotype control (open histograms). The viral protein M2 was used as a positive control for viral infection. The results are representative of two independent experiments.

FIG 2

The host cellular protein A5 is translocated to the cell surface. (A) A549 cells were either left uninfected or infected with IAV A/WSN/33 virus (MOI of 1). At 24 hpi, cellular A5 or viral HA proteins were visualized by immunofluorescence microscopy, using anti-A5 and anti-HA specific antibodies, respectively. The nuclei were stained with DAPI (4′,6′-diamidino-2-phenylindole), and the merged images are shown (original magnification, ×189). The results are representative of two independent experiments. Please note the presence of A5 labeling in the cytoplasm in uninfected cells, which is largely absent in the infected ones (arrows) but rather detected at the plasma membrane (stars). (B) A549 cells were either left uninfected or infected with A/WSN/33 virus (MOI of 10). At 24 hpi, flow cytometry analysis was performed using an anti-A5 antibody (closed histograms) or an isotype control (open histograms). Labeling of A5 was performed either on unpermeabilized cells, showing cell surface A5 proteins, or on permeabilized cells, showing total A5 proteins (left panel). Quantification of the mean fluorescence intensity of A5 expression ± the SD from five independent experiments is shown on the right panel. *, P < 0.05 (NI versus WSN).

FIG 3

Kinetic of cell surface expression of A5 after IAV infection and its expression in DRM fractions. (A) Time course experiment of cell surface expression of A5 upon infection of A549 cells with A/WSN/33 virus (MOI of 10⁻² in the presence of trypsin). Expression of the viral M2 protein was used as a positive control of viral infection. (B) A549 cells were either left uninfected or infected with A/WSN/33 virus (MOI of 10) for 16 h. Cells were then lysed, and the DRM domains were isolated by sucrose gradient ultracentrifugation. After centrifugation, 1-ml fractions were collected from the top of the tube and characterized by Western blot analysis (fractions 1 to 10). Blots were probed with anti-ERK antibody (ERK), cholera toxin B subunit (GM1), and anti-HA (HA₀-HA₂), anti-M2 (M2), and anti-A5 (A5) antibodies. Fractions 3 to 5 correspond to the DRMs, whereas the soluble fractions correspond to fractions 7 to 10. The results are representative of two independent experiments.

FIG 4

Cellular A5 protein is incorporated into IAV particles. (A) A/PR/8/34, A/Udorn/72, and A/WSN/33 viruses, produced on MDCK cells, were purified by sucrose ultracentrifugation and analyzed by Western blotting with anti-A5, anti-M2, and anti-ERK antibodies. Aliquots of total proteins from MDCK cells either left uninfected or infected for 16 h with A/PR/8/34 strain were used as controls. The molecular mass is indicated in kilodaltons. (B) Electron microscopic immunogold labeling was performed on purified virions using A5-specific antibodies or isotype control. Scale bar, 50 nm. The results presented in both panels are representative of three independent experiments.

FIG 5

Packaged A5 inhibits IFN-γ receptor signaling. (A) Macrophage-differentiated THP-1 cells were infected with A/WSN/33 virus (MOI of 1), and virus titers were determined in the supernatants of the cells at the indicated time points postinoculation. (B) Macrophage-differentiated THP-1 cells were treated with different doses of human rIFN-γ for 5 min at 37°C. The cells were lysed, and Stat phosphorylation was analyzed by Western blotting with an anti-phospho Stat antibody (p-Stat). Tubulin was used as a control for loading. (C and D) Differentiated THP-1 cells were incubated for 5 min with purified A/WSN/33 particles (MOI of 1), which were either pretreated (AV-V) or not pretreated (V) with an anti-A5 antibody. The cells were then either left unstimulated or stimulated with IFN-γ (1,000 IU). (C) After 5 min, the cells were lysed, and Stat phosphorylation was analyzed by Western blotting. (D) Alternatively, IP-10 release was evaluated in the supernatant at 3 h poststimulation by classical ELISA. *, P < 0.05 (between “−” versus “V” and “V” versus “AV-V”). The results in panels A to D are representative of at least two independent experiments. (E) Differentiated THP-1 cells were incubated for 5 min with purified A/WSN/33 particles (MOI of 1), which were either pretreated (AV-V) or not pretreated (V) with an anti-A5 antibody. The cells were then analyzed for virus binding by flow cytometry with an anti-HA antibody (left panel). The MFI for HA staining was obtained from three replicates (right panel). (F) Infectious titers of V and AV-V preparations.

