Mx proteins are interferon (IFN) type I (α/β)- and type III (λ)-induced effector proteins with intrinsic antiviral activity. Mammalian Mx proteins show different subcellular localizations and distinct yet partially overlapping viral specificities. However, the precise mechanism(s) of antiviral action are still unresolved. Human MxA accumulates in the cytoplasm and inhibits a wide variety of RNA and DNA viruses, among them influenza A virus (IAV). In contrast, MxB, the second human Mx protein, localizes via its amino (N) terminus to the outer nuclear membrane at or near nuclear pores and inhibits the nuclear import of incoming human immunodeficiency viruses (HIV) and herpesviruses, but not that of IAV. Here, we evaluated whether the antiviral specificity of MxB is determined by its subcellular localization. For this purpose, we redirected MxB to the nucleus or cytoplasm by either attaching a nuclear localization signal to its N terminus or by exchanging the N terminus of MxB with that of MxA. Interestingly, ectopic expression of these MxB variants in the nucleus or in the cytoplasm rendered the host cells resistant to IAV, revealing that the capacity of MxB to block IAV replication critically depends on the site where the protein accumulates in the infected cell. Furthermore, coimmunoprecipitation (co-IP) assays demonstrated that MxB physically interacted with the nucleoprotein (NP) of IAV. Taken together, the data indicate that the subcellular localization of the MxB protein plays a pivotal role in determining its antiviral specificity. [ABSTRACT FROM AUTHOR]