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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2013 Feb 11;110(9):3525–3530. doi: 10.1073/pnas.1216655110

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Jiri Vlach 1, Jamil S Saad 1,1
PMCID: PMC3587261  PMID: 23401539

Abstract

Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P2. There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P2 binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P2 and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.

Keywords: Gag targeting, myristoyl switch

A critical step in the late phase of HIV type-1 (HIV-1) infection is targeting of the virally encoded Gag proteins to the plasma membrane (PM) for assembly (1–6). Membrane targeting is mediated by the myristoylated N-terminal matrix (MA) domain. The myristoyl (myr) group functions in concert with a group of conserved basic residues to facilitate membrane anchoring and assembly of Gag (1, 2, 7). The finding that Gag binds membranes more efficiently than the isolated MA protein led to the hypothesis that the myr group is exposed in Gag and sequestered in the MA protein, a hypothesis that has come to be known the “myristoyl switch mechanism” (6, 8–12). NMR structural studies confirmed that the myr group can adopt sequestered [myr(s)] and exposed conformations even in the isolated MA protein and in the absence of membranes (13). Structural and biophysical studies on HIV-1 MA revealed that the myr switch is modulated by several factors including protein concentration, pH, and inclusion of the capsid protein (13, 14).

Proper targeting of HIV-1 Gag to the PM is dependent on specific interactions with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] (15–18), a cellular phospholipid localized in the inner leaflet of the PM (19–21). We have shown that PI(4,5)P2 binds directly to HIV-1 MA, inducing a conformational change that triggers myr exposure (22). Structural data have revealed that the 2′-acyl chain of PI(4,5)P2 is buried into MA, which led to the hypothesis that PI(4,5)P2 can function as both an allosteric trigger for myr exposure and as a direct membrane anchor (22). Direct interactions of Gag and MA with PI(4,5)P2 have also been detected by mass spectrometric protein footprinting and surface plasmon resonance (SPR) methods (23, 24). The involvement of the acyl chain of PI(4,5)P2 in MA and Gag binding has been confirmed by SPR studies (24).

In addition to PI(4,5)P2, other phospholipids have been implicated in efficient Gag binding to membranes (17, 25–29). Increasing amounts of phosphatidylserine (PS) increases Gag’s affinity to membranes (15, 16, 29), and MA has been shown to preferentially bind to membranes containing PS (10, 28–33). HIV-1 Gag and MA proteins are able to bind efficiently to liposomes made of PS and phosphatidylcholine (PC) (16, 28). Targeting of murine leukemia virus (MLV) Gag to the PM is also mediated by PI(4,5)P2 and PS (34). Collectively, these results suggest that binding of Gag and MA to membranes is not only dependent on PI(4,5)P2 but is also enhanced by other lipids.

The lipid distribution of the PM bilayer is highly asymmetric. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylethanolamine (PE) and PS and contains small quantities of PC, PI(4,5)P2, and phosphatidylinositol-(3,5)-trisphosphate (Fig. 1A). The outer layer of the PM, however, is enriched in sphingomyelin (SM) and PC (35–39). Compared with the host cell, selective lipids such as SM, PS, and PI(4,5)P2 are enhanced by approximately two- to threefold in the viral membrane (37, 38). Because Gag is anchored and therefore captured by its interaction with the available phospholipids, the intracellular targeting of Gag is likely to be determined by the relative strength of its interaction with the dominant lipid composing each subcompartment. The intrinsic differences in the polar head and acyl chain characteristics of PC, PS, PE, and PI(4,5)P2 may also suggest that both electrostatic and hydrophobic interactions govern Gag binding to the PM. Shortly after submission of this manuscript, Dick et al. (40) reported that efficient binding of HIV-1 Gag and MA is not only dependent on inclusion of PI(4,5)P2 or nature of the phospholipid (PS vs. PC), but is also sensitive to the hydrophobic environment of the bilayer (acyl chains and cholesterol). These results were recapitulated for MLV and Rous sarcoma virus (RSV) Gag proteins. Taken together, it appears that Gag binding to the inner leaflet of the PM is more complex than initially thought and is likely to be mediated by a network of interactions between the MA domain and various membrane lipids and/or contents such as cholesterol.

Fig. 1.

Fig. 1.

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MA–lipid interactions as detected by NMR. (A) Lipids present in the inner leaflet of the PM (n = 18 or 20 carbons). (B) Overlay of 2D 1H-15N HSQC spectra upon titration of a 100-μM sample of HIV-1 MA with di-C6-PC [at di-C6-PC (mM) = 0 (black), 1 (red), 2 (magenta), 5 (green), 10 (blue), and 20 (orange)]. Spectra (Left) show signals corresponding to the binding site residues that exhibited substantial chemical shift changes. At CMC, a subset of signals (Right) shift toward their corresponding positions in the myr(–)MA protein (gray), indicating myr exposure. (C) Surface representation of the MA protein (Protein Data Bank ID code 2H3I) showing the proximity of PI(4,5)P2 and PS/PE/PC binding sites in green and orange, respectively. Myristoyl group is shown in red. (D) Overlay of 2D 1H-13C HMQC spectra obtained for MA containing a 13C-labeled myr group in the unbound (black) and di-C6-PC-bound (red) states. For comparison, a spectrum of free myristic acid is shown in blue.

In summary, in vivo and in vitro studies indicate that binding of Gag to the PM is not only dependent on lipid composition and nature of the polar head but is also sensitive to the hydrophobic nature of the bilayer. Here, we used NMR methods to discern the molecular determinants of MA binding to major membrane glycerophospholipids (PS, PC, and PE). Results show that these lipids interact directly with MA, that the myr group is readily exposed upon association with membrane models (micelles and bicelles), and that the 2′-acyl chain of PS/PC/PE is buried in a hydrophobic pocket that is distinct from the PI(4,5)P2 binding site. Our findings led us to propose a model for HIV-1 Gag assembly on the PM.

