NEDDylation of PB2 Reduces Its Stability and Blocks the Replication of Influenza A Virus

Post-translational modifications of viral proteins play important roles in regulating viral replication. Here we demonstrated that the PB2 of influenza A virus (IAV) can be modified by NEDD8. We revealed that E3 ligase HDM2 can promote PB2 NEDDylation. Overexpression of either NEDD8 or HDM2 can inhibit IAV replication, while knockdown of HDM2 has the opposite effect. Then we identified residue K699 in PB2 as the major NEDDylation site. We found that NEDDylation deficient PB2 mutant (PB2 K699R) has a longer half-life than wild-type PB2, indicating that NEDDylation of PB2 reduces its stability. We generated an IAV mutant in which PB2 was mutated to PB2 K699R (WSN-PB2 K699R) and examined the replication of WSN and WSN-PB2 K699R viruses in both MDCK and A549 cells and found that the replication of WSN-PB2 K699R was more efficient than wild-type WSN. In addition, we observed that overexpression of NEDD8 significantly inhibited the replication of WSN, but not WSN-PB2 K699R. The infection assay in mice showed that WSN-PB2 K699R exhibited enhanced virulence in mice compared to WSN, suggesting that NEDDylation of PB2 reduced IAV replication in vivo. In conclusion, we demonstrated that NEDDylation of PB2 by HDM2 negatively regulates IAV infection.

Scientific RepoRts | 7:43691 | DOI: 10.1038/srep43691 deconjugation of NEDD8 from its substrates 23,24 . NEDDylation is now accepted widely to be an important modification mechanism to regulate protein activity, stability, cellular localization, and protein-protein interactions. Furthermore, it has been shown that NEDDylation plays important roles during the life cycle of several viruses, and several viral proteins were found to be NEDDylated 25 . For human immunodeficiency virus type-1 (HIV-1), NEDD8 acts through p27 and β -catenin pathways to disrupt adipogenesis and consequent lipodystrophy in patients affected by HIV infection under HAART therapy 26,27 . For Kaposi's sarcoma-associated herpesvirus, NEDDylation is essential for viral latency because it is critical for the viability of KSHV infected lymphoma cells and for the replication of viral genome 28 . However, the relevance of NEDDylation modification with IAV is not well understood.
In this study, we demonstrate that the PB2 of IAV can be NEDDylated at residue K669 by HDM2 during infection. NEDDylation of PB2 reduces its stability and in turn inhibits viral replication, whereas mutation of the PB2 NEDDylation site favors viral replication and enhances virulence in mice. We provide evidence that NEDDylation modification plays an important role in regulating the replication of IAV.

Results
PB2 is conjugated to NEDD8. In recent years several ubiquitin-like (UBL) proteins, such as SUMO1 and ISG15, have been identified to regulate IAV replication. In order to determine whether vRNP proteins can be modified by NEDD8, we transfected HEK293T cells with FLAG-tagged PB1, PB2, PA or NP expression plasmids together with His-NEDD8 expression plasmid, and then performed the His-pulldown assay. As shown in Fig. 1a, NEDDylated protein bands can be observed in cells expressing FLAG-PB2 and His-NEDD8, a few weak bands were seen from cells expressing PA and PB1 with His-NEDD8 and there was no band in NP expressed cells, indicating that PB2 is obviously modified by NEDD8. Interestingly, we confirmed that PB2 can also be ubiquitinated, which is consistent with previous report 29 (Fig. 1b). However, we did not observe that PB2 can be modified by SUMO1.
We then examined whether PB2 can be modified by endogenous NEDD8. HEK293T cells were transfected with FLAG-PB2 expression plasmid and the cell lysates were subjected to immunoprecipitation with FLAG antibody, followed by immunoblotting with NEDD8 antibody. PB2 can also be NEDDylated by endogenous NEDD8 (Fig. 1c).
