Derivation and characterization of an HIV-1 mutant that rescues IP₆ binding deficiency

A critical step in the HIV-1 replication cycle is the assembly of Gag proteins to form virions at the plasma membrane. Virion assembly and maturation are facilitated by the cellular polyanion inositol hexaphosphate (IP₆), which is proposed to stabilize both the immature Gag lattice and the mature capsid lattice by binding to rings of primary amines at the center of Gag or capsid protein (CA) hexamers. The amino acids comprising these rings are critical for proper virion formation and their substitution results in assembly deficits or impaired infectiousness. To better understand the nature of the deficits that accompany IP₆ binding deficiency, we passaged HIV-1 mutants that had substitutions in IP₆ coordinating residues to select for compensatory mutations.

We found a mutation, a threonine to isoleucine substitution at position 371 (T371I) in Gag, that restored replication competence to an IP₆-binding-deficient HIV-1 mutant. Notably, unlike wild-type HIV-1, the assembly and infectiousness of resulting virus was not impaired when IP₆ biosynthetic enzymes were genetically ablated. Surprisingly, we also found that the maturation inhibitor Bevirimat (BVM) could restore the assembly and replication of an IP₆-binding deficient mutant. Moreover, using BVM-dependent mutants we were able to image BVM-induced assembly of individual HIV-1 particles assembly in living cells.

Overall these results suggest that IP₆-Gag and Gag-Gag contacts are finely tuned to generate a Gag lattice of optimal stability, and that under certain conditions BVM can rescue IP₆ deficiency. Additionally, our work identifies an inducible virion assembly system that can be utilized to visualize HIV-1 assembly events using live cell microscopy.