Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

doi:10.1128/JVI.02122-06

J Virol. 2007 February; 81(3): 1472–1478.

Published online 2006 November 15. doi: 10.1128/JVI.02122-06

PMCID: PMC1797500

Human Immunodeficiency Virus Type 1 Matrix Protein Assembles on Membranes as a Hexamer

Ayna Alfadhli, Doug Huseby,^† Eliot Kapit, Dalbinder Colman, and Eric Barklis^*

Vollum Institute and Department of Molecular Microbiology and Immunology, Oregon Health & Sciences University, Portland, Oregon

^*Corresponding author. Mailing address: Vollum Institute and Department of Molecular Microbiology and Immunology, Oregon Health & Sciences University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098. Phone: (503) 494-8098. Fax: (503) 494-6862. E-mail: barklis/at/ohsu.edu.

^†Present address: Division of Biological Sciences, University of California at Davis, Davis, CA.

Received September 27, 2006; Accepted November 5, 2006.

This article has been cited by other articles in PMC.

Abstract

The membrane-binding matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) structural precursor Gag (PrGag) protein oligomerizes in solution as a trimer and crystallizes in three dimensions as a trimer unit. A number of models have been proposed to explain how MA trimers might align with respect to PrGag capsid (CA) N-terminal domains (NTDs), which assemble hexagonal lattices. We have examined the binding of naturally myristoylated HIV-1 matrix (MyrMA) and matrix plus capsid (MyrMACA) proteins on membranes in vitro. Unexpectedly, MyrMA and MyrMACA proteins both assembled hexagonal cage lattices on phosphatidylserine-cholesterol membranes. Membrane-bound MyrMA proteins did not organize into trimer units but, rather, organized into hexamer rings. Our results yield a model in which MA domains stack directly above NTD hexamers in immature particles, and they have implications for HIV assembly and interactions between MA and the viral membrane glycoproteins.

The N-terminal matrix (MA) domain of the human immunodeficiency virus type 1 (HIV-1) precursor Gag (PrGag) protein serves two important virus assembly functions: it helps direct PrGag to assembly sites at the plasma membrane or multivesicular body, and it interacts with the cytoplasmic domain of the viral envelope (Env) protein complex SU/TM (gp120/gp41) to facilitate the assembly of wild-type Env proteins into assembling virus particles (12, 16, 17, 29, 43, 45, 55, 59). During PrGag translation, the N-terminal methionine of MA is replaced by the saturated, 14-carbon fatty acid myristic acid (tetradecanoic acid) through the action of the cellular enzyme N-myristoyltransferase (NMT) 9, 56). Evidence supports a myristoyl switch model for HIV, in which the MA myristate is partially buried until PrGag oligomerization, when its hydrocarbon tail is exposed, fostering membrane binding and virus assembly (5, 15, 22, 47, 49, 54, 56, 61). Moreover, recent experiments indicate that phosphatidylinositol 4,5-bisphosphate binding to MA acts as a trigger to initiate this process (46, 51). Accumulated data also imply that HIV assembly occurs not at random plasma membrane or multivesicular body sites but preferentially at cholesterol-rich regions, and cholesterol depletion can have deleterious effects on virus replication (19, 24, 28, 41, 44).

Several lines of investigation have shown that MA assembles into trimers in solution. Morikawa et al. (35, 36) demonstrated that bacterially expressed MA, as well as MACA proteins composed of the HIV-1 PrGag MA plus capsid (CA) domains, oligomerizes as trimers. Similarly, myristoylated MA (MyrMA) and myristoylated MACA (MyrMACA) associate as trimers in solution, and trimerization is coupled with exposure of the MA myristyl group (56). These observations are consistent with the determination that simian immunodeficiency virus and HIV MA proteins form trimers in three-dimensional (3D) crystals (23, 48). Moreover, in X-ray crystal structures, the exposed basic residues of MA trimers align in a way that could mediate binding to acidic phospholipids at the cytoplasmic faces of cellular membranes (23, 48). Not surprisingly, a number of investigators have found that retroviral matrix domains preferentially bind to membranes containing the net negatively charged phospholipid phosphatidylserine (PS) versus the neutral, zwitterionic phospholipid phosphatidylcholine (PC) (11, 14, 53, 57, 60, 61).