Results

Direct MA Binding to PS, PE, and PC.

In a typical mammalian cell, glycerophospholipids PS, PE, and PC contain saturated 1′ and unsaturated 2′ 18-carbon fatty acid chains (Fig. 1A) (41–43). Lipids with long chains (greater than nine carbons) are capable of forming bilayers (or liposomes) in aqueous solution, whereas those with short chains (C4–C8) can aggregate into micelles with critical micelle concentration (CMC) ranging from millimolar to micromolar (44–46). Titration of native PC, PS, and PE into a 15N-labeled MA sample led to severe broadening and loss of 1H-15N signals in the 2D heteronuclear single-quantum coherence (HSQC) NMR spectra. Therefore, soluble PS, PE, and PC lipids with truncated 1′- and 2′-acyl chains were used (Fig. 1A; n = 6, 7, and 8). Similar lipids have been successfully used in previous NMR studies (47–49).

Representative 1H-15N HSQC NMR data obtained upon titration of HIV-1 MA with di-C6-PC (DHPC), -PS, and -PE are shown in Fig. 1B and Fig. S1. The most pronounced chemical shift perturbations were observed for a subset of signals corresponding to residues R39, L41, E42, R43, A45, V46, L50, L51, E52, C57, I60, L61, Q63, E73, E74, and L75. Titration data were well fit by a single-molecule binding model with dissociation constant (Kd) values of ∼2–6 mM (Table S1). The striking similarity of Kd values for di-C6-PS, -PE, and -PC suggests that lipid binding is not dependent on the character of the polar head. Mapping of the binding site of all three lipids on the MA protein structure revealed several exposed basic and acidic residues surrounding a hydrophobic cavity formed by residues L41, F44, V46, I60, L64, and L75 (Fig. 1C). This region defines a unique binding site that is distinct from, but adjacent to, the PI(4,5)P2 binding site. This finding is surprising because this region has not been previously implicated in membrane binding. Similar results have been obtained for the myr(–)MA protein, indicating that binding of PS, PC, and PE is not linked to protein myristoylation (Figs. S1 and S2). To test whether other major PM lipids bind to MA, we performed NMR titrations on a 15N sample of myr(–)MA with an SM analog having a natural sphingosine and a short N-acyl chain (C6-SM). No significant chemical shift changes were observed in the HSQC spectrum, indicating no direct binding (Fig. S2). The alternate possibility is that the difference in acyl chain lengths in SM vs. PC, PS, or PE explains the difference in their binding. Our results offer structural evidence of direct interactions between PS, PE, and PC and a retroviral MA protein, suggesting that these phospholipids may play a role in Gag-membrane association through direct interactions with a polar-hydrophobic region on the MA domain.

Lipid Binding to MA Triggers Myristoyl Exposure at CMC.

As mentioned above, PS, PE, and PC with short chains are capable of forming micelles in solution (44–46). The CMC is strongly dependent on the length of acyl chains (∼10–15 mM for di-C6-PS, -PE, and -PC) (46, 50, 51). Interestingly, our NMR data obtained for MA and myr(–)MA at 10 mM di-C6-PS, -PE, and -PC show no further chemical shift changes for 1H-15N resonances corresponding to residues located in the binding site. However, at 10–20 mM di-C6-PC and -PE a second subset of 1H-15N resonances corresponding to residues 2–18, 49, 52, 53, and 89 of MA shifted dramatically toward the corresponding signals of myr(–)MA (Fig. 1B and Fig. S2). These resonances were not perturbed in the HSQC spectra of myr(–)MA at >10 mM lipid concentrations. Such changes in MA and not myr(–)MA are indicative of myr exposure (13, 14, 22). Heavy protein precipitation was observed for di-C6-PS at >8 mM, which precluded collection of NMR data at CMC. Two-dimensional 1H-13C heteronuclear multiple-quantum coherence (HMQC) data of MA containing a 13C-labeled myr group confirmed exposure of the myr group at CMC of di-C6-PC (Fig. 1D). The 1H-13C signals of the myr group in the free MA protein (black) are identical to those reported previously for the myr(s) form (13, 52) and are very different from those observed for free myristic acid (blue) (52). For the MA:di-C6-PC complex, the 1H-13C signals of the myr group (red) shift toward the corresponding signals of the free molecule, again indicating exposure of the myr group. 1H-13C HMQC data obtained for 13C-labeled MA bound to di-C6-PC at CMC show that 1H-13C signals corresponding to key residues (e.g., L8 and L85) that are sensitive to the position of the myr group shifted toward those of the myr(–)MA protein, indicating myr exposure (Fig. S3). Taken together, our data demonstrate that formation of micelles facilitates myr exposure in MA.

Binding Affinity of Lipids to MA Is Strongly Dependent on the Acyl Chain Length.

The finding that PE, PS, and PC bind to a hydrophobic region in the MA protein may suggest that the acyl chains of lipids are involved in the interaction. To examine how the acyl chain length affects lipid binding to MA, we collected 2D NMR data on MA and myr(–)MA as titrated with lipids bearing longer chains (di-C7-PC, di-C8-PC, di-C8-PS, and di-C8-PE). The 1H-15N signals that exhibited chemical shift perturbations were very similar to those observed for di-C6 lipids, indicating that lipids bind to the same region. However, the binding affinity increased substantially upon increasing the length of acyl chains (Table S1). For example, the binding affinity of PS to MA increased by 55-fold upon increasing the length of the acyl chains from C6 to C8. A very similar trend has been observed for PC and PE lipids. No additional residues exhibited 1H and 15N chemical shift changes upon increasing the chain length, suggesting that the additional methylene groups interact with the hydrophobic chains in the pocket. The strong relationship between the binding affinity and acyl chain length, independent of the polar head character, suggests that MA binding to lipids is governed by hydrophobic interactions.

