Duck PIAS2 Promotes H5N1 Avian Influenza Virus Replication Through Its SUMO E3 Ligase Activity

Abstract

The protein inhibitor of the activated STAT2 (PIAS2) has been implicated in many cellular processes and can also regulate viral replication in mammals. However, the role of PIAS2 in the highly pathogenic avian influenza virus (HPAIV) H5N1 replication in ducks is still unclear. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, we identified that duck PIAS2 (duPIAS2) was one protein that interacted with the nucleoprotein (NP) from the H5N1 HPAIV strain of DK212. Through confocal microscopy images and Co-IP assay, we confirmed NP could interact with duPIAS2. Overexpression of duPIAS2 in primary duck embryo fibroblast (DEF) cells was shown to promote DK212 replication, and knockdown of duPIAS2 could repress DK212 replication. We further found duPIAS2 could promote NP SUMOylation through duck SUMO1 (duSUMO1), and the potential SUMOylation sites of NP were at lysines 7, 48, and 87. Furthermore, duPIAS2 promoted the replication of DK212, here relying on the activity of its SUMO E3 ligase. Duck SENP1 (duSENP1), a deSUMOylation enzyme, could repress NP SUMOylation and also inhibit DK212 replication. Together, we identified duPIAS2 could interact with NP and that duPIAS2 promoted H5N1 HPAIV replication, which might be related to NP SUMOylation.

Keywords: H5N1 avian influenza virus; duck; nucleoprotein; protein inhibitor of activated STAT2; protein–protein interactions.

FIGURE 1

duPIAS2 is identified as a DK212 NP-interacting protein. (A–C) pCAGGS-DK212-NP-Flag or the control vector pCAGGS-eGFP-Flag was transfected into DEF cells. After 36 h, the cells were lysed and immunoprecipitated using anti-Flag agarose beads. The immunoprecipitated protein complexes were identified using LC-MS/MS. (A) Basic statistics for the LC-MS/MS data from pCAGGS-DK212-NP-Flag group. (B) A partial of the identified proteins interacted with DK212 NP using LC-MS/MS. (C) Mass spectrometry of the representative peptides of DK212 NP (upper panel) and duPIAS2 (lower panel). (D,E) duPIAS2 interacted with DK212 NP in transfected cells. HEK 293T cells were transfected with pCAGGS-duPIAS2-HA and pCAGGS-DK212-NP-Flag. After 36 h, the cells were lysed and immunoprecipitated using anti-Flag agarose beads (D) or anti-HA agarose beads (E). The interaction was detected by Western blot using an anti-HA and anti-Flag antibody.

FIGURE 2

duPIAS2 and DK212 NP were colocalized in transfected cells. HEK 293T cells were cultured on coverslips in 24-well plates until 70% confluence and were transfected with pCAGGS-NP-AsRed and pCAGGS-duPIAS2-eGFP. After 36 h, the transfected cells were fixed with cold methanol, stained with DAPI, and visualized by confocal laser scanning microscope.

FIGURE 3

Relative expression of duPIAS2 in DEF cells infected with DK212. DEF cells were cultured in six-well plates until 90% confluence and infected with 10 and 100 TCID₅₀/mL of DK212. At 12 h (A) and 24 h (B) post-infection, the expression of duPIAS2 mRNA was measured by qRT-PCR. duPIAS2 mRNA levels were expressed as relative mRNA indexes, calculated as index (duPIAS2 mRNA copy number/GAPDH mRNA copy number) of test group by index of control group. The experiment was repeated three times. The error bars represent the means and SDs (n = 3) and are compared using a student’s t-test. ^nsP > 0.05, *P < 0.05; **P < 0.01, ****P < 0.0001.

FIGURE 4

duPIAS2 promotes the replication of DK212. (A) DEF cells were transfected with empty vector or pCAGGS-duPIAS2-HA. At 24 h post-transfection, the cells were infected with 100 TCID₅₀ of DK212. The culture supernatants were harvested for measurement of the viral TCID₅₀ at 12, 24, 36, and 48 h post-infection, respectively. (B) DEF cells were transfected with three shRNA targeting duPIAS2 or negative control (shNC). After 36 h, the transfected cells were measured using qRT-PCR for the expression of endogenous duPIAS2. (C) DEF cells were transfected with shPIAS2-1945 or shNC. After 24 h, 100 TCID₅₀/well DK212 was used to infect shRNA-treated DEF cells. Supernatants were collected at 12, 24, 36, and 48 hpi, and viral titers were measured by TCID50 assay. The experiment was repeated three times. The error bars represent the means and SDs (n = 3) and are compared using a student’s t-test. ^nsP > 0.05, *P < 0.05; **P < 0.01.

FIGURE 5

duPIAS2 promotes DK212 NP SUMOylation by duSUMO1. (A) HEK 293T cells were transfected with pCAGGS-DK212-NP-Flag, SUMOylation-related expression plasmids, pCAGGS-duPIAS2-HA, or pCAGGS-duPIAS2mu-HA. (B) HEK 293T cells were transfected with pCAGGS-DK212-NP-Flag, SUMOylation-related expression plasmids, pCAGGS-duSUMO1-Myc, or pCAGGS-duSUMO1mu-Myc. (C) HEK 293T cells were transfected with pCAGGS-DK212-NP-Flag, SUMOylation-related expression plasmids, pCAGGS-duSUMO2-Myc, or pCAGGS-duSUMO2mu-Myc. (D) HEK 293T cells were transfected with pCAGGS-DK212-NP-Flag, SUMOylation-related expression plasmids, or pCAGGS-duPIAS-HA. After 36 h, the transfected cells were lysed and immunoprecipitated with anti-Flag agarose beads. The cellular lysates and immunoprecipitation complexes were analyzed by immunoblotting with the indicated antibodies.

FIGURE 6

K7, K48, and K87 of DK212 NP were potential SUMOylation site. (A) HEK 293T cells were transfected with SUMOylation-related expression plasmids and individual specific K-to-R mutant constructs of NP and NPwt. (B) HEK 293T cells were transfected with SUMOylation-related expression plasmids and NP, K7R, K48R, K87R, K273R, or K325R mutant NP to further confirm the SUMOylation sites of NP. After 36 h, the transfected cells were lysed and immunoprecipitated with anti-Flag agarose beads. The cellular lysates and immunoprecipitation complexes were analyzed by immunoblotting with the indicated antibodies.

FIGURE 7

The SUMO E3 activity of duPIAS2 is necessary to promote DK212 replication. (A) duSENP1 inhibited the SUMOylation of DK212 NP promoted by duPIAS2. HEK 293T cells were transfected with pCAGGS-DK212-NP-Flag, SUMOylation-related expression plasmids, and pCAGGS-duSENP1-V5. After 36 h, the transfected cells were lysed and immunoprecipitated with anti-Flag agarose beads. (B) DEF cells were transfected with pCAGGS-duPIAS2-HA, pCAGGS-duPIAS2mu-HA, pCAGGS-duSENP1-V5, or pCAGGS. At 24 h post-transfection, the cells were infected with 100 TCID₅₀ of DK212. The culture supernatants were harvested for measurement of their viral TCID₅₀ at 12, 24, 36, and 48 h post-infection, respectively. The experiment was repeated three times. The error bars represent the means and SDs (n = 3) and are compared using two-way ANOVA. ^nsP > 0.05, *P < 0.05; **P < 0.01; ****P < 0.0001.