A protein-fragment complementation screen reveals VP1pX interactions with ESCRT proteins

To better understand how pX functions in eHAV release, we screened the entire 1D capsid protein (VP1pX) for interactions with ESCRT-associated proteins using a sensitive Gaussia princeps luciferase (GLuc) protein-fragment complementation assay that is capable of detecting weak and/or transient protein-protein interactions [26–28]. Unprocessed VP1pX protein, fused as ‘bait’ to the C-terminal half of GLuc (‘GLuc2’), was transiently co-expressed in 293T cells with a panel of 24 ESCRT-associated proteins fused as ‘prey’ to the N-terminus of GLuc (‘GLuc1’) (Fig 1B and 1C). A robust interaction signal was generated with ALIX, and to a lesser extent with proteins associated with ESCRT-0 (HRS and STAM2) and ESCRT-I (TSG101) (Fig 1D top). Subsequent screens using isolated VP1 and pX polypeptides as bait suggested ALIX interacts primarily with pX, whereas the interaction with HRS and TSG101 mapped to VP1 (Fig 1D bottom, middle). Additional studies with GLuc2-VP2 bait were consistent with the previously reported ALIX-interacting YPx₃L late domains in VP2 [16], although the signal was not reduced by single Ala substitutions of Tyr or Leu residues in each of the domains (Fig 1E). By contrast, VP3 showed no evidence of interacting with ALIX. These data are consistent with ESCRT-recruitment signals residing in VP1pX as well as VP2, with pX interacting either directly or indirectly with ALIX as reported previously [24].

pX drives extracellular release of computationally-designed nanocage assemblies

HAV replication is slow and inefficient in cell culture, complicating the functional analysis of viral proteins. Thus, to assess the role of pX in eHAV release, we fused the pX sequence to the C-terminus of a 203 amino acid de novo designed protein that self-assembles into a 60-copy, 25 nm wire cage dodecahedron [29] (Fig 2A). These engineered protein nanocages (EPN) have been shown to be secreted from cells within vesicles by an ESCRT-dependent process when the protein is fused at its C-terminus to the p6^Gag protein of human immunodeficiency virus (HIV-1) that contains late domains interacting with TSG101 and ALIX [30]. Secretion is also dependent upon the EPN protein being linked at its N-terminus to residues 2–6 of Gag that contains a myristoylation signal mediating association with membranes [30]. Fusing pX to the C-terminus of the nanocage protein (‘EPN-pX’) in lieu of p6^Gag did not interfere with nanocage assembly, and resulted in nanocages being visibly present within membrane-limited vesicles released from transfected 293T cells (Fig 2B). Detergent treatment was required to immunoprecipitate EPN-pX released by the transfected cells, consistent with its sequestration within vesicles (Fig 2C). Furthermore, abundant EPN-pX could be recovered from beneath a 20% sucrose cushion following centrifugation of extracellular fluids, confirming the particulate nature of the secreted nanocage (Fig 2D, lanes 1 and 13). A 6.7 Å 3D structure of these nanocages determined by cryo-electron microscopy was very similar to that reported previously [29] (S1A–S1C Fig). However, additional density was evident in the vicinity of the C-terminus of the nanocage protein, facing towards the inside of the nanocage and consistent with pX (S1D and S1E Fig). Release was suppressed by co-expression of a dominant-negative VPS4A mutant (E228Q) (Fig 2D, compare lanes 1 and 7), confirming ESCRT-dependence [30]. Nanoparticle tracking analysis revealed a small increase in the median size of EVs released by 293T cells expressing EPN-pX (127 nm) compared with control cells (110 nm) (S1F and S11G Fig), but little change in overall EV concentration (S1H Fig). Thus, EPN-pX expression minimally perturbed the background release of exosomes and other microvesicles from the cells.

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Fig 2. pX mediates ESCRT-dependent extracellular release of assembled EPN-pX nanocages and noncytopathic p16 virus.

(A) (top) EPN-pX, in which pX is fused to the C-terminus of the nanocage protein with an intervening Myc tag. (bottom) pX sequence in EPN-pX and related mutants. The pentamer assembly domain (PAD) [21] and YxxL motif are indicated. Δ1, Δ2 and Δ3 deletions were described previously [23]. (B) Representative electron micrograph showing multiple EPN-pX nanocages (white arrows) within a membrane-bound vesicle (black arrow) in extracellular fluids of transfected 293T cells. Scale bar = 20 nm. The inset shows nanocages in extracellular fluids treated with CHAPS. Scale bar = 30 nm. (C) pX and Myc immunoblots of anti-pX immunoprecipitates of extracellular fluids, with and without NP-40 treatment, versus lysate from EPN-pX transfected cells. (D) Myc immunoblot of released EPN protein recovered following centrifugation through a 20% sucrose cushion, with soluble and insoluble intracellular EPN proteins expressed by cells transfected with EPN-pX and related constructs shown below the solid horizontal line. Cells were co-transfected with vectors expressing eGFP (control, lanes 1–6) or the E228Q VPS4A mutant (lanes 7–12). (E) Quantitation of nanocage protein release normalized to intracellular protein expression. Results are means ±S.E.M. from 3 independent experiments. ****p<0.0001 by ANOVA with Sidak’s multiple comparison test. (F) Protein-fragment complementation assay with (left) VP1pX and (right) pX GLuc2 baits with and without YxxL/A substitutions and GLuc1 prey fused to ESCRT components. Results are means ± S.D. from two independent experiments, each with 3 technical replicates. ***p<0.001 by two-way ANOVA with Sidak’s multiple comparison test. (G) Extracellular HAV RNA determined by RT-PCR [genome equivalents (GE) per mL supernatant fluids] and intracellular HAV RNA [GE per μg total RNA], and calculated extracellular/intracellular RNA ratios, 12 days after transfection of cells with p16 or p16-YxxL/A RNA; GE = genome equivalents. Data are from n = 6 cultures from two transfections, and are representative of multiple independent experiments. **p<0.01 calculated by Mann-Whitney test.

https://doi.org/10.1371/journal.ppat.1010543.g002

EPN-pX was released from cells as efficiently as EPN fused to p6^Gag (S2A Fig). Since EPN-p6^Gag release requires both TSG101-interacting ‘PTAP’ and ALIX-interacting ‘YPx₃L’ late domains of p6^Gag [30] (S2A Fig), this indicates that pX is capable of functionally replacing both p6^Gag late domains. By contrast, EPN was not released when fused to HAV VP2 sequence containing tandem YPx₃L late domains that interact only with ALIX [16] (S2B Fig).

