It has been reported that AIP4 is involved in M1 ubiquitination (Su et al. 2013). To investigate whether AIP4 is the specific E3 ubiquitin ligase of M1, the 293T cells were transfected with Myc-M1 and HA-Ub, along with Flagtagged AIP4 or other E3 ubiquitin ligases, such as TRIM25 and Smurf1. We observed that only AIP4 promoted M1 ubiquitination (Fig. 1A). We next examined the type of ubiquitin chain modification on M1 by AIP4. Flag-AIP4 and Myc-M1 were co-transfected with HA-Ub, HA-Ub-K48, or HA-Ub-K63 in 293T cells. As shown in Fig. 1B, AIP4 mediated K48-linked M1 ubiquitination.
Figure 1. AIP4 mediates K48-linked ubiquitination of M1. (A) Immunoblot analysis of lysates of 293T cells transfected with Flag-vector, Flag-AIP4, Flag-TRIM25, or Flag-Smurf-1, along with Myc-M1 and HA-Ub for 36 h, followed by immunoprecipitation (IP) with anti-M1 antibody. (B) Immunoblot analysis of lysates of 293T cells transfected with HA-Ub, HA-Ub-K48 or HA-Ub-K63, along with FlagAIP4 and Myc-M1 for 36 h, followed by IP with anti-M1 antibody.
It is well established that the K48-linked polyubiquitin chains lead to the proteasome degradation of target protein (Finley 2009). Therefore, we examined whether AIP4 affects the stability of M1. 293T cells, co-transfected with Myc-M1 and Flag-AIP4, were treated with CHX, along with DMSO, MG132 or NH4Cl. The results showed that the proteasome inhibitor MG132 blocked the degradation of M1 promoted by AIP4, whereas DMSO and the lysosome inhibitor NH4Cl did not (Fig. 2A), indicating that AIP4 promoted the ubiquitin–proteasome degradation of M1. Additionally, we further investigated the stability of M1 regulated by AIP4 upon influenza A virus infection. Flag-AIP4 or Flag-vector was transfected in 293T cells, then infected with the influenza virus A/WSN/1933(H1N1). As expected, AIP4 promoted the degradation of M1 (Fig. 2B). Collectively, these results revealed that AIP4 promoted ubiquitin–proteasome degradation of M1.
Figure 2. AIP4 affects the stability of M1. (A) Immunoblot analysis of lysates of 293T cells transfected with Myc-M1 and Flag-AIP4 for 24 h, then treated with 100 μg/mL CHX, along with 10 μmol/L MG132, 10 μmol/L NH4Cl or DMSO for 9 h (left). The relative density of M1 was normalized to β-actin (right). (B) Immunoblot analysis of lysates of 293T cells transfected with Flag-AIP4 or Flag-vector for 24 h, then infected with the influenza virus A/WSN/1933(H1N1) (MOI = 1) for 9 h (left). The relative density of M1 was normalized to β-actin (right). Data are shown as mean ± SD (n = 3). ***P < 0.001 (unpaired, two-tailed student's t test).
The E3 ubiquitin ligase is known to be covalently linked to the K residue of target proteins. Thus, the K residues of IAV M1 were analyzed based on the influenza sequence database (Fig. 3A). To determine the ubiquitination sites of AIP4, ten lysine residues (K21, K35, K47, K57, K95, K98, K102, K104, K113, and K187) were selected for further studies. We first constructed the M1 mutants (K to R), among which the adjacent K95/K98 and K102/K104 were constructed to have dual mutation. The 293T cells were transfected with the wild-type (WT) and mutant M1, together with Flag-AIP4 and HA-Ub. The Co-IP assay showed that only K102R/K104R blocked AIP4-mediated M1 ubiquitination (Fig. 3B), indicating that K102 and K104 are crucial for M1 ubiquitination induced by AIP4. Furthermore, we also found that the K35R mutant significantly increased the ubiquitination of M1. The precise mechanism needs to be further explored. To assess the effect of K102 and K104 on AIP4-mediated M1 stability, 293T cells were transfected with Flag-AIP4, along with WT-M1 or mutant M1 (K102R/K104R). As shown in Fig. 3C, the degradation of M1 was largely blocked by K102R/K104R, suggesting that K102 and K104 is crucial to the ubiquitin–proteasome degradation of M1 mediated by AIP4.
Figure 3. AIP4 regulates M1 ubiquitination at K102 and K104. (A) Schematic diagram representing the functional domains of M1. K21, K35, K47, K57, K95, K98, K102, K104, K113, and K187 of M1 were displayed. NEP, nuclear export protein. NLS, nuclear localization signal. (B) Immunoblot analysis of lysates of 293T cells transfected with the indicated plasmids for 36 h, followed by IP with anti-M1 antibody. (C) Immunoblot analysis of lysates in lysates of 293T cells transfected with the indicated plasmids for 36 h.
