Structural basis for HIV-1 capsid adaption to a deficiency in IP6 packaging

Abstract

Inositol hexakisphosphate (IP6) promotes HIV-1 assembly by stabilizing the immature Gag lattice and becomes enriched within virions, where it is required for mature capsid assembly. Previously, we identified Gag mutants that package little IP6 yet assemble particles, though they are non-infectious due to defective capsid formation. Here, we report a compensatory mutation, G225R, in the C-terminus of capsid protein (CA) that restores capsid assembly and infectivity in these IP6-deficient mutants. G225R also enhances in vitro assembly of CA into capsid-like particles at far lower IP6 concentrations than required for wild-type CA. CryoEM structures of G225R CA hexamers and lattices at 2.7 Å resolution reveal that the otherwise disordered C-terminus becomes structured, stabilizing hexamer-hexamer interfaces. Molecular dynamics simulations support this mechanism. These findings uncover how HIV-1 can adapt to IP6 deficiency and highlight a previously unrecognized structural role of the CA C-terminus, while offering tools for capsid-related studies.

Fig. 1. Characterization of Gag mutants defective in IP6 binding and a compensatory mutation G225R.

a–d Representative tomographic slices of WT, K227A, K158A/K227A (KAKA) and KAKA/T8I VLPs, respectively. Scale bar, 50 nm. e–h CryoET STA structures of immature Gag hexamers from WT, K227A, KAKA and KAKA/T8I VLPs superimposed with the model (PDB: 7ASH). Dashed gray lines mark the height position for IP6. i Specific infectivity of KAKA and KAKA/T8I in the presence and absence of G225R measured in TZM-bl cells at 36–48 h post-infection. Data are the mean of at least three independent biological replicates for each mutant, and error bars depict ± SEM. Precise n for each group: WT = 24, KAKA = 19, KAKA/G225R = 6, KAKA/T8I = 24, KAKA/T8I/G225R = 14, A77V/KAKA/T8I/G225R = 7, G225R = 4. Replicates that did not produce a measurable signal were assigned a value of 0 and are plotted at the base on the y-axis for visualization. Statistical analysis was performed using GraphPad Prism. Statistical significance was determined by a two-tailed one-sample Student’s t test with a hypothetical value set to 100 when comparing groups to WT. All other comparisons were made using two-tailed unpaired Student’s t tests. (p-value summary: >0.05 = not significant; <0.05 = *; <0.01 = **; <0.001 = ***; <0.0001 = ****). Precise p-values for each comparison: WT vs. KAKA P < 0.0001, WT vs. KAKA/G225R P < 0.0001, WT vs. KAKA/T8IP < 0.0001, WT vs. KAKA/T8I/G225R P < 0.0001, WT vs. A77V/KAKA/T8I/G225R P < 0.0001, WT vs. G225R P < 0.0001, KAKA vs KAKA/G225R P = 0.0390, KAKA/T8I vs KAKA/T8I/G225R P < 0.0001, KAKA/T8I vs A77V/KAKA/T8I/G225R P < 0.0001, KAKA/T8I/G225R vs G225R P = 0.4633. j Representative replication kinetics of KAKA and KAKA/T8I mutants in MT-4 cells showing a delay in viral replication relative to WT. k Representative re-passage of KAKA and KAKA/T8I in fresh MT-4 cells showing an increase in replication kinetics relative to the initial passage. Source data are provided as a Source Data file.

Fig. 2. G225R does not restore IP6 recruitment in IP6 deficient mutants.

