Calcium Contributes to Polarized Targeting of HIV Assembly Machinery by Regulating Complex Stability

Abstract

Polarized or precision targeting of protein complexes to their destinations is fundamental to cellular homeostasis, but the mechanism underpinning directional protein delivery is poorly understood. Here, we use the uropod targeting HIV synapse as a model system to show that the viral assembly machinery Gag is copolarized with the intracellular calcium (Ca²⁺) gradient and binds specifically with Ca²⁺. Conserved glutamic/aspartic acids flanking endosomal sorting complexes required for transport binding motifs are major Ca²⁺ binding sites. Deletion or mutation of these Ca²⁺ binding residues resulted in altered protein trafficking phenotypes, including (i) changes in the Ca²⁺–Gag distribution relationship during uropod targeting and/or (ii) defects in homo/hetero-oligomerization with Gag. Mutation of Ca²⁺ binding amino acids is associated with enhanced ubiquitination and a decline in virion release via uropod protein complex delivery. Our data that show Ca²⁺–protein binding, via the intracellular Ca²⁺ gradient, represents a mechanism that regulates intracellular protein trafficking.

Introduction

Directional delivery of protein complexes to their destinations is fundamental to cellular homeostasis, but mechanisms underpinning intracellular protein trafficking are not well-understood. Cell polarity demands a robust yet simple framework to control intracellular cargo movements. This network is vital for cells to expedite responses across biological processes, including (i) firing of neurotransmitters through neurological synapses,^1,2 (ii) dispatching exosomes via exocytosis,^3,4 and (iii) mounting immune responses through immunological synapses.^5,6 In the context of immunity, immune cells may undergo rapid reversing of cell polarity (via reorientation of the microtubule organizing center [MTOC]) to engage with target cells.^5,6 While organelle targeting signals or association with “destination-specific proteins” could explain how proteins reach destinations via hitchhiking “existing” delivery vehicles, these narratives do not, however, account for the logistics of how these delivery systems initiate or redirect cargos toward organelles located in the opposite end of the cells in the first place. As viruses rely on cellular machineries to propagate, the polarized assembly and release of HIV via virological synapses may shed light on the underlying mechanisms of intracellular protein trafficking.

The polarity of cells can be defined based on the relative positioning of the nucleus and MTOC, where the latter is generally found on one side of the nucleus that consists of a higher abundance of endoplasmic reticulum (ER). In fibroblasts, neurons, and epithelial and endothelial cells, the MTOC localizes in front of the nucleus in the direction of migration (leading edge).⁷ In contrast, the MTOC in immune cells is generally found in the uropod (retracting end) and reorients itself to sit between the attachment interface (synapse) and the nucleus upon cell–cell engagement.^5,6 Regardless of cell types, MTOC and enrichment of ER are found on the side of the secretory apparatus where vesicles or viruses are released. Due to Ca²⁺ storage in the ER and mitochondrial regulation,⁸⁻¹³ both an overall and subcellular local Ca²⁺ gradient exists within cells.⁸⁻¹³ Another common thread among synapse biology and exocytosis events is the involvement of the multivesicular bodies (MVBs) pathway and endosomal sorting complexes required for transport (ESCRT) machineries, which are also exploited by many viruses for assembly and release.^14,15

Results

Uropod Targeting and Oligomerization of HIV Gag Are Associated with Ca²⁺ Gradient