The full-length M1 sequences of 1, 429 H1N1, 195 H2N2, 1, 097 H3N2, 403 H5N1, 57 H7N7, 81 H7N9, and 437 H9N2 IAV isolates from the Influenza Virus Database of NCBI were analyzed by multiple sequence alignment. Sequence homology alignments demonstrated that the K102 and K104 residue are highly conserved among all the analyzed IAVs (Table 1).
Table 1. Conservation of K102 and K104 on M1 among different subtypes of influenza A viruses.
Our previous study has demonstrated that CypA is involved in the ubiquitin–proteasome degradation of IAV M1 (Liu et al. 2012), but the precise mechanism is yet to be elucidated. Since AIP4 mediates the ubiquitination of M1, we speculated that CypA might regulate AIP4-mediates M1 ubiquitination. The Co-IP assay results showed that overexpression of CypA in 293T cells inhibited M1 ubiquitination (Fig. 4A). It has been reported that both AIP4 and CypA can interact with M1 (Liu et al. 2009; Su et al. 2013). Therefore, we speculated that CypA might affect the interaction between AIP4 and M1. Co-IP assay was performed using anti-Flag beads to detect the interaction between Flag-AIP4 and Myc-M1 WT or Myc-M1 K102R/K104R in CypA-knockdown 293T cells (293T/CypA-) or CypA-overexpressing 293T/CypA-cells. We observed that CypA greatly decreased the interaction between M1 WT and AIP4, whereas the interaction between M1 K102R/K104R and AIP4 could hardly be affected by overexpressing CypA (Fig. 4B). Furthermore, we examined whether CypA regulates the stability of M1. The 293T/CypA-cells were transfected with Myc-M1 and Flag-AIP4, along with Flag-CypA or Flag-vector, then treated with CHX. The result showed that CypA suppressed AIP4-mediated M1 degradation (Fig. 4C). These results taken together suggested that CypA inhibited AIP4-induced M1 ubiquitination at K102 and K104 by inhibiting the interaction of AIP4 with M1.
Figure 4. CypA inhibits AIP4-mediated M1 ubiquitination by impairing the interaction between AIP4 and M1. (A) Immunoblot analysis of lysates of 293T cells transfected with the indicated plasmids for 36 h, followed by IP with anti-M1 antibody. (B) Immunoblot analysis of lysates of 293T/CypA-cells transfected with the indicated plasmids for 36 h, followed by IP with anti-Flag beads. (C) Immunoblot analysis of lysates in 293T/CypA- cells transfected with Myc-M1 and Flag-AIP4, along with Flag-CypA or Flag-vector for 24 h, then treated with 100 lg/mL CHX for the indicated durations (left). The relative density of M1 was normalized to β-actin (right).
To investigate the effect of CypA-regulated M1 ubiquitination on the replication of IAV, we first attempted to generate a recombinant WSN virus expressing M1 K102R/K104R using the 12-plasmid reverse genetics system, but K102R/K104R did not result in any recovery of infectious virus in five independent attempts (data not shown), indicating that the ubiquitination of K102 and K104 are critical for IAV replication.
K102 and K104 are located within the NLS and NEP binding domains of M1 (Shimizu et al. 2011; Cao et al. 2012). Thus, we speculated that the ubiquitination of K102 and K104 might play a critical role in the cellular localization of M1. To examine whether CypA or K102R/K104R affect the cellular localization of M1, 293T/CypA-cells were transfected with Myc-M1 WT and Myc-M1 K102R/K104R respectively, and co-transfected with or without Flag-CypA. The results of immunofluorescence assay (IFA) showed that M1 WT displayed both nuclear and cytoplasmic localization at 12 hours post-transfection (h.p.t.), and the majority of cytoplasmic distribution at 24 and 36 h.p.t.. While M1 K102R/K104R had more nuclear distribution at all three time points than M1 WT in both 293T/CypA-cells and CypA-overexpressing 293T/CypA-cells. Additionally, M1 WT in CypA-overexpressing 293T/CypA-cells displayed greater nuclear distribution at 24 h.p.t., compared to that in 293T/CypA-cells (Fig. 5). These data revealed that both K102R/K104R and CypA resulted in nuclear retention of M1, suggesting that CypA played an important role in regulating the cellular localization of M1 via inhibiting the ubiquitination of M1 at K102 and K104.
Figure 5. CypA regulates the cellular distribution of M1. (A) Cellular location of M1 determined by immunofluorescence assays. 293T/ CypA-cells were transfected with Myc-M1 WT (WT) and Myc-M1 K102R/K104R (KK) respectively, and co-transfected with or without Flag-CypA for the indicated times. The localization of Myc-M1 (green) and Flag-CypA (red) was determined using immunofluorescence assays with anti-Myc monoclonal antibody and anti-Flag monoclonal antibody. The nuclei were stained with DAPI (blue). Scale bars, 10 lm. (B) The cellular distribution of M1 in cells. At least 100 cells from each group were scored as predominantly nuclear (N), nuclear and cytoplasmic (N + C), or predominantly cytoplasmic (C), and the percentage of cells is shown. Error bars represent the standard error of the mean from at least three independent experiments.