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HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

Abstract

During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription1. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a ‘molecular iris’ formed by the amino-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target.

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Figure 1: HIV-1 capsid hexamers have a pore at the six-fold symmetry axis.
Figure 2: The HIV-1 capsid pore is strongly electropositive and recruits dNTPs with rapid association and dissociation kinetics.
Figure 3: R18 is crucial for nucleotide recruitment, reverse transcription and infectivity.
Figure 4: HIV-1 reverse transcription is inhibited by blockade of the capsid pore.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor files have been deposited in the Protein Data Bank under accession numbers 5HGK, 5HGL, 5HGM, 5HGN, 5HGO, and 5JPA.

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Acknowledgements

This work was funded by the Medical Research Council (UK; U105181010), the European Research Council (281627 -IAI), the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013), ERC grant agreement number 339223 and the National Institute for Health Research University College London Hospitals Biomedical Research Centre. G.J.T. was supported by a Wellcome Trust Senior Biomedical Research Fellowship, D.A.J. by an NHMRC Early Career Fellowship (CJ Martin) (GNT1036521) and AJP by a Research Fellowship from Emmanuel College, Cambridge. We thank L. McKeane for her help designing figures.

Author information

Affiliations

Authors

Contributions

D.A.J. performed the majority of the protein production, crystallization experiments and analysis; the fluorescence anisotropy binding experiments; differential scanning fluorimetry; chimaeric virus production and associated infectivity and RT measurements; and TRIM5 abrogation assay. W.A.M. performed the HIV core preparation and endogenous RT experiments. L.H. performed R18G and H12Y infectivity and RT characterizations. A.J.P. crystallized and collected diffraction data from CAhexamer in the open state. L.C.J. performed the stopped-flow kinetics experiments. G.J.T. and L.C.J. supervised the project. The paper was primarily written by D.A.J. and L.C.J. All authors discussed the results and implications and commented on the manuscript at all stages.

Corresponding authors

Correspondence to Greg J. Towers or Leo C. James.

Additional information

Reviewer Information Nature thanks H. Bayley, P. Cherepanov, M. Yeager and T. Yeates for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 dATP binds to the R18 pore at the centre of the capsid hexamer.

a, b, 2Fo − Fc density (grey mesh) contoured at 1.0σ about R18 for the unbound (a) and dATP-bound (b) CAhexamer structures. Fo − Fc omit density (green mesh) contoured at 3.0σ is shown for the dATP-bound structure. c, dATP lies on the crystallographic six-fold axis and clear rotationally averaged density is observed only for the triphosphate group.

Extended Data Figure 2 Controls for dNTP-binding experiment.

a, Titration of CAhexamer into 2 nM fluorescein-labelled dTTP in the presence of 1 mM (physiological) or 5 mM inorganic phosphate. Under the 1 mM conditions, there is no significant effect on hexamer binding to dTTP. At 5 mM, apparent affinity is decreased to 851 nM, demonstrating that inorganic phosphate can compete for the pore. However, given that the intracellular [dNTP] is approximately 100 μM, under intracellular conditions dNTP binding would dominate. b, Titration of CAhexamer into BODIPY-labelled rGTP-γ-S and fluorescein-labelled dTTP. Each binds with the same affinity, which suggests that the R18 pore is unable to discriminate between ribose and deoxyribose nucleoside triphosphates. The difference in the magnitude of the fluorescence anisotropy signals is due to differences in fluorophore excited state lifetimes. KD values are indicated by a dotted line. All measurements were performed in quadruplicate and reported as mean ± s.d.

Extended Data Figure 3 DSF melt curves.

The left-hand panels show the ratio of tryptophan fluorescence emission at 350 nm and 330 nm as a function of temperature. The right-hand panels show the first derivative of the same data, the peak of which is used to determine the Tm value. a, b, Effect of dATP and DTT on wild-type CAhexamer. c, d, Effect of dATP and DTT on R18G CAhexamer. e, f, Effect of each dNTP on wild-type CAhexamer. g, h, Comparison of the effects of carboxybenzene compounds on wild-type CAhexamer. i, j, Comparison of the effects of hexacarboxybenzene on wild-type and R18G CAhexamer.