Uropod targeting or virological synapse formation is conserved among retroviruses,¹⁶⁻²⁰ and this process is associated with the oligomerization of the retroviral Gag protein.²¹ Using HIV virological synapse as a model system, our fluorescent imaging analyses show that the subcellular distributions of HIV Gag (imCherry, modified from ref (22)) in peripheral blood lymphocytes (PBLs) are copolarized and overlapped with intracellular Ca²⁺ gradient (fluo-4) both in the elongated and round PBLs (Figures 1a,b and S1). The median area distribution of detectable HIV Gag represented 4.2 and 3.7% of cell areas in the elongated (virological synapses displaying) PBLs and the round (less-extended virological synapse forming) PBLs (Figures 1c and S1), whereas detectable Ca²⁺ occupied 22.0 and 25.9% areas of corresponding PBLs, respectively (Figures 1c and S1). Surface plasmon resonance (SPR) analyses showed recombinant HIV Gag specifically binds to Ca²⁺ with an estimated SPR dissociation constant (K_d) of 9.18 μM (Figures 1d,e and S2 and S3) but has undetectable SPR binding with other cations (Na⁺, K⁺, Mg²⁺, and Zn²⁺; Figures 1e and S3). Isothermal titration calorimetry (ITC) has been used to record the thermodynamics of HIV Gag oligomerization events among low-order Gag oligomers.²³ ITC showed that the inclusion of divalent cations resulted in energetically favorable reactions (ΔG < 0) during low-order Gag oligomerization over monovalent cations, where Ca²⁺ stimulated the highest change in enthalpy (ΔH) across all cations tested (Figures 1f,g and S4). The Ca²⁺-induced enhancement (for both ΔG and ΔH) on the low-order Gag oligomerization in ITC was strengthened in the presence of nucleic acids (Figures 1h,i and S4). Charge detection mass spectrometry (CDMS) is a single molecule technique that can quantify high-order oligomerization (∼4 MDa [120mers] of hepatitis B viral proteins) in vitro.^24,25 CDMS analyses showed that in vitro high-order oligomerization of HIV Gag (with a median of 7–12 MDa [125–220mers]) is promoted by Ca²⁺ (Figures 1j and S5) with optimal Ca²⁺ concentrations ([Ca²⁺]) at 0.1–1.0 mM (Figures 1k and S5) that mirrors the high end of intracellular Ca²⁺ gradient. The lower “overall” 100 nM cytoplasmic [Ca²⁺] is in sharp contrast with the [Ca²⁺] needed to induce HIV Gag oligomerization. Whether it is known as “sparks” in muscle cells,²⁶⁻²⁸ “puffs” in oocytes,²⁹ or “syntillas” in neurons,³⁰ high [Ca²⁺] can be transiently released locally via intracellular organelles, such as mitochondria, ER, and acidic vacuoles.³¹ The local [Ca²⁺] in these storage compartments can reach 100–800 μM,³¹ which is sufficient to support the oligomerization of HIV Gag during its trafficking to virological synapses. Our time-lapse live imaging showed that stochastic Ca²⁺ sparks occur among T-lymphocytes, and the two selected T-lymphocytes displayed spikes of Ca²⁺ signals (via fluo-4 complex) at ∼90 s with a slow decay of the Ca²⁺ signal (Figure 1l and Videos S1–S6). A combination of (i) super-resolution microscopy, (ii) 3D volume imaging, (iii) time-lapse video, and (iv) alternative Ca²⁺ dye to distinguish the flux (blink) of Ca²⁺ will be needed to define the details of the temporal and spatial relationship between local Ca²⁺ release and intracellular trafficking of Gag.

Figure 1.