EPN-pX release requires an association with intracellular membranes

In addition to self-assembly and ESCRT recruitment, the release of EPN-p6^Gag requires a myristoylation signal at its N-terminus that drives its association with membranes [30]. EPN-pX release was similarly dependent on this myristoylation signal (S2C Fig), indicating that pX does not by itself mediate an association with intracellular membranes. The VP4 capsid protein of some picornaviruses is N-terminally myristoylated and plays an important role in membrane penetration during cell entry [31]. By contrast, HAV VP4 is small (only 21–23 amino acids) and lacks a myristoylation signal, yet associates with membranes through its N-terminus [32]. EPN-pX constructs in which the N-terminal 9 or 11 residues of VP4 replaced the Gag myristoylation signal of HIV-1 were actively released, albeit not as efficiently as EPN-pX with the Gag signal (S2D Fig). Treating cells with IMP-1088, an inhibitor of N-myristoyltransferases (NMT1 and NMT2) [33], reduced the release of EPN-pX containing the N-terminal Gag signal, but had little impact on EPN-pX constructs containing N-terminal VP4 sequence (S2D Fig, lanes 6–10, inset). These results support previous studies indicating VP4 associates with membranes independently of myristoylation [32], and suggest VP4 could provide the membrane association required for ESCRT-mediated capsid export. However, VP4 is located internally within the mature, extracellular HAV capsid [19]. Dynamic ‘breathing’ is known to occur in the capsids of other picornaviruses [34], but it is uncertain whether this would be sufficient to externalize VP4 and allow it to drive an association of the HAV capsid with membranes.

A YxxL motif in pX is crucial for nanocage export and eHAV release

Deleting the N-terminal 27 amino acid pentamer assembly domain of pX [21] (‘EPN-ΔPAD’) had neglible impact on EPN-pX release, whereas deleting the C-terminal 44 amino acids, leaving only the pentamer assembly domain, completely abolished release (‘EPN-PAD’, Fig 2A, 2D, and 2E). This localizes the responsible export signal to the C-terminal half of pX. Three deletions within this sequence that were shown previously to limit HAV spread in cell culture [23] had variable effects on EPN-pX release. Two 15-amino acid deletions (‘Δ1’ and ‘Δ2’) completely ablated release, whereas a 9-amino acid deletion (‘Δ3’) near the C-terminus of pX reduced secretion by 30 ±9.8% (Fig 2A, 2D, and 2E). The sequence deleted in the Δ1 mutant contains a YxxL motif (YKEL beginning with Tyr⁸⁰⁰) resembling late domains found in arenavirus and paramyxovirus proteins that facilitate budding of these enveloped viruses [35–37] (Fig 2A). Replacing the residues in this motif with 4 serial Ala substitutions eliminated EPN-pX release (‘YxxL/A’ mutant, Fig 2A, 2D, and 2E), and also reduced interactions of Gluc2-VP1pX and GLuc2-pX bait with GLuc1-ALIX prey in the protein fragment complementation assay (Fig 2F). Consistent with these results, a mutant virus with similar Ala substitutions in the YxxL motif (‘p16-YxxL/A’ virus) replicated well, but was severely impaired in egress resulting in its accumulation within infected cells (Fig 2G). Intracellular viral RNA copy numbers were increased approximately 10-fold, whereas the ratio of extracellular to intracellular viral RNA was reduced by approximately 60-fold. Taken collectively, these data support the existence of a potent export signal centered within the pX sequence that is essential for release of quasi-enveloped virus.

The pX export signal is functionally conserved in bat hepatoviruses

To gain a fuller understanding of the pX sequence required for viral release, we compared the pX sequence in human HAV (Hepatovirus A) with pX sequences in 8 other non-primate hepatovirus species [38]. Although pX is highly conserved among Hepatovirus A strains from humans and nonhuman primates, it is very divergent in hepatoviruses identified in seals, rodents, bats and other small mammals [39,40] (S3, and S4A Figs). Nonetheless, a constraint-based sequence alignment revealed a YxxL motif in 21 of 24 hepatovirus sequences recovered from 18 different mammalian host species (S3 Fig). In 2 other viruses, the Leu residue in the motif was replaced with similarly hydrophobic Val or Ala residues. Multiple residues downstream of the YxxL motif were also highly conserved in sequences from 22 of these hepatoviruses, revealing a broadly conserved pX segment extending between Tyr800 of the YxxL motif and Leu819 in human HAV (S3 Fig). This conserved pX segment spans the Δ1 and Δ2 deletions that ablated EPN-pX release (Fig 2A and 2D), and exists in 8 of 9 hepatovirus species (all but Hepatovirus G). Infectious hepatoviruses have yet to be isolated in cell culture from species other than Hepatovirus A, but the sequence conservation evident within this segment of pX suggests it has a similar role in capsid quasi-envelopment in other hepatovirus species.

To test this hypothesis, we replaced the pX sequence in EPN-pX with the pX sequence of M32 virus (Hepatovirus H), which was recovered from the African straw-colored fruit bat Eidolon helvum, and SMG18520 virus (‘SMG’, Hepatovirus C), recovered from the long-fingered bat Miniopterus manavi in Madagascar [39]. These bat pX sequences share only 39–42% amino acid identity (56–58% similarity) with pX of Hepatovirus A (Figs 3A and S4A). Despite lower expression of soluble proteins, both EPN-M32pX and EPN-SMGpX were released from cells with 50–80% of the efficiency of EPN-pX (calculated from immunoblot intensities of released versus soluble intracellular protein) (Fig 3B). As with EPN-pX, EPN-M32pX release was reduced or eliminated by co-expression of the dominant negative VPS4A-E228Q mutant, indicating that release was ESCRT-dependent (S4B Fig). ALIX was also increased in abundance in extracellular, particulate EPN-M32pX and EPN-pX fractions recovered from beneath a sucrose cushion (S4B Fig, lanes 2,3), indicating that ALIX is recruited to these nanocage assemblies during export. Taken collectively, these data indicate that pX-directed nonlytic virus release is an ancient and functionally conserved attribute of hepatoviruses recovered from mammalian hosts separated by millions of years of evolution.

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Fig 3. A conserved centrally-located pX peptide sequence, ExpD, mediates ALIX-dependent nanocage release.