While HIV-1 MA associates in solution and in 3D crystals as trimers, the capsid domains of retroviruses tend to organize into hexamer rings (3, 4, 6, 7, 25, 27, 30, 31, 32, 34, 37, 39, 40, 63). These observations have prompted several researchers to incorporate MA trimers into PrGag hexamer models by postulating that trimers form nodes which interconnect hexamers and that two MA members from each of three trimer units position themselves in a p3 fashion over CA hexamers (15, 48). However, it should be noted that the locations of basic patches on retrovirus MA monomers are not conserved (38), that the distances between MA and CA PrGag domains are not conserved (8), that some retrovirus matrix proteins show dimer units in 3D crystals (21, 50), and that electrostatic calculations suggest that binding to PC/PS membranes may be more stable for monomers than for trimers (38).

To examine the association of HIV-1 MA and PrGag proteins to membranes, we have analyzed the assembly of MyrMA and MyrMACA proteins on lipid monolayers. Remarkably, we observed that MyrMACA and MyrMA proteins showed nearly identical 2D crystal lattice projection structures when assembled on PS-containing lipid monolayers. Both of the structures were composed of hexamer rings, and neither structure showed any evidence of membrane-bound matrix trimer units. Our results support a model in which MA hexamers rather than trimers are a fundamental unit of HIV particles.

MATERIALS AND METHODS

Protein purification.

MyrMA and MyrMACA proteins were expressed in Escherichia coli strain BL21 (DE3)/pLysS (Novagen) along with yeast NMT from pET-11a-based vectors kindly provided by Michael F. Summers (University of Maryland Baltimore County) (56). Proteins were expressed and purified under conditions which yield only traces of unmyristylated species (56). The purified proteins were desalted by buffer exchange in Sephadex G25 spin columns in 10 mM sodium phosphate (pH 7.8) and were concentrated to 0.2 to 0.3 mg/ml by sealing in dialysis tubing and extracting liquid in a bed of Spectra Gel (Spectrum) for 1 to 2 h at 4°C, after which the concentrated proteins were supplemented with β-mercaptoethanol (1 mM final concentration), aliquoted, and stored at −80°C under nitrogen gas. Protein purities were evaluated, after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 3, 25, 32), by Coomassie blue staining (1, 25) and immunoblotting (4, 25). Immunoblot detection of MyrMACA proteins used a primary anti-HIV CA monoclonal antibody (Hy183) (1), while for MyrMA proteins, a sheep antiserum (no. 286; lot 1DV-010) to MA obtained from Micheal Phelan via the NIAID AIDS Research and Reference Reagent Program was employed. Myristoylation of MyrMA proteins was confirmed by mass spectrometry through the Oregon Health & Sciences University Proteomics Core Facility.

Membrane binding assays.

