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

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 a,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 c and S1), whereas detectable Ca2+ occupied 22.0 and 25.9% areas of corresponding PBLs, respectively (Figures c and S1). Surface plasmon resonance (SPR) analyses showed recombinant HIV Gag specifically binds to Ca2+ with an estimated SPR dissociation constant (Kd) of 9.18 μM (Figures d,e and S2 and S3) but has undetectable SPR binding with other cations (Na+, K+, Mg2+, and Zn2+; Figures e 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 f,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 h,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 j and S5) with optimal Ca2+ concentrations ([Ca2+]) at 0.1–1.0 mM (Figures k 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 l 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 Pr55Gag-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 Pr55Gag-imCherry (red) and Ca2+ (green) in elongated and round PBLs expressing Pr55Gag are shown. Medians are highlighted (n = 75 cells per arm). (d) Ca2+ binds to Pr55Gag in SPR. (e) SPR estimated dissociation constant (Kd) between cations and Pr55Gag are listed (n > 3). (f) ITC binding profiles between Pr55Gag and Ca2+ or other cations (Mg2+, Zn2+, Na+, and K+) (n > 3). (g) ITC thermodynamic parameters between Pr55Gag and cation interaction are listed (n > 3). (h) ITC profiles of Pr55Gag 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 Pr55Gag 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 Pr55Gag containing 4:1 molar ratio of Pr55Gag/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 Pr55Gag 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), Pr55Gag-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 a) 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 b). SPR revealed that Gag–Gag homodimerization was strengthened up to 7-fold in the presence of Ca2+ with SPR Kd (Figures c and S6), but this Ca2+-induced homodimerization effect disappeared when the p6 domain was removed (Figures c and S6). The ITC-detected Ca2+ binding during low-order Gag oligomerization also vanished in the absence of p6 (Figures d,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 p15NC-SP2-p6 and p7NC (lacking the p39MA-CA-Sp1 domains, Figure a) have mapped that the p6Gag domain directly contributes to Ca2+ binding (Figures d,e and S7).

Figure 2

p6Gag is an important determinant of Ca2+ binding and enhances Gag–Gag interactions. (a) Schematic shows domains in Pr55Gag and its derivatives (Pr50GagΔp6, p15NC-SP1-p6, and p7NC). (b) p6Gag contributes to in vitro Ca2+-induced high-order Gag oligomerization in CDMS (n > 3). (c) Ca2+ promotes SPR-detected homodimerization of Pr55Gag but not with Pr50GagΔp6 (n > 3). (d) ITC Ca2+ binding profiles of Pr55Gag (and its derivatives) show that p6Gag contributes to Ca2+ interaction. Zoom-In ITC profiles are presented for p15NC-SP2-p6 and p7NC (n > 3). (e) ITC thermodynamic parameters are obtained between Pr55Gag (or its derivatives) and Ca2+ (n > 3). (f–i) Distribution of Pr50GagΔp6-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 Pr50GagΔp6-imCherry (red) and Ca2+ (green) in elongated and round PBLs expressing Pr50GagΔp6 are shown. Medians are highlighted (n = 75 cells per arm). Statistical distribution analyses against Pr55Gag are with two-sample Kolmogorov–Smirnov test.

