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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
Source data are provided with this paper.
References
Dick, R. A. et al. Inositol phosphates are assembly co-factors for HIV-1. Nature 560, 509–512 (2018).
Mallery, D. L. et al. IP6 is an HIV pocket factor that prevents capsid collapse and promotes DNA synthesis. eLife 7, e35335 (2018).
Mallery, D. L. et al. Cellular IP6 levels limit HIV production while viruses that cannot efficiently package IP6 are attenuated for infection and replication. Cell Rep. 29, 3983–3996 (2019).
Ricana, C. L., Lyddon, T. D., Dick, R. A. & Johnson, M. C. Primate lentiviruses require inositol hexakisphosphate (IP6) or inositol pentakisphosphate (IP5) for the production of viral particles. PLoS Pathog. 16, e1008646 (2020).
Sowd, G. A. & Aiken, C. Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells. PLoS Pathog. 17, e1009190 (2021).
Qiu, D. et al. Analysis of inositol phosphate metabolism by capillary electrophoresis electrospray ionization mass spectrometry. Nat. Commun. 11, 6035 (2020).
Letcher, A. J., Schell, M. J. & Irvine, R. F. Do mammals make all their own inositol hexakisphosphate? Biochem. J. 416, 263–270 (2008).
Mallery, D. L. et al. A stable immature lattice packages IP6 for HIV capsid maturation. Sci. Adv. 7, eabe4716 (2021).
Xu, C. et al. Permeability of the HIV-1 capsid to metabolites modulates viral DNA synthesis. PLoS Biol. 18, e3001015 (2020).
Jacques, D. A. et al. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis. Nature 536, 349–353 (2016).
Lanman, J., Sexton, J., Sakalian, M. & Prevelige, P. E.Jr. Kinetic analysis of the role of intersubunit interactions in human immunodeficiency virus type 1 capsid protein assembly in vitro. J. Virol. 76, 6900–6908 (2002).
Kucharska, I. et al. Biochemical reconstitution of HIV-1 assembly and maturation. J. Virol. 94, e01844-19 (2020).
Endres, D. & Zlotnick, A. Model-based analysis of assembly kinetics for virus capsids or other spherical polymers. Biophys. J. 83, 1217–1230 (2002).
Chi, H. et al. Targeted deletion of Minpp1 provides new insight into the activity of multiple inositol polyphosphate phosphatase in vivo. Mol. Cell. Biol. 20, 6496–6507 (2000).
Clift, D. et al. A method for the acute and rapid degradation of endogenous proteins. Cell 171, 1692–1706 (2017).
Dostalkova, A. et al. In vitro quantification of the effects of IP6 and other small polyanions on immature HIV-1 particle assembly and core stability. J. Virol. 94, e00991-20 (2020).
Marquez, C. L. et al. Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis. eLife 7, e34772 (2018).
Loeb, D. D. et al. Complete mutagenesis of the HIV-1 protease. Nature 340, 397–400 (1989).
Marquez, C. L. et al. Fluorescence microscopy assay to measure HIV-1 capsid uncoating kinetics in vitro. Bio Protoc. 9, e3297 (2019).
Renner, N. et al. A lysine ring in HIV capsid pores coordinates IP6 to drive mature capsid assembly. PLoS Pathog. 17, e1009164 (2021).
Veiga, N. et al. The behaviour of myo-inositol hexakisphosphate in the presence of magnesium(II) and calcium(II): protein-free soluble InsP6 is limited to 49 μM under cytosolic/nuclear conditions. J. Inorg. Biochem. 100, 1800–1810 (2006).
Obr, M. et al. Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer. Nat. Commun. 12, 3226 (2021).
Azevedo, C. & Saiardi, A. Extraction and analysis of soluble inositol polyphosphates from yeast. Nat. Protoc. 1, 2416–2422 (2006).
Zennou, V., Perez-Caballero, D., Gottlinger, H. & Bieniasz, P. D. APOBEC3G incorporation into human immunodeficiency virus type 1 particles. J. Virol. 78, 12058–12061 (2004).
Naldini, L. et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267 (1996).
Price, A. J. et al. Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly. PLoS Pathog. 10, e1004459 (2014).
Vermeire, J. et al. Quantification of reverse transcriptase activity by real-time PCR as a fast and accurate method for titration of HIV, lenti- and retroviral vectors. PLoS ONE 7, e50859 (2012).
Waheed, A. A., Ono, A. & Freed, E. O. Methods for the study of HIV-1 assembly. Methods Mol. Biol. 485, 163–184 (2009).
Toohey, K., Wehrly, K., Nishio, J., Perryman, S. & Chesebro, B. Human immunodeficiency virus envelope V1 and V2 regions influence replication efficiency in macrophages by affecting virus spread. Virology 213, 70–79 (1995).
Wehrly, K. & Chesebro, B. p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents. Methods 12, 288–293 (1997).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
Nguyen, H. L. et al. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies. Protein Expr. Purif. 116, 59–65 (2015).
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.
Author information
Authors and Affiliations
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
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
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.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
(A–C) 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. (E–G) 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.
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.
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.
Source data
Source Data Fig. 1
Statistical source data.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Statistical source data.
Source Data Fig. 3
Unprocessed western blots.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Fig. 7
Statistical source data.
Source Data Extended Data Fig. 1
Statistical source data.
Source Data Extended Data Fig. 3
Statistical source data.
Source Data Extended Data Fig. 8
Statistical source data.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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 30, 370–382 (2023). https://doi.org/10.1038/s41594-022-00887-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41594-022-00887-4
This article is cited by
-
Enrich and switch: IP6 and maturation of HIV-1 capsid
Nature Structural & Molecular Biology (2023)
