Human MxA protein is an interferon-induced member of the dynamin superfamily of large GTPases. MxA inhibits the multiplication of several RNA viruses, including Thogoto virus, an influenza virus-like orthomyxovirus transmitted by ticks. Previous studies have indicated that GTP binding is required for antiviral activity, but the mechanism of action is still unknown. Here, we have used an in vitro cosedimentation assay to demonstrate, for the first time, a GTP-dependent interaction between MxA GTPase and a viral target structure. The assay is based on highly active MxA GTPase as effector molecules, Thogoto virus nucleocapsids as viral targets, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) as a stabilizing factor. We show that MxA tightly interacts with viral nucleocapsids by binding to the nucleoprotein component. This interaction requires the presence of GTPgammaS and is mediated by domains in the carboxyl-terminal moiety of MxA. We propose that GTP-bound MxA adopts an antivirally active conformation that allows interaction with viral nucleocapsids, thereby impairing their normal function.