If the Ca2+–p6Gag interaction is important for intracellular trafficking of HIV Gag, removal of p6Gag 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 f,h–j and S8). In contrast, the intracellular Ca2+ distributions were generally more condensed within a smaller fractional area of Pr50GagΔp6 expressing cells than Pr55Gag expressing cells, a phenotype that is more noticeable in the less extended virological synapse forming round PBLs (Figures g,j and S8). In elongated PBLs, an increase in Gag signal was detected in Pr50GagΔp6- over Pr55Gag-expressing cells (Figures f,h–j and S8), while no difference in overall Gag signal was found between Pr50GagΔp6 and Pr55Gag round PBLs (Figures g,j and S8). The signal strength ratio of Gag to Ca2+ was consistently higher in Pr50GagΔp6- than in Pr55Gag-expressing cells across all PBLs (Figures f–j and S8). The altered Ca2+–Gag distribution relationship between Pr50GagΔp6- and Pr55Gag-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 p6Gag to generate the Pr55Gag p6-7aa mutant (Figure a [red conservation scores]). Imaging analyses showed that Pr55Gag p6-7aa occupied half of the fractional area and exhibited half of the signal intensity as seen with wild-type Pr55Gag (Figures b–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 Pr55Gag p6-7aa-expressing cells in comparison with those of wild-type Pr55Gag-expressing cells (Figures b–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 Pr55Gag point mutants displayed between 3- and 15-fold reduction in Ca2+ SPR binding (Figures e and S10), and the enhancement of Ca2+-induced Gag–Gag homodimerization in SPR was either reduced or eliminated in 5 out of 6 recombinant Pr55Gag point mutants (Figures e and S10). Fluorescent imaging and SPR data supported a role of p6Gag 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 Pr55Gag E461A that registered indistinguishable thermodynamic properties compared to wild-type Pr55Gag, 3 out of 6 mutants (Pr55Gag E460A, Pr55Gag E482A, and Pr55Gag D496A) showed no ITC detectable Ca2+ binding in low-order Gag–Gag homo-oligomerization in vitro (Figures f,g and S11). While mutant Pr55Gag E468A and Pr55Gag 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 f,g and S11). Circular dichroism showed that Pr55Gag E468A and Pr55Gag E477A maintained the same overall α-helix and β-sheet contents of Pr55Gag (Figure h). These data support a role for E/D amino acid residues flanking the ESCRT motifs in the HIV p6Gag as Ca2+ binding sites during HIV Gag trafficking. Production of recombinant Pr55Gag with more than one Ca2+ binding site mutations was not successful thus far, making it difficult to further dissect the biophysical properties Pr55Gag containing combined Ca2+ binding site mutations using cell-free assays.

Figure 3

Conserved p6Gag E/D residues influence Ca2+-Pr55Gag interactions. (a) Nine E/D residues in p6Gag 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 . Both PTAP and LXXLF motifs are denoted with a gray background. Conservation scores are in red. (b,c) Distribution of Pr55Gag 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 Pr55Gag p6-7aa-imCherry (red) and Ca2+ (green) in elongated and round PBLs expressing Pr55Gag p6-7aa are shown. Medians are highlighted (n = 75 cells per arm). Statistical distribution analyses against Pr55Gag are with a two-sample Kolmogorov–Smirnov test. (e) SPR estimated dissociation constants (Kd) are Ca2+ induced homodimerization impacted by Ca2+ binding site mutations (n > 3). (f) ITC Ca2+ binding profiles of Pr55Gag p6Gag E/D point mutants are shown (n > 3). (g) ITC thermodynamic parameters are obtained between Pr55Gag p6Gag E/D point mutant and Ca2+ (n > 3). (h) CD spectra of recombinant Pr55Gag E468A and Pr55Gag E477A are shown (n > 3).

Figure 4

p6Gag Ca2+ binding sites contribute to homo/hetero-oligomerization of proteins. (a) Ubiquitination of Pr55Gag is associated with Ca2+ binding site mutations (n > 3). (b) Deletion of p6Gag and mutations of p6Gag Ca2+ binding sites reduces particle release (n > 3). (c,d) Deletion of p6Gag and mutations of p6Gag Ca2+ binding sites are associated with defects in Pr55Gag processing and Pr160GagPol packaging. Increasing concentrations of indinavir (at 0, 0.5, 5, 50 μM) are used to slow down Pr160GagPol-mediated proteolytic processing (n > 3). (e) Relative infectivity of p6Gag Ca2+ binding site mutants against wild-type control are shown for T-cell line and PBLs. (f) Virion protein profiles among wild-type (NL4-3WT) and mutant HIV (NL4-3E454G, NL4-3E460G, NL4-3E461G, NL4-3E468G, NL4-3E477G, NL4-3E482G, NL4-3E496G, NL4-3E468G+E477G, and NL4-3E482G+E496G). HIV patient sera is the source of antibodies (n > 3). (g,h) Virion protein profiles of dual mutants (NL4-3E468G+E477G and NL4-3E482G+E496G) in comparison with NL4-3WT. Virion proteins are probed with anti-p24CA (g) and anti-p66/51RT (h) antibodies. (i) Virion-associated Pr160GagPol are compared between NL4-3WT and NL4-3E468G+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-p24CA (n > 3). (j) SPR analyses of Ca2+–Pr68GagPR binding and effects of Ca2+ on Pr68GagPR homodimerization (n > 3). (k) Coimmunoprecipitation on the stability of Pr55Gag/Pr160GagPol complexes in the presence of EGTA and quantifications of relative Pr55Gag/Pr160GagPol ratio (n > 3).

