F. STEINER¹, P. NIGG², L. MURER^1,3, M. CRAMERI¹, E. MORITZ¹, J. PAVLOVIC<sup>1

1</sup>Institute of Medical Virology, University of Zurich, Zurich, Switzerland, ²Friedrich Miescher Institute for Biomedical Research, University of Basel, Basel, Switzerland, ³Institute of Virology, University of Freiburg, Freiburg, Germany

Mx proteins belong to the family of dynamin-like, large GTPases and are primarily active against negative-stranded RNA viruses, including influenza A (IAV). Human MxA is able to form higher order oligomeric structures, however, the mode of action of MxA remains to be fully elucidated. Increasing evidence suggests that MxA requires auxiliary cellular factors for its antiviral activity. Recently, we have shown that MxA interacts with the DEAD-box helicase UAP56, which is an essential factor for efficient replication of IAV. There is increasing evidence that NP represents the viral target of MxA. For instance, the resistance phenotype of human IAV segregates with a discrete cluster of surface-exposed amino acids in the NP. We showed that the dimeric form of MxA is able to form stable complexes with viral NP as well as UAP56 suggesting that the dimeric form of MxA plays a central role in antiviral function. In line with these findings, co-immunoprecipitation (Co-IP) experiments revealed that NPs of several MxA-sensitive or resistant strains exhibit different binding affinities to dimeric MxA. Furthermore, we observed that binding of MxA to UAP56 is greatly enhanced in the presence of NP.
Currently we characterize the interactions of MxA, NP and UAP56 by Co-IP and a tripartite split-GFP system. In particular, we investigate the effect of (i) GTPase activity mutants of MxA, (ii) ATPase and RNA binding activity mutants of UAP56 as well as (iii) NP mutants with altered MxA sensitivity. Furthermore, we assess the influence of these mutations on MxA antiviral activity.