We first verified the interaction between NS1 and PARP10 with co-localization, co-immunoprecipitation and GST-pull down. Cell co-localization found that the presence of NS1 could induce the PARP10 protein localized in cytoplasm to migrate from cytoplasm to nuclei, indicating that NS1 could change localization and function of PARP10. As PARP10 mainly localized in nuclei under the action of nuclear export inhibitor, we supposed that NS1 might inhibit the nuclear export of PARP10 in nuclei, and make it remain in the nuclei. Further study found that NS1 acts on Glu-rich region and PARP domain of PARP10, and Glu-rich region contains potential nuclear export signal and two ubiquitin interaction motifs (UIM). Some studies report that UIM play certain regulating role in nuclear export and import in some proteins [26–28]. The interaction between NS1 and PARP10 might block nuclear export signal (NES) and UIM of PARP10. As NS1 has two nuclear export signals, NS1 and PARP10 are co-localized in nuclei under the action of nuclear export signal of NS1. NS1's action on catalytic domain of PARP10 may affect the enzymatic activity of PARP10. It is reported that NS1 can promote virus replication through interacting with many proteins of the host and interrupting the normal expression regulation of host cells. Expression profiles of human and mouse tissues show that PARP10 is a widely expressed protein , indicating that PARP10 has wide and fundamental biological functions, and may play certain role in some basic pathways. Therefore, the interaction between NS1 and PARP10 may involve some basic biological functions of cells, and also involve some general protein molecules in signal transduction and protein expression regulation.
Individual NS1 protein expression and PARP10 knock-down did not have significant effect on cell cycle in A549 cells, but the NS1 protein expression and PARP10 knock-down together would significantly induce cell arrest in G2-M stage, with percentage of cells in G2-M stage increased from the previous 10%-45%, consistent to the cell proliferation result. When PARP10 siRNA transcription plasmids, NS1 expression plasmids and PARP10 expression plasmids were co-transfected, it was found that the percentage of cells in G2-M stage saw significant decrease, back to the percentage of cells transfected by empty vector, but the percentage of cells in G1-S stage grew from less than 10%-20%, indicating that co-transfection promoted cells progress into S stage. However, there was a contradictory result that the percentage of cells in G2-M stage when NS1 protein expression only was not above the percentage of empty vector, this may be due to the PARP10 expression level. When PARP10 expression was inhibited significantly, the cells would be apt to G2-M stage. When PARP10 expression was inhibited slightly, the cells would be not apt to G2-M stage. Therefore, this also indicated that NS1 protein of AIV interacted with various proteins to change cell cycle and facilitate AIV infection.
AIV could have quick proliferation in MDCK cells and induce significant pathological changes, but MDCK cells have a low transfection rate, and are not suitable for this study. As AIV is quite selective for hosts, to better detect PARP10's effect on virus replication, we explored the proliferation of H5N1 AIV in A549, BHK21 and COS7 cells. It was found that the virus replication had significant growth in BHK21 cells, but slower proliferation in the other two. As AIV had effective replication in BHK21 cells and the log growth period of the virus was between 36 h and 48 h, BHK21 cells were used as host cells of AIV.
After host cells were decided, we explored the effect of BHK21 cells on virus proliferation through PARP10 over expression or knock-down, with 48 h after the infection as starting point of the detection. The analysis of PFU showed that PARP10 over expression induced virus replication decrease, while PARP10 expression inhibition induced virus replication growth, indicating that AIV replication is regulated by PARP10 protein molecule, and PARP10 expression inhibition can promote virus replication.
In summary, PARP10 can interact with NS1, and the interaction can affect cell cycle and virus replication. NS1 might inhibit activity of host cells and promote virus proliferation through the interaction with PARP10. The findings provide clue and foundation for virus replication mechanism in cells.