Structures of myr(–)MA Bound to PC and PS.

Complications arising from protein precipitation and micelle formation precluded collection of high-quality NMR data on the myristoylated MA protein in complex with di-C8-PS, -PE, and -PC lipids. Further, titration of myr(–)MA with di-C8-PE at protein concentrations >200 μM and ∼3:1 lipid:MA led to sample precipitation and NMR signal broadening, which precluded detection of intermolecular NOEs for structural determination. Thus, NMR data for structure determination were obtained for myr(–)MA in complex with di-C8-PS and di-C8-PC. Representative slices of the 13C-edited/12C-double-half-filtered NOESY data for the myr(–)MA:di-C8-PC complex are shown in Fig. 2. A subset of unambiguous intermolecular NOEs have been observed between the 2′-acyl chain of di-C8-PC and the hydrophobic side chains of L41, F44, V46, I60, L64, L75, and L78. Identical intermolecular NOE cross-peaks have also been observed for myr(–)MA:di-C8-PS. No detectable NOE cross-peaks were observed between MA and the glycerol group, polar head, or 1′-acyl chain of either di-C8-PC or di-C8-PS lipids. Superposition of the 20 lowest-penalty structures calculated for myr(–)MA complexes with di-C8-PC and di-C8-PS (see statistics Table S2) show that the 2′-acyl chain is buried into a hydrophobic cavity formed by helices II, IV, and V. The 1′-acyl chain and polar head are disordered (Fig. S4). Representative low-energy models for myr(–)MA bound to di-C8-PC and di-C8-PS show favorable hydrophobic contacts between the 2′-acyl chain and side chains of residues L41, F44, V46, I60, L64, L75, and L78 (Fig. 3). Our data present marked evidence for direct interactions of MA with major PM lipids via the 2′-acyl chain.

Fig. 2.

Fig. 2.

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Three-dimensional 13C-edited/12C-double-half-filtered NOE data obtained for the myr(–)MA:di-C8-PC complex showing unambiguously assigned intermolecular NOEs (dashed lines) between di-C8-PC and key residues of a 13C-labeled protein sample. Solid lines denote diagonal 1H-13C doublets.

Fig. 3.

Fig. 3.

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Structures of myr(–)MA bound to di-C8-PC (A and C) and di-C8-PS (B and D). The 2′-acyl chain of PS/PC is buried in a hydrophobic cavity in MA, whereas the 1′-acyl chain is exposed. Favorable electrostatic interactions can occur between the acidic polar head of PS and the R43 side chain.

Two Distinct Lipid-Binding Sites on MA.

Previous studies have shown that di-Cn-PI(4,5)P2 (n = 4 or 8) binds to a cleft formed by helices II and V and the β hairpin of MA (22). Here, we show that the PC/PS/PE binding pocket is in close proximity to the PI(4,5)P2 binding site. To assess how PI(4,5)P2 binds to MA when prebound to a lipid mixture of PS, PE, and PC lipids that mimics the membrane lipid distribution, we performed NMR titration in which di-C4-PI(4,5)P2 was titrated into a 15N-labeled MA sample saturated with a mixture of di-C7-PC, di-C6-PS, and di-C6-PE (molar ratio 1:1:2). PI(4,5)P2-dependent chemical shift changes (Fig. S5) and the binding affinity (Kd = 132 ± 49 μM) in the presence of PS/PC/PE lipids are almost identical to those observed previously for PI(4,5)P2 only (Kd = 150 ± 30 μM) (22), indicating that PI(4,5)P2–MA and PS/PE/PC–MA interactions are independent events and do not influence each other in terms of mechanism or strength.

Binding of MA to a Membrane Bilayer Model (Bicelles).

Results described above led to the following questions: Do lipids with long (i.e., native) acyl chains interact with MA via the same region? Is it possible for the 2′-acyl chain to flip out of the membrane bilayer? Is the hydrophobic pocket deep enough to accommodate a native-like 2′-acyl chain? We attempted to address these questions by devising two approaches. First, we designed a membrane bilayer model (bicelles) made of dimyristoylphosphatidylcholine (DMPC) and DHPC as described (Fig. 4A) (53, 54). Interactions of MA and myr(–)MA with DMPC/DHPC bicelles were assessed by 2D 1H-15N HSQC NMR (Fig. 4 and Fig. S6). Binding of DMPC/DHPC to MA and myr(–)MA led to substantial chemical shift changes for a subset of amide signals corresponding to residues R39, L41, E42, R43, A45, V46, L50, L51, E52, C57, I60, L61, Q63, E73, E74, and L75 (Fig. S6). These changes are similar to those observed when MA is bound to lipids with shorter chains. For MA, a second subset of signals corresponding to residues 2–18, 49, 52, 53, and 89 also shifted significantly toward the corresponding signals of myr(–)MA, indicating myr exposure (Fig. 4B and Fig. S5). However, two major differences are observed in the HSQC spectra when MA is bound to DMPC/DHPC bicelles vs. di-C6-PC micelles. First, 1H-15N signals were relatively broad and weak for MA–bicelles, a possible consequence of the large size and slow tumbling of the protein–bicelle complex. Second, the amide signals corresponding to residues 2–18, 49, 52, 53, and 89 exhibited more dramatic chemical shift changes when MA was bound to bicelles vs. when bound to micelles, suggesting that the myr group is more readily exposed upon binding to a membrane bilayer. Despite these differences, we do not rule out the possibility that DHPC lipids bind to the hydrophobic region of MA in a bicellar environment. Signal broadening in the NMR spectra of MA–bicelle precluded detection of unambiguous NOEs between DMPC and MA. Taken together, these results indicate that MA binds to bicelles in a mode that is similar to that observed for micelles.