(A) Wild-type and mutant pX sequences of human p16 virus aligned with bat virus pX sequences of M32 (Hepatovirus H), recovered from Eidolon helvum, and SMG18520 (‘SMG’, Hepatovirus C), recovered from Miniopterus manavi [39]. The N-terminal pentamer assembly domain is highlighted in blue, central ExpD sequence in red, and C-terminal Pro-Arg motif in yellow. Below is shown a WebLogo for ExpD derived from an alignment of 22 pX sequences from different mammalian hepatoviruses (see S3 Fig). Note that the Δ1a deletion in the human pX sequence extends the Δ1 deletion (Fig 2) to Leu805 with a GlySer insertion resulting from the cloning strategy. (B) Immunoblots showing released EPN-pX, EPN-M32pX, and EPN-SMGpX nanocages recovered from beneath a sucrose cushion, and related soluble and insoluble intracellular EPN proteins. EPN-Δ1a was included as a negative control. Quantitation is shown on the right, with mean p16 and bat pX nanocage release normalized to intracellular protein expression, ± S.E.M., n = 2–5. (C) Alanine scanning of conserved amino acid residues within EPN-pX using the nanocage release assay. Shown below are immunoblots of released and intracellular EPN proteins, and at the bottom quantitation of immunoblots in 3 independent experiments. Brackets indicate residues within the YxxL motif. (D) Immunoblots showing released and soluble/insoluble intracellular EPN-pX and EPN-ExpD proteins following transfection with ALIX (PDCD6IP)-targeting (+) or non-targeting (-) scrambled siRNAs. Immunoblotting of soluble intracellular proteins was with anti-Myc and anti-ALIX antibodies. Quantitation of EPN-ExpD release is shown on the right.

https://doi.org/10.1371/journal.ppat.1010543.g003

To better define the export signal in pX, we created single Ala substitutions at residues in EPN-pX that are conserved in both the human and bat viruses. Substitutions at Tyr800, Leu803, Arg804, Leu805, Gly808, and Arg811 (Hepatovirus A numbering) substantially ablated nanocage release, as did substituting Arg for the conserved Ala815 (Fig 3C). Fusing the conserved sequence at the center of pX (residues 797–820) to EPN (‘EPN-ExpD’) resulted in release of the nanocage with 64 ±26% s.d. the efficiency of EPN-pX (Fig 3D), which is comparable to EPN-Δ3 (Fig 2E). Like EPN-pX, EPN-ExpD release was reduced by siRNA-mediated depletion of ALIX (Fig 3D). These results thus define a conserved ESCRT-interacting export signal comprised of YxxLR[L/M]xxGxxRxxxA at the center of pX that is both necessary and sufficient for ALIX-dependent nanocage release.

Label-free quantitative proteomic analysis of the C-terminal pX interactome

The ability of pX to functionally substitute for both the TSG101- and ALIX-interacting late domains of p6^Gag in driving EPN release (S2A Fig) suggests it interacts not only with ALIX, but also with other ESCRT components. To assess this, we used label-free quantitative (LFQ) proteomics to analyze proteins precipitated by antibody targeting the Myc tag in EPN from lysates of 293T cells expressing EPN-pX versus EPN-PAD, which lacks the C-terminal 44 amino acid sequence of pX and is not released (S5A and S5B Fig). Triplicate samples of the two precipitates were subjected to tryptic digestion and peptide fragments identified by mass spectrometry. A total of 2192 cellular proteins were identified in the two sample sets. Normalized LFQ intensities and sequence coverage were comparable in each sample for peptides derived from the N-terminal pX sequence common to both EPN-pX and EPN-PAD, indicating similar anti-Myc precipitation efficiencies during preparation of the samples (S5C and S5D Fig). Eighty-seven cellular proteins were enriched greater than 4-fold in EPN-pX versus EPN-PAD precipitates, suggesting they interact directly or indirectly with the C-terminus of EPN-pX (Fig 4A and S1 Table). As expected, these proteins included ALIX. However, the ALIX paralog, HD-PTP (encoded by PTPN23), was equally enriched in the complex pulled down with EPN-pX (Figs 4A and S5E and S5F). HD-PTP shares structural homology with ALIX, interacts with ESCRT-0 (STAM2) and ESCRT-III (CHMP4B), and is known to contribute to the sorting of ubiquitylated cargo into MVEs [41–43].

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Fig 4. pX interacts directly with the Bro1 domain of ALIX.

(A) EPN-pX constructs expressed in 293T cells for label-free quantitative (LFQ) proteomic analysis of the pX interactome. Below is a volcano plot showing differential abundance of proteins (LFQ intensities) identified in anti-Myc precipitates of EPN-pX versus EPN-PAD lysates. Differences in normalized LFQ peptide intensity are plotted versus significance (p value). Lines represent lower limits of FDR <0.01 and <0.05. Each protein is represented by a single data point with proteins of interest labelled. (B) Co-immunoprecipitation of HA-tagged ALIX with EPN-pX. Proteins in lysates of 293T cells transfected with EPN-pX or EPN-Δ1a were immunoprecipitated with anti-pX, followed by blotting with (top) anti-HA (ALIX) and (bottom) anti-pX. Whole cell lysates are on the left. IP: immunoprecipitation; IB, immunoblot. (C) Merged, single-channel Airyscan fluorescent images of a cell transfected with EPN-pX and HA-ALIX expression vectors, showing pX (green), ALIX (HA, red) and LAMP1 (magenta). At the bottom are shown enlarged dual- and single-channel recordings of the region delineated by the yellow rectangle in the merged image at the top. (D,E) Co-immunoprecipitation of HA-tagged ALIX or the indicated ALIX domain fragments (top) with EPN-pX expressed in 293T cells. (F) Co-immunoprecipitation of bacterially-expressed pX and 6XHis-tagged ALIX Bro1 protein (residues 1–392) mixed in buffer containing 0.7% BSA and 0.07% Tween-20. The input mixture (lane 1) was precipitated with the indicated antibodies, followed by immunoblotting as shown. IgG = isotype control. (G) Biotin-tagged ExpD peptide pulldown assay. Recombinant ALIX Bro1 protein was incubated with synthetic biotin-tagged ExpD peptide (top), affinity isolated on streptavidin beads, and probed with anti-ALIX. Competitor peptides, representing ExpD, the Y800A ExpD mutant, and the C-terminus of CHMP4 [44] (top) were added to the mixture at molar ratios decreasing from 1 to 0.125 relative to the biotin-tagged peptide. At the bottom is shown a quantitative analysis of Bro1 pulled down by biotin-tagged ExpD, normalized to pulldown in the absence of competitor peptide. Results shown are means ± s.e.m. from n = 3 independent experiments.

https://doi.org/10.1371/journal.ppat.1010543.g004

An additional 268 proteins were over 2-fold enriched in EPN-pX precipitates (S1 Table). Among these were the ESCRT-III associated proteins IST1 (IST1 homolog), which we identified previously in extracellular eHAV virions and is required for efficient eHAV release [7], and VTA1 (vacuolar protein sorting-associated protein VTA1), which regulates VPS4 ATPase and helps to coordinate disassembly of ESCRT-III complexes [45] (Figs 4A and S5E and S5F). Interestingly, the NEDD4 family E3 ubiquitin ligase ITCH (E3 ubiquitin-protein ligase Itchy homolog), which regulates ESCRT recruitment during budding of multiple enveloped viruses [46,47], was also present in the complex, suggesting it could play a role in EPN-pX release (Figs 4A and S5E and S5F). Consistent with eHAV originating from MVEs, proteins over-represented in the EPN-pX precipitate were highly enriched for components of exosomes and lysosomes (S5G Fig).

