186: Role of GTP-mediated structural changes of MxA on its antiviral function against influenza A

Abstract

Mx proteins are interferon type I induced effector proteins and members of the family of dynamin-like GTPases. Human MxA protein exerts antiviral activity against a broad range of viruses including influenza A. The GTPase activity of MxA is essential for its proper antiviral function. Based on extensive mutational biochemical analyses we proposed a model where Mx proteins can assemble into highly ordered oligomers but exert their antiviral activity as monomers. Moreover, we have shown that human MxA physically interacts with the cellular DExD/h-box RNA helicases UAP56 and URH49 that are required for efficient replication of influenza A virus. Recently Gao and coworkers determined the crystal structure of MxA, revealing a globular GTP-binding domain and a four helical bundle stalk domain. In vitro, MxA multimerizes into large ring-like structures, involving three interaction interfaces. Mutations in the interfaces of MxA interfere with the oligomer formation, but, depending on the mutation, some MxA variants are still able to form monomers, dimers or tetramers. To date it is still a matter of debate whether MxA exerts antiviral activity in form of monomers, dimers and/or higher oligomeric structures. In order to assess whether GTP-binding has an effect on the stoichiometry of MxA we purified recombinant wildtype MxA and various mutants thereof and assessed the number of MxA protomers in the presence or absence GTP-γS by multi angle laser light scattering (MALLS). Surprisingly, we observed that in the presence of GTP-γS wildtype MxA converted from oligomers to tetramers and dimers. Similarly, addition of GTP-γS to the tetrameric mutant MxA (R640A) resulted in its dimerization. Furthermore, dimeric and monomeric MxA variants did not alter their stoichiometry upon addition of GTP-γS. Further, we evaluated the antiviral activity of wild type MxA and MxA variants employing an influenza A minireplicon system and influenza A virus infection of MxA expressing cells. The data clearly indicated that certain tetrameric, dimeric or monomeric variants of MxA were still able to efficiently inhibit virus replication while MxA mutants deficient for GTP binding were inactive. We currently test whether binding of UAP56 by MxA is required for its antiviral activity.