Calcium cation binds specifically to HIV Gag and promotes Gag–Gag assembly. (a,b) Distribution of Pr55^Gag-imCherry (red) and Ca²⁺ (green) in elongated (a) and round (b) PBLs is shown. DIC, single fluorescent and merged images are included. Scale bar 10 μm. (c) Fractional areas and detectable signals of Pr55^Gag-imCherry (red) and Ca²⁺ (green) in elongated and round PBLs expressing Pr55^Gag are shown. Medians are highlighted (n = 75 cells per arm). (d) Ca²⁺ binds to Pr55^Gag in SPR. (e) SPR estimated dissociation constant (K_d) between cations and Pr55^Gag are listed (n > 3). (f) ITC binding profiles between Pr55^Gag and Ca²⁺ or other cations (Mg²⁺, Zn²⁺, Na⁺, and K⁺) (n > 3). (g) ITC thermodynamic parameters between Pr55^Gag and cation interaction are listed (n > 3). (h) ITC profiles of Pr55^Gag binding with cations in the presence of 20 μM of 20mers DNA oligonucleotides (4× 5′-GAGAA-3′) are shown (n > 3). (i) ITC thermodynamic parameters between Pr55^Gag and cation in the presence of 20 μM of 20 mers DNA oligonucleotides (4× 5′-GAGAA-3′) are shown (n > 3). (j) CDMS of the assembly reaction of Pr55^Gag containing 4:1 molar ratio of Pr55^Gag/DNA oligonucleotides (4× 5′-GAGAA-3′), plus 2 μM IP5 and 1 mM cationic cofactors, such as Na⁺, K⁺, Zn²⁺, Mg²⁺, and Ca²⁺. Ion counts are normalized to 100; the y-axis is truncated to reveal the broad extent of oligomerization (n > 3). (k) CDMS quantification of Pr55^Gag oligomerization as a function of Ca²⁺ concentration from the absence of Ca²⁺ (front) to 25 mM Ca²⁺ (rear). Only ions above 500 kDa are shown, and counts are normalized to 100 (n > 3). (l) Distribution of Ca²⁺ (green), mitochondria (magenta), Pr55^Gag-imCherry (red), and nucleus (blue) in CD4+ T-lymphocytes from three different time points are shown. DIC, single fluorescent and merged images are included. Scale bar 10 μm.

C-Terminus p6 Domain of HIV Gag Is a Determinant of Ca²⁺-Associated In Vitro Gag–Gag Interaction and Intracellular Trafficking

The capsid (CA) domain within HIV Gag (Figure 2a) drives viral assembly by facilitating homo-oligomerization of Gag molecules,^32,33 and in vitro assembly of virus-like-particles can occur in the absence of p6.³⁴ Deletion of p6 from recombinant HIV Gag drastically reduced the quantity and the size of CDMS-detectable Ca²⁺-induced high-order HIV Gag oligomers (Figure 2b). SPR revealed that Gag–Gag homodimerization was strengthened up to 7-fold in the presence of Ca²⁺ with SPR K_d (Figures 2c and S6), but this Ca²⁺-induced homodimerization effect disappeared when the p6 domain was removed (Figures 2c and S6). The ITC-detected Ca²⁺ binding during low-order Gag oligomerization also vanished in the absence of p6 (Figures 2d,e and S7). As the energy exchange detected in ITC low-order Gag oligomerization is contributed in part by CA–CA-based interactions,²³ ITC analyses with p15^NC-SP2-p6 and p7^NC (lacking the p39^MA-CA-Sp1 domains, Figure 2a) have mapped that the p6^Gag domain directly contributes to Ca²⁺ binding (Figures 2d,e and S7).

Figure 2.

p6^Gag is an important determinant of Ca²⁺ binding and enhances Gag–Gag interactions. (a) Schematic shows domains in Pr55^Gag and its derivatives (Pr50^GagΔp6, p15^NC-SP1-p6, and p7^NC). (b) p6^Gag contributes to in vitro Ca²⁺-induced high-order Gag oligomerization in CDMS (n > 3). (c) Ca²⁺ promotes SPR-detected homodimerization of Pr55^Gag but not with Pr50^GagΔp6 (n > 3). (d) ITC Ca²⁺ binding profiles of Pr55^Gag (and its derivatives) show that p6^Gag contributes to Ca²⁺ interaction. Zoom-In ITC profiles are presented for p15^NC-SP2-p6 and p7^NC (n > 3). (e) ITC thermodynamic parameters are obtained between Pr55^Gag (or its derivatives) and Ca²⁺ (n > 3). (f–i) Distribution of Pr50^GagΔp6-imCherry (red) and Ca²⁺ (green) in elongated (f,h,i) and round (g) PBLs are shown. DIC, single fluorescent and merged images are included. Scale bar 10 μm. (j) Fractional areas and detectable signals of Pr50^GagΔp6-imCherry (red) and Ca²⁺ (green) in elongated and round PBLs expressing Pr50^GagΔp6 are shown. Medians are highlighted (n = 75 cells per arm). Statistical distribution analyses against Pr55^Gag are with two-sample Kolmogorov–Smirnov test.

