Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

Significance

The HIV matrix domain of Gag is known to play a key role in the incorporation of the envelope glycoprotein into viral particles. Here we provide evidence that matrix is organized as a trimer in HIV-1 particles, and that the ability of matrix to form trimers is essential for the packaging of the envelope glycoprotein. The requirement for matrix trimerization depends on steric constraints between matrix and the cytoplasmic tail of envelope and matrix. This represents a novel model of envelope incorporation in this family of viruses, and suggests that the matrix trimer interface may represent a novel drug target to inhibit the production of infectious viral particles and combat the spread of AIDS.

Abstract

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.

The assembly and budding of retroviruses involve a series of regulated steps, driven primarily by the viral Gag protein (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and budding occur predominantly at the plasma membrane. The HIV-1 Gag protein is expressed as a 55-kDa polyprotein, comprising four major domains and two spacer peptides (SPs). The major domains are matrix (MA), capsid (CA), nucleocapsid (NC), and p6; the spacer peptides are known as SP1 and SP2 and are located between CA-NC and NC-p6, respectively. Assembly and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral particles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3).

In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 Env is synthesized as a 160-kDa precursor that traffics to the plasma membrane via the Golgi apparatus, where it is processed to form the surface glycoprotein gp120 and the transmembrane glycoprotein gp41 (reviewed in ref. 4). The processed Env glycoproteins remain noncovalently associated as a heterodimer; the Env spike is a homotrimer of these dimers (5, 6). On the surface of the viral particle, gp120 binds the viral receptor CD4 and the chemokine coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and coreceptor triggers structural changes in gp41 that lead to fusion of the viral and target cell membranes. The fusion activity of gp41 is conferred by the ecto- and transmembrane domains of the protein (7). A third domain, the cytoplasmic tail (CT), is dispensable for fusion but plays important roles in Env trafficking and cell signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a very long CT composed of ∼150 amino acids. In contrast, most other retroviruses encode Env CTs that are ∼25–35 amino acids in length (4). The reasons for the greater length of lentivirus Env CTs are not fully understood. For HIV-1, the CT is required for Env incorporation into particles in physiologically relevant cell types, such as peripheral blood mononuclear cells, monocyte-derived macrophages, and most T-cell lines (8, 9). Recent work has shown that the CT mediates an interaction with Rab11 family-interacting protein 1c (FIP1c), which in turn interacts with Rab14 and appears to direct trafficking of HIV-1 Env to sites of assembly; it is likely that interactions with FIP1c contribute to the observed requirement for the CT in Env incorporation (10, 11). It is, however, unclear why lentiviral CTs are so large, because far smaller Env CTs also contain essential functional trafficking and signaling motifs (12).

A consequence of the large lentiviral CT is the potential for, or inevitability of, interactions with the MA domain of Gag during particle assembly. The Gag protein forms a hexameric lattice, driven primarily by CA–CA interactions. Whereas the structure of CA in mature particles has been studied extensively, with many high-resolution structures now available (13–16), and recently progress has been made in determining the structure of the immature Gag lattice (17), the organization of MA in particles has proven difficult to address directly, with no long-range order discernable for the MA shell. The structure of HIV-1 MA has been solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either approach; however, crystallography suggests a trimeric arrangement for both HIV-1 and simian immunodeficiency virus (SIV) MA proteins (19, 20), whereas NMR reveals only structures for the monomer, with no evidence for higher-order interactions (18). A third approach visualized 2D lattices of MA or MA-CA, using myristylated proteins on a synthetic lipid bilayer with a composition intended to mimic that of the plasma membrane at sites of assembly (21). Under these conditions, MA was seen to arrange as hexamers of trimers, although the low resolution of this approach precluded more-detailed structural analysis. A hexamer-of-trimers arrangement for MA would be compatible with the most recently suggested model for the immature CA lattice, which proposes a hexamer-of-trimers arrangement for the CA amino-terminal domain (CA-NTD), which lies immediately below MA (17).

The available structures of MA can be used to place the data acquired through molecular and genetic approaches into context. Mutations have been identified in MA that prevent the incorporation of Env into particles (22–26). The majority of these mutations map to the tips of the MA trimer (27), a region of the protein that lies around the central aperture of the hexamer of trimers. These findings implicate this central aperture as the site of Env incorporation in the particle. This idea is further supported by the observation that, in cell lines permissive for packaging of CT-truncated Env, removal of the CT relieves the inhibition of Env incorporation imposed by the MA mutations (22, 25, 28). The ability to rescue Env incorporation by removing the CT suggests that steric hindrance of Env incorporation may be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data showing that a mutation at the trimer interface was able to rescue the Env incorporation defects imposed by several MA mutations and a deletion in the Env CT (24). These data suggest that the MA trimer interface regulates the ability to rescue mutants that are defective for Env incorporation.