Next, we investigated whether PB2 was NEDDylated during IAV infection. We infected A549 cells with the A/WSN/33 (H1N1; WSN) virus and harvested the cell lysates for immunoprecipitation with PB2 antibody, followed by immunoblotting with NEDD8 antibody. The data showed that NEDDylated PB2 can be detected in virus infected cells (Fig. 1d), indicating that viral PB2 can be modified by NEDD8.
To quantify the ratio of NEDDylated PB2 in infected cells, A549 cells were infected with WSN virus. Then the cells were subjected to immunoprecipitation with NEDD8 antibody with 1/20 of total cell lysates as input, followed by immunoblotting with PB2 antibody (Fig. 1e). The intensity of NEDDylated PB2 and input PB2 was quantified. The ratio of NEDDylated PB2 was approximately 3.9% of total PB2. We next examined if PB2 proteins derived from other IAV strains can also be modified by NEDD8. Expression plasmids for PB2 from four IAV subtypes (A/WSN/1933 (H1N1); A/Guangdong/ST798/2008 (H3N2); A/Anhui/1/2013 (H7N9); and A/HK/2108/2003 (H9N2)) were cotransfected with pEF-His-NEDD8 into HEK293T cells, and cell lysates were subjected to His-pulldown assay as described previously. The data demonstrated that PB2 from these IAV strains can also be NEDDylated (Fig. 1f).
To quantify the ratio of NEDDylated versus total PB2 in transfected cells, HEK293T cells were transfected with FLAG-PB2 along with or without His-NEDD8. Cell lysates were then subjected to His-pulldown assay with 1/10 of lysates as input (Fig. 1g). The intensity of NEDDylated and input PB2 was quantified, and the ratio of NEDDylated PB2 was determined to be approximately 9.3% of total PB2. HDM2 is the NEDDylation E3 ligase for PB2. To identify the E3 ligase responsible for the NEDDylation of PB2, we transfected HEK293T cells with FLAG-PB2 and His-NEDD8 together with plasmids expressing one of the known E3 ligases (HDM2, XIAP, TRIM40, Smurf1, RNF111, c-CBL, SCCRO or RBX1) 30 , and performed the His-pulldown assay. The results showed that HDM2 can significantly enhance the intensity of PB2 NEDDylation (Fig. 2a). We then generated a HDM2 C464A mutant, which lacks E3 ligase activity 31 , and repeated the experiment. The data indicated that HDM2 C464A can no longer promote the NEDDylation of PB2 (Fig. 2b).
Since HDM2 is also known to be a ubiquitin E3 ligase, we wondered whether it specifically promotes PB2 NEDDylation. To address this question, we transfected HEK293T cells with plasmids expressing His-ubiquitin or His-NEDD8 together with HDM2 or XIAP as a negative control. The cell lysates were subjected to His-pulldown assay. As shown in Fig. 2c, HDM2 only promotes the NEDDylation, but not ubiquitination, of PB2. We next examined whether HDM2 can interact with PB2 by a co-immunoprecipitation assay. The data showed that HDM2 can bind to PB2 (Fig. 2d). To examine the effect of endogenous HDM2 on PB2 NEDDylation, we performed the HDM2 knockdown experiments. As shown in Fig. 2e, NEDDylation of PB2 was reduced in HEK293T cells treated with two sets of HDM2 siRNAs. These results demonstrated that HDM2 functions as an E3 ligase to mediate the NEDDylation of PB2.