The ExpD sequence binds the Bro1 domain of ALIX

We confirmed that pX interacts with ALIX by showing that HA-ALIX co-immunoprecipitates with EPN-pX, but not EPN-pX mutants in which the YxxL motif was deleted or mutated (EPN-Δ1a and EPN-YxxL/A), when co-expressed in 293T cells (Figs 4B and S6A). Moreover, the degree to which EPN-pX mutants containing Ala or Arg substitutions of conserved ExpD residues co-immunoprecipitated with ALIX correlated well with the impact of these substitutions on EPN-pX release (Spearman r = 0.836, S6B Fig). Super-resolution fluorescence imaging also revealed striking co-localization of ectopically expressed ALIX and EPN-pX in 293T cells, both on internal membranes and at buds on the plasma membrane (Figs 4C and S6C). By contrast, there was little to no colocalization of ALIX with the EPN-Δ1a mutant (S6D Fig).

ALIX consists of an N-terminal, boomerang-shaped Bro1 domain, a central ‘V’ domain bent into a V-like conformation, and a C-terminal proline-rich ‘PRD’ domain [15,48,49] (Fig 4D). Co-immunoprecipitation experiments with various ALIX deletion mutants indicated that EPN-pX interacts with the N-terminal Bro1 domain rather than the V domain, with which the p6^Gag YPx₃L late domain interacts [50] (Fig 4D). The Bro1 domain of ALIX also co-immunoprecipitated efficiently with FLAG-tagged VP1pX expressed in 293T cells, whereas immunoprecipitation of the V domain was weak and inconsistent (S6E Fig). These results stand in sharp contrast to those in a previous report that suggested pX interacts with the V domain of ALIX [24]. While deletion of the N-terminal 134 ALIX residues had no effect, further deletion of residues 134–209, which span helices 4–6 near the center of the Bro1 structure [48], ablated the interaction with EPN-pX (Fig 4E). To ascertain whether the pX interaction with ALIX is direct or indirect, possibly bridged by other proteins in the complex, we expressed the 8 kDa pX sequence in E. coli and purified it to >95% homogeneity. This recombinant pX efficiently formed a complex with a second recombinant protein representing the Bro1 domain of ALIX (residues 1–392, also produced in E. coli) that could be precipitated by antibody to either ALIX or pX (Fig 4F). We also synthesized a 24 amino acid peptide representing the ExpD sequence conjugated at its N-terminus to biotin. This biotin-linked ExpD peptide bound the recombinant Bro1, allowing its recovery on streptavidin-coated beads (Fig 4G). Importantly, the biotin-tagged peptide interaction was efficiently blocked by a non-biotin-conjugated ExpD peptide, but not a related peptide with a single Ala substitution at Tyr800 (Y800A) (Fig 4G). Binding was also blocked by a peptide representing the C-terminal 20 residues of the ESCRT-III protein, CHMP4B (Fig 4G), which is known to interact with and contact helices 6–7 of ALIX Bro1 [44]. Taken collectively, these results show the ExpD segment of pX interacts directly with the Bro1 domain of ALIX, overlapping at least partially the CHMP4B binding site. Nonetheless, although pX binds the Bro1 domain of ALIX, overexpression of the entire ALIX protein, including the V and C-terminal PRD domains, was required to enhance EPN-pX release from 293T cells (S6F Fig).

pX binds the Bro1 domain of HD-PTP

HD-PTP is an ALIX paralog with a similar multi-domain structure within its N-terminal 714 residues. It acts non-redundantly with ALIX at endosomes in MVE formation [43,51]. The N-terminal Bro1 domains of ALIX (residues 5–360) and HD-PTP (residues 10–362) share 32.8% amino acid identity (49.9% similarity). Co-immunoprecipitation experiments with HA-tagged HD-PTP indicated that it forms a complex with EPN-pX when co-expressed in 293T cells, but not with EPN-PAD or EPN-YxxL/A (Fig 5A), confirming the proteomics results shown in Fig 4A. Similar experiments with the EPN-pX mutants containing single amino acid substitutions demonstrated an impact on HD-PTP co-immunoprecipitation similar to the impact on ALIX co-immunoprecipitation, with the exception of the Arg replacement of Ala815 which did not reduce HD-PTP co-immunoprecipitation (Fig 5B, compare with S6A Fig). As with ALIX, co-immunoprecipitation experiments indicated pX interacts with the Bro1 domain of HD-PTP (Fig 5C). Similarly, the recombinant pX protein was efficiently precipitated with antibody to HD-PTP (and vice versa) when mixed with purified recombinant HD-PTP Bro1 domain expressed in E. coli (Fig 5D). We also carried out pulldown experiments using the biotin-tagged ExpD peptide and HD-PTP Bro1 protein expressed in vitro in a coupled transcription/translation reaction (Fig 5E). As with ALIX Bro1 (Fig 4G), ExpD peptide pulldown of HD-PTP Bro1 was efficiently blocked by free ExpD and CHMP4 peptides, but not the ExpD Y800A mutant. These results indicate that pX interacts with the Bro1 domain of HD-PTP in a manner similar to its interaction with ALIX. Super-resolution fluorescence imaging was consistent with this, showing that ectopically-expressed HA-tagged HD-PTP colocalized with EPN-pX on the plasma membrane as well on internal membranes of cells (Fig 5F and 5G).

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Fig 5. pX interacts directly with the Bro1 domain of the ALIX paralog, HD-PTP.