Liposome flotation binding assays followed previously described procedures (11), using liposomes prepared from stock solutions in chloroform (42) of cholesterol (Sigma), 1,2-dioleoyl-sn-glycerol-3-phoshocholine (PC) (Avanti), and 1,2-diacyl-sn-glycero-3-phospho-l-choline (PS) (Avanti). Lipids were dried in glass vials with a stream of nitrogen gas, supplemented with liposome buffer (10 mM HEPES [pH 7.4], 50 mM NaCl, 0.002% sodium azide), and suspended by 20 30-s rounds of sonication in a Branson 1210 bath sonicator with incubations on ice between each sonication round. Final liposome lipid concentrations were 0.4 mg/ml cholesterol and 1.6 mg/ml of either PC or PS, and liposomes were stored for up to 1 month under nitrogen at −20°C. Monolayer membrane binding assays were modified from the monolayer binding assays described by Zuber et al. (63). One-hundred-microliter samples containing 200 to 300 nM protein in monolayer buffer (25 mM sodium phosphate [pH 7.8], 50 to 125 mM NaCl) were deposited into 15-mm-wide, 1.5-mm-deep glass depression (Boerner) slides. Protein samples were overlaid with 5 μl lipid mixes in 1:1 chloroform-hexane, and lipid mixes were composed of either 2.5 μg phospholipid or 0.5 μg cholesterol plus 2.0 mg phospholipid. The phospholipids (Avanti) used were 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phospho-l-serine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate, and 1,2-diacyl-sn-glycero-3-phosphoinositol. After 1-h incubations at 25°C, lipid monolayers and bound proteins were lifted onto 13-mm-diameter Parafilm M (Pechiney Plastic Packaging) circles. To do so, Parafilm circles were held at their edges with forceps, and their freshly exposed faces were touched to monolayer surfaces so that no intervening air bubbles remained. The Parafilm circles with attached monolayers then were placed for 1 to 2 s on top of 100-μl fresh monolayer buffer drops in glass depression slides as a washing step. Finally, the Parafilm circles were transferred to 1.5-ml Eppendorf tubes, and bound samples were extracted into 100 μl of IPB (20 mM Tris [pH 7.5], 1 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 0.02% sodium azide) by vortexing three times for 10 s each. For each experiment, companion unbound samples (50 μl) were mixed with 50 μl IPB to permit comparison of bound and unbound fractions from each binding incubation. Bound and unbound samples were subjected to SDS-PAGE and immunoblotting for detection of MyrMA and MyrMACA proteins, and protein levels in each fraction were quantitated as described above. Note that because proteins form denatured monolayers at the air-buffer interface in the absence of lipids, controls for monolayer membrane binding assays employed lipid mixture variations rather than incubations without lipid. Note also that in control experiments with glutathione S-transferase, no glutathione S-transferase was detected in the fractions bound to either PC-cholesterol or PS-cholesterol membranes (data not shown).

Electron microscopy (EM).

Crystallizations were performed with 7.5 μl of MyrMA or MyrMACA protein plus 2.5 μl of 4× crystallization buffer (50 mM sodium phosphate [pH 7.4], 10 mM sodium acetate [pH 7.6], 200 mM NaCl, 40% [vol/vol] glycerol, 5 mM β-mercaptoethanol). Ten-microliter drops were deposited onto glass slides, overlaid with 1 μl of lipid mix (200 μg/ml PS plus 50 μg/ml cholesterol in 1:1 chloroform-hexane), and incubated for 4 to 16 h in sealed 15-cm-diameter plastic dishes humidified with blotter paper wetted with 4 ml water. For negative staining, samples were stained with 1.33% (wt/vol) uranyl acetate. For cryosamples, monolayers were plunge-frozen into liquid ethane by using a Gatan cryoplunge apparatus and stored under liquid nitrogen until being viewed.

Samples were viewed on a Philips CM120 transmission electron microscope under low-dose conditions at defocus values of between 200 nm and 1,500 nm. Negatively stained samples were viewed at ambient temperature, while cryosamples were imaged at −176°C to −180°C on a Gatan 626 cryostage. Images were recorded at 14-bit grayscale in Gatan digital micrograph 3 (DM3) format on a 1,024- by 1,024-pixel Gatan 794 charge-coupled device multiscan camera at 5.24 Å/pixel. Gatan digital micrograph 3.4.0 software was used to calculate power spectra and to convert DM3 format raw images and power spectra to eight-bit grayscale TIF images.

Image analysis.

Image analysis steps followed previously described procedures (3, 4, 30, 31, 32, 34). All tagged image file format (TIF) images were converted to Medical Research Council (MRC) format and processed using the ICE suite of MRC programs (10). Boxed images were Fourier transformed using the 2DFFT function of integrated crystallographic environment (ICE), transform (TNF) files were examined using SPECTRA (20), and reflections were indexed using the SPECTRA interface. Indexed lattices were unbent, and amplitudes and phases (APH) format text files were produced by the LATREF, UNBENDA, and MNBOX functions of SPECTRA (20). APH files were contrast transfer function (CTF) (2, 10) corrected via CTF_DETERMINE and CTF_APPLY, as necessary.

