Structural and mechanistic bases for a potent HIV-1 capsid inhibitor

Abstract

The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.

Fig. 1.. Multimodal mechanism of action of GS-6207.

(A) Chemical structure of GS-6207. (B) Antiviral activities and cytotoxicity of GS-6207 (also see Table S1). (C) Effects of GS-6207 on formation of total reverse transcripts, 2-LTR circles and proviruses. Error bars indicate SD for three independent experiments. (D) Effect of GS-6207 on the number of post-fusion HIV-1 cores in the cytoplasm (also see Fig. S2). (E) Inhibition of nuclear import of HIV-1 (see Fig. S3A). (F) Effect of GS-6207 on HIV-1 infectivity (see Fig. S3B). (G) GS-6207 increases the stability of isolated HIV-1 cores in vitro (see Fig. S4). Error bars in (D-G) represent SEM from 4 fields of view for a representative experiment out of 2 independent experiments (***p < 0.0001). (H) Effects of GS-6207 on the stability of recombinant CA tubes. Only pelleted fractions of CA from each reaction are shown. CA tubes were assembled in 2 M NaCl in the absence (upper image) or presence of GS-6207 (lower image) and then either directly pelleted (lane 1) or exposed to low ionic strength (150 mM NaCl) buffer for increasing periods of time (0, 1, 4, 24, 48 and 96 h shown in lanes 2–7) and then pelleted.

Fig. 2.. Structural basis for GS-6207 interaction with CA hexamer.

(A) X-ray crystal structure of GS-6207 (orange) bound to the pre-stabilized CA_{A14C/E45C/W184A/M185A} hexamer (PDB ID: 6VKV). GS-6207 binds at the pocket formed by two adjoining CA subunits CA1 (light grey) and CA2 (pale yellow). Relative positioning of CA1-NTD, CA2-NTD, and CA2-CTD are indicated. (B) Cartoon representation of the structure indicating GS-6207’s interactions with the two subunits that form the binding pocket, CA1 and CA2. Hydrogen bonds are denoted by black dashed lines. (C) The main helices (αH2*, αH3, αH4, αH8 and αH9) that interact with GS-6207 are indicated. (D) Reported resistance mutations (green) for GS-CA1 (1) are shown in the context of GS-6207 bound to CA1-NTD.

Fig. 3.. Cryo-EM structure of GS-6207 stabilized CA tubes.

(A) Cryo-EM image of A92E CA tubes stabilized by GS-6207 in 150 mM NaCl. Inset shows a subset of the averages obtained by 2D clustering of tube segments. (B) 6.3 Å resolution Cryo-EM map from helical processing of GS-6207 stabilized A92E CA tubes. (C) Diagram showing the pseudo-two-fold symmetric arrangement of monomers in a tube hexamer. (D) Atomic model of a hexamer in the GS-6207 stabilized CA tube generated by rigid-body fitting of six copies of the X-ray structure of a GS-6207 bound CA monomer into the RASTR map. (E) A portion of a tube showing interactions between seven hexamers. Coloring corresponds to HDX protection levels. The cyan, green and orange lines indicate three helical directions. (F) Close up of αH9-αH9 interactions involving a central hexamer. All six H9 helices in the central hexamer (in dark blue) were superimposed. αH9 helices in neighboring hexamers are shown in cyan, green and orange, matching the coloring of helical directions in (E). The absence of true two-fold symmetry results in slight differences in the positioning of the two helices along a specific helical direction. The visible side chain is Glu180.