Characterization of Influenza A Virus Protein
Interactions
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Cal Ham
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The focus of my research over the last six weeks has been three proteins from the Influenza A Virus. These three viral proteins are suspected of forming a complex that carries replicated virus's to the infected cell's membrane prior to lysis. Our goal was to prove whether or not these three proteins interacted. In the future we might possibly be able to engineer a drug that could block the interactions and thus stop viral replication.
The three proteins that we experimented with were the M1, M2 and NS2 proteins. M1 is the matrix protein and is the most abundant viral protein. It underlies the viral lipid membrane and provides rigidity to the viral membrane. M2 is an integral membrane protein. It is an ion channel embedded in the membrane of the virus. Most of the RNA coding for the M2 protein is for the cytoplasmic tail that projects into the cytoplasm. Strangely enough removal of the tail does not effect ion channel activity. One of the reasons that we believe there to be a link between M1 and M2 is because of the research done with the 14C2 antibody. This antibody recognizes M2 and stops viral replications when administered. Virus's that are resitant to 14C2 have mutations in the M1 and M2 proteins. From this it can be reasonably deduced that there is a link between the two. We believe that the removable tail is the site where this linkage occurs since virus's do not tend to keep aroud unnecesary proteins. The third protein is the NS2 protein. It was originaly thought to be a non-structural protein but is now believed to be the link between M1 and M2.
In order to determine whether or not these three proteins do in fact interact we used the Mammalian Two-Hybrid System assay. This assay is based on the concept that eukaryotic cells can have two separate transcription activation proteins that must interact with each other before transcription takes place. Simply put, we inserted two of our experimental proteins at the ends of the two transcriptional proteins and see if transcription takes place. If it does that means that the two experimental proteins did interact. If they do not interact with each other then transcription will not take place.
To do all of this, we obtained the RNA sequence from the Influenza A virus that coded for each protein and cloned it into two plasmids. One plasmid coded for one activation protein and the other coded for the second activation protein. We then transfected these plasmids into eukaryotic cells and looked for expression of our reportor gene.
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Washington University - Biology
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