Unit cell size parameters were calculated from the positions of 1,0 and 0,1 in power spectra and were averaged from eight stained zero-tilt images for both MyrMA and MyrMACA crystals. Space group analysis to 20-Å resolution was calculated using ALLSPACE (10, 20) in 3° phase origin search steps, and average phase residuals for space group fits also derive from eight images for both MyrMA and MyrMACA. For merging of untilted, stained image files to obtain average 2D projection images, p6 symmetry was applied, and files with the lowest phase residuals and highest number of comparisons in the p6 space group were chosen as references. Merging was performed using the TILT_ORG (33) interface running ORIGTILT_B (10) on seven MyrMA or MyrMACA APH files. Merges to 18-Å resolution used reflections of IQ = 5 (10) or better and phase origin search steps of 3°. Merge completeness and phase residual values were determined as described previously (32). For calculation of average projection maps, amplitudes and phases for each p6 reflection were vector averaged using APH_EDIT (33). For image rendering, averaged p6 reflection values were used to generate a corresponding p6-symmetrized p1 APH file, which was back transformed using APH_EDIT running the MRC CREATE_TNF, FFTRANS, and ICE_SKEW commands (10, 33). Projection averages were displayed as TIF images with proteins in white and using the entire 256-value gray scale generated in back transformations.

With cryoimages of MyrMA crystals, TIF, MRC, TNF, and APH files were prepared as described above. Fourier-filtered back transformations of cryoimages were obtained using APH_EDIT as for stained projection averages, but without any symmetry constraints (p1). Cryoimage back transformations are displayed as TIF images with proteins in white, and contour maps were prepared by combining the 256 gray scale values into 32 bins and displaying the 20 highest contours. For comparison with the X-ray crystal structure of HIV-1 MA, Protein Data Bank (PDB) entry 1HIW (23) was used directly to generate a projection view of the trimer, while the hexagonal model was prepared with a monomer extracted from the trimer unit.

RESULTS

In the absence of membranes, HIV-1 MyrMA and MyrMACA proteins appear to exist as monomers or trimers in solution (35, 36, 56), consistent with the 3D crystallization of HIV-1 MA in trimer units (23, 48). However, CA proteins from several different retroviruses have been shown to assemble higher-order structures as hexamers, with no evidence for trimer subunits (3, 4, 6, 27, 30, 31, 32, 34). Moreover, N-terminally histidine-tagged (His-tagged) proteins composed of the HIV-1 MA, CA, and nucleocapsid (NC) domains assemble on nickel-chelating lipids in a hexamer lattice (25), again with no clear evidence for trimers. Since it is possible that the His tag-nickel-chelating lipid interaction could influence the manner in which proteins oligomerize on membranes, we decided to examine the binding of myristoylated HIV-1 MA and MACA proteins to membranes.

For expression and purification of MyrMA and MyrMACA proteins, we followed protocols shown to produce only trace amounts of unmyristoylated species (56). As shown in Fig. Fig.1,1, expression of MyrMA and MyrMACA proteins and purification by virtue of their C-terminal His₆ tags (56) yielded single bands on Coomassie blue-stained SDS-polyacrylamide gels (Fig. (Fig.1,1, lanes B and C). Although the mobility of MyrMA protein was slightly lower than expected for its predicted molecular mass of 15,744.8 Da, mass spectrometry analysis measured the molecular mass at 15,745.6 Da, confirming our isolation of the full-length myristoylated protein. Additional immunoblot analysis of SDS-PAGE-fractionated proteins using MA and CA specific primary antibodies verified the identities of MyrMA and MyrMACA.

FIG. 1.

Purification of MyrMA and MyrMACA proteins. MyrMA (lane B) and MyrMACA (lane C) proteins were coexpressed with yeast NMT in bacteria and purified under conditions shown to yield only trace amounts of unmyristoylated products. The purified proteins were (more ...)

HIV-1 MA has been reported to bind preferentially to membranes containing PS versus PC, presumably via basic residues in its β-sheet and second helix (23). However, HIV assembly preferentially occurs at cholesterol-rich membrane regions (24, 28, 41, 44). Thus, as a precursor to EM studies, we used liposome flotation binding assays to verify that MyrMA and MyrMACA proteins bind to membranes composed of 20% (wt/wt) cholesterol and 80% PS (data not shown). As an alternative assay, we modified the monolayer membrane binding assay described by Zuber et al. (63), which mimics the methodology utilized in lipid monolayer 2D crystallizations (3, 4, 25, 30, 31, 32, 34). For these assays, proteins are incubated in buffer beneath a lipid monolayer (Fig. (Fig.2A).2A). After incubations, membrane monolayers and bound proteins are lifted onto hydrophobic Parafilm discs, after which bound and unbound protein levels are measured by immunoblotting of samples fractionated by SDS-PAGE. Using the monolayer membrane binding assay, we found less than 25% of the MyrMA protein in incubations bound to PC-cholesterol membranes, while greater than 50% of the protein bound to PS-cholesterol membranes (Fig. (Fig.2B).2B). Similarly, significantly more MyrMACA bound to PS-cholesterol membranes than to PC-cholesterol membranes (Fig. (Fig.2B).2B). We also observed that MyrMACA bound well to membranes containing the acidic phospholipid phosphatidic acid but bound poorly to phosphatidylethanolamine and phosphatidylinositol (data not shown).

