Retroviruses use different IP6 binding mechanisms to alter the properties of their capsids.

HIV-1 uses the metabolite inositol hexakisphosphate (IP6) as a host factor to assemble its capsid, but whether this strategy is unique to lentiviruses or represents a common feature of retroviral capsids remains unclear. Here we show that IP6 binding is conserved across diverse retroviruses but occurs through distinct capsid sites and mechanisms, and influences viral behaviour. In contrast to HIV-1, the beta-retrovirus Mason-Pfizer Monkey Virus (MPMV) and the gamma-retrovirus Murine Leukaemia Virus (MLV) bind IP6 at the threefold lattice interface between capsomers rather than within capsomer pores. Cryo-EM structures of core-like particles reveal that two lysine residues from each capsomer coordinate IP6 between either two discrete three-lysine rings (MPMV) or a single heterogeneous six-lysine ring (MLV). MPMV and MLV are largely insensitive to IP6 availability in producer cells, but this binding mode renders them highly dependent on IP6 in target cells - the opposite of the dependency pattern of HIV-1. The way in which retroviruses use IP6 to build their capsids alters their dependence on the metabolite at different stages of the replicative cycle and in key capsid behaviours, such as assembly and stability.