a–d Representitive tomographic slices of KAKA/T8I/G225R, KAKA/G225R, A77V/KAKA/T8I/G225R and G225R VLPs with slice thickness 4.02 nm (top) and their corresponding cryoET STA structures of immature Gag hexamer (bottom). Scale bar, 100 nm. e Cell lysates and concentrated virus lysates were collected after a 24 h incubation following media change after co-transfection of pNL4–3 with 500 ng of empty vector (EV) or MINPP1 expression vector (IPMK KO and IPPK KO). Cell and virus Gag levels were quantified by western blot, and virus production efficiency was calculated as described in the methods. Data are the mean of 4 independent biological replicates for all groups ±SEM. Statistical analysis was performed using GraphPad Prism. Statistical significance was determined by a two-tailed one-sample Student’s t test with a hypothetical value set to 100 when comparing groups to WT. All other comparisons were made using two-tailed unpaired Student’s t tests. (p-value summary: >0.05 = not significant; <0.05 = *; <0.01 = **; <0.001 = ***; <0.0001 =  ****. Precise p-values for each comparison—WT (Parental vs IPMK P < 0.0001; Parental vs. IPPK P < 0.0001), KAKA (Parental vs. IPMK P = 0.1473; Parental vs. IPPK P = 0.1402), KAKA/T8I (Parental vs. IPMK P = 0.0107; Parental vs. IPPK P = 0.1094), KAKA/G225R (Parental vs. IPMK P = 0.2634; Parental vs. IPPK P = 0.0482), KAKA/T8I/G225R (Parental vs. IPMK P = 0.0594; Parental vs. IPPK P = 0.0378), A77V/KAKA/T8I/G225R (Parental vs. IPMK P = 0.9613; Parental vs. IPPK P = 0.5511), G225R (Parental vs. IPMK P = 0.0001; Parental vs. IPPK P = 0.0002). f Virus from cells transfected as above was collected 24 h post-transfection and assessed for specific infectivity on TZM-bl cells. Infectivity was normalized to the infectivity of WT produced from parental cells. Data are the mean of 4 independent biological replicates for KAKA/G225R and 5 independent biological replicates for all other groups. Replicates that did not produce a measurable signal were assigned a value of 0 and are plotted at the base on the y-axis for visualization. Statistical analysis was performed as in (e). Precise p-values for each comparison—WT (Parental vs. IPMK P < 0.0001; Parental vs. IPPK P < 0.0001), KAKA (Parental vs. IPMK P = 0.0153; Parental vs. IPPK P = 0.0234), KAKA/T8I (Parental vs. IPMK P = 0.0266; Parental vs. IPPK P = 0.0322), KAKA/T8I/G225R (Parental vs. IPMK P = 0.0031; Parental vs. IPPK P = 0.0040), A77V/KAKA/T8I/G225R (Parental vs. IPMK P = 0.0025; Parental vs. IPPK P = 0.0021), G225R (Parental vs. IPMK P = 0.0007; Parental vs. IPPK P = 0.0007). g A Gallery of distinct morphologies of HIV-1 particles produced from HEK-293 cells, shown in tomographic slices. Particle morphologies are classified as indicated. The slice thickness is 4.36 nm. Scale bar, 50 nm. h Distribution of particle morphologies of WT (n = 442) and KAKA (n = 162), KAKA/T8I (n = 181), KAKA/G225R (n = 152), KAKA/T8I/G225R (n = 66), A77V/KAKA/T8I/G225R (n = 687) mutant Gag virions from one independent virus production. Source data are provided as a Source Data file.

Fig. 3. G225R increases the efficiency of capsid formation in vitro.

a–e Sup-pellet assays for the in vitro assembly of CA WT, KAKA, G225R, KAKA/G225R, and A77V/KAKA/G225R at 0, 5, 15, 50, 150 μM and 5 mM IP6 concentrations (Three independent experiments were conducted with similar results for each mutant). f Negative stain images of CA WT, KAKA, G225R, KAKA/G225R and A77V/KAKA/G225R with IP6 concentrations from left to right are 0, 5, 50 and 150 µM, respectively (More than 10 micrographs were imaged for each condition). The numbers of total assemblies (g) and assembled tubes (h) and cones (i) in each micrograph of WT and mutant CA at different IP6 concentrations (Data are the mean of 5 independent micrographs for each condition and error bars depict ±STD). Scale bars, 100 nm. Source data are provided as a Source Data file.