(A) Co-immunoprecipitation of HA-tagged HD-PTP with EPN-pX. Proteins in lysates of cells expressing EPN-pX, EPN-PAD, or EPN-YxxL/A (Fig 2A) were precipitated with anti-Myc antibody and immunoblotted with antibody to HD-PTP. (B) Co-immunoprecipitation assays of HD-PTP with EPN-pX mutants containing single amino acid substituitions of conserved residues in the ExpD sequence. See Fig 3C. (C) Co-immunoprecipitation assays of EPN-pX with HD-PTP, HD-PTP with L202D and I206D substitutions in the Bro1 domain that eliminate CHMP4 binding (Bro1-mt) [43], and various HD-PTP domain fragments. PRR, proline-rich region; PTP, protein tyrosine phosphatase; PEST, “rich in proline, glutamate, serine and threonine” [51] (D) Co-immunoprecipitation of bacterially-expressed pX and recombinant HD-PTP Bro1 protein (residues 1–360) produced in E. coli. (E) Biotin-tagged ExpD peptide pulldown assay of HD-PTP Bro1 domain (residues 1–360) produced by in vitro transcription/translation in rabbit reticulocyte lysate. See legend to Fig 4G for details. (F) Merged low magnification (top left) and merged and single-channel enlarged super-resolution Airyscan fluorescence images of the area demarcated by the yellow lines showing pX (green), HD-PTP (HA, red), and LAMP1 (magenta) in a 293T cell expressing EPN-pX and HA-HD-PTP. Arrows indicate sites of pX colocalization with HD-PTP. (G) Similar low magnification (top image) and enlarged super-resolution images showing co-localization of pX and HD-PTP along the plasma membrane.

https://doi.org/10.1371/journal.ppat.1010543.g005

HD-PTP is required for ESCRT-dependent release of EPN-pX and eHAV

RNAi-mediated depletion of HD-PTP significantly suppressed EPN-pX release from transfected 293T cells (Fig 6A). Release was rescued by overexpression of HD-PTP, confirming the specificity of the knockdown and a requirement for HD-PTP in extracellular release of the nanocage (Fig 6B, lanes 2 versus 3). EPN-pX release was rescued to a lesser extent by overexpression of an HD-PTP mutant (Bro1-mt) with L202D and I206D substitutions in the Bro1 domain that eliminate CHMP4 binding [43], and not at all by a Bro1 deletion mutant (ΔBro1) (Fig 6B, lanes 4 and 5). Interestingly, overexpressing the Bro1 domain only, or Bro1-V (residues 1–714), also rescued EPN-pX release (Fig 6B, lanes 6 and 7). The fact that EPN-pX release is suppressed by depleting either ALIX (Fig 3D) or HD-PTP (Fig 6A) indicates that these Bro1 domain proteins function in a coordinate rather than redundant fashion to mediate release. Nonetheless, overexpression of HD-PTP, or the Bro1 or Bro1-V domains of HD-PTP, substantially rescued EPN-pX release from 293T cells with CRISPR-Cas9 mediated ALIX depletion (ALIX-KO cells) (Fig 6C, lanes 8–14). Unlike ALIX, which is distributed broadly throughout the cell and supports multiple ESCRT-related processes, HD-PTP appears to function selectively at endosomes [43, 51]. The rescue of EPN-pX release from ALIX-KO cells by HD-PTP may have resulted from nonphysiologic localization of the ectopically-expressed protein, as fluorescence microscopy demonstrated its presence at the plasma membrane as well as at internal locations within transfected cells (Fig 5G).

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Fig 6. HD-PTP is functionally required for EPN-pX and HAV release.

(A) EPN-pX release from 293T cells with RNAi-mediated knockdown of HD-PTP (encoded by PTPN23) (see legend to Fig 2D for details of release assay). siCtrl = scrambled siRNA pool. Quantitative analysis on the right with release normalized to release from siCtrl-transfected cells. n = 3 independent experiments. p-value by t-test with Welch’s correction. (B) Rescue of EPN-pX release by overexpression of HD-PTP, HD-PTP with L202D and I206D substitutions (Bro1-mt), or HD-PTP deletion mutants (see Fig 5C) in HD-PTP-depleted 293T cells (lanes 2–7), and HD-PTP-depleted CRISPR-Cas9 engineered 293T ALIX knockout (ALIX-KO) cells (lanes 9–14). siRNA: ‘+’ = siHD-PTP, ‘-’ = scrambled siCtrl. EV = empty vector. (C) (left to right) Ratio of extracellular/intracellular HAV RNA, extracellular HAVl RNA, and intracellular HAV RNA, 48 hrs after cell-free virus infection of Huh-7.5 cells with and without prior RNAi depletion of HD-PTP. Data shown are technical replicates from one of 3 experiments with similar results. GE = genome equivalent. (D) Merged and single-channel super-resolution Airyscan fluorescent microscopy images of pX (green, labelled with monoclonal antibody), endogenous ALIX (red), and endogenous HD-PTP (magenta) in an 18f virus-infected Huh-7 cell. A 3D reconstruction is shown on the top left with a sectional view to the right. Below are single- and dual-channel images shown in two projections (X-Y and X-Z). Arrows in the middle panels indicate a prominent site of pX colocalization with both ALIX and HD-PTP. (E) Quantitative estimates of pX-ALIX volume colocalization in Z-stack images: the first column shows the percentage of pX volume (voxels with pX signal above threshold) containing ALIX signal above threshold; the second column shows the percent ALIX volume containing pX signal. (n = 8 cells). See Methods for details. (F) Quantitative volume analysis of pX colocalization with HD-PTP similar to that in panel E. (G) Voxel colocalization of ALIX with HD-PTP (left) and HD-PTP with ALIX (right) in HAV-infected and uninfected Huh-7.5 cells. Adjusted p-values determined by ANOVA with Kruksal-Wallis test. n = 17–27 cells from multiple experiments.

https://doi.org/10.1371/journal.ppat.1010543.g006

As with the nanocage protein, HD-PTP depletion significantly reduced the release of virus from infected Huh-7.5 hepatoma cells (Fig 6C). This defect in release was reversed by overexpression of HD-PTP (Fig 6C). The involvement of both ALIX and HD-PTP in virus release was further supported by super-resolution fluorescence microscopy of virus-infected cells, which revealed pX and capsid antigen in close proximity to endogenously expressed ALIX and HD-PTP (Figs 6D and S7A and S7B). Quantitative analysis of recorded 3D Z-stack images indicated that 60% ± 5 s.e.m. of the volume occupied by fluorescence signal specific to pX also contained signal above background specific for endogenous ALIX (Fig 6E). Much less colocalization was evident with endogenous HD-PTP, with only 4.7% ± 0.5% of the pX signal sharing the same volume with HD-PTP (Fig 6F). As might be expected, only a minor fraction of either endogenous ALIX (9.3% ± 2.5 s.e.m.) or HD-PTP (1.6 ± 0.6%) colocalized with the viral protein. Colocalization occurred primarily on internal membranes (Figs 6D and S7A and S7B), and not on the plasma membrane as observed with ectopically expressed HA-tagged proteins (Fig 5G). Importantly, ALIX fluorescence strongly co-localized with foci of HD-PTP colocalization with pX (i.e., three-way colocalization), and there were highly statistically significant increases in the percentage of ALIX volume colocalizing with HD-PTP, and HD-PTP volume with ALIX, in infected versus uninfected cells (Fig 6G).

