Highlights d MX2 interacts with the HIV-1 capsid via N-terminal and GTPase (G) domains d The G-domain interaction enhances MX2 binding to the viral capsid d The MX2 short isoform is not antiviral and binds the capsid through its G domain d The MX2 short isoform suppresses the antiviral activity of the long isoform SUMMARYMyxovirus resistance 2 (MX2/MXB) is an interferon (IFN)-induced HIV-1 restriction factor that inhibits viral nuclear DNA accumulation. The amino-terminal domain of MX2 binds the viral capsid and is essential for inhibition. Using in vitro assembled Capsid-Nucleocapsid (CANC) complexes as a surrogate for the HIV-1 capsid lattice, we reveal that the GTPase (G) domain of MX2 contains a second, independent capsid-binding site. The importance of this interaction was addressed in competition assays using the naturally occurring non-antiviral short isoform of MX2 that lacks the amino-terminal 25 amino acids. Specifically, these experiments show that the G domain enhances MX2 function, and the foreshortened isoform acts as a functional suppressor of the full-length protein in a G-domain-dependent manner. The interaction of MX2 with its HIV-1 capsid substrate is therefore multi-faceted: there are dual points of contact that, together with protein oligomerization, contribute to the complexity of MX2 regulation.