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.