Quantification of MxA mRNA isoform and regulation of MxA gene by Herpes Simplex Virus type I

• Main Authors: Yi-Yuan Chen, 陳奕源
• Other Authors: Chia-Chi Ku
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/29651544841057609447

碩士 === 臺灣大學 === 免疫學研究所 === 98 === Herpes simplex virus 1 (HSV-1) is a member of human alphaherpesviruses which has evolved different strategies to evade type I interferon (IFN) response. Mammalian Mx proteins are dynamin-like GTPases that are induced by interferon (IFN) α/β and have antiviral activity against RNA viruses. Our previous studies have found that HSV-1 induces an alternatively spliced MxA isoform (varMxA) to enhance viral replication in primary human fibroblast in the absence of IFN triggering. In this study experiments have been designed to investigate expression of MxA isoforms in IFN-treated versus HSV-1 infected fibroblasts as well as the effect of HSV-1 on MxA promoter activity in luciferase reporter system. MxA isoform-specific primers were designed and used to measure the expression of MxA and varMxA transcripts in IFN-treated or HSV-1 infected human fibroblasts by real-time PCR analysis. The results showed that IFN-α treatment induced both MxA and varMxA expression at least 100 folds and remained stable over the 24h-course of treatment. Whereas HSV-1 infection resulted in degradation of housekeeping genes such as β-actin and GAPDH, expression of MxA and varMxA transcripts remained stable and increased slightly over an interval of at least 10h and started to decline at 24h after infection. These data suggested that either IFN-α or HSV-1 stimulated the expression of MxA and varMxA mRNA. The importance that MxA isoforms were not degraded but rather selectively remained expression in HSV-1 infected cells is deserved for further investigation. HSV-1 mediated MxA promoter activity was evaluated by transient transfection of melanoma cells with a luciferase reporter plasmid containing MxA promoter region (-533 to -1). HSV-1 infection result in the activation of MxA promoter by 5 folds was not dependent on ISRE (IFN-stimulated response element) binding since disruption of ISRE1 and ISRE2 binding sites did not eliminate MxA promoter activity after HSV-1 infection. However, HSV-1 mediated luciferase activity from MxA promoter mutant constructs with deletion in the frangment from -533 to -250 was significantly reduced to basal level. Site-directed mutagenesis at ICP4/TFIID putative binding site at -317 to -306 also showed a reduced luciferase activity either by HSV-1 infection or by co-transfection with a plasmid expressing ICP4. These experiments have demonstrated that ICP4 was able to trans activate MxA gene. Whether the effect requires the coorperation with cellular transcription factor TFIID remains to be elucidated.