FIG 6

Packaged A5 inhibits IFN-γ receptor signaling. (A) Western blot analysis of virions produced from 293T cells transfected with nontargeted siRNA (Ctl siRNA v) or specific siRNA targeting A5 (A5 siRNA v), using an anti-A5 antibody. Anti-HA antibody was used as a positive control for virus detection. (B) Infectious titers of Ctl siRNA v and A5 siRNA v preparations. (C) Western blot analysis of control siRNA v and A5 siRNA v, using a polyclonal anti-influenza virus antibody. (D to F) Differentiated THP-1 cells (D) or HeLa cells (E) were incubated for 5 min with Ctl siRNA v or A5 siRNA v (MOI of 1). Cells were then either left unstimulated or stimulated with IFN-γ (, IU). After 5 min, the cells were lysed, and Stat phosphorylation was analyzed by Western blotting. (F) Alternatively, IP-10 release was evaluated in the supernatant at 3 or 24 h poststimulation by classical ELISA. *, P < 0.05 (between “−” versus “Ctl siRNA v” and “Ctl siRNA v” versus “A5 siRNA v”). The results are representative of at least two independent experiments.

FIG 7

Packaged A5 does not inhibit IFN-α receptor signaling. (A) Differentiated THP-1 cells were incubated with Ctl siRNA v or A5 siRNA v (MOI of 1) for 5 min at 37°C or for 30 min at 4°C. (B) The cells were then analyzed for virus binding by flow cytometry with an anti-HA antibody, and the MFI of HA staining was obtained from three triplicates. (C) Alternatively, cells were incubated with the virus for 30 min at 4°C and with a shift to 37°C to allow virus internalization. Labeling of HA was performed either on unpermeabilized cells, showing cell surface-bound viruses (left panel), or on permeabilized cells, showing total viruses, including the cell surface and internalized ones (right panel). (D) The MFI of HA staining was obtained from three triplicates. (E and F) Differentiated THP-1 cells were incubated for 5 min with Ctl siRNA v or A5 siRNA v (MOI of 1). The cells were then either left unstimulated or stimulated with IFN-α (1,000 IU) or IFN-γ (1,000 IU). (E) After 5 min, the cells were lysed, and Stat1 phosphorylation was analyzed by Western blotting. (F) Alternatively, IP-10 release was evaluated in the supernatant at 3 h poststimulation by classical ELISA. *, P < 0.05 (between “−” versus “Ctl siRNA v” and “Ctl siRNA v” versus “A5 siRNA v”). The results are representative of two independent experiments (B and C).

FIG 8

Packaged A5 inhibits the antiviral activity mediated by IFN-γ in vitro. PMA-differentiated THP-1 macrophages were infected with purified A/WSN/33 particles, in which A5 was previously masked (AV-V) or not masked (V) with anti-A5 antibody (A), or the supernatant of A/WSN/33-infected 293T cells, in which expression of A5 was downregulated by siRNA (A5 siRNAv) or not downregulated (Ctl siRNAv) (B). All viruses were used at an MOI of 1. The cells were either left in the presence or in the absence of rIFN-γ. Infectious virus titers were then evaluated in the supernatant of the cells at 24 hpi. The results represent mean virus titers ± the SD from three independent experiments. *, P < 0.05 (between “V” versus “AV-V” and “Ctl siRNA v” versus “A5 siRNA v”). The results are representative of three independent experiments.

FIG 9

Packaged A5 inhibits the antiviral activity mediated by IFN-γ in vivo. (A) Mice were infected with purified A/PR/8/34 virus (500 PFU) and treated with the indicated quantities of mouse rIFN-γ by intranasal administration. At 2 days postinfection, virus titers were evaluated in the lungs by classical plaque assay. (B) Mice (n = 5 per group) were treated with 8 × 10⁴ IU of rIFN-γ and infected with purified A/PR/8/34 viruses, in which A5 was previously blocked with anti-A5 antibody (AV-V) or not blocked (V). At 2 days postinfection, lung virus titers were evaluated by plaque assay. *, P < 0.05 (between “V” and “AV-V”). The results are representative of two independent experiments.