Minnell, J. J.; Punch, E.; Vanzo, T.; Safari, R.; Cronin, N. B.; Cherepanov, P.; Maertens, G. N.

Integrase catalyses the insertion of a DNA copy of the retroviral genome into host cell chromatin. Human T-cell lymphotropic virus type 1 (HTLV-1) and other deltaretroviral integrases associate with protein phosphatase 2A holoenzymes containing B56 regulatory subunits (PP2A-B56). Here, we show that integrase mutants defective in binding to most B56 isoforms retain intrinsic DNA strand transfer activity but are impaired in establishing infection. Using single-particle cryo-EM, we determined the structure of the simian T-cell lymphotropic virus type 1 (STLV-1) intasome in a 0.5-MDa complex with two copies of the heterotrimeric PP2A-B56{gamma} holoenzyme at 2.8 [A] resolution. The structure reveals that, in addition to engaging B56, integrase forms direct contacts with the catalytic subunit of PP2A and sterically occludes the phosphatase active site, preventing substrate access. Consistent with these findings, we show that the HTLV-1 intasome suppresses PP2A catalytic activity in a manner dependent on the integrase LxxIxE short linear motif. We further demonstrate that pharmacological inhibition of PP2A does not impair HTLV-1 infection. Together, our results indicate that recruitment of PP2A-B56 by the intasome serves a structural rather than catalytic function.