FIG. 2.

Monolayer membrane binding assays. (A) Binding of MyrMA and MyrMACA proteins to membrane monolayers was performed by incubation of proteins in buffer beneath membrane monolayers formed at the air-buffer interface. After incubations, monolayers and bound (more ...)

To analyze how MyrMA and MyrMACA proteins organize on membranes, we examined the arrangements of the proteins assembled on membranes by transmission EM, using lipid monolayer 2D crystallization methods (3, 4, 25, 30, 31, 32, 34). In a procedure similar to monolayer membrane incubations (Fig. (Fig.2A),2A), MyrMA and MyrMACA proteins were incubated beneath membranes composed of 80% PS and 20% cholesterol, but rather than being lifted onto Parafilm discs, the membranes and attached proteins were lifted onto lacey EM grids and processed for EM imaging. Because we previously have shown that N-terminally His-tagged HIV-1 CA and MACANC proteins assemble into hexagonal lattices on nickel-chelating lipids (4, 25, 32), we anticipated that the more natural binding of myristoylated MyrMACA proteins to PS-cholesterol membranes also might yield hexagonal protein arrays. However, because little information was available concerning the membrane arrangement of MA, it was unclear whether or how MyrMA proteins might organize themselves.

Interestingly, EM analysis of negatively stained images indicated that both MyrMACA and MyrMA formed ordered arrays on PS-cholesterol membranes (Fig. (Fig.3).3). MyrMACA arrays were difficult to detect directly from images and occasionally showed small aggregates adhering to the monolayers (Fig. (Fig.3A).3A). In contrast, MyrMA arrays rarely had adherent aggregates and tended to show discernible patterns (Fig. (Fig.3D).3D). Surprisingly, calculated diffraction patterns (Fourier transforms) yielded remarkably similar power spectra for the two proteins, with a hexagonal arrangement of reflections readily apparent by eye (Fig. 3B and E). Unit cell dimensions, averaged from eight images for each protein, were consistent with those of a hexagonal lattice (Table (Table1).1). However, the unit cell lengths of 93.5Å for MyrMACA and 96.2 Å for MyrMA were larger than what has been observed for tightly packed HIV-1 CA or immature virus-like particles and were close to that observed with loosely packed CA tubes and sheets or the cores of mature virions (6, 27, 32). To assess the space group symmetry for membrane-bound arrays of MyrMA and MyrMACA proteins, the phases of predicted symmetry-related reflections were compared and evaluated by calculating phase residuals, where a 0° residual indicates a perfect match, while a 90° residual is a random match (10, 20). Our analysis indicated that both MyrMACA and MyrMA 2D crystals gave excellent matches with both p3 and p6 space groups (Table (Table11).

FIG. 3.

Comparison of MyrMACA and MyrMA membrane-bound projection structures. MyrMACA (A to C) and MyrMA (D to F) proteins were assembled onto lipid monolayers composed of 4:1 PS-cholesterol, lifted onto lacey carbon grids, and negatively stained. (A and D) Crystalline (more ...)

TABLE 1.