If the Ca²⁺–p6^Gag interaction is important for intracellular trafficking of HIV Gag, removal of p6^Gag should alter the relationship between Ca²⁺ and HIV Gag during virological synapse formation. In virological synapse displaying elongated PBLs, cell imaging analyses showed that the p6-deleted HIV Gag had a more dispersed distribution and occupied a greater fractional area when compared with that of wild-type HIV Gag (Figures 2f,h–j and S8). In contrast, the intracellular Ca²⁺ distributions were generally more condensed within a smaller fractional area of Pr50^GagΔp6 expressing cells than Pr55^Gag expressing cells, a phenotype that is more noticeable in the less extended virological synapse forming round PBLs (Figures 2g,j and S8). In elongated PBLs, an increase in Gag signal was detected in Pr50^GagΔp6- over Pr55^Gag-expressing cells (Figures 2f,h–j and S8), while no difference in overall Gag signal was found between Pr50^GagΔp6 and Pr55^Gag round PBLs (Figures 2g,j and S8). The signal strength ratio of Gag to Ca²⁺ was consistently higher in Pr50^GagΔp6- than in Pr55^Gag-expressing cells across all PBLs (Figures 2f–j and S8). The altered Ca²⁺–Gag distribution relationship between Pr50^GagΔp6- and Pr55^Gag-expressing cells could be related to a nonsynchronous lateral movement of the Ca²⁺ gradient and viral proteins during the re-establishment of virological synapses from the virus-laden uropod.¹⁶ Our data support the notion that a relationship exists between the intracellular Ca²⁺ and the p6 domain of HIV Gag that contributes to the intracellular trafficking of HIV Gag for directional release.

Conserved Glutamic/Aspartic Acids Flanking HIV ESCRT Binding Motifs Are Ca²⁺ Binding Sites Involved in HIV Gag Trafficking

Ca²⁺ often acts as a coordination point that interacts with multiple oxygen atoms from the carbonyl group of amino acids with negatively charged side chains (such as glutamic (E)/aspartic (D) acids) to stabilize intra- or intermolecule interactions.³⁵⁻³⁷ Point mutations were introduced into 7 out of 9 of the most conserved E/D residues within the p6^Gag to generate the Pr55^Gag p6-7aa mutant (Figure 3a [red conservation scores]). Imaging analyses showed that Pr55^Gag p6-7aa occupied half of the fractional area and exhibited half of the signal intensity as seen with wild-type Pr55^Gag (Figures 3b–d and S9). Consistent with a role of direct Ca²⁺ binding in HIV Gag trafficking, both the fractional area occupied and the signal intensity displayed of Ca²⁺ were significantly lower in Pr55^Gag p6-7aa-expressing cells in comparison with those of wild-type Pr55^Gag-expressing cells (Figures 3b–d and S9). A single alanine point mutation was independently introduced into 6 of the putative Ca²⁺ binding sites within the recombinant HIV Gag. Biophysical analyses showed that all 6 recombinant Pr55^Gag point mutants displayed between 3- and 15-fold reduction in Ca²⁺ SPR binding (Figures 3e and S10), and the enhancement of Ca²⁺-induced Gag–Gag homodimerization in SPR was either reduced or eliminated in 5 out of 6 recombinant Pr55^Gag point mutants (Figures 3e and S10). Fluorescent imaging and SPR data supported a role of p6^Gag E/D residues in Ca²⁺ binding to facilitate intracellular trafficking. The individual contributions of these mutations on Ca²⁺ binding during low-order Gag oligomerization were independently examined by ITC. Apart from Pr55^Gag E461A that registered indistinguishable thermodynamic properties compared to wild-type Pr55^Gag, 3 out of 6 mutants (Pr55^Gag E460A, Pr55^Gag E482A, and Pr55^Gag D496A) showed no ITC detectable Ca²⁺ binding in low-order Gag–Gag homo-oligomerization in vitro (Figures 3f,g and S11). While mutant Pr55^Gag E468A and Pr55^Gag E477A retained their in vitro ITC-detectable Ca²⁺ binding, the thermodynamic properties of these Ca²⁺–Gag homo-oligomer interactions were reversed from a wild-type exothermic reaction to endothermic reactions in these mutants (Figures 3f,g and S11). Circular dichroism showed that Pr55^Gag E468A and Pr55^Gag E477A maintained the same overall α-helix and β-sheet contents of Pr55^Gag (Figure 3h). These data support a role for E/D amino acid residues flanking the ESCRT motifs in the HIV p6^Gag as Ca²⁺ binding sites during HIV Gag trafficking. Production of recombinant Pr55^Gag with more than one Ca²⁺ binding site mutations was not successful thus far, making it difficult to further dissect the biophysical properties Pr55^Gag containing combined Ca²⁺ binding site mutations using cell-free assays.

