Interaction between Influenza A virus PA protein and cellular protein Siva-1
| Autor: | Shao-Yu Tsai, 蔡紹宇 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 Influenza A virus contains an RNA-dependent RNA polymerase that composed of viral proteins PA, PB1 and PB2. Among them, PA has been shown to play important roles in vRNA synthesis and to activate viral mRNA synthesis by promoting PB1’s endonuclease activity. To further investigate the role of PA in virus life cycle, our lab set to identify cellular factors that may interact with PA by using yeast two-hybrid system. We found that Siva-1, a protein that has an apoptotic activity, is one of the potential PA-interacting cellular proteins. To further confirm the interaction between PA and Siva-1, full-length Siva-1 was fused with Gal4 activation domain to generate pGAD-GH -Siva-1. This plasmid was then co-transformed with pGBDU-C1-PA into yeast to verify the interaction between PA and Siva-1 in a two-hybrid assay. We found that PA indeed can interact with Siva-1. We also demonstrated that PA can interact with Siva-1 in vivo and in vitro by co-immunoprecipitation and GST pull-down assays and that PA may interact with the death domain homology region (DDHR) of Siva-1. To study the role of Siva-1 in influenza A virus replication, influenza A virus was used to infect Siva-1-knockdown NPC-TW04 and H1299 cells. By using plaque assays, we found that the production of influenza A virus was significantly increased in the Siva-1 knockdown cells, suggesting that Siva-1 plays an inhibitory role in influenza A virus multiplication. To further study whether Siva-1 could affect the replication of influenza A viral genome, a replication-reporter system that can be used to measure the replication of influenza A viral genome was used. We found that the replication rate of influenza viral genome is similar in Siva-1 knockdown cells and control cells, indicating that Siva-1 has no effect on the replication of influenza viral genome. Taken together, these data suggest that Siva-1 may affect the multiplication of influenza A virus at stages other than the replication of viral genome. Siva-1 has been reported to have an apoptotic activity. To study the functional outcome of PA-Siva-1 interaction, we tested whether PA could affect the apoptotic activity of Siva-1. We found that Siva-1, as reported, could induce apoptosis and that PA could more or less block apoptosis induced by Siva-1. Viruses are well known to encode anti-apoptotic proteins to facilitate their own multiplication. Whether the interaction between PA and Siva-1 could prevent apoptosis and facilitate virus multiplication during influenza A virus infection remains to be determined. Our data, though not prove, point to this possibility. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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