Cells and virus

H1 Hela and human hepatocyte-derived Huh-7.5 [64] cells were mycoplasma-free and cultured as described previously [65]. HEK293T (293T) cells were procured from the ATCC (CRL-3216). Viruses used in these studies were derived from modified infectious molecular clones of low (p16) or high cell culture-passage (18f) HM175 strain HAV, pT7-p16.2 (GenBank KP879217.1) and pT7-18f.2 (KP879216.1), respectively, containing 40-nt long 3’ poly-A tails [66, 67]. The 18f-NLuc reporter virus expressing nanoluciferase has been described previously [68].

Plasmids and oligonucleotides

Plasmids encoding de novo designed protein nanocages fused to wildtype or mutant HIV p6^Gag sequence (EPN01N-based vectors) and VPS4A(E228Q) [29,30], ALIX [50], and expression vectors for Gaussia princeps luciferase fragments [27] have been described previously. pHA-IST1 (#131619), pRK5-HA-Ubiquitin-WT (#17608) and pRK5-HA-Ubiquitin-K0 (#17603) were from Addgene. Vectors expressing the nanocage fused to wild-type or mutant pX sequences, ALIX mutants, and GLuc2-VP2 mutants were constructed using PCR-based strategies. DNA encoding a nanocage fragment fused to pX sequences of Eidolon helvum M32 virus (GenBank KT452714.1) and Miniopterus manavi SMG18520 virus (KT452742.1) was synthesized by Genewiz (South Plainfield, NJ) and ligated into pEPN-pX at EcoR1 and Afl2 sites to produce pEPN-M32pX and pEPN-SMGpX, respectively. GLuc2-VP1pX, -VP1 and -pX were constructed using Gateway cloning. pT7-16.2 mutants with Ala and Arg substitutions in pX were constructed by splicing synthetic cDNA from Genewiz into Sac1 and PflM1 sites. All final constructs were verified by DNA sequencing. Oligonucleotides used in this study are listed in S2 Table.

Antibodies

A murine monoclonal antibody to pX (clone 59.7.1) was raised against bacterially-expressed 6xHis-tagged HM175 pX protein (RRID:AB_2868525, 1:1000). Other antibodies used in this study are listed in S3 Table. Species-specific Alexa Fluor conjugated secondary antibodies were purchased from Thermo Fisher Scientific.

Chemical reagents

IMP-1088 was purchased from Cayman Chemical (Cat# 25366–1). Iodixanol (Opti-Prep Density Gradient Medium) was purchased from Millipore-Sigma (Cat# D1556). SMARTPool ON-TARGETplus and non-targeting control #2 siRNAs were purchased from Dharmacon.

siRNA and CRISPR/Cas9 protein depletion

For RNAi depletion of cellular proteins, cells were transfected with 50 nM ON-TARGETplus siRNA (set of 4) or ON-TARGETplus Non-targeting Control siRNA (Control #2, ‘siCtrl’) (Dharmacon), using the Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Four different siRNA sequences were used and the siRNA providing the most efficient knockdown was selected for subsequent experiments. CRISPR/Cas9 genetic knockout was carried out using pre-cloned lentivirus vectors from Applied Biological Materials (Richmond, Canada). Dual sgRNA/CRISPR/Cas9 expressing lentivirus was produced by co-transfecting sgRNA-expressing vectors and non-targeting control (Applied Biological Materials) and 3rd Generation Packaging System Mix (Applied Biological Materials) into 293T cells using the FuGENE-HD transfection reagent (Promega). Supernatant fluids were harvested at 72 h, passed through a 0.22-μm syringe filter and transduced into 293T cells. Puromycin (5 mg/mL) was added to the medium 72 hrs later for antibiotic selection.

In vitro transcription and RNA and DNA transfections

Genome-length RNA (5 μg) was transcribed in vitro from plasmid DNA and electroporated into 5x10⁶ Huh-7.5 cells as described previously to generate virus [69]. DNA transfections were carried out with FuGENE HD (Promega) according to the manufacturer’s recommended protocol. Briefly, 293T cells (5×10⁵ per well in 6-well plates) were transfected with 500 ng of EPN vectors, or co-transfected with an equal amount of empty or VPS4A(E228Q) expression vector. For co-immunoprecipitation experiments, 2.5×10⁶ 293T cells, seeded previously in 10-cm dishes, were co-transfected with 500 ng EPN vector and 5 μg empty or HA-tagged ALIX, or HD-PTP expression vectors. For proteomic analysis, similarly seeded 293T cells were transfected with 2.5 μg EPN-pX or EPN-PAD vector.

Isopycnic gradient centrifugation of virus

Isopycnic gradient analysis of intracellular virus and virus released into cell culture supernatant fluids was carried out as previously described [1,16]. Cell culture supernatant fluids were centrifuged at 1,000 x g for 10 min at 4°C to remove debris, further clarified by 2x centrifugation at 10,000 x g for 30 min, and concentrated by ultracentrifugation at 100,000 x g for 1 hr at 4°C. The resulting pellet was resuspended in PBS, loaded onto a preformed 8–40% iodixanol (Opti-Prep) step gradient, and centrifuged at 141,000 x g in a Superspin 630 rotor for 48 hrs at 4°C in a Sorvall Ultra-80 ultracentrifuge. For gradient analysis of intracellular virus, infected cells were subjected to freeze-thaw lysis in 1X PBS. The lysate was clarified by centrifugation for 5 min at 10,000 x g and loaded onto an 8–40% iodixanol gradient and centrifuged as described above. Approximately 20 fractions were manually collected from the top of the gradients. Density of the fractions was determined using a refractometer. RNA was extracted from fractions using the RNeasy kit (Qiagen), followed by RT-qPCR quantitation of HAV RNA.

RT-qPCR quantitation of viral RNA

Total RNA was extracted from infected cells using the RNeasy Kit (Qiagen) and used in cDNA synthesis reactions with the SuperScript III First-Strand Synthesis System (Invitrogen). Primers targeting the 5’ untranslated region of the HAV genome were used to quantify HAV RNA genome equivalents (GE) in a SYBR Green Real-Time qPCR assay (BioRad, Cat# 1725121) against a synthetic RNA standard [8].

