Human MxA protein belongs to the family of dynamin-like large GTPases and exerts antiviral activity primarily against negative-stranded RNA viruses, including influenza A (IAV). MxA is able to form higher order oligomeric structures. However, the mode of action of MxA remains unknown. There is increasing evidence that MxA targets the IAV nucleoprotein (NP) in conjunction with UAP56, a cellular DEAD-box RNA helicase, which is required for efficient IAV replication due to its chaperone activity for viral NP. In order to better understand the molecular mechanism of action of MxA we analyzed in detail the interplay between MxA, UAP56 and the viral target NP.
By performing co-immunoprecipitation (co-IP) as well as split-GFP experiments we were able to show that NP binds (the dimeric form of) MxA as well as UAP56. In order to narrow down the interaction domains of MxA and UAP56 with NP, we introduced N- and/or C- terminal deletions and point mutations to identify the binding regions of the trimeric complex. Furthermore, we were able to identify the residues in NP important for binding the MxA-UAP56 complex.
From a mechanistic point of view, we are investigating the importance of the oligomeric state of MxA as well as its GTPase function. We have evidence that a functional G-domain is important for proper localization of MxA but is dispensable for its binding to UAP56. The oligomeric state on the other hand is crucial for the binding of MxA to NP since this binding is increased if MxA is rendered dimeric.
We are additionally testing whether binding of MxA to UAP-NP dimers leads to the sequestration of UAP56 and hence inhibition of IAV replication.