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.

Figure 1. Altering IP composition in producer cells changes HIV-1 particle production, Gag processing and infectivity.

(A) Simplified biosynthetic scheme for IP6. (B-C) 293Ts, CRISPR KOs for IPMK or IPPK[3], or KOs over-expressing full-length (FM1), plasma-membrane targeted (PM1) or cytosol-targeted (DM1) Minpp1 were grown in tritiated inositol. IP species were extracted and separated by SAX-HPLC as previously described[31]. (B) The counts per minute (CPM) of each inositol species normalised to total lipid is shown. Error bars depict mean CPM ± SEM from at least two independent experiments. An unpaired t test against 293Ts was used for statistical analysis of IPs in each cell line and only significant differences indicated (P = 0.05 (*), < 0.005 (**)). (C) The proportion of each IP species as a fraction of the total IP2-IP6 concentration. (D) HIV-1 production, as measured by RT activity, expressed as a percentage of virus production in 293T cells. An unpaired t test against 293T EV was used for statistical analysis and significant differences indicated (P = 0.05 (*), < 0.005 (**)). (E) Gag cleavage efficiency in purified virions, calculated as the percentage of p24 (CA), p41 and Pr55Gag. (F-H) Infectivity of viruses from (D) plotted against quantity of input virus. Error bars depict the SEM from three independent experiments. Nonlinear regression was used to compare Y-intercepts against 293T EV and significant differences indicated (P < 0.05 (*)). (I) Infection of 293Ts, CRISPR KOs for IPMK or IPPK, or KOs over-expressing cytosol-targeted (ΔM1) Minpp1 by HIV-1. Error bars depict the SEM from three independent experiments.

Figure 2. HIV-1 particles produced in IP5/IP6-depleted cells display aberrant morphology and lack a condensed capsid.

(A-C) Cryo-ET on indicated HIV-1 mutants produced in IPMK or IPPK KO cells with Minpp1 overexpression. Tilt series were collected and reconstructions performed to assess capsid morphology. A total of 131 WT, 34 IPPK + FM1 and 48 IPMK + FM1 particles were analysed. (A) Virions were classified into the indicated categories: Immature (pink), Mature Conical (dark blue), Mature Tubular (light blue), Mature Irregular (green). Slices through representative tomograms of the virions are shown together with quantification. Scale bars, 100 nm. (B) Virions with mature lattices were further subdivided into: Multiple Cores (green), Single Cores (cyan), Cores with additional closed structure (orange), Cores with additional open structure (light orange), Multilayered Cores (blue). Slices through example tomograms of the virions are shown together with quantification. Scale bars, 100 nm. (C) All particles that were categorized as VSV-G positive (as indicated by the spikes on the surface of the particles) on the grid but did not contain a clear assembled lattice were categorized into: Virions (black), Partial density (gray), Empty (orange), Structured density (blue), Filament structures (dark pink), Partial density (light pink). Slices through representative tomograms of the virions are shown together with quantification. Scale bars, 100 nm. (D) Thin-section electron-microscopy of HIV-1 virions produced in 293T cells or IPMK KO cells over-expressing Minpp1. Scale bar = 120 nm.

Figure 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.

(A) In vitro assembly of immature particles using recombinant DMA-CANC protein. 7.5 μM RNA was added to 75 μM DMA-CANC and assembly monitored through light scattering changes at 350 nm at 37 °C. Indicated IPs were added at equimolar DMA-CANC concentration or 12.5 μM, to achieve a stoichiometry with immature hexamers of 6:1 or 1:1, respectively. Representative of two experiments. (B) EM images of negative-stained samples of the final assembly reactions shown in (A). Scale bars are 200 nm. (C) In vitro assembly reaction of immature particles as in (A) except IP6 was added at stoichiometric ratios with respect to immature hexamers of 1:1 (12.5 μM), 0.5:1 (6.25 μM) or 0.25:1 (3.12 μM). Once assembly had plateaued, additional IP6 was added to achieve 1:1 stoichiometry, resulting in similar final yields by light scattering. Representative of two experiments. (D) In vitro assembly of mature particles using 150μM recombinant CA protein and 2.5 mM of the indicated IP. EM images of negative-stained samples of the final assembly reactions are shown to the right. Size bars are 200 nm. Representative of at least three experiments. (E) Thermostability of in vitro assembled particles with 7.5 μM RNA, 75 μM ΔMA-CANC and 12.5 μM IP was measured by differential scanning fluorimetry (DSF). The change in melt temperature (ΔTm) was calculated with respect to the thermostability of unassembled ΔMA-CANC protein. An unpaired t test against unassembled ΔMA-CANC was used for statistical analysis and significant differences indicated (P < 0.0005 (***)). (F) In vitro assembled particles as in (E) but with an additional condition including 375 μM Tartrate rather than IP and unassembled DMA-CANC were incubated with recombinant HIV-1 protease for the indicated times and analysed by SDS PAGE and western blot. The probable cleavage products, based on size, are indicated. Triangles point to additional cleavage products that are not present in particles assembled with IP6.