Analysis of MyrMACA and MyrMA 2D crystals^a

Given that phase data for both MyrMACA and MyrMA membrane-bound crystals were compatible with p3 and p6 symmetry but that p6 calculations used two to three times as many symmetry comparisons, we assumed p6 symmetry in the reconstruction of averaged 2D projection maps. Merging data from seven image files for each protein produced averages that were complete to 18 Å, with low phase residuals, indicative of good data matching (10): we did not attempt higher-resolution reconstructions due to the rapid fall-off in calculated reflection intensities. Not surprisingly, our MyrMACA projection map is reminiscent of maps generated for HIV-1 CA, His-tagged CA, and His-tagged MACANC (Fig. (Fig.3C).3C). Hexamers of protein units, depicted in white, are in turn organized in a hexagonal lattice; at this resolution with our negatively stained samples, presumed capsid C-terminal domain interconnections between hexamer units (27, 31) were not clear. Unexpectedly, the MyrMA projection map appeared nearly identical to the MyrMACA map, with hexamer units in a hexagonal lattice (Fig. (Fig.3F).3F). Although HIV-1 MA crystallizes in 3D as a trimer (23) and MyrMA trimerizes in solution (35, 36, 56), no MA trimer units were visible.

Because the lack of trimer units in membrane-bound MyrMA projection maps ran counter to expectation, several additional measures were taken. As protein controls, crystallizations were performed with several different MyrMA preparations, and unbound proteins from 2D crystallization setups were collected, subjected to SDS-PAGE, and immunoblotted to verify protein size and homogeneity (data not shown). Additionally, each of the data sets used in the MyrMA reconstruction was back transformed, assuming no symmetry constraints (p1), and evaluated to ensure that the projection map (Fig. (Fig.3F)3F) accurately represented the data with regard to the hexamer rings, the hexagonal lattice, and the absence of trimer units (data not shown).

We also were successful in obtaining several unstained EM images of MyrMA arrays taken under cryo conditions. Although these crystals were small, high-quality reflections (IQ ≤ 5) (10) occurred out to 20-Å resolution. Importantly, calculated diffraction patterns were hexagonal and gave unit cell sizes similar to those of the stained MyrMA crystals (a and b = 94.2 ± 1.2 Å; γ = 120.1 ± 1.2°). Back transformation with no symmetry constraints (p1) from indexed Fourier transforms of MyrMA cryo-EM crystals gave projection maps (Fig. (Fig.4A)4A) similar to the stained MyrMA 2D projection map (Fig. (Fig.3F),3F), after contrast flipping. As depicted, MyrMA proteins (in white) organize in hexamer rings in a hexagonal lattice, and this observation demonstrates that staining did not introduce any gross reconstruction artifacts. A contour map of the unstained sample back transformation (Fig. (Fig.4B)4B) emphasizes the hexamer nature of membrane-bound MyrMA protein associations, showing a cage of proteins surrounding trigonally symmetric and hexagonally symmetric cage holes. Comparison of the contour map with a scaled MA trimer viewed from above its putative membrane binding side (Fig. (Fig.4C)4C) revealed the difficulty of fitting a trimer unit into the projection structure. In contrast, rotation of six MA monomers around an axis yielded a hexamer unit that aligned neatly onto our projection map (Fig. (Fig.4C).4C). The implications of our observations are discussed below.

FIG. 4.

Projection structure of unstained, membrane-bound MyrMA. MyrMA proteins were assembled onto lipid monolayers composed of 4:1 PS-cholesterol, lifted onto lacey carbon grids, plunge-frozen in liquid ethane, and imaged at −176°C to −180°C (more ...)