Extended Data Figure 4 Alignment of selected retrovirus capsid sequences bordering the electropositive pore.

The position equivalent to R18 in HIV-1 is marked with an arrow.

Extended Data Figure 5 Confirmation of CAhexamer chimaera assemblies.

a, Non-reducing SDS–PAGE of CAhexamer wild-type:R18G chimaera samples demonstrates that the recombinant proteins had reassembled into hexamers. Molecular weight standards (kDa) are presented in the first lane. For gel source data, see Supplementary Fig. 1. b, Comparison of 1:5 homohexamer mix and the equivalent chimaera. There is a six-fold loss of apparent Kd for the wild-type:R18G mix, as expected for a six-fold dilution of wild type with a non-binding mutant. In contrast, the 1:5 chimaera chimaera has a 58-fold decrease in Kd, demonstrating that chimaeric hexamers had indeed formed. All measurements were performed in quadruplicate and reported as mean ± s.d.

Extended Data Figure 6 Effects of HIV-1 CA R18G on viral infectivity.

a, R18G is capable of abrogating TRIM5α-mediated restriction. Rhesus TRIM5α provides a potent block to infection of HIV in FRhK-4 cells. Titration of a non-GFP-expressing virus can compete for TRIM5α-binding and relieve the restriction of a GFP-expressing virus only if it delivers an assembled capsid into the cytoplasm. R18G abrogates restriction but W184A/M185A, which is incapable of forming assembled capsids due to loss of the CTD–CTD dimerization interface, does not. Reported values are mean of triplicate ± s.d. b, Binomial distribution model for the relative proportion of capsid hexamers carrying a discrete number of glycines at position 18 at defined bulk ratios of wild-type:R18G. c, Six models (dotted lines) predicting the effect of replacing arginine 18 with glycines. Each model assumes that a different number of glycines is required to render the pore defective. The data from wild-type:R18G chimaeric virus measurements (solid line) are consistent with a model in which four or more arginines (that is, two or fewer glycines, green) are required to maintain a functional pore.

Extended Data Figure 7 ERT assay.

a, HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3–7 were pooled for further experiments. b, Endogenous reverse transcriptase activity for strong-stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c, dNTPs were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to −80 °C and levels of strong-stop were quantified. d, Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTPs in the presence of DNase I. e, Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or benzonase. f, Effect of carboxybenzene compounds on recombinant reverse transcriptase activity. All measurements were performed in triplicate and reported as mean ± s.e.m.

Extended Data Figure 8 Comparison of wild-type and H12Y crystal structures.

The H12Y monomer (in the context of the hexamer, purple) superposes on the wild type (green) with r.m.s.d. of 0.2471 Å. Residues 4–9 of the H12Y structure have been modelled in two alternate conformations, owing to flexibility towards the tip of the hairpin.

Extended Data Table 1 Details of structures used for β-hairpin analysis
Extended Data Table 2 Crystallographic data collection and refinement statistics

Supplementary information

Supplementary Data

The file contains the gel source data for Extended Data Figure 5a. (PDF 891 kb)

Structural morph between the closed and open states of CAHexamer

On the left the protein is represented in cartoon format, coloured according to secondary structure. The sidechain of L6 is shown as sticks to emphasise that it is this residue that results in pore closure. On the right P1, H12, T48, Q50, and D51 are represented as sticks to show that the movement of the β-hairpin is driven by the formation of a salt-bridge between H12 and D51. Distances shown are in Ångstroms. (MP4 9234 kb)

Pore opening exposes R18

Surface representation of the morph depicted in Supplementary Video 1. The β-hairpin and R18 are coloured yellow and blue, respectively. (MP4 10614 kb)

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Jacques, D., McEwan, W., Hilditch, L. et al. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis. Nature 536, 349–353 (2016). https://doi.org/10.1038/nature19098

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