In this study, we sought to develop a system that would allow us to directly determine whether MA forms trimers in virions and, if so, whether MA trimerization plays a role in Env incorporation. By using a combination of biochemical, genetic, and virological approaches, we demonstrate the presence of MA trimers in replication-competent HIV-1 particles, and show a strong correlation between loss of MA trimerization and impaired Env incorporation. These MA trimerization-defective mutants could be rescued by removal of the long Env CT, demonstrating that the requirement for MA trimerization in Env incorporation is linked to the presence of the long gp41 CT. This report both demonstrates the existence of MA trimers in infectious HIV-1 particles and establishes the importance of this structure for Env incorporation.

Results

Detection of MA Trimers in Replication-Competent HIV-1 Particles.

In our previous study, we proposed that residues Gln62 and Ser66 are positioned in the putative MA trimer such that Gln62 of chain A is less than 5 Å from Ser66 of chain C [Protein Data Bank (PDB) ID code 1HIW]. We described genetic evidence for interaction between these positions, and found that these residues could be mutated to arginine, either singly or together, without causing major defects in viral replication (24). The tolerance for arginine suggested that the introduction of physicochemically similar lysine residues at positions 62 and 66 should likewise be tolerated; furthermore, the presence of primary amines in the lysine side chain could potentially allow for the formation of intratrimer cross-links following treatment with glutaraldehyde (Fig. 1A). Having generated a 62QK/66SK double mutant, we confirmed its replication competence in Jurkat T cells, where only a small delay relative to WT replication was observed (Fig. 1B). We then performed a cross-linking assay. Viral particles were harvested and treated with very low (0.0001–0.0064%) concentrations of glutaraldehyde, which, as a bifunctional cross-linker, is able to react with two primary amine groups. We were able to detect both dimers and trimers of MA in particles, but only in the 62QK/66SK mutant (Fig. 1C); at these concentrations of glutaraldehyde, no cross-linking of WT MA was observed. Likewise, no MA dimers or trimers were observed when the cross-linking assay was performed using an MA mutant with only one residue substituted for lysine (62QK) (Fig. S1). We repeated the trimerization assay using the T-cell lines Jurkat and MT4 and observed MA trimerization in both, suggesting that MA trimerization probably occurs independent of the cell type used to generate the virions (Fig. 1D). The dimeric and trimeric MA species were observed to migrate slightly more rapidly by SDS/PAGE than their predicted molecular weights, probably because the cross-linking sites are centrally located in the MA molecule, giving the cross-linked proteins a more compact form than a simple linear polypeptide. To confirm that the appearance of oligomeric MA species was dependent on the presence of an intact particle, we treated particles with the nonionic detergent Triton X-100 and the strongly denaturing anionic detergent SDS. In mature particles, in which MA exists as a mature protein separate from Gag, both detergents completely disrupted the MA trimers (Fig. 1E). In immature particles, in which MA is present as a domain of the Gag polyprotein, treatment with SDS but not Triton X-100 disrupted MA oligomers (Fig. 1E). These data demonstrate the presence of MA trimers in replication-competent HIV-1 particles.

Fig. 1.

Visualization of trimeric MA in HIV-1 particles. (A) Two residues near the trimer interface, Gln62 and Ser66, were mutated to lysines to permit cross-linking with glutaraldehyde. 62QK, dark blue; 66SK, light blue. (B) Jurkat cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution at positions 62 and 66. At 2-d intervals, the cells were split and samples of media were assayed for reverse transcriptase (RT) activity. (C) HeLa cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation at 76,000 × g. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Positions of monomeric MA and MA dimers and trimers are indicated. Positions of molecular mass markers are shown (Left). The asterisk indicates the position of a band presumed to be p55Gag. (D) 293T cells were transfected with the pNL4-3 molecular clone or a mutant bearing the double-lysine substitution and a plasmid expressing vesicular stomatitis virus G glycoprotein (VSV-G). After 48 h, supernatants were filtered and infectious particles were titered on TZM-bl cells. These supernatants were used to infect Jurkat and MT4 cells. At 48 h postinfection, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Positions of monomeric MA and MA dimers and trimers are indicated. The asterisk indicates the position of a band presumed to be p55Gag. (E) Mature particles were produced as described in C; immature particles were generated using clones with an inactive PR. Before treatment with glutaraldehyde, the resuspended particles were treated with PBS (control), Triton X-100 (Tx100), or SDS. Detergents were prepared as 10× solutions in water before use. Cross-linking and analysis were then performed as described above. Conditions under which cross-linking is possible are indicated by a red box.

Fig. S1.

Trimer cross-linking requires two lysine residues. HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting (WB) for MA and Gag. At 0.01% glutaraldehyde, nonspecific cross-linking is apparent for both MA and CA.

Loss of MA Trimerization Impairs Env Incorporation.

