| Summary: | 碩士 === 慈濟大學 === 醫學生物技術研究所 === 99 === To search for cellular factors modulating influenza A virus replication, ddRT-PCR analysis was performed to detect the cellular genes differentially expressed after influenza A virus WSN33 infection in A549 cells. Three up-regulated genes (PRPF8, RPL35, DBI) after virus infection were detected and PRPF8 gene was future analyzed in this study. The up-regulation of PRPF8 after virus infection was confirmed using Western blotting analysis. To determine the effect of PRPF8 gene on influenza A virus replication, siRNA technology was used to knock-down PRPF8 gene expression in A549 cells and the effect on viral replication was done using Western blotting analysis to detect viral protein and plague assay to analyze the viral particles. Our results showed that reduction of PRPF8 expression did inhibit virus replication. PRPF8 protein interacts with several other spliceosomal proteins, snRNAs, and the pre-mRNA, and thereby organizes the active site(s) of the spliceosome. The effect of PRPF8 on virus replication was analyzed using real time RT-PCR to detect the relative mRNA level of M1 versus M2 and that of NS1 versus NS2. The mRNA ratio of M1 versus M2 is much more in the PRPF8 knockdown cells than in the control cells. Therefore, PRPF8 could facilitate influenza A virus replication possibly through helping the mRNA splicing. Plasmids expressing individual influenza A virus gene products were transfected separately into A549 cells. NS1 protein was found to up-regulate the PRPF8 expression. Neither N-terminus (1-73 a.a.) nor C-terminus (74-237 a.a.) of NS1 protein alone could up-regulate the PRPF8 expression. Molecular mechanisms regarding how influenza A virus up-regulates PRPF8 gene expression and how PRPF8 affects virus replication will be addressed in the future.
|
|---|