Figure 4. HIV-1 can become independent of IP6 for immature particle assembly.

(A) Virus release efficiency of Gag mutants, calculated as the percentage of particle-associated p24 (CA) as a fraction of total (cell- + particle-associated Gag) normalised to WT virus. Error bars depict the SEM from at least three independent experiments. An unpaired t test against WT was used for statistical analysis and significant differences indicated [P < 0.005 (**)]. (B) Gag cleavage efficiency in purified virions, calculated as the percentage of p24 (CA), p41 and Pr55Gag. (C) Infectivity of Gag mutants normalised to the quantity of input virus [per 30 pg of reverse transcriptase (RT)]. Error bars depict the SEM from three independent experiments. An unpaired t test against WT was used for statistical analysis and significant differences indicated [P = 0.005 (**), < 0.0005 (***)]. (D) In vitro assembly of immature particles using recombinant DMA-CANC protein [comprising capsid (CA) and nucleocapsid (NC) domains]. 7.5 μM RNA was added to 75 μM ΔMA-CANC and assembly monitored through light scattering changes at 350 nm. EM images of negative stained samples of the final assembly reactions. Scale bars are 200 nm. Representative of two experiments. (E) The thermostability of in vitro assembled particles was measured by differential scanning fluorimetry and expressed as a change in melt temperature (Tm) compared to unassembled ΔMA-CANC. An unpaired t test against unassembled ΔMA-CANC was used for statistical analysis and significant differences indicated [P < 0.0005 (***)]. (F) Kinetics of immature particle assembly of ΔMA-CANC mutants from (E), using 10 μM RNA and 100 μM CANC. Representative of at least two experiments.

Figure 5. HIV-1 virions lacking enriched IP6 assemble mature capsids with low efficiency and which have reduced stability.

(A) Viruses produced in cells supplemented with tritiated inositol were purified and inositol phosphate species extracted and fractioned by SAX-HPLC. Data were analysed as previously described[8] and the counts per minute (CPM) of IP6 shown as a fraction of total CPM in the sample. Error bars depict mean CPM ± SEM from at least two independent experiments. An unpaired t test against WT was used for statistical analysis and significant differences indicated [P < 0.0005 (***)]. (B-D) Cryo-ET on indicated HIV-1 mutants. Tilt-series were collected and reconstructions performed to assess capsid morphology. A total of 163 KAKA and 187 KAKA/T8I particles were analysed. (B) Virions produced by the indicated Gag mutants were classified into the indicated categories: Ambiguous (orange), Immature (pink), Mature Conical (dark blue), Mature Tubular (light blue), Mature Irregular (green). (C) Slices through tomograms show representative examples of the viral morphologies in (B). Scale bars, 100 nm. (D) Virions with mature lattices were further subdivided into: Multiple Cores (green), Single Cores (cyan), Cores with additional closed structure (orange), Cores with additional open structure (light orange), Multilayered Cores (blue). (E) Schematic diagram of a viral particle in the kinetic TIRF assay detecting capsid uncoating. HIV particles are loaded with low levels of EGFP using a cleavable fusion protein with EGFP and Vpr. These EGFP-loaded HIV particles are immobilised then permeabilised in the presence of AF568-labelled CypA. Fluorescence traces are recorded at the locations of individual HIV particles by TIRF microscopy. Permeabilisation of the viral membrane (step 1) with a pore-forming protein leads to loss of the EGFP signal and concomitant binding of AF647-CypA molecules to the capsid. Note that any capsid internalized EGFP is not detected above background. Capsid uncoating (step 2) is detected as the loss of the AF647-CypA signal and capsid lifetime is calculated as the time difference (Δt) between permeabilization and uncoating. (F-G) Capsid survival curves (G) constructed from the lifetimes of all particles in the field of view reveal an unstable subpopulation that decays away within the first few minutes (no or short-lived AF647-CypA signal with a half-life of < 2 min) and a stable subpopulation with slow uncoating kinetics (long-lived AF647-CypA signal). The fraction of stable capsids for WT and KAKA mutant in the absence and presence of IP6 is shown (G), with the calculated half-life of the stable fraction obtained by fitting of survival curves (F) in the presence or absence of 100 μM IP6. A one-way ANOVA was used for statistical analysis and significant differences indicated [P < 0.0001 (****)].