Figure 3.

Conserved p6^Gag E/D residues influence Ca²⁺-Pr55^Gag interactions. (a) Nine E/D residues in p6^Gag are identified. Seven out of nine conserved E/D residues are highlighted in rainbow shadow, and the same color scheme is used for both this figure and Figure 4. Both PTAP and LXXLF motifs are denoted with a gray background. Conservation scores are in red. (b,c) Distribution of Pr55^Gag p6-7aa-imCherry (red) and Ca²⁺ (green) in elongated (b) and round (c) PBLs is shown. DIC, single fluorescent and merged images are included. Scale bar 10 μm. (d) Fractional areas and detectable signals of Pr55^Gag p6-7aa-imCherry (red) and Ca²⁺ (green) in elongated and round PBLs expressing Pr55^Gag p6-7aa are shown. Medians are highlighted (n = 75 cells per arm). Statistical distribution analyses against Pr55^Gag are with a two-sample Kolmogorov–Smirnov test. (e) SPR estimated dissociation constants (K_d) are Ca²⁺ induced homodimerization impacted by Ca²⁺ binding site mutations (n > 3). (f) ITC Ca²⁺ binding profiles of Pr55^Gag p6^Gag E/D point mutants are shown (n > 3). (g) ITC thermodynamic parameters are obtained between Pr55^Gag p6^Gag E/D point mutant and Ca²⁺ (n > 3). (h) CD spectra of recombinant Pr55^Gag E468A and Pr55^Gag E477A are shown (n > 3).

Ca²⁺ Binding Stabilizes HIV Gag Assembly Complexes for Directional Trafficking and Release

Reduction of fluorescent signals of Pr55^Gag p6-7aa suggests that the stability of the HIV protein complex (such as the homo-oligomerization of Gag) might be compromised without wild-type Ca²⁺ binding (Figures 3b–d and S9). As ubiquitination is both important in HIV biology^38,39 and a post-translational protein degradation system,⁴⁰ we examined the impact of changes in Ca²⁺ binding on the ubiquitination of HIV Gag protein complexes. Immunoprecipitation of Pr55^Gag showed that an increased level of ubiquitination was detected in Ca²⁺-binding-repressed Pr55^Gag p6-7aa (Figure 4a). As p6^Gag is a major segment for HIV Gag ubiquitination,^38,41 deletion of p6^Gag has reduced detectable ubiquitinated Pr50^GagΔp6 (Figure 4a). Virological assays were used as a surrogate to quantify the functional impacts of interfering with Ca²⁺ interactions on directional trafficking of proteins in cells. Particle release from a protease inactive (PR[-] via PR^D25G mutation) and an envelope negative immature virus-like particle (VLP) system (HIV_NL GagPol PR[-], modified from ref (42)) was used for direct comparison for uropod targeting of HIV Gag oligomeric complexes. Equivalent volumes of viral particle supernatants were pelleted for Western blot analyses. A lower number of pelleted particles was detected upon in-frame deletion of p6^Gag (HIV_NL GagPol Gag^Δp6 PR[-]) (Figure 4b), which was in part due to ESCRT-related membrane arrest of particle release. Lower levels of immature Pr55^Gag p6-7aa VLPs (HIV_NL GagPol Gag^{p6-7aa (E/D-G)} PR[-]) supported the notion that Ca²⁺ binding site mutations were defective in uropod particle targeting (Figure 4b), implying that the Ca²⁺-mediated Gag–Gag homo-oligomerization is a determinant of directional trafficking of HIV protein complexes in cells. Previous analyses with a different combination of glutamic acids mutations in p6^Gag reported a similar ubiquitination-mediated degradation of HIV Gag.⁴³

