STRUCTURAL INSIGHTS INTO HIV-1 GAG AND ITS CLEAVAGE PRODUCTS

Abstract

HIV-1 initially assembles as a non-infectious 'immature' virus particle. Upon budding from the host cell, it undergoes a critical reorganization known as maturation, forming an infectious and fusion-competent ‘mature’ virus. This process is initiated by proteolytic cleavage of the Gag polyprotein into its constituent domains: Matrix (MA), Capsid (CA), spacer peptide 1 (SP1), Nucleocapsid (NC), spacer peptide 2 (SP2), and p6. The first part of this thesis concerns the structure of mature CA. Cleavage of Gag induces a dramatic structural rearrangement of CA, transitioning from a broadly spherical immature hexameric lattice into a structurally distinct mature lattice with a characteristic conical ultrastructure. Unlike the immature CA lattice, the mature core accommodates variable curvature by incorporating pentameric vertices. The core plays a central role in the post-entry infection pathway, orchestrating transport into the nucleus via interactions with host factors. In this work, I determine the cryo-EM structure of the mature CA hexameric lattice from in-vitro assembled CA cores, including a range of curvature and pentamer-contacting contexts (2.6-3.9 Å). Additionally, I report the CA pentamer structure within the lattice at 3.1-3.5 Å resolution, enabling visualisation of residue-level detail for the first time. Together, these reconstructions offer a comprehensive description of mature CA lattice-forming contacts, both canonical and seemingly aberrant, improving understanding of curvature accommodation and lattice formation. Further structures determined from cores coated with co-factor-derived peptides reveal two structural switches that regulate co-factor binding in distinct ways. The ability of the core to fine-tune co-factor binding as a function of curvature may explain the apparent fitness advantage of its distinct conical morphology. In the second part of this thesis, I investigate the structure and maturation of MA. Like CA, membrane-bound MA forms distinct hexagonal lattices in immature and mature viruses. Unlike CA, fundamental gaps in MA maturation remain, including its trigger, mechanism, and function. Here, I determine the cryo-EM structure of the mature MA lattice (3.1 Å) from intact virus particles, defining amino acids mediating matrix layer assembly. The MA maturation state is further determined for cleavage mutants, showing that the two cleavages flanking SP2 are the only Gag cleavages required for MA maturation. Surprisingly, SP2 directly binds MA, consistent with the mature MA lattice reconstruction rather than lipid PI(4,5)P2, as previously hypothesized. The observation that SP2 triggers MA maturation is the first assigned function of this highly conserved viral component. Lastly, I show that MA residue R22 is critical for regulating MA maturation and serves as a sensor of SP2 binding. Structural determination of the MA lattice in MAR22A virions reveals SP2-bound MA in an immature configuration. A residue-level reconstruction of the immature MA lattice (3.8 Å) is finally reported and described from this mutant.