Overexpression of HDM2 and NEDD8 inhibits IAV replication. To examine whether NEDDylation affects IAV replication, we transfected A549 cells with His-NEDD8 or pEGFP-C2 as a control, and infected the cells with IAV at an MOI of 0.5. The cells were harvested at 6 h post-infection, and then subjected to analysis by real-time PCR and immunoblotting. The levels of vRNA and mRNA of M1 were found to be reduced in NEDD8-overexpressing cells (Fig. 3a). The levels of M1, NP and PB2 proteins were also decreased in cells overexpressed NEDD8 compared to that in control cells (Fig. 3b). Since HDM2 is the E3 ligase for PB2 NEDDylation, we transfected A549 cells with pCMV Myc-HDM2 or control plasmids and then infected these cells with IAV ) were separately co-transfected with or without pEF-His-NEDD8 into HEK293T cells. After 48 h, cell lysates were harvested for His-pulldown assay followed by immunoblotting with anti-Myc antibody. TCL: total cell lysate. (g) HEK293T cells were transfected with FLAG-PB2 together with or without His-NEDD8, and cell lysates were collected for His-pulldown assay. 1/10 of total cell lysates was used as input. The intensity of NEDDylated PB2 and input PB2 were measured by Quantity One. *Unmodified PB2 non-specifically pulleddown by anti-NEDD8 antibody.  (A/WSN/33 (H1N1)). Cell lysates were harvested for immunoblotting and supernatants were collected for plaque assay to determine virus titers. The immunoblotting data indicated that the amount of M1, NP and PB2 proteins were also reduced in HDM2 transfected cells compared to that in control cells (Fig. 3c), and virus titers were significantly lower in HDM2 overexpressed cells than that of control cells (Fig. 3d).
To examine the effect of endogenous HDM2 on viral replication, we performed the HDM2 knockdown and rescue experiments. We transfected A549 cells with si-HDM2 alone or together with pCMV-Myc-HDM2, and then infected the cells with IAV. The cell lysates were harvested for immunoblotting and supernatants were collected for plaque assay. The data showed that the protein levels of NP and PB2 (Fig. 3e) and viral titers (Fig. 3f) were higher in HDM2 knockdowned cells than that in control cells, while Myc-HDM2 can rescue the effect of HDM2 knockdown on viral replication. Taken together, these data suggested that HDM2 negatively regulates IAV replication.
K699 is the major NEDDylation site in PB2. We 18 conserved lysines were found. We generated PB2 mutants containing individual mutations of these lysines to arginines, and examined their NEDDylation levels in HEK293T cells. The level of NEDDylation in the K699R mutant was dramatically reduced compared to that of wild type and other PB2 mutants (Fig. 4a), which suggested that residue K699 is the major NEDDylation site. Since PB2 can also be modified by ubiquitin, we compared the level of ubiquitination of wild-type PB2 (PB2-WT) and mutant PB2 K699R (PB2-K699R). As shown in Fig. 4b, the level of ubiquitination of PB2-WT and PB2-K699R are comparable, suggesting that K699 is not a ubiquitination site. Furthermore, we performed mass-spectrometry (MS) analysis to determine whether PB2 K699 was modified. We transfected HEK293T cells with pCMV-Myc-PB2 (aa 600-759), together with plasmids expressing His-NEDD8. The Myc-PB2 (aa 600-759) was purified with anti-Myc agarose beads and subjected to MS analysis. The data indicated that the peptide GFLILGK (699) EDRR of PB2 was GlyGly-modified (Fig. 4c). Taken together, these data demonstrate that PB2 K699 is the major NEDDylation site in PB2.
NEDDylation reduces PB2 stability. To examine whether NEDDylation of PB2 influences its stability, we compared the half-life of PB2-WT and PB2-K699R. We transfected HEK293T cells with pCMV-FLAG-PB2 or pCMV-FLAG-PB2 K699R with or without pEF-His-NEDD8, and then treated the cells with cycloheximide (CHX) for 1 and 2 h. The total cell lysates were harvested for immunoblotting to determine the amount of FLAG-PB2 and FLAG-PB2 K699R (Fig. 5a). The relative levels of FLAG-PB2 and FLAG-PB2 K699R from 2 independent experiments were quantified (Fig. 5b). The data indicated that the level of PB2-WT was decreased in cells treated with CHX and reduced more dramatically in NEDD8 overexpressed cells, whereas the level of PB2-K699R did not decrease even in cells overexpressing His-NEDD8.