Figure 4.

p6^Gag Ca²⁺ binding sites contribute to homo/hetero-oligomerization of proteins. (a) Ubiquitination of Pr55^Gag is associated with Ca²⁺ binding site mutations (n > 3). (b) Deletion of p6^Gag and mutations of p6^Gag Ca²⁺ binding sites reduces particle release (n > 3). (c,d) Deletion of p6^Gag and mutations of p6^Gag Ca²⁺ binding sites are associated with defects in Pr55^Gag processing and Pr160^GagPol packaging. Increasing concentrations of indinavir (at 0, 0.5, 5, 50 μM) are used to slow down Pr160^GagPol-mediated proteolytic processing (n > 3). (e) Relative infectivity of p6^Gag Ca²⁺ binding site mutants against wild-type control are shown for T-cell line and PBLs. (f) Virion protein profiles among wild-type (NL4-3^WT) and mutant HIV (NL4-3^E454G, NL4-3^E460G, NL4-3^E461G, NL4-3^E468G, NL4-3^E477G, NL4-3^E482G, NL4-3^E496G, NL4-3^E468G+E477G, and NL4-3^E482G+E496G). HIV patient sera is the source of antibodies (n > 3). (g,h) Virion protein profiles of dual mutants (NL4-3^E468G+E477G and NL4-3^E482G+E496G) in comparison with NL4-3^WT. Virion proteins are probed with anti-p24^CA (g) and anti-p66/51^RT (h) antibodies. (i) Virion-associated Pr160^GagPol are compared between NL4-3^WT and NL4-3^E468G+E477G particles that have been produced with increasing concentrations of the viral protease inhibitor indinavir (at 0, 0.5, 5, 50 μM). Virion proteins are probed with anti-p24^CA (n > 3). (j) SPR analyses of Ca²⁺–Pr68^GagPR binding and effects of Ca²⁺ on Pr68^GagPR homodimerization (n > 3). (k) Coimmunoprecipitation on the stability of Pr55^Gag/Pr160^GagPol complexes in the presence of EGTA and quantifications of relative Pr55^Gag/Pr160^GagPol ratio (n > 3).

The principle of direct Ca²⁺ binding to regulate homo-oligomerization of proteins during trafficking should be applicable to hetero-oligomerization of protein complexes, that is, Ca²⁺-dependent interaction between Gag and non-Gag proteins. The copackaging of virion-associated proteins provides an opportunity to interrogate the role of Ca²⁺ binding on hetero-oligomerization of proteins during the trafficking and the release of HIV particles. HIV GagPol (Pr160^GagPol) is a well-characterized cotrafficking and copackaging virion-associated protein.^32,33,44 Pr160^GagPol represents 10% of the total virion protein⁴⁵ and is understood to be packaged into Gag particles via interactions across the mutually shared CA domain between Gag and GagPol.^32,33 Unlike the p6^Gag in Pr55^Gag, the amino acids of p6^Pol in Pr160^GagPol have a different protein sequence due to overlapping reading frames. Pr50^GagΔp6 and Pr55^Gag p6-7aa were introduced into protease active HIV-1_NL GagPol constructs.⁴² The proteolytic processing of Pr50^GagΔp6 was compromised due to deletion of both p6^Pol and part of PR (Figure 4c, lane 2 vs lane 6). Site-specific mutations of E/D to G mutations in Pr55^Gag p6-7aa via codon modifications have not altered the amino acid sequences of p6^Pol of HIV_NL GagPol Gag^{p6-7aa (E/D-G)}. The higher ratio of p25/24 CA doublets in Pr55^Gag p6-7aa particles compared to that in wild-type Pr55^Gag particles suggested that a defect of virion protein maturation may exist in the mutant HIV_NL GagPol Gag^{p6-7aa (E/D-G)} (Figure 4c, lane 2 vs lane 10). Western blot analyses of pelleted particles produced under increased concentrations of the protease inhibitors indinavir (IDV) supported the notion that the defects in Pr55^Gag p6-7aa particles could be related to reduction in virion packaging of Pr160^GagPol (Figure 4c,d [anti-CA] and [anti-RT], respectively).