Fig. 4. CryoEM structure of CA KAKA/G225R hexamer.

a A representative cryoEM micrograph of in vitro assembled KAKA/G225R capsid with 100 μM IP6 (8443 micrographs were collected). b The overview KAKA/CA G225R density map at 2.7 Å resolution superimposed with the refined molecular model (PDB 9I8I), with CA NTD colored blue and CA CTD colored gold. c The central slice of KAKA/G225R CA density map. d The central slice of KAKA/G225R CA hexamer density map superimposed with the refined model with IP6 density colored pink. R18 sidechain is indicated. e The top view of KAKA/G225R CA hexamer density map superimposed with the refined model, showing β-hairpins in an open conformation. f Comparison of β-hairpin among HIV-1 CA hexamers from crystal of WT (M) (PDB 5HGN, gold) and HIV-1 (O) (PDB 7T12, pink), cryoEM of WT CLP (PDB 7URN, grey) and KAKA/G225R (PDB 9I8I, blue). g Comparison of CA NTD-CTD interfaces among HIV-1 KAKA/G225R, HIV-1 WT (M) and HIV-1 (O). h An enlarged view of NTD-CTD interfaces. Helices H4 from CA1 NTD and H8 from CA2 CTD are labeled. i Comparison of CA monomers among HIV-1 KAKA/G225R (PDB 9I8I, blue), HIV-1 (M) WT (PDB 5HGN, gold) and HIV-1 (O) (PDB 7T12, pink). Inset: an enlarged view of the C-terminus, additionally overlapped with an NMR HIV-1 WT CTD_144-231 model (PDB 2KOD, grey). The last resolved residue G223 in the cryoEM structure (blue) is marked. Scale bar, 50 nm.

Fig. 5. CryoEM structures of CA KAKA/G225R tri-hexamer.

a The overview KAKA/CA G225R tri-hexamer density map at 3.63 Å resolution superimposed with the refined molecular model (PDB 9I8I). Three hexamer models are colored blue, green and purple. The dimer and trimer interfaces are marked with red and black dashed circles. b Comparison of dimer interfaces between WT (PDB 6SKN, green) and KAKA/G225R (PDB 9I8I, blue). c Comparison of trimer interfaces between WT (PDB 6SKN, green) and KAKA/G225R (PDB 9I8I, blue). Two 3D classes of CA KAKA/G225R tri-hexamer maps, superimposed with model (PDB 9I8I), viewed from CTD side (inside of capsid). Class 2 e distinguishes from Class 1 d by additional densities (segmented in red) extending from H11 to the dimer interface, which is overlaid with an MDFF model of the C-terminus (220–231). f Interactions observed between C-terminal region (220–231, red) and residues at the dimer interface in unbiased molecular dynamics simulation of the KAKA/G225R CA trimer of dimers superimposed with the density map. Interacting residues are labeled.

Fig. 6. KAKA/G225R CA dimer interfaces compared to assembly interfaces from full size capsid cone.

a All cases of capsomer interfaces in the full WT capsid (left) overlayed on the KAKA/G225R CA trimer of dimers (right). NTD is colored tan on hexamers and green on pentamers; CTD is colored dark blue. b Distance and orientation measurements of the CTD structural elements at the dimer interface for the KAKA/G225R CA trimer of dimers (ToD) or the WT capsomer interfaces involving hexamers (H) and pentamers (P). Bar plots represent mean values over all dimers in a trimer of dimers (n = 3), and error bars represent standard deviation. Helices for a WT hexamer-hexamer-hexamer are colored in teal, and helices for the KAKA/G225R CA ToD are colored in purple. Dimer interface residues M185 and W184 are visualized in licorice representation. Source data are provided as a Source Data file.

Fig. 7. A schematic model for the role of G225R in HIV-1 mature capsid assembly.

a The assembly WT mature capsid with high level of IP6 enrichment through the immature Gag assembly. b The assembly KAKA/G225R mature capsid with low level of cytosolic IP6, as the mutant is defective in IP6 packaging by immature Gag. The assembly of mutant mature capsid is facilitated by the interactions with the CA C-terminal fragment which is otherwise unstructured and flexible.