Figure 6. Simultaneously reducing cellular IP6 and the ability of HIV-1 to enrich it into virions amplifies infectivity defects.

(A) Virus release efficiency of Gag mutants from either 293T cells or IPMK KOs overexpressing FM1, calculated as the percentage of particle-associated p24 (CA) as a fraction of total (cell- + particle-associated Gag) normalised to WT virus. Error bars depict the SEM from at least three independent experiments. An unpaired T test was used for statistical analysis and only significant differences indicated [P = 0.05 (*), = 0.005 (**), < 0.0005 (***)]. Blue asterisks refer to significant differences to WT virus produced in 293Ts, whilst green asterisks refer to significant differences to WT virus produced in IPMK KO + FM1 cells. (B) Gag cleavage efficiency of WT, KAKA and KAKA/T8I purified virions, calculated as the relative amount of p24 (CA), p41 and Pr55Gag as a percentage of total Gag. (C) Thin-section electron-microscopy of KAKA mutant HIV-1 virions produced in 293T cells or IPMK KO cells expressing FM1. Scale bar = 120 nm. (D) Infectivity of Gag mutants normalised to the quantity of input virus as determined by RT assay and relative to WT HIV-1. Error bars depict the SEM from three independent experiments. An unpaired T test was used for statistical analysis and significant differences indicated [P = 0.05 (*), = 0.005 (**), < 0.0005 (***)]. Blue asterisks refer to significant differences from WT virus infection of 293Ts.

Figure 7. KAKA virions produced in IP6-low cells have fewer and less-stable capsids.

(A) Cryo-ET comparison of KAKA and WT virus produced in IPMK KO + FM1 cells. Tilt-series were collected and reconstructions performed to assess capsid morphology. A total of 48 WT and 24 KAKA particles were analysed. Virions were classified into the indicated categories: Immature (pink), Mature Conical (dark blue), Mature Tubular (light blue), Mature Irregular (green). Slices through representative tomograms of the virions are shown together with quantification. Scale bars, 100 nm. (B) TIRF microscopy on WT virions produced in 293T cells and KAKA mutants produced in IPMK KO + FM1 cells. Virions were adhered to Ibidi slides and treated with SLO in the presence or absence of IP6. Samples were fixed, permeabilised and labelled with VSV-G and p24 antibody. Virions from three independent images for each condition were analysed for mean fluorescence object intensity above threshold (see Supplementary Figure 8). A one-way ANOVA was used for statistical analysis and significant differences indicated [P = 0.01 (**), < 0.0001 (****)]. Right panel shows representative images of virions used in the analysis. Scale = 20μm.

Figure 8. The HIV-1 immature lattice enriches IP6 into virions to catalyse mature capsid assembly.

The immature lattice does not intrinsically need IP6 for assembly but instead acts as a ‘net’ to capture IP6 from producer cells and enrich it into virions. Virions that have budded from the cell undergo maturation during which the immature lattice is cleaved by the viral protease. This results in the liberation of CA protein from Gag and the release of IP6 from its binding site in the immature Gag lattice. The newly freed IP6 promotes the assembly of CA into capsomers (predominately hexamers but also pentamers), which are used to construct the conical capsid characteristic of mature HIV-1 particles. Because maturation happens inside virions, separated from the cell, if there are insufficient IP6 molecules packaged into virions then stable mature capsids cannot form and the resulting HIV-1 particles fail to become infectious.