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HIV-1 is dependent on its immature lattice to recruit IP6 for mature capsid assembly

Abstract

HIV-1 Gag metamorphoses inside each virion, from an immature lattice that forms during viral production to a mature capsid that drives infection. Here we show that the immature lattice is required to concentrate the cellular metabolite inositol hexakisphosphate (IP6) into virions to catalyze mature capsid assembly. Disabling the ability of HIV-1 to enrich IP6 does not prevent immature lattice formation or production of the virus. However, without sufficient IP6 molecules inside each virion, HIV-1 can no longer build a stable capsid and fails to become infectious. IP6 cannot be replaced by other inositol phosphate (IP) molecules, as substitution with other IPs profoundly slows mature assembly kinetics and results in virions with gross morphological defects. Our results demonstrate that while HIV-1 can become independent of IP6 for immature assembly, it remains dependent upon the metabolite for mature capsid formation.

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Fig. 1: Altering IP composition in producer cells changes HIV-1 particle production, Gag processing and infectivity.
Fig. 2: HIV-1 particles produced in IP5/IP6-depleted cells display aberrant morphology and lack a condensed capsid.
Fig. 3: Immature particles assemble more slowly with smaller IPs, are less stable and have altered Gag processing relative to particles assembled in the presence of IP5 or IP6.
Fig. 4: HIV-1 can become independent of IP6 for immature particle assembly.
Fig. 5: HIV-1 virions lacking enriched IP6 assemble mature capsids with low efficiency and which have reduced stability.
Fig. 6: Simultaneously reducing cellular IP6 and the ability of HIV-1 to enrich it into virions amplifies infectivity defects.
Fig. 7: KAKA virions produced in IP6-low cells have fewer and less stable capsids.
Fig. 8: The HIV-1 immature lattice enriches IP6 into virions to catalyze mature capsid assembly.

Data availability

Source data are provided with this paper.

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Acknowledgements

This work was supported by the Medical Research Council (MRC UK; U105181010), a Wellcome Trust Investigator Award (200594/Z/16/Z), a Wellcome Trust Collaborator Award (214344/A/18/Z) to L.C.J. and an NHMRC grant (APP1182212) to T.B. Research in the Freed laboratory is supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health. A.K. was supported in part by an Intramural AIDS Research Fellowship. A.S. is supported by MRC UK grant no. MR/T028904/1. We are grateful to the MRC-LMB Electron Microscopy Facility for access and support with electron microscopy sample preparation and data collection, the MRC-LMB Light Microscopy facility, in particular J. Boulanger, for help with TIRF image analysis and the MRC-LMB Visual Aids department. We thank V. Shah for help with particle production and TIRF assays at UNSW.

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Contributions

The study was conceived by N.R., A.K., D.L.M., A.S., E.O.F. and L.C.J. The manuscript was written by L.C.J. with contributions from all authors. Experiments were performed by N.R., A.K., D.L.M., A.A., A.S., K.M.R.F. and T.B. Analysis was carried out by all authors.

Corresponding authors

Correspondence to Eric O. Freed or Leo C. James.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Alan Engelman, Hans-Georg Kräusslich and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Beth Moorefield, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Immature VLPs assemble at 100-fold lower IP6 concentrations than mature capsids.

(A) In vitro mature assembly kinetics with 75 µM CA and 50–1500 µM IP6. (B) Maximum assembly from (A) at different IP6 concentrations fit to equation I: Y = Amin + (Xh)*(Amax-Amin)/(EC50MAh + Xh); where Amin and Amax is the minimum and maximum assembly, EC50MA is the effective concentration for half-maximal mature assembly and h is the Hill slope. Fitting gave an EC50MA of 250 ± 40 µM. Error bars depict mean Amax ± SD of N = 3 independent experiments. (C) EM images of negatively stained samples of the final assembly reaction from (A) using 1.5 mM IP6. Size bars are 200 nm. (D) In vitro immature VLP assembly with 75 µM ΔMA-CANC and a range of IP6 concentrations. (E) Data from (D) were fitted as in (B) to give an EC50IA of 3 ± 1 µM. Error bars depict mean Amax ± SD of N = 3 independent experiments. (F) EM images of negatively stained samples of the final assembly reaction from (C) using 14 µM IP6. Size bars are 100 nM.

Source data

Extended Data Fig. 2 Gag processing of WT HIV-1 produced in cells with different IP profiles.

