Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor.

Through a screen that combines functional and evolutionary analyses, we identified tripartite motif protein (Trim69), a poorly studied member of the Trim family, as a negative regulator of HIV-1 infection in interferon (IFN)-stimulated myeloid cells. Trim69 inhibits the early phases of infection of HIV-1, but also of HIV-2 and SIVMAC in addition to the negative and positive-strand RNA viruses vesicular stomatitis virus and severe acute respiratory syndrome coronavirus 2, with magnitudes that depend on the combination between cell type and virus. Mechanistically, Trim69 associates directly to microtubules and its antiviral activity is linked to its ability to promote the accumulation of stable microtubules, a program that we uncover to be an integral part of antiviral IFN-I responses in myeloid cells. Overall, our study identifies Trim69 as the antiviral innate defense factor that regulates the properties of microtubules to limit viral spread and highlights the cytoskeleton as an unappreciated battleground in the host-pathogen interactions that underlie viral infections.