Influenza is a highly contagious respiratory disease, which can have severe impacts on human health. Influenza type B is traditionally known as the seasonal flu and is the main source for annually occurring influenza outbreaks. The Non-Structural protein 1 of influenza B (NS1B) is a highly conserved protein that the influenza virus produces post infection. NS1B is hypothesized to inhibit the innate immune system via interactions with the RIG-I activation pathway. NS1B has been known to bind dsRNA via its N-terminal domain (NS1B-NTD) for decades, but recently a second RNA binding site was discovered on the C-terminal domain of NS1B (NS1B-CTD). Due to the high conservation of NS1B, its ability to inhibit the innate immune system, and the recent discovery of a second RNA binding domain, this dissertation research focused on the biological function of this second RNA binding site. We discovered a surprising novel blunt-end binding orientation of the NS1B-CTD by dsRNA. We then looked at the connection between RIG-I’s well-known ability to detect and bind triphosphorylated-5’ hairpin RNA (3P-5’-hpRNA) with a much higher affinity than OH-5’ hairpin RNA (OH-5’-hpRNA). We discovered similar binding affinity changes and characteristics with NS1B-CTD and the 3P-5’-hpRNA/OH-5’-hpRNA. When the second RNA binding site in NS1B was mutated in transgenic influenza B viruses, we observed reduction in the ability of the virus to suppress Rig-I activation, as Rig-I induced phosphorylation of IRF3 was no longer suppressed flowing virus infection. Our results suggests that the function of the second RNA binding site in the CTD of wildtype NS1B is to outcompete RIG-I for its RNA substrates, typically 5’ triphosphorylaed vRNA molecules. Based on these studies we propose that NS1B-CTD acts as a sensory domain with high specificity for vRNA molecules, which form a “panhandle dsRNA duplex structure” with a unique 3P-5’ modification not found in host cells. This interaction functions to prevent activation of Rig-I, and the innate host immune response.
|Advisor:||Montelione, Gaetano T|
|Commitee:||Roth, Monica, Arnold, Eddy, Khare, Sagar|
|School:||Rutgers The State University of New Jersey, School of Graduate Studies|
|Department:||Chemistry and Chemical Biology|
|School Location:||United States -- New Jersey|
|Source:||DAI-B 81/3(E), Dissertation Abstracts International|
|Keywords:||Influenza, NS1, RIG-I, RNA protein complex, SAXS, Structural biology|
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