Western blot of purified HIV-1 particles run on a capillary-based protein detection system. Viruses were produced in 293Ts or CRISPR KOs for IPMK or IPPK, or these cells overexpressing either full-length (FM1) or plasma-membrane targeted Minpp1 (PM1).

Extended Data Fig. 3 In vitro assembly kinetics of immature particles using different IPs.

(AC) In vitro assembly of 75 µM ΔMA-CANC using IPs at indicated concentrations at 25 °C. (D) Negative stain EM images of immature particles from A-C. Scale bar = 200 nm. (EG) In vitro assembly of immature VLPs at indicated ΔMA-CANC and IP concentrations sufficient to maintain 1:1 stoichiometry (1 IP per 1 hexamer) at 25 °C. (H) In vitro assembly with purified ΔMA-CANC at 75 µM hexamer and 12.5 µM (1:1 stoichiometry) IP3 or IP6 at 37 °C. At the indicated time point, an additional 12.5 µM of IP3 or IP6 was added. When additional IP6 but not IP3 is added to the reaction there is a renewed increase in light scattering indicative of further assembly. (I) As with (H), but at the indicated time point excess IP3 is added (500 µM) leading to a resumption in assembly to IP6-stimulated levels.

Source data

Extended Data Fig. 4 Schematic of Gag processing by HIV-1 protease.

The indicated domains of Gag are shown, together with the cleavage products during normal processing. The ΔMA-CANC construct is the starting material used for in vitro assembly and proteolysis experiments throughout this work. The sequential order of proteolytic cleavage and the MW of the cleavage products are shown. The black lines indicate the order of cleavage. Some products are rarely observed under normal conditions, such as ΔMA-CA which represents a species in which SP1, (encoding part of the six helix bundle), is prematurely liberated.

Extended Data Fig. 5 IP6 binding to immature hexamers.

Model of IP6 binding to immature HIV-1 hexamers. IP6 (orange & yellow sticks) is coordinated by two rings of lysines at position K227 (yellow sticks) and K158 (green sticks). The axial phosphate in the inositol ring is labeled (2’PO4) along with the six helix bundle (6HB) that is formed by the SP1 domain in Gag. Based on PDB 6BHR.

Extended Data Fig. 6 Gag processing of HIV-1 mutants.

Western blot of purified viruses of indicated Gag mutants run on a capillary-based protein detection system.

Extended Data Fig. 7 Gag processing of HIV-1 mutants produced in cells with different IP profiles.

Western blot of purified viruses of indicated Gag mutants run on a capillary-based protein detection system. Viruses were produced in 293 T cells or 293 T cells in which Minpp1 was over-expressed (FM1), kinase KO cells IPMK or IPPK, or kinase KO cells overexpressing Minpp1.

Extended Data Fig. 8 TIRF microscopy on WT or D25A protease mutant virions.

(A, B) Virions were produced in 293T cells and adhered to Ibidi slides followed by fixation, permeabilization and antibody labeling. Virions were labeled with antibodies against p24 (in magenta) and VSV-G (in cyan) and imaged by TIRF microscopy. (A) Representative images of WT and D25A protease mutant virions. Yellow circles highlight examples of protease mutant capsids that co-localize with VSV-G. Scale = 5 μm. Right panels show representative images of masks used for particle analysis. Scale = 20 μm. (B) Analysis of mean p24 fluorescence intensity from three representative images of WT and D25A virions. Based on D25A data, the minimum threshold for mature capsid fluorescence was taken as 330. N = 3 biologically independent samples. (C) Virions either non-permeabilized or permeabilized and incubated with VSV-G antibody in the presence or absence of SLO and IP6. Scale = 5 μm. (D) Analysis of mean VSV-G fluorescence intensity of WT and KAKA virions under different conditions. N = 3 biologically independent samples.

Source data

Supplementary information

Supplementary Information

Supplementary Table 1 Calculated IP5 and IP6 levels in modified 293T cell lines. Based on quantified levels in 293T cells as published (33247133) and relative IP levels measured from IPs extracted from modified cells grown in tritiated inositol, as shown in Fig. 2b.

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Renner, N., Kleinpeter, A., Mallery, D.L. et al. HIV-1 is dependent on its immature lattice to recruit IP6 for mature capsid assembly. Nat Struct Mol Biol (2023). https://doi.org/10.1038/s41594-022-00887-4

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