Fig. 4.

Fig. 4.

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Interaction of MA with bicelles. (A) Bicelles were made of DMPC and DHPC. (B) Overlay of 2D 1H-15N HSQC spectra of MA in the free state (black) and as bound to bicelles (red). Signals of N-terminal residues shift toward the corresponding signals of myr(–)MA (green), indicating myr exposure.

In the second approach, we have performed structure calculations on a MA:PS complex using 18-carbon saturated (stearoyl) 1′- and unsaturated (oleoyl) 2′-acyl chains (18:0/18:1) based on the NMR restraints obtained for the myr(–)MA:di-C8-PS complex. We have also included PI(4,5)P2 with stearoyl and arachidonoyl chains at the 1′- and 2′ positions of the glycerol group, respectively, using NMR restraints obtained previously for the myr(–)MA:di-C8-PI(4,5)P2 complex (Fig. 5) (22). The lowest-penalty structure shows that the 2′-acyl chain of PS is accommodated well in the hydrophobic pocket (Fig. 5 and Fig. S4B). The terminal methyl and four CH2 groups of the 2′-acyl chain are buried in the pocket. The long kinked chain renders the acidic polar head closer to the protein surface and in juxtaposition to the R43 side chain, and also allows for additional CH2 groups to make favorable contacts with F44 and the surface of the protein (Fig. 5 and Fig. S4B). The lowest-energy model of myr(–)MA:di-C8-PS:PI(4,5)P2 has been used to construct a “trio engagement model” for MA anchored to a membrane bilayer (Fig. 5). In this model, the 2′-acyl chains of PS (PC or PE) and PI(4,5)P2 are buried in the protein whereas the 1′-acyl chains and the myr group are embedded in the membrane bilayer.

Fig. 5.

Fig. 5.

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A membrane binding model of MA:PS:PI(4,5)P2 based on the NMR data obtained for MA complexes with di-C8-PI(4,5)P2 (22) and di-C8-PS. Di-C8 chains of PS were substituted with 18-carbon saturated (stearoyl) 1′- and unsaturated (oleoyl) 2′-acyl chains (18:0/18:1), and di-C8 chains of PI(4,5)P2 were substituted with stearoyl and arachidonoyl chains at the 1′ and 2′ positions of the glycerol group (18:0/20:4), respectively. The hydrophobic cavity is deep enough to accommodate a lipid with an 18-carbon acyl chain (Fig. S4). Favorable electrostatic interactions occur between the acidic polar head of PS and the R43 side chain. This trio engagement model shows how MA is anchored to the PM via the 1′-acyl chains of PI(4,5)P2 and PS, and the myr group, which all together bracket a basic patch projecting toward the polar leaflet of the membrane.

Discussion

For over two decades, biochemical, in vitro, and genetic studies have focused on how retroviral Gag proteins bind to membranes, but only recently have the structural and molecular determinants of Gag assembly begun to emerge. It is widely accepted that efficient association of HIV-1 Gag with membranes is dependent on the myr group, a conserved basic region in MA, capsid multimerization, and PI(4,5)P2 (1–6, 12, 15–18, 28, 33). A report published shortly after submission of this manuscript has shed new light on the molecular requirements for Gag assembly. Dick et al. (40) provided evidence that efficient Gag binding to membranes is sensitive not only to the net negative charge, but also to the hydrophobic environment of the bilayer (i.e., acyl chains and cholesterol). In PC/PS liposome models, Gag and MA strongly preferred lipids with both acyl chains unsaturated over those with one chain unsaturated. These results suggest that the role of PM lipids and the interplay between lipid composition and Gag assembly are more complex than initially thought.

Here, we present structural details on how HIV-1 MA interacts with three major PM lipids (PS, PE, and PC). Several points have emerged: (i) MA interacts directly with soluble analogs of PS, PE, and PC via a region that is distinct from the PI(4,5)P2 binding site; (ii) the 2′-acyl chain of PS, PE, and PC inserts in a preexisting hydrophobic cavity, whereas the 1′-acyl chain is exposed; (iii) the myr group is readily exposed when MA is bound to membranes (micelles or bicelles) in the absence of PI(4,5)P2; (iv) MA binds to PI(4,5)P2 and PS/PE/PC in a concurrent fashion; and (v) SM, a sphingolipid localized exclusively on the outer leaflet of the PM, does not interact with MA.

We have previously reported structural evidence of a unique binding mode by which the 2′-acyl chain of PI(4,5)P2 is buried into HIV-1 MA (22), an observation that led us to suggest that PI(4,5)P2 can function as both an allosteric trigger for myr exposure and as a direct membrane anchor. The finding that the MA domain of Gag interacts directly with PS, PE, and PC suggests that these lipids play a more important role than previously implicated. Indeed, Gag and MA were found to associate efficiently with PS/PC liposomes even in the absence of PI(4,5)P2 (16, 28). The importance of PS/PC lipids in Gag assembly can be masked by RNA binding to the basic region of MA (16). However, Gag binding to PS/PC liposomes in the absence of RNA is as efficient as that in PS/PC/PI(4,5)P2 liposomes (16). Moreover, our finding that the myr group in MA is exposed upon association with bicelles and micelles indicates that a membrane-like environment is sufficient to trigger this event. Consistent with this observation, encapsulation of the HIV-1 MA protein in reverse micelles also led to exposure of the myr group independent of PI(4,5)P2 binding (55).