Fine mutational analyses were performed to separate out Ca²⁺ binding site mutations that induced complex instability of Gag homo-oligomers (reduction in virion particle release) from the potential defect of Gag–GagPol hetero-oligomerization (suppression in virion packaging of Pr160^GagPol). Seven single-point mutations and two double-point mutations were introduced into infectious HIV_NL4-3. One mutant (HIV^{Gag E468G+E477G}) was identified to be noninfectious in both T-cell line (MT2) and PBLs (Figure 4e). Although all mutants have wild-type levels of particle release (i.e., no detectable functional impact on Gag–Gag homo-oligomerization), HIV^{Gag E468G+E477G} exhibited virion protein processing profile defects (Figure 4f) reminiscent of HIV_NL GagPol Gag^{p6-7aa (E/D-G)} (Figure 4c,d, [anti-CA] and [anti-RT], respectively). The lack of functional impact from the seven single-point mutants (Figure 4e,f) and one double-point mutant (HIV^{Gag E482G+D496G}, Figure 4e,f) suggests the redundant nature of these Ca²⁺ binding sites in viral replication, which is consistent with the known role of multiple contact points that Ca²⁺-based interactions often need to stabilize protein complexes. Western blot virion analyses of the double-point mutant (HIV^{Gag E468G+E477G}) particles with specific anti-HIV antibodies confirmed a defect in virion Pr55^Gag processing (Figure 4g) and a reduction of virion-associated polymerase–reverse transcriptase (Figure 4h). Unlike Pr55^Gag p6-7aa particles (Figure 4b), the defects in HIV^{Gag E468G+E477G} were not associated with impeded viral particle release (i.e., homo-oligomerization of Gag). Inclusion of virion protease inhibitor IDV during particle production confirmed that HIV^{Gag E468G+E477G} was defective in virion Pr160^GagPol packaging (Figure 4i), showing that the suppression of Ca²⁺ binding via Pr55^Gag can lead to reduced hetero-oligomerization of Pr55^Gag–Pr160^GagPol complexes during directional trafficking to the uropod for virion release. To illustrate that Pr160^GagPol can directly interact with Ca²⁺, a recombinant Pr160^GagPol surrogate, Pr68^GagPR, was made by engineering mutations in both the Pr160^GagPol frameshift site and the protease active site to express Pr68^GagPR, consisting of the natural Pr160^GagPol domains from p17^MA to p12^PR[-]. SPR analyses showed that Pr68^GagPR bound to Ca²⁺ specifically without detectable binding against other cations tested (Figures 4j and S12). To directly examine whether Ca²⁺ can stabilize Pr55^Gag–Pr160^GagPol complexes (hetero-oligomerization), coimmunoprecipitation analyses of immature virus-like particles were done with the divalent cation chelating agent EGTA (or EDTA) via an anti-FLAG antibody and C-terminus FLAG-tagged Pr160^GagPol containing virion particles. A dose-dependent reduction of detectable Pr55^Gag was seen in the presence of increasing concentrations of EGTA, Ca²⁺ preferred chelating agent, or EDTA (Figures 4k and S12), supporting the notion that direct Ca²⁺ interaction is a mechanism that regulates hetero-oligomerization of HIV Pr55^Gag–Pr160^GagPol complexes during trafficking for directional virion release.