DISCUSSION

The matrix domains of retrovirus Gag proteins tend to have surface patches of basic amino acids (38). For HIV-1 MA, these basic residues, along with the N-terminal myristate, appear to mediate PrGag binding to membranes containing the acidic phospholipid PS but not to membranes composed of the neutral, zwitterionic phospholipid PC (11, 14, 53, 57, 60, 61). Our studies support this binding model for PC and PS (Fig. (Fig.2),2), and characterization of conditions suitable for binding of MyrMA and MyrMACA to PS-containing lipid monolayers permitted us to examine how these proteins organize on membranes. Consistent with our observations of N-terminally His-tagged HIV-1 MACANC proteins assembled on nickel-chelating lipids (25), myristoylated MACA on PS-cholesterol membranes organized into hexamer rings (Fig. (Fig.33 and and4).4). Previous analyses of retrovirus capsid protein assemblies imply that capsid NTDs contribute to the observed hexamer units and are interconnected via C-terminal domain contacts (3, 4, 25, 27, 30, 31, 32, 34, 37, 63). Unexpectedly, HIV-1 MyrMA organized onto membranes in a hexamer arrangement almost identical to that of the MyrMACA lattice (Fig. (Fig.33 and and4).4). This was especially surprising given the predilection of HIV-1 MA to trimerize both in solution and in 3D crystals (23, 35, 36, 56). Nevertheless, our data clearly show that MyrMA organizes onto PS-cholesterol membranes in a hexagonal fashion, whether or not symmetry is imposed (Table (Table1;1; Fig. Fig.33 and and4).4). Moreover, simple visual inspection of scaled HIV-1 MA trimers demonstrates that they are incompatible with the membrane-bound MyrMA lattice. In contrast, a hexamer MA model is fitted readily to our MyrMA projection structure (Fig. (Fig.4).4). While we have not performed molecular dynamics calculations to assess how a hexamer model might alter MA trimer interface contacts, it is noteworthy that calculations predict a membrane-bound MyrMA trimer to be less stable than three membrane-bound monomers (38). Thus, there are no prevailing energetic arguments to suggest that hexamers are not the most favorable organizational form for HIV on membranes.

What are the implications of the similarity between MyrMA and MyrMACA 2D crystals? One obvious conclusion is that when precursor Gag proteins assemble on membranes, MA domains stack directly above NTD hexamers. In this sort of arrangement, residues that have been identified genetically to interact with the HIV-1 transmembrane envelope protein (TM) and its cytoplasmic tail (12, 13, 16, 17, 59) are oriented towards threefold, clover leaf-shaped symmetric MyrMA lattice cage holes between hexamer units, rather than the hexamer holes themselves (Fig. (Fig.44 and and5).5). This organization would seem well suited to accommodate envelope protein trimers (52, 58, 62), although estimates as to the spacing between glycoprotein knobs on virus surfaces (15) imply that only a subset of these cage holes will be occupied by TM cytoplasmic tails.

FIG. 5.

Locations of MA mutations that impair envelope protein incorporation into virions. (A) A hexamer model of MA was produced using a monomer extracted from the HIV-1 MA trimer structure from PDB entry 1H1W (23) and is viewed from its putative membrane-binding (more ...)

Because our experiments demonstrate that myristoylated HIV MA proteins are capable of maintaining a membrane-bound lattice in the absence of other Gag proteins, they imply that this organization may be maintained in some fashion in mature HIV-1 virions. Such a lattice has proven difficult to visualize in electron micrographs of mature virions (6, 8). However, some support for a mature retrovirus MA lattice may be taken from atomic force microscopy analyses of mature murine leukemia virus particles (26) and from the observation that cellular expression of simian immunodeficiency virus MA in the absence of other viral proteins resulted in the assembly and budding of virus-like particles (18). At 93 to 96 Å, the unit cell dimensions of both our MyrMA and MyrMACA assemblies are greater than what has been observed in tightly packed membrane-bound HIV-1 His-tagged CA arrays, His-tagged MACANC arrays, and PrGag proteins in virus-like particles (7, 25, 32). One explanation for this is that in-plane protein-protein packing may be constrained in our system by MA rather than CA and that the packing of MA is less dense than that of CA in virions, as it is located towards the periphery of virus particles. Thus, the observed unit cell sizes for MyrMA and MyrMACA extrapolate to a packing density of one protein per 13.1 nm², in reasonable agreement with the 11.5-nm² value calculated for MA in HIV-1 particles (8). We believe our results will prove helpful in understanding how HIV-1 proteins associate with membranes and with each other.

Acknowledgments

We are grateful to Chun Tang and Michael F. Summers (University of Maryland, Baltimore County) for providing the plasmids for bacterial expression of the myristoylated MA and MACA proteins and to Cory Bystrom and Debra McMillen (Oregon Health & Sciences University Proteomics Core Facility) for mass spectrometry analysis.

This work was supported by NIGMS, National Institutes of Health grant GM060170 to E.B. A.A. gratefully acknowledges support from American Foundation for AIDS Research postdoctoral fellowship grant 106523-35-RFNT and the National Institutes of Health (NIAID training grant AI007472).

Footnotes

Published ahead of print on 15 November 2006.

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