Another surprising result in this study is the finding that the 2′-acyl chain of PS, PE, and PC is buried in a hydrophobic pocket on MA, which may suggest that PS, PE, and PC can function as membrane anchors to increase the avidity and further stabilize Gag association with the PM. Similar sequestration of the 2′-acyl chain of PI(4,5)P2 by MA led us to propose a potential mechanism for the lateral targeting of PI(4,5)P2:Gag complex to lipid raft microdomains (22). Because membrane rafts are known to preferentially incorporate saturated fatty acids (56, 57), our previous and current findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the saturated 1′-acyl chains of PI(4,5)P2 and PS/PE/PC, and the myr group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane (Fig. 5).

Interestingly, several hydrophobic residues in the PS/PE/PC binding site (F44, I60, L64, and L75) are almost strictly conserved in ∼4,000 HIV-1, 98 HIV-2, and 662 SIV isolates (www.hiv.lanl.gov). Previous studies have shown that substitution or insertion of residues in the PC/PS/PE binding site can have diverse effects on virus infectivity, particle production, and assembly. For example, insertion of LELE between residues E40 and L41 or Q63 and L64 abolished virus infectivity (58). Likewise, mutation of I60 blocked virus replication and profoundly impaired infectivity (59). A Gag F44H mutant, however, produced WT-like virions (60). It is possible, though, that replacement of F44 with His does not greatly alter the binding pocket. Other mutations in proximity to the binding pocket (R39E/R43E) led to an increase in cytoplasmic Gag and produced less than 1% of WT virus titer (60). Careful analysis of mutants within the PS/PC/PE binding site should be sought because it is likely that substitutions of these highly conserved residues may affect protein stability and thus its function, as was observed for the MA R39E/R43E mutant (60).

Gaps in understanding of retroviral assembly still exist. We previously found that the site of HIV-2 assembly in vivo can be also manipulated by enzymes that regulate PI(4,5)P2 localization (61). PI(4,5)P2 binds to HIV-2 MA in a manner that is essentially identical to that of HIV-1 MA; however, structural data revealed that the myr group in HIV-2 MA is tightly sequestered and does not exhibit concentration- or PI(4,5)P2-dependent exposure (61). Another recent study on Mason-Pfizer monkey virus MA also revealed that the myr group is tightly sequestered and that PI(4,5)P2 binding to MA does not trigger myr exposure (62). The MA proteins of RSV and equine infectious anemia virus (EIAV) lack myr modification; Gag binding to the PM is mediated by electrostatic interactions (63–65). Recent studies revealed that RSV Gag association with membranes is not dependent on PI(4,5)P2, and neither membrane localization of Gag nor release of virus-like particles was affected by phosphatase-mediated depletion of PI(4,5)P2 in transfected avian cells (29). In liposome flotation experiments, RSV Gag had no specificity for PI(4,5)P2 but required acidic lipids (e.g., PS) for liposome binding (29). The precise molecular mechanism of EIAV Gag assembly is still unclear. In vitro and NMR studies revealed that EIAV MA interacts with PI(4,5)P2 as well as other phosphoinositides normally localized on endocytic membranes (66, 67). EIAV Gag was detected on the PM and in endocytic compartments, suggesting that targeting to peripheral and internal membranes is critical for assembly and release (67). Further, it has been recently shown that PI(4,5)P2 is not required for human T-lymphotropic virus type (HTLV-1) Gag association with the PM (68). Taken together, the mechanisms of retroviral assembly seem to be complex and may proceed via multiple steps that require the involvement of several lipids.

Our findings shed light on a potential role for major PM phospholipids as membrane anchors and may provide insight into a possible alternative mechanism for Gag assembly in retroviruses lacking the myr group or PI(4,5)P2 requirement. Although our studies are conducted with MA, they are likely to be applicable to Gag due to the very similar structural properties of MA in the isolated form and as part of Gag. There are many questions that have yet to be answered: What is the order of Gag binding to the PM? Which occurs first? How do various lipids contribute to the overall binding? Based on the current understanding, membrane selection seems to be the first step because targeting of HIV-1 Gag to the PM is critically dependent on PI(4,5)P2 (15, 18). Once at the membrane, several factors work in synergy to ensure stable and efficient association with the PM. These include anchoring of myristoyl and lipid acyl chains into membrane, Gag multimerization, electrostatic interactions with acidic phospholipids, and possibly interactions with other cellular proteins (69). A recent report has proposed an alternative mechanism for Gag binding to the PM (70). It was suggested that Gag traps acidic phospholipids via ionic interactions, thereby inducing what is called acidic lipid-enriched microdomains. In-depth understanding of the precise role of lipids in virus assembly and replication and elucidation of the molecular requirements of Gag–membrane interaction may aid in the development of new antiviral therapeutic strategies.

Materials and Methods

Sample preparation, lipid titrations, NMR methodology, and structure calculations are described in SI Materials and Methods. The N-terminal Met, which is absent in the MA protein, is designated as residue 1. In contrast, other studies considered the N-terminal Gly of MA as residue 1 (6, 16, 59).