Protein-fragment complementation assay

The Gaussia princeps luciferase protein-fragment complementation assay was carried out as described previously [26,27]. In brief, mixtures of plasmids encoding prey (A) or bait (B) protein, each fused to a fragment of the Gaussia luciferase protein (GLuc1 or GLuc2, respectively), or control vectors, were co-transfected in triplicate in 96-well plates of 293T cells. Twenty-four hrs posttransfection, cells were lysed and assayed for luciferase activity (Promega). Normalized luminescence ratios (NLR) were calculated as the average signal generated in cells co-transfected with GLuc1-A and GLuc2-B, divided by the average signal in wells transfected with GLuc1-A and an empty GLuc2 vector, and those transfected with GLuc2-B and an empty GLuc1 vector. Positive bait-prey protein interactions were identified by NLR values exceeding those obtained with a large panel of control prey proteins [27]. Each interaction pair was assessed in at least two independent experiments.

Nanocage release assay

Release was assayed as previously described [30]. In brief, 24 h after transfection of 10⁶ Huh-7.5 or 293T cells with nanocage vectors, supernatant fluids were collected, clarified by centrifugation at 8,000 rpm for 10 min, and then centrifuged through a 200 μL 20% sucrose cushion in an Eppendorf 5424R centrifuge at full speed for 90 min. Samples were carefully removed from the bottom of the cushion and suspended in 50 μL Laemmli sample buffer, incubated at 95°C for 5 min, then resolved in a 4–15% gradient SDS–polyacrylamide pre-cast gel (4561086, BioRad). Proteins were transferred to a polyvinylidene fluoride membrane by semi-dry transfer using a Transblot Turbo apparatus (BioRad). Membranes were blocked in Odyssey Blocking Buffer (LI-COR Biosciences) and probed with a 1:1000 dilution of primary antibodies overnight. The membrane was washed with 0.05% Tween-20 and probed with a 1:10,000 dilution of donkey anti-goat secondary antibodies conjugated with IRDye 800 (LI-COR Biosciences) for 1 h at room temperature. Excess secondary antibodies were removed by washing with 0.05% Tween-20, and protein bands were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences). Direct quantitation was accomplished by measuring the near-infrared fluorescence intensity of individual protein bands.

Co-immunoprecipitation analysis

Pierce Protein A/G Agarose and magnetic Dynabeads Protein G beads (both from Thermo Scientific) were used for co-immunoprecipitation and protein pull-down proteomic analysis respectively. Cell lysates were prepared from 10-cm dish cultures with lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 250 mM NaCl, 10% glycerol, 5 mM EDTA, 1× complete protease inhibitor and 1× PhoSTOP, 0.5% NP-40). The clarified lysates were then used for immunoprecipitation according to the manufacturer’s protocols.

Nanoparticle tracking analysis

The size and concentration of EVs released by 293T cells were monitored by laser scattering video microscopy using a ZetaView device (Particle Metrix) with a particle cut-off size of 1000 nm [70]. Cells were propagated in media supplemented with exosome-free fetal calf serum, and supernatant fluids clarified by low-speed centrifugation prior to analysis.

Purification of engineered protein nanocages (EPNs)

Ten 175 cm² flasks of HEK293T cells were transfected with pEPN-pX using polyethylenimine [71]. At 48 hrs, clarified culture medium (10,000 g, 10 minutes) was centrifuged at 150,000 g (r_max) for 1 hr at 12°C and the pellet was resuspended in 12 mL PBS. The sample was centrifuged again at 150,000 g (r_max) for 1 hr at 12°C and resuspended in 200 μL PBS. It was then loaded on a 5–35% iodixanol density gradient in PBS and centrifuged at 300,000 g (r_max) for 4 hrs at 12°C. The diffuse band of EPNs in the central region of the gradient was collected and diluted in PBS before being centrifuged at 150,000 g (r_max) for 2 hrs at 12°C. The pellet was resuspended in 20 μL PBS and stored at 4°C.

Cryo-EM sample preparation, data collection and processing

The EPN suspension was applied to glow-discharged copper support R2/2 holey carbon TEM grids (Quantifoil) and vitrified in liquid ethane/propane using a Cryoplunge 3 (Gatan) with a blot time of 4–5 s at >90% humidity. Grids were screened using a Glacios TEM operating at 200 keV and a data set of 1803 movies was acquired through EPU (Thermo Fisher FEI) at a nominal magnification of 92 kX and a nominal defocus range of -0.7 to -3 μm using a Falcon-III detector (ThermoFisher FEI) in integrating mode. Each acquired image had a total dose of 2.3 e^–/Ų distributed over 20 frames. Movies were aligned and summed using MotionCor2 [72] before CTF correction using CTFFIND4 [73] on non-dose-weighted image sums. The resulting corrected micrographs were then processed using Relion3.0 [74]. Promising particles were manually selected through the Relion3.0 GUI and extracted particles were filtered by classification with Relion3.0 (1830 particles). Three-dimensional reconstruction was performed using a symmetry-free ab initio model derived using Relion’s Initial Model module as a reference with icosahedral symmetry. The final three-dimensional map was masked and B-factor applied via the post-processing module in Relion. A calibrated pixel size was derived from fitting of the previously solved nanocage structure (PDB: 5KPN) (27) and determined as 1.51 Å/pix. The cryo-EM coordinates of the nanocage structure have been deposited to the Protein Data Bank with the identifier 8A8N, and to the EMDataResource as EMD-15234.

Label-free quantitative proteomics

Lysates were prepared from three independent 10-cm dish cultures of 293T cells transfected with pEPN-pX and pEPN-PAD with lysis buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 250 mM NaCl, 10% glycerol, 5 mM EDTA, 1× complete protease inhibitor and 1× PhoSTOP, 0.5% NP-40) followed by immunoprecipitation with anti-Myc antibody coupled to magnetic beads (Thermo Scientific). The protein samples were separated by SDS-PAGE, extracted from segments cut from the gel, destained, reduced and alkylated and then subjected to tryptic digestion. The peptides were extracted and desalted on home-made C18 stage-tips, then dissolved in 0.1% formic acid and analyzed on a Q-Exactive HF-X coupled with an Easy nanoLC 1200 (Thermo Fisher Scientific, San Jose, CA). Peptides were loaded onto a nanoEase MZ HSS T3 Column (100Å, 1.8 μm, 75 μm x 150 mm, Waters). Analytical separation of all peptides was achieved with 45-min gradient. A linear gradient of 5 to 30% buffer B over 29 min and 30% to 45% buffer B over 6 min was executed at a 300 nl/min flow rate, followed by a ramp to 100% buffer B in 1 min and a 9-min wash with 100% buffer B, where buffer A was aqueous 0.1% formic acid, and buffer B was 80% acetonitrile and 0.1% formic acid. LC-MS experiments were also carried out in a data-dependent mode with full MS (externally calibrated to a mass accuracy of <5 ppm and a resolution of 60,000 at m/z 200) followed by high energy collision-activated dissociation-MS/MS of the top 15 most intense ions with a resolution of 15,000 at m/z 200. High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 27 eV in the presence of nitrogen bath gas atoms. Dynamic exclusion was 20.0 seconds. Each of the three biological replicates was subjected to two replicate technical LC-MS analyses.