NEDDylation of PB2 hinders the replication of IAV. To understand the biological significance of
NEDDylation of PB2 K699 in the propagation of IAV, we generated an IAV mutant in which PB2 was mutated to PB2 K699R (WSN-PB2 K699R) by taking the approach of reverse genetics. Then we compared the proliferation kinetics of WSN with WSN-PB2 K699R. We infected MDCK cells with WSN or WSN-PB2 K699R and harvested the supernatants at different time points (6,12,24, and 36 h post-infection) for plaque assay. WSN-PB2 K699R replicated much more efficiently than WSN in MDCK cells, resulting in higher virus titers. At 12 h, 24 h and 36 h post infection, the titers of WSN-PB2 K699R were significantly higher than that of WSN (Fig. 6a), This result suggested that NEDDylation of PB2 K699 restricted optimal virus growth.
Furthermore, we examined the replication of wild type WSN virus and WSN-PB2 K699R mutant in A549 cells transfected with NEDD8 expressing plasmid or control plasmid. We analyzed virus titers in the supernatant by plaque assay and compared protein levels of M1, PB2 and NP by immunoblotting. The data showed that the titers of WSN were lower than that of WSN-PB2 K699R. More importantly, we found that the titers of WSN were significantly reduced in the NEDD8-overexpressing cells while the titers of WSN-PB2 K699R were comparable between control and NEDD8-overexpressing cells (Fig. 6b). Consistently, immunoblotting data indicated that the protein levels of M1, NP and PB2 of WSN but not WSN-PB2 K699R in NEDD8-overexpressing cells were greatly decreased compared to that in control cells (Fig. 6c). Furthermore, we generated the vRNP reporter 293 T cells (293T-IAV-Luc), in which the luciferase gene driven by NP promoter was integrated in the genome of cells. We infected the 293T-IAV-Luc cells with WSN or WSN-PB2 K699R and harvested the cell lysates for luciferase assay. The data showed that luciferase activity in WSN infected cells were much lower than that in WSN-PB2 K699R infected cells (Fig. 6d). These results strongly suggested that NEDDylation of PB2 at K699 suppressed the replication of IAV.

WSN-PB2 K699R virus possesses higher virulence than WSN.
We next assessed the impact of NEDDylation of PB2 on virus pathogenicity in mice. We infected BALB/c mice intranasally with WSN or WSN-PB2 K699R at a dose of 10000 PFU, measuring the body weights and monitoring for survival daily after challenge. Mice infected with WSN-PB2 K699R exhibited more severe weight loss compared to those infected with WSN (Fig. 7a). Also, WSN-PB2 K699R caused 30% mortality as early as at 5 day post-infection (dpi), and 67% mortality at 6 dpi, which were higher than that caused by WSN (Fig. 7b). We also examined the viral titers in the lungs of infected mice at both 3 and 5 dpi. Consistently, viral titers in lungs of mice infected with WSN-PB2 K699R were higher than that of WSN infected mice at both time points (Fig. 7c). In addition, we performed histological analysis of the lungs from infected mice at 3 and 5 dpi. Both viruses caused bronchus-alveolar epithelial degeneration and interstitial pneumonia, as well as focal or diffuse lung damage. The peribronchiolar and perivascular areas of infected mice were infiltrated by numbers of lymphocytes and plasma cells. However, the alveolar damage and interstitial inflammatory infiltration in WSN-PB2 K699R mice were much more severe than that of WSN infected mice. Moreover, necrosis was observed in the lung tissues of mice infected with WSN-PB2 K699R (Fig. 7d). These results illustrate that WSN-PB2 K699R causes more severe pathogenicity in mice than that of WSN, suggesting that preventing NEDDylation of PB2 promotes virus propagation and enhances virulence. . The experiment is repeated twice and results were found to be comparable, while all samples were assayed in triplicate. The presented data is the mean from one experiment. Cell lysates were harvested and subjected to immunoblotting with indicated antibodies (c). (d) 293T-IAV-Luc cells were infected with WSN or WSN-PB2 K699R at an MOI of 0.1 for 12 h. The cell lysates were harvested for luciferase assay. The experiment was repeated twice and results were found to be consistent. The presented data is the mean of three biological samples from one experiment. *p < 0.05.