Supplementary Material

Supporting Information
supp_110_9_3525__index.html (7.1KB, html)

Acknowledgments

We thank Michael Summers for providing the yeast N-myristoyltransferase expression molecular clone and Peter Prevelige for critical review of the manuscript. This work was supported by National Institutes of Health Grant 1R01AI087101 and Grant P30CA13148 from the Center for AIDS Research and Comprehensive Cancer Center at the University of Alabama at Birmingham.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. V.M.V. is a guest editor invited by the Editorial Board.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 2LYA and 2LYB); the NMR chemical shifts have been deposited in the BioMagResBank, www.bmrb.wisc.edu (accession nos. 18715 and 18716).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1216655110/-/DCSupplemental.

References

  • 1.Ganser-Pornillos BK, Yeager M, Sundquist WI. The structural biology of HIV assembly. Curr Opin Struct Biol. 2008;18(2):203–217. doi: 10.1016/j.sbi.2008.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Adamson CS, Freed EO. Human immunodeficiency virus type 1 assembly, release, and maturation. Adv Pharmacol. 2007;55:347–387. doi: 10.1016/S1054-3589(07)55010-6. [DOI] [PubMed] [Google Scholar]
  • 3.Jouvenet N, et al. Plasma membrane is the site of productive HIV-1 particle assembly. PLoS Biol. 2006;4(12):e435. doi: 10.1371/journal.pbio.0040435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Li H, Dou J, Ding L, Spearman P. Myristoylation is required for human immunodeficiency virus type 1 Gag-Gag multimerization in mammalian cells. J Virol. 2007;81(23):12899–12910. doi: 10.1128/JVI.01280-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Ghanam RH, Samal AB, Fernandez TF, Saad JS. Role of the HIV-1 matrix protein in Gag intracellular trafficking and targeting to the plasma membrane for virus assembly. Front Microbiol. 2012;3:55. doi: 10.3389/fmicb.2012.00055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Ono A, Freed EO. Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus. J Virol. 1999;73(5):4136–4144. doi: 10.1128/jvi.73.5.4136-4144.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Ono A. HIV-1 assembly at the plasma membrane: Gag trafficking and localization. Future Virol. 2009;4(3):241–257. doi: 10.2217/fvl.09.4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hermida-Matsumoto L, Resh MD. Human immunodeficiency virus type 1 protease triggers a myristoyl switch that modulates membrane binding of Pr55(gag) and p17MA. J Virol. 1999;73(3):1902–1908. doi: 10.1128/jvi.73.3.1902-1908.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Spearman P, Horton R, Ratner L, Kuli-Zade I. Membrane binding of human immunodeficiency virus type 1 matrix protein in vivo supports a conformational myristyl switch mechanism. J Virol. 1997;71(9):6582–6592. doi: 10.1128/jvi.71.9.6582-6592.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Zhou W, Resh MD. Differential membrane binding of the human immunodeficiency virus type 1 matrix protein. J Virol. 1996;70(12):8540–8548. doi: 10.1128/jvi.70.12.8540-8548.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Paillart J-C, Göttlinger HG. Opposing effects of human immunodeficiency virus type 1 matrix mutations support a myristyl switch model of gag membrane targeting. J Virol. 1999;73(4):2604–2612. doi: 10.1128/jvi.73.4.2604-2612.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Bouamr F, Scarlata S, Carter CA. Role of myristylation in HIV-1 Gag assembly. Biochemistry. 2003;42(21):6408–6417. doi: 10.1021/bi020692z. [DOI] [PubMed] [Google Scholar]
  • 13.Tang C, et al. Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc Natl Acad Sci USA. 2004;101(2):517–522. doi: 10.1073/pnas.0305665101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Fledderman EL, et al. Myristate exposure in the HIV-1 Matrix Protein is modulated by pH. Biochemistry. 2010;49:9551–9562. doi: 10.1021/bi101245j. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chukkapalli V, Hogue IB, Boyko V, Hu W-S, Ono A. Interaction between HIV-1 Gag matrix domain and phosphatidylinositol-(4,5)-bisphosphate is essential for efficient Gag-membrane binding. J Virol. 2008;82:2405–2417. doi: 10.1128/JVI.01614-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Chukkapalli V, Oh SJ, Ono A. Opposing mechanisms involving RNA and lipids regulate HIV-1 Gag membrane binding through the highly basic region of the matrix domain. Proc Natl Acad Sci USA. 2010;107(4):1600–1605. doi: 10.1073/pnas.0908661107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Chukkapalli V, Ono A. Molecular determinants that regulate plasma membrane association of HIV-1 Gag. J Mol Biol. 2011;410(4):512–524. doi: 10.1016/j.jmb.2011.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO. Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc Natl Acad Sci USA. 2004;101(41):14889–14894. doi: 10.1073/pnas.0405596101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Martin TFJ. PI(4,5)P(2) regulation of surface membrane traffic. Curr Opin Cell Biol. 2001;13(4):493–499. doi: 10.1016/s0955-0674(00)00241-6. [DOI] [PubMed] [Google Scholar]
  • 20.Behnia R, Munro S. Organelle identity and the signposts for membrane traffic. Nature. 2005;438(7068):597–604. doi: 10.1038/nature04397. [DOI] [PubMed] [Google Scholar]
  • 21.McLaughlin S, Murray D. Plasma membrane phosphoinositide organization by protein electrostatics. Nature. 2005;438(7068):605–611. doi: 10.1038/nature04398. [DOI] [PubMed] [Google Scholar]
