The interaction between Influenza A viral PB2 protein and cellular factor HNRPM

• Main Authors: Pei-Yun Chen, 陳沛芸
• Other Authors: Won-Bo Wang
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/78851614388217988298

碩士 === 國立臺灣大學 === 微生物學研究所 === 97 === PB2, a component of influenza A RNA polymerase complex, plays an important role in influenza A viral transcription and replication. In the process of searching for cellular proteins that interact with PB2, we accidentally found that PB2 could interact with cellular HNRPM protein, an mRNA splicing factor, by using co-immunoprecipitation assay. This finding is consistent with the previous report that HNRPM can be pull-down by PA-PB2-PB1-TAP tag complex. To further confirm that PB2 can interact with HNRPM, GST pull-down assays were performed. We found that PB2 indeed can interact with HNRPM and this interaction was mediated through medial region (amino acids 281-511) of PB2. Since both PB2 and HNRPM can bind RNA, it is important to rule out the possibility that the interaction is mediated through RNA. We thus treated the cell extract with RNase A before performing GST pull-down assays. We found that HNRPM could still be pull-down by GST-PB2, indicating that PB2 can interact with HNRPM even in the absence of RNA. We also found that the HNRPM and PB2 were co-localized in the nucleus in the immunofluorescence assays. To test the effect of HNRPM on influenza A viral replication, we generated HNRPM knock-down cell line by using lentivirus expressing shRNA against HNRPM. We found that influenza A viral replication was slightly decreased in HNRPM knock-down cell line, indicating that HNRPM is involved in influenza A viral replication. This conclusion was further supported by the finding that the viral RNA-dependent RNA replication rate was decreased in the viral replication reporter assay. To test the effect of PB2 on the alternative splicing activity of HNRPM, we transfected the FGFR2 minigene, HNRPM , and PB2 plasmids into 293T cells. Through semi-quantitative PCR, we found that low level of PB2 could slightly stimulate HNRPM activity whereas high level of PB2 could inhibit HNRPM activity. To test whether HNRPM was involved in the regulation of influenza A M mRNA splicing, we infected HNRPM-knockdown NPC-TW04 cells and the parental cells with influenza A virus and quantitated the level of M1 mRNA, M2 mRNA, and mRNA3 in the infected cells. We found that the relative proportion of M2 mRNA was decreased in HNRPM knock-down cells, indicating that HNRPM is involved in regulating M mRNA splicing. How HNRPM affects M mRNA splicing and what role HNRPM may play in the influenza A viral transcription and replication require further investigation.