Raw proteomics data processing and analysis

Mass spectra were processed, and peptide identification carried out using MaxQuant software version 1.6.10.43 (Max Planck Institute, Germany). Protein database searches were against the UniProt human protein sequence database (UP000005640), EPN protein and HAV pX sequences. A false discovery rate (FDR) for both peptide-spectrum match (PSM) and protein assignment was set at 1%. Search parameters included up to two missed cleavages at Lys/Arg on the sequence, oxidation of methionine, and protein N-terminal acetylation as a dynamic modification. Carbamidomethylation of cysteine residues was considered as a static modification. Peptide identifications were reported by filtering of reverse and contaminant entries and assigning to their leading razor protein. The label-free quantitation (LFQ) was carried out using MaxQuant. Data processing and statistical analysis were performed on Perseus (Version 1.6.0.7). Protein quantitation of technical replicates, and two-sample t-test statistics with a threshold p-value of 0.01, were used to report statistically significant protein abundance fold-changes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD022107.

Recombinant pX production and purification

E. coli BL21 (DE3) was transformed with plasmid DNA encoding maltose-binding protein fused to pX with an intervening tobacco etch virus (TEV) cleavage sequence, and grown at 37°C until OD600 = 1.0. Cells were induced with 1 mM IPTG and allowed to grow overnight at 37°C. Cells were harvested by centrifugation and the pellet resuspended in buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 1mM DTT, cOmplete Protease Inhibitor cocktail (Roche)). A French press was used to lyse cells and the cell debris pelleted by centrifugation at 15,000 rpm. Supernatant was passed over an amylose resin column (New England Biolabs) and the column washed with 10 column volumes of buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA). Protein bound to the amylose column was eluted using elution buffer (20mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 10 mM maltose). The eluate from the amylose column purification was digested with TEV protease and subjected to anion exchange chromatography (SOURCE 15Q column, GE Healthcare) and bound proteins eluted with an increasing NaCl gradient. Fractions were analyzed by SDS-PAGE and fractions containing pX protein were pooled and concentrated using a Vivaspin 20 concentrator (3000 MWCO, Sartorius). The final protein preparation was visualized on SDS PAGE and concentration determined using the bicinchoninic acid (BCA) protein assay.

Super-resolution Airyscan fluorescence microscopy

DNA-transfected 293T or 18f-NLuc virus-infected Huh-7.5 cells were visualized in 35mm glass bottom dishes with 14mm bottom wells (Cellvis, Cat# D35-14-1.5-N). Cells were fixed with 4% paraformaldehyde for 12 mins, washed twice with PBS and incubated with a blocking/permeabilization solution containing 5% goat serum and 0.1% saponin in PBS for 1 hr at room temperature. Cells were stained for 1 hr at room temperature with primary antibody diluted in PBS with 1% BSA and 0.1% saponin, then washed with PBS containing 0.05% saponin followed by staining with appropriate secondary antibodies for 1 hr. Nuclei were counterstained with Hoechst 33342 (Invitrogen) for 10 mins at room temperature. Slides were then washed three times with PBS containing 0.05% saponin, and where appropriate incubated for 3 min with a 1:2000 dilution of CellMask Deep Red (Thermo Fisher) in PBS. Imaging data were recorded in super-resolution mode on a laser-scanning confocal Zeiss 880 microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with an Airyscan detector and controlled by Zen Blue 3.0 software. Airyscan super-resolution recording enhances resolution in all directions by a factor of 1.7. The microscope was mounted on a Zeiss Inverted Axioobserver Z1 base equipped with a Definite Focus unit to ensure focus plane stability. The Plan-Apochromat 63X/1.40 Oil DIC M27 objective (Zeiss) was used for all imaging. For each image plane, fluorescence signals were recorded sequentially at different wavelengths in super-resolution mode using appropriate laser excitation and filter sets: for DAPI/ Hoechst, excitation 405nm, emission filter BP 420–480 + BP 495–550; for Alexa488, excitation 488nm, emission filter BP 420–480 + BP 495–550; for Alexa568/594, excitation 561nm, emission filter BP 420–480 + BP 495–620; and, for Alexa647, excitation 633nm, emission filter BP 570–620 + LP 645. Fields of view were selected randomly maintaining identical imaging conditions for different cells in each experiment. The number of X-Y pixels on the frame was set to “optimal” in the Zeiss software controller, with XY pixel size 0.043 μm. The distance between images in the Z direction during Z-stack recording was 0.160 μm. Immediately after recording, the raw data was processed by Zen Blue software (Zeiss) to produce a super-resolution image with 16-bit depth.

Post-imaging analysis

Imaris software (version 9.6, Bitplane, Zurich) was used for 3D visualization of recorded Airyscan images and to analyze colocalization of fluorescent signals. Following background subtraction, the Imaris colocalization module was used to identify the percentage of 3D image volume within which fluorescence signals were colocalized. Intensity thresholds of fluorescence signals were selected for each fluorescence channel separately using a semi-automatic procedure with the condition that the threshold should not be less than 10% of maximum channel intensity. Separate channels were created for colocalization results obtained from the analysis of each pair of fluorescent signals (e.g., pX or HAV with HD-PTP; and pX or HAV with ALIX). The percentage volume colocalization was then determined for these newly created channels and the fluorescence signal from a third channel, allowing identification of colocalization of all three signals above threshold. The positions and shapes of structures labeled with specific antibodies were visualized by rendering the surfaces of 3D fluorescent signals based on their intensities.

Statistical analysis

Unless indicated otherwise, significance was assessed by unpaired t test or ANOVA. All statistical calculations were carried out using with Prism 8.4.3 software (GraphPad). Significance values are shown as ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.