  • 22.Saad JS, et al. Structural basis for targeting HIV-1 Gag to virus assembly sites on the plasma membrane. Proc Natl Acad Sci USA. 2006;103:11364–11369. doi: 10.1073/pnas.0602818103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Shkriabai N, et al. Interactions of HIV-1 Gag with assembly cofactors. Biochemistry. 2006;45(13):4077–4083. doi: 10.1021/bi052308e. [DOI] [PubMed] [Google Scholar]
  • 24.Anraku K, et al. Highly sensitive analysis of the interaction between HIV-1 Gag and phosphoinositide derivatives based on surface plasmon resonance. Biochemistry. 2010;49(25):5109–5116. doi: 10.1021/bi9019274. [DOI] [PubMed] [Google Scholar]
  • 25.Waheed AA, Freed EO. Lipids and membrane microdomains in HIV-1 replication. Virus Res. 2009;143(2):162–176. doi: 10.1016/j.virusres.2009.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Waheed AA, Freed EO. The role of lipids in retrovirus replication. Viruses. 2010;2(5):1146–1180. doi: 10.3390/v2051146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Ono A. HIV-1 assembly at the plasma membrane. Vaccine. 2010;28(Suppl 2):B55–B59. doi: 10.1016/j.vaccine.2009.10.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Dalton AK, Ako-Adjei D, Murray PS, Murray D, Vogt VM. Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain. J Virol. 2007;81(12):6434–6445. doi: 10.1128/JVI.02757-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chan J, Dick RA, Vogt VM. Rous sarcoma virus gag has no specific requirement for phosphatidylinositol-(4,5)-bisphosphate for plasma membrane association in vivo or for liposome interaction in vitro. J Virol. 2011;85(20):10851–10860. doi: 10.1128/JVI.00760-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Alfadhli A, Still A, Barklis E. Analysis of human immunodeficiency virus type 1 matrix binding to membranes and nucleic acids. J Virol. 2009;83(23):12196–12203. doi: 10.1128/JVI.01197-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ehrlich LS, Fong S, Scarlata S, Zybarth G, Carter C. Partitioning of HIV-1 Gag and Gag-related proteins to membranes. Biochemistry. 1996;35(13):3933–3943. doi: 10.1021/bi952337x. [DOI] [PubMed] [Google Scholar]
  • 32.Scarlata S, Ehrlich LS, Carter CA. Membrane-induced alterations in HIV-1 Gag and matrix protein-protein interactions. J Mol Biol. 1998;277(2):161–169. doi: 10.1006/jmbi.1997.1615. [DOI] [PubMed] [Google Scholar]
  • 33.Zhou W, Parent LJ, Wills JW, Resh MD. Identification of a membrane-binding domain within the amino-terminal region of human immunodeficiency virus type 1 Gag protein which interacts with acidic phospholipids. J Virol. 1994;68(4):2556–2569. doi: 10.1128/jvi.68.4.2556-2569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Hamard-Peron E, et al. Targeting of murine leukemia virus gag to the plasma membrane is mediated by PI(4,5)P2/PS and a polybasic region in the matrix. J Virol. 2010;84(1):503–515. doi: 10.1128/JVI.01134-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.van Meer G. Cellular lipidomics. EMBO J. 2005;24(18):3159–3165. doi: 10.1038/sj.emboj.7600798. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Buckland AG, Wilton DC. Anionic phospholipids, interfacial binding and the regulation of cell functions. Biochim Biophys Acta. 2000;1483(2):199–216. doi: 10.1016/s1388-1981(99)00188-2. [DOI] [PubMed] [Google Scholar]
  • 37.Brügger B, et al. The HIV lipidome: A raft with an unusual composition. Proc Natl Acad Sci USA. 2006;103(8):2641–2646. doi: 10.1073/pnas.0511136103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Chan R, et al. Retroviruses human immunodeficiency virus and murine leukemia virus are enriched in phosphoinositides. J Virol. 2008;82(22):11228–11238. doi: 10.1128/JVI.00981-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Boon JM, Smith BD. Chemical control of phospholipid distribution across bilayer membranes. Med Res Rev. 2002;22(3):251–281. doi: 10.1002/med.10009. [DOI] [PubMed] [Google Scholar]
  • 40.Dick RA, Goh SL, Feigenson GW, Vogt VM. HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes. Proc Natl Acad Sci USA. 2012;109(46):18761–18766. doi: 10.1073/pnas.1209408109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Yabuuchi H, O’Brien JS. Positional distribution of fatty acids in glycerophosphatides of bovine gray matter. J Lipid Res. 1968;9(1):65–67. [PubMed] [Google Scholar]
  • 42.van Meer G, de Kroon AIPM. Lipid map of the mammalian cell. J Cell Sci. 2011;124(Pt 1):5–8. doi: 10.1242/jcs.071233. [DOI] [PubMed] [Google Scholar]
  • 43.Spector AA, Yorek MA. Membrane lipid composition and cellular function. J Lipid Res. 1985;26(9):1015–1035. [PubMed] [Google Scholar]
  • 44.Walde P, Blöchliger E, Morigaki K. Circular dichroic properties of phosphatidylcholine micelles. Langmuir. 1999;15(7):2346–2350. [Google Scholar]
  • 45.Ranganathan R, Vautier-Giongo C, Bakshi MS, Bales BL, Hajdu J. Phospholipid containing mixed micelles: Characterization of diheptanoyl phosphatidylcholine (DHPC) and sodium dodecyl sulfate and DHPC and dodecyl trimethylammonium bromide. Chem Phys Lipids. 2005;135(1):93–104. doi: 10.1016/j.chemphyslip.2005.02.004. [DOI] [PubMed] [Google Scholar]
  • 46.Hauser H. Short-chain phospholipids as detergents. Biochim Biophys Acta. 2000;1508(1-2):164–181. doi: 10.1016/s0304-4157(00)00008-3. [DOI] [PubMed] [Google Scholar]
  • 47.Kutateladze TG, et al. Multivalent mechanism of membrane insertion by the FYVE domain. J Biol Chem. 2004;279(4):3050–3057. doi: 10.1074/jbc.M309007200. [DOI] [PubMed] [Google Scholar]