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

Reduction of fluorescent signals of Pr55Gag 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 b–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 Pr55Gag showed that an increased level of ubiquitination was detected in Ca2+-binding-repressed Pr55Gag p6-7aa (Figure a). As p6Gag is a major segment for HIV Gag ubiquitination,[38,41] deletion of p6Gag has reduced detectable ubiquitinated Pr50GagΔp6 (Figure a). 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 PRD25G mutation) and an envelope negative immature virus-like particle (VLP) system (HIVNL 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 p6Gag (HIVNL GagPol GagΔp6 PR[-]) (Figure b), which was in part due to ESCRT-related membrane arrest of particle release. Lower levels of immature Pr55Gag p6-7aa VLPs (HIVNL GagPol Gagp6-7aa (E/D-G) PR[-]) supported the notion that Ca2+ binding site mutations were defective in uropod particle targeting (Figure b), 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 p6Gag reported a similar ubiquitination-mediated degradation of HIV Gag.[43]

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 (Pr160GagPol) is a well-characterized cotrafficking and copackaging virion-associated protein.[32,33,44] Pr160GagPol represents 10% of the total virion protein[45] 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 p6Gag in Pr55Gag, the amino acids of p6Pol in Pr160GagPol have a different protein sequence due to overlapping reading frames. Pr50GagΔp6 and Pr55Gag p6-7aa were introduced into protease active HIV-1NL GagPol constructs.[42] The proteolytic processing of Pr50GagΔp6 was compromised due to deletion of both p6Pol and part of PR (Figure c, lane 2 vs lane 6). Site-specific mutations of E/D to G mutations in Pr55Gag p6-7aa via codon modifications have not altered the amino acid sequences of p6Pol of HIVNL GagPol Gagp6-7aa (E/D-G). The higher ratio of p25/24 CA doublets in Pr55Gag p6-7aa particles compared to that in wild-type Pr55Gag particles suggested that a defect of virion protein maturation may exist in the mutant HIVNL GagPol Gagp6-7aa (E/D-G) (Figure c, 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 Pr55Gag p6-7aa particles could be related to reduction in virion packaging of Pr160GagPol (Figure c,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 Pr160GagPol). Seven single-point mutations and two double-point mutations were introduced into infectious HIVNL4-3. One mutant (HIVGag E468G+E477G) was identified to be noninfectious in both T-cell line (MT2) and PBLs (Figure e). Although all mutants have wild-type levels of particle release (i.e., no detectable functional impact on Gag–Gag homo-oligomerization), HIVGag E468G+E477G exhibited virion protein processing profile defects (Figure f) reminiscent of HIVNL GagPol Gagp6-7aa (E/D-G) (Figure c,d, [anti-CA] and [anti-RT], respectively). The lack of functional impact from the seven single-point mutants (Figure e,f) and one double-point mutant (HIVGag E482G+D496G, Figure e,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 (HIVGag E468G+E477G) particles with specific anti-HIV antibodies confirmed a defect in virion Pr55Gag processing (Figure g) and a reduction of virion-associated polymerase–reverse transcriptase (Figure h). Unlike Pr55Gag p6-7aa particles (Figure b), the defects in HIVGag 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 HIVGag E468G+E477G was defective in virion Pr160GagPol packaging (Figure i), showing that the suppression of Ca2+ binding via Pr55Gag can lead to reduced hetero-oligomerization of Pr55Gag–Pr160GagPol complexes during directional trafficking to the uropod for virion release. To illustrate that Pr160GagPol can directly interact with Ca2+, a recombinant Pr160GagPol surrogate, Pr68GagPR, was made by engineering mutations in both the Pr160GagPol frameshift site and the protease active site to express Pr68GagPR, consisting of the natural Pr160GagPol domains from p17MA to p12PR[-]. SPR analyses showed that Pr68GagPR bound to Ca2+ specifically without detectable binding against other cations tested (Figures j and S12). To directly examine whether Ca2+ can stabilize Pr55Gag–Pr160GagPol 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 Pr160GagPol containing virion particles. A dose-dependent reduction of detectable Pr55Gag was seen in the presence of increasing concentrations of EGTA, Ca2+ preferred chelating agent, or EDTA (Figures k and S12), supporting the notion that direct Ca2+ interaction is a mechanism that regulates hetero-oligomerization of HIV Pr55Gag–Pr160GagPol 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 p6Gag 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 transport[46] and synaptic vesicle exocytosis[47] 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.