Discussion

The intracellular landscape afforded by the local and overall Ca²⁺ gradient may provide part of the traffic control accounting for the logistics of directional protein–complex movement within the intracellular terrain. Our data provide supportive evidence that both the formation and the trafficking of HIV protein complexes are facilitated in part by direct Ca²⁺ binding. Manipulation of Ca²⁺ binding has resulted in changes in the precision of movement during directional targeting of viral proteins, including association with the post-translational ubiquitination protein degradation regulatory system.⁴⁰ In contrast to the well-established Gag oligomerization domain in CA that is known to be critical in Gag–Gag homo-oligomerization and Gag–GagPol hetero-oligomerization,^32,33 our identified Ca²⁺ binding sites in p6^Gag that facilitate HIV complex formation were previously not known to be involved in mediating protein oligomerization (homo/hetero). In the context of retroviruses, these data fundamentally change our understanding of retroviral assembly and virion packaging of retroviral GagPol. Moreover, our demonstrated roles of Ca²⁺ to stabilize both homo-oligomerization and hetero-oligomerization in HIV protein complexes could be a generic mechanism that extends beyond HIV biology, given that similar putative Ca²⁺ binding sites are highly conserved amino acids flanking ESCRT binding motifs within retroviral Gag plus the fact that ESCRT machineries are utilized by many viruses to facilitate viral assembly and release.^14,15 Although a role of direct Ca²⁺ binding has not been previously shown to mediate protein trafficking, the contributions of Ca²⁺ to the processes involving ESCRT-mediated vesicle transport⁴⁶ and synaptic vesicle exocytosis⁴⁷ are well-documented. Since a single Ca²⁺ cation has the capacity to interact with multiple carbonyl oxygens in amino acids with negatively charged side chains thereby stabilizing (or destabilizing) intra- and interprotein interactions,³⁵ it is plausible that a series of Ca²⁺-mediated interactions can exert rigorous control to regulate intracellular protein movement. In the context of HIV and ESCRT-dependent viruses, such a mechanism may allow viral complexes to detour away from the MVB–lysosome degradation pathway for viral particle assembly and release. In a manner analogous to the execution principles in decision-making procedures that are well-known in computational binary processing, whereby a series of on/off events can work together to execute complex tasks with a high degree of accuracy and efficiency, this direct Ca²⁺ binding trafficking strategy has the likelihood to be broadly applicable to the regulation of protein trafficking across all cells that have an intracellular Ca²⁺ gradient.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.1c00563.

• Additional materials and methods details and Figures S1–S12 (PDF)

• Time-lapse videos of CD4+ T-lymphocytes expressing HIV Gag with 10 s intervals for 10 min that are separated into single fluorescent 326 channels: DIC (AVI), calcium (AVI), mitochondria (AVI), Gag-imCherry (AVI), nucleus 327 (AVI), and merged (AVI) (scale bar is 10 μm)

Author Contributions

Co-first authors: C.K., B.L.S., J.L. (these authors contribute equally). Conceptualization: J.M. Methodology: C.K., B.L.S., J.L., C.A.L., C.-H.L., W.J.Mc.K., C.J.D., J.M. Investigation: C.K., B.L.S., J.L., C.A.L., C.-H.L., C.J.D., J.M. Visualization: C.K., B.L.S., J.L., C.A.L., C.-H.L., C.J.D., J.M. Funding acquisition: B.L.S., M.P.J., M.F.J., J.M. Project administration: J.M. Supervision: M.F.J., J.M.Writing of the original draft: C.K., B.L.S., J.M. Writing, review and editing: C.K., B.L.S., J.L., C.A.L., C.-H.L., W.J.Mc.K., C.J.D., M.P.J., M.F.J., J.M.

Advance Queensland IRF AQIRS050-2020 (B.L.S.); US National Science Foundation CHE-1531823 (M.F.J.); Australian National Health and Medical Research Council (NHMRC) principal Research Fellowship 1138466 (M.P.J.), Project Grant 1121697 (J.M.).

The authors declare no competing financial interest.