Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM

Abstract

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but high-resolution information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryo-electron tomography and single particle analysis cryo-electron microscopy to determine high-resolution structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a new method of in vitro lattice assembly that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate (IP6) in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (Lenacapvir). Our work reveals new structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and single particle analysis as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.

Arg18 substitutions reveal the capacity of the HIV-1 capsid protein for non-fullerene assembly

“…The differing configurations of the TVGG motif in the WT CA hexamer and canonical pentamer also determine the capacity of the differing NTD-CTD interfaces to bind ligands that contain phenylalanine-glycine (FG) motifs; only the hexamer but not the pentamer is configured to bind FG [11,12,26]. Since the alternative R18L pentamer contains a hexamer-like TVGG configuration and NTD-CTD interface, we predicted that it should be also capable of binding FG ligands.…”

“…Sub-tomogram lattice mapping is a template-based approach, and so it is possible that rare instances of octahedral arrangements may have been missed in prior studies. However, IP 6 induces and stabilizes the canonical HIV-1 CA pentamer by coordinating the native Arg18 ring [11,26]. We believe that the mM-level concentrations of IP 6 inside virions [38] would disfavor the alternative pentamer configuration during native capsid assembly.…”

IP6 and PF74 affect HIV-1 Capsid Stability through Modulation of Hexamer-Hexamer Tilt Angle Preference

“…Of the 120 HP interfaces found in the 3J3Y (7) and 3J3Q (7) whole capsid structures, one HP pair was chosen through a combination of its interface tilt/twist angles as well as the presence of its nonbonded interactions (see Supplemental Information). Holo-pentamer structures were not included in this study due to unavailability of structures and recent findings (10,55) in which pentamers were not found to bind any substituents. HH IP6 .…”

Passage of the HIV capsid cracks the nuclear pore

“…Finally, the complete capsid was built by replacing each bead with atomic models of hexamers (PDB ID: 8ckv 74 ) and pentamers (PDB ID: 8g6l 75 ) rotated and translated according to the lattice (Figure S5A6). Missing loop residues were modeled using Swiss model 77 .…”

“…Hexamers and pentamers were represented as beads. The six neighboring beads of hexamers, and the five of pentamers were connected by harmonic springs κ ( r – r 0 ) 2 /2 with r the pair distance, r 0 = 9.3 nm the distance between hexamers obtained by placing atomic models 74 , 75 in the experimental STA of the hexamer which included the first neighbor, and κ = 3 kJ⋅mol -1 ⋅nm -2 the spring constant. In addition, the beads were softly tethered to their initial position by a potential kd 2 /2 with d the distance and k = 1.0 kJ⋅mol -1 ⋅nm -2 the spring constant.…”