Structural maturation of the matrix lattice is not required for HIV-1 particle infectivity.

HIV-1 assembly is initiated by the binding of Gag polyproteins to the inner leaflet of the plasma membrane, mediated by the myristylated matrix (MA) domain of Gag. Subsequent to membrane binding, Gag oligomerizes and buds as an immature, non-infectious virus particle, which, upon cleavage of the Gag precursor by the viral protease, transforms into a mature, infectious virion. During maturation, the MA lattice underlying the viral membrane undergoes a structural rearrangement and the newly released capsid (CA) protein forms a mature capsid that encloses the viral genome. While it is well established that formation of the mature capsid is essential to particle infectivity, the functional role of MA structural maturation remains unclear. Here, we examine MA maturation of an MA triple mutant, L20K/E73K/A82T, which exhibits distinct biochemical behaviours. The L20K/E73K/A82T mutant is a revertant derived by propagating the L20K mutant, which exhibits reduced infectivity and increased association of the Gag polyprotein with membranes. L20K/E73K/A82T replicates similarly to wild type but retains the increased Gag membrane binding properties of L20K. L20K/E73K/A82T MA also sediments to high-density fractions in sucrose gradients after detergent treatment under conditions that fully solubilize WT MA, suggesting enhanced MA-MA interactions. Cryo-electron tomography with subtomogram averaging reveals that the immature MA lattice of L20K/E73K/A82T closely resembles the wild type. However, mature virions of the triple mutant lack a detectable MA lattice, in stark contrast to both the wild type and L20K mutant. All-atom molecular dynamics simulations suggest that this absence results from destabilized inter-trimer interactions in the mature L20K/E73K/A82T MA. Furthermore, introducing additional mutations designed to disrupt the mature MA lattice does not impair particle infectivity. These findings suggest that an ordered, membrane-associated mature MA lattice is not essential for HIV-1 infectivity, providing new insights into the structural plasticity of the matrix during maturation and its functional role in the viral lifecycle.