Discussion
The NEDD8 protein is a type of ubiquitin-like protein (UBL) and shares the greatest similarity to ubiquitin among all of the UBLs, and can be conjugated to substrates in a process known as NEDDylation 21 . NEDDylation plays pivotal roles in regulating cellular processes as well as the viral life cycle 32 . Ubiquitin and other ubiquitin-like proteins, such as SUMO and ISG15, have been found to regulate the IAV life cycle 16,[33][34][35] . However, whether NEDDylation modification is involved in the replication of IAV is not known. In this study, we demonstrated that PB2 can be modified by NEDDylation, which reduces its stability and inhibits the replication of IAV in vitro and in vivo.
PB2 is an 87-kDa basic cap-binding protein involved in the initiation of viral transcription as well as viral replication. The N-and C-terminal regions of PB2 contain independent NP binding sites, located between residues 1-269 and 580-683, respectively. The interaction of PB2 with NP affected the activity of RNPs, and may be involved in regulating the switch from viral transcription to replication [36][37][38] . Although we did not observe that PB2 NEDDylation influences the association of PB2 with NP, we cannot rule out the possibility that NEDDylation of PB2 could affect the formation of vRNP complexes. It has been reported that two regions of PB2 (aa 448-496 and aa 736-739) were required for the nuclear localization of influenza virus PB2 39,40 . It will be interesting to further analyze whether PB2 NEDDylation affects its subcellular localization in addition to influence its stability.
Recently, the crystal structures of influenza viral polymerase were solved 41,42 . PB2 K699 is exposed at the external loop of the trimeric viral polymerase, and is likely to be modified by NEDD8. According to the structure of PB2 and other viral polymerase subunits, the PB2 K699R mutation would not affect the tertiary structure of PB2 or the trimeric viral polymerase. However, the NEDDylation of PB2 K699 may influence the structure of PB2 and consequently interfere with the function of vRNP.
It has been reported that some mutated influenza viruses grow faster in vitro than wild type viruses, but peak lung viral titers in vivo were not observed to be different among these viruses 43 . We compared WSN and WSN-PB2 K699R growth kinetics in cell culture, in addition to virulence in a mouse model. We observed that WSN-PB2 K699R replicated better than WSN in MDCK cells, and consistently WSN-PB2 K699R causes more severe pulmonary damage and results in higher lethality rates. In addition, the viral titers in the lungs of mice infected with WSN-PB2 K699R were higher than animals in the WSN group. Our results demonstrate that NEDDylation of PB2 influences viral replication both in vitro and in vivo. In order to know whether PB2 K699 is conserved or not among the different influenza virus strains, we compared the sequences of 3500 publically-available influenza virus strains in the NCBI database (http://www.uniprot.org/uniprot/?query= pb2+ AND+ influenza+ virus&sort= score). The data showed that PB2 K699 is highly conserved and present in 3399 out of 3500 strains, and 95 strains are with PB2 R699, indicating that the virus strains with PB2 K699 are predominant. We postulate that the virus with PB2 K699R mutation may proliferate better in cultured cells due to avoiding been , respectively (10000 PFU each). The body weight (a) and survival (b) of mice were monitored daily. At 3 and 5 dpi, the lungs of infected mice (n = 3) were collected to measure the virus titer (c) and as well as histopathological analysis (d). Representative histological images of lung tissues from mock-challenged mice (left panels), mice treated with WSN (middle panels) or WSN-PB2 K699R (right panels) were shown. All Experiments for the plague assay were repeated twice and results were found to be comparable. The presented data were expressed as the mean from one experiment. *p < 0.05; **p < 0.01; ***p < 0.001.