  • 48.Lee SA, et al. Molecular mechanism of membrane docking by the Vam7p PX domain. J Biol Chem. 2006;281(48):37091–37101. doi: 10.1074/jbc.M608610200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Lee SA, et al. Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch. Proc Natl Acad Sci USA. 2005;102(37):13052–13057. doi: 10.1073/pnas.0503900102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Kleinschmidt JH, Tamm LK. Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy. Biophys J. 2002;83(2):994–1003. doi: 10.1016/S0006-3495(02)75225-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Koppaka V, Wang J, Banerjee M, Lentz BR. Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution. Biochemistry. 1996;35(23):7482–7491. doi: 10.1021/bi952063d. [DOI] [PubMed] [Google Scholar]
  • 52.Ghanam RH, Fernandez TF, Fledderman EL, Saad JS. Binding of calmodulin to the HIV-1 matrix protein triggers myristate exposure. J Biol Chem. 2010;285(53):41911–41920. doi: 10.1074/jbc.M110.179093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Vold RR, Prosser RS, Deese AJ. Isotropic solutions of phospholipid bicelles: a new membrane mimetic for high-resolution NMR studies of polypeptides. J Biomol NMR. 1997;9(3):329–335. doi: 10.1023/a:1018643312309. [DOI] [PubMed] [Google Scholar]
  • 54.Glover KJ, et al. Structural evaluation of phospholipid bicelles for solution-state studies of membrane-associated biomolecules. Biophys J. 2001;81(4):2163–2171. doi: 10.1016/s0006-3495(01)75864-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Valentine KG, et al. Reverse micelle encapsulation of membrane-anchored proteins for solution NMR studies. Structure. 2010;18(1):9–16. doi: 10.1016/j.str.2009.11.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Brown DA, London E. Structure of detergent-resistant membrane domains: does phase separation occur in biological membranes? Biochem Biophys Res Commun. 1997;240(1):1–7. doi: 10.1006/bbrc.1997.7575. [DOI] [PubMed] [Google Scholar]
  • 57.Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, Brown DA. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts: Many raft proteins are acylated, while few are prenylated. J Biol Chem. 1999;274(6):3910–3917. doi: 10.1074/jbc.274.6.3910. [DOI] [PubMed] [Google Scholar]
  • 58.Reicin AS, et al. Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: Effects on virion particle assembly, release, and infectivity. J Virol. 1995;69(2):642–650. doi: 10.1128/jvi.69.2.642-650.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Freed EO, Orenstein JM, Buckler-White AJ, Martin MA. Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol. 1994;68(8):5311–5320. doi: 10.1128/jvi.68.8.5311-5320.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Cannon PM, et al. Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17. J Virol. 1997;71(5):3474–3483. doi: 10.1128/jvi.71.5.3474-3483.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Saad JS, et al. Structure of the myristylated HIV-2 MA protein and the role of phosphatidylinositol-(4,5)-bisphosphate in membrane targeting. J Mol Biol. 2008;382:434–447. doi: 10.1016/j.jmb.2008.07.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Prchal J, Srb P, Hunter E, Ruml T, Hrabal R. The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding. J Mol Biol. 2012;423(3):427–438. doi: 10.1016/j.jmb.2012.07.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Provitera P, Bouamr F, Murray D, Carter C, Scarlata S. Binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions. J Mol Biol. 2000;296(3):887–898. doi: 10.1006/jmbi.1999.3482. [DOI] [PubMed] [Google Scholar]
  • 64.Dalton AK, Murray PS, Murray D, Vogt VM. Biochemical characterization of rous sarcoma virus MA protein interaction with membranes. J Virol. 2005;79(10):6227–6238. doi: 10.1128/JVI.79.10.6227-6238.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Erdie CR, Wills JW. Myristylation of Rous sarcoma virus Gag protein does not prevent replication in avian cells. J Virol. 1990;64(10):5204–5208. doi: 10.1128/jvi.64.10.5204-5208.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Chen K, et al. Solution NMR characterizations of oligomerization and dynamics of equine infectious anemia virus matrix protein and its interaction with PIP2. Biochemistry. 2008;47(7):1928–1937. doi: 10.1021/bi701984h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Fernandes F, et al. Phosphoinositides direct equine infectious anemia virus gag trafficking and release. Traffic. 2011;12(4):438–451. doi: 10.1111/j.1600-0854.2010.01153.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Inlora J, Chukkapalli V, Derse D, Ono A. Gag localization and virus-like particle release mediated by the matrix domain of human T-lymphotropic virus type 1 Gag are less dependent on phosphatidylinositol-(4,5)-bisphosphate than those mediated by the matrix domain of HIV-1 Gag. J Virol. 2011;85(8):3802–3810. doi: 10.1128/JVI.02383-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Cooper J, et al. Filamin A protein interacts with human immunodeficiency virus type 1 Gag protein and contributes to productive particle assembly. J Biol Chem. 2011;286(32):28498–28510. doi: 10.1074/jbc.M111.239053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 70.Kerviel A, Thomas A, Chaloin L, Favard C, Muriaux D. Virus assembly and plasma membrane domains: Which came first? Virus Res. 2012 doi: 10.1016/j.virusres.2012.08.014. 10.1016/j.virusres.2012.08.014. [DOI] [PubMed] [Google Scholar]

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