| Summary: | 碩士 === 國立陽明大學 === 微生物及免疫學研究所 === 99 === Influenza A virus cause seasonal epidemic of disease worldwide every year. In March and early April 2009, a novel influenza A virus H1N1 emerged in the United States and Mexico, which was named pandemic H1N1 2009 influenza A virus (2009 H1N1). Tracing its phylogeny, scientists found its genome was reassorted from human, avain and swine flu.
The viral protein PB1-F2 is another translated product which ribosome possesses a leaky scanning when it is scanning on the open reading frame (ORF) of PB1. Its length is about 87~90 a.a. PB1-F2 had multiple functions which were reported: it can induce apoptosis and enhance the viral pathogenesis in animal model; some references reported the stability of PB1-F2 was very bad. 2009 H1N1 PB1-F2 had three stop codons at 12, 58, 87 positions in its original PB1-F2 ORF, resulting in the production of a truncated protein that is 11 a.a. It was reported that 2009 H1N1 had a stronger pathogenesis and better propagation compared to the else seasonal H1N1 flu.
To determine 2009 H1N1 possess thees published function when expressing the full-length PB1-F2? I chose the models of viral infection and single gene overexpression to find out the possible function of 2009 H1N1 PB1-F2. After 2009 H1N1 which could express its PB1-F2 was produced by site-specific mutagenesis and reverse genetics, and then operate viral infection in U937. The results revealed 2009 H1N1 PB1-F2 maybe inhibit apoptosis based on the cellular survival rate and the expression patterns of partial apoptotic marker. The data of cDNA apoptosis array exhibited 2009 H1N1 PB1-F2 could inhibit Bcl-2 mRNA expression. Because the data in the continued experiment analyzed by real-time PCR were no difference, Bcl-2 was not a main reason which contibuted this phenomenon. But I find out the 2009 H1N1 PB1-F2 expression could decrease the amount of total cellular mRNA accidentally. In single gene overexpressing model, confirming 2009 H1N1 PB1-F2 is extremely unstable because its expression must accompany with proteasome inhibition, e.g. MG132 treatment, and 2009 H1N1 PB1-F2 could likely decrease IFN-β mRNA expression in this system. Because MG132 could strongly decrease IFN-β expression, we must search for another model to confirm this possibility.
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