Scientific RepoRts | 7:43691 | DOI: 10.1038/srep43691 NEDDylated, but on the other hand it may not adapt to host as well as the strain with PB2 K699 which may limit it to spread out. However this assumption needs further investigated.
Taken together, this study revealed that NEDDylation of PB2 by HDM2 negatively regulates influenza A viral replication. Our results suggested that NEDDylation modification on viral protein could be a host antiviral manner. These findings will contribute to our understanding of NEDDylation and its role in regulating viral infection.

Cell Culture, Virus Strains and Reagents. Human embryonic kidney HEK293T cells (HEK293T),
Madin-Darby canine kidney cells (MDCK), and human lung adenocarcinoma epithelial cells (A549) were obtained from China Infrastructure of Cell Line Resources and maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin G and 100 g/ml streptomycin).
His-pulldown Assay. His-pulldown assays were performed to examine the post-translational modification of proteins. Briefly, HEK293T cells in 10 cm dish were transfected with pEF-His-NEDD8 and the indicated plasmids for 48 h. Cells were harvested and lysed in denaturing lysis buffer (6 M guanidinium-HCl, 0.1 M Na 2 HPO 4 and NaH 2 PO 4 , 0.01 M Tris-HCl, pH 8.0, 5 mM imidazole and 10 mM β -mercaptoethanol) for 30 min at room temperature. 70 μ l of Ni + beads were added to the lysate for 4 h and then washed with washing buffer. The samples were eluted with 30 μ l of elution buffer (0.15 M Tris-HCl, pH 6.7, 30% Glycerol, 5% SDS, 500 mM imidazole) and analyzed by SDS-PAGE and immunoblotting.
Immunoprecipitation. HEK293T cells in 10-cm dishes were transfected with the indicated plasmids for 48 h. Cells were collected and lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA) supplemented with protease inhibitor (Roche) for 15 min at 4 °C, and then centrifuged at 13400 × g for 25 min. The supernatants were incubated with 10 μ l of the indicated antibody agarose slurry (Sigma) for 4 h at 4 °C, and then washed three times with lysis buffer. The samples were dissolved in SDS loading buffer and subjected to immunoblotting.
Generation of Recombinant Viruses. The reverse-genetics systems for the generation of recombinant IAV (WSN) have been described previously 44,46 . For the generation of recombinant WSN viruses, HEK293T cells were cotransfected with eight viral genome-expressing plasmids (pHH21 PB2 for wild type WSN virus, or pHH21 PB2-K699R for WSN-PB2 K699R virus) and four pCAGGS protein expression plasmids encoding the viral polymerase subunits PB1, PB2 and PA (pCAGGS PB2 for wild type WSN virus, or pCAGGS PB2-K699R for WSN-PB2 K699R virus) as well as NP. At 48 h post-transfection, recombinant viruses were passaged to fresh MDCK cells for virus amplification and rescue. Viral Growth Kinetics. A549 cells were infected with wild-type A/WSN/33 (H1N1) or mutant virus WSN-PB2 K699R at an MOI of 0.01. At 1 h post infection, the cells were cultured in serum-free medium. The supernatants were harvested at the indicated times, and virus titers were determined by plaque assay on MDCK cells.
Plaque Assay. MDCK monolayer cells in 12-well dishes were washed twice with PBS, followed by infection with serial 10-fold dilutions of virus in serum-free DMEM medium supplemented with 4 μ g/ml of TPCK-trypsin (Sigma), and incubated at 37 °C for 1 h. Afterwards, the cells were washed with PBS and overlaid with Modified Eagle's Medium (MEM) containing 1% agarose (AMRESCO) and 2 μ g/ml of TPCK-trypsin. After 2 days, visible plaques were counted and viral titers were calculated.