Our previous work revealed that mutation of the highly conserved Thr69 to Arg (69TR) blocked HIV-1 Env incorporation apparently by a mechanism distinct from that of MA mutants previously described to block Env incorporation (24). Examination of the MA trimer crystal structure suggested that the 69TR mutation might sterically hinder MA trimerization (Fig. 2A). We further explored the consequences of introducing mutations at this position through vertical scanning mutagenesis, and found that polar and hydrophobic residues (69TA, 69TN, and 69TV) were well-tolerated, whereas introduction of charged residues (69TD, 69TK, and 69TR) resulted in a loss of viral replication (Fig. 2B). The 69TW mutant possessed an intermediate phenotype, suggesting that a large side chain impaired function, but was less inhibitory than the charged side chains. Further examination of these mutants showed that virus assembly and release were efficient in all cases, but Env incorporation and infectivity were severely impaired in those mutants bearing charged residues at position 69 (Fig. 2C). MA trimerization was then examined in a subset of mutant viruses that were either competent (69TA and 69TW) or impaired (69TD and 69TR) for Env incorporation (Fig. 2D). The mutants that were able to incorporate Env contained trimeric MA, whereas those unable to incorporate Env lacked MA trimerization in this assay.

Fig. 2.

Mutation of Thr69 can inhibit MA trimerization and Env incorporation. (A) Model of the MA trimer interface showing threonine (WT) and arginine side chains in red at position 69 on chain A. Side chains of Gln58, Ile59, and Gln62 on chain B are shown in dark gray. (B) Jurkat cells were transfected with the pNL4-3 molecular clone or mutants bearing substitutions at position 69. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. (C) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the Gag processing intermediate Pr41Gag (p41), the mature CA protein, and gp41 are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from four independent experiments are shown, ± SEM. (D) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured, and the amount of trimeric MA was determined as a percentage of the total MA for that lane and expressed relative to WT. Averages from three independent experiments are shown, ± SEM.

Similar data were generated with an additional set of mutants in which cysteine instead of lysine was introduced at positions Gln62 and Ser66, permitting spontaneous disulfide-bridge formation between MA molecules in particles. However, although these mutants were replication-competent, this approach yielded only dimers, which were lost in the presence of the MA trimer-defective mutation 69TR (Fig. S2). We suspect this failure to cross-link trimers may be due to the shorter side chain of cysteine, relative to lysine, and the absence of the glutaraldehyde, limiting the chances of simultaneously cross-linking three MA monomers; consequently, we did not pursue this approach further.

Fig. S2.

Visualization of MA dimers by disulfide-bridge formation. (A) Jurkat cells were transfected with the pNL4-3 molecular clone or a mutant bearing a double-cysteine substitution at positions 62 and 66. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. (B) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in nonreducing Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured and the amount of trimeric MA was expressed as a percentage of the total MA for that lane. Averages from two independent experiments are shown, ± SEM.

We expanded the analysis of MA trimer formation through a series of conservative and nonconservative mutations at, or near, the trimer interface predicted by the crystal structure (Fig. 3). Although several polar residues were sufficiently close together to potentially form hydrogen bonds at the trimer interface (Asn46, Gln58, and Gln68), we found no evidence that such interactions contributed to trimer formation (Fig. 3A). In contrast, nonconservative mutations at positions 44, 71, and 74 inhibited MA trimerization. Again, mutants that were deficient for MA trimerization were also impaired for Env incorporation and infectivity, whereas the conservative 74LV mutation retained both MA trimerization and Env incorporation (Fig. 3B). These results suggest that a small cluster of hydrophobic interactions appears to be required for the formation of the MA trimer, whereas the peripheral hydrophilic residues are largely dispensable (Fig. 3 C and D). We additionally confirmed that the trimer interface mutants that were deficient for MA trimerization, Env incorporation, and infectivity were also deficient for replication in Jurkat cells (Fig. 4). As previously observed with the Thr69 mutants, trimer-deficient mutants (44AE, 71SR, 71SW, 74LE, and 74LG) were unable to replicate in Jurkat cells (Fig. 4). Finally, we introduced trimer-defective MA mutations in the context of 62QK/66SK into a PR(−) backbone. This allowed us to repeat the trimerization assay in immature particles; again, the introduction of the trimer-defective mutations 74LE and 74LG inhibited MA trimerization (Fig. 5). These data demonstrate a strong and consistent correlation between Env incorporation and formation of WT MA trimers. At this point, we cannot exclude the possibility that the failure to detect trimers could be due to a more subtle change in MA structure that is refractory to both glutaraldehyde cross-linking and Env incorporation rather than a complete loss of MA trimerization.

Fig. 3.

Identification of MA trimer interface residues. (A) HeLa cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured, and the amount of trimeric MA was determined as a percentage of the total MA for that lane and expressed relative to WT. Averages from three to five independent experiments are shown, ± SEM. (B) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the mature CA protein, and gp41 are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from three to six independent experiments are shown, ± SEM. (C and D) Top-down (C) and side-view (D) models of the MA trimer interface, indicating in red positions at which mutations inhibited trimerization (labeled) and in green positions at which mutations did not affect MA trimerization.

Fig. 4.

Replication of MA trimer-defective mutants. Jurkat cells were transfected with the HIV-1 mutants indicated. At 2-d intervals, the cells were split and samples of media were assayed for RT activity. Mutations of (A) Ala44, (B) Ser71, and (C) Leu74 were analyzed.

Fig. 5.

MA trimer-defective mutants block MA trimerization in immature particles. 293T cells were transfected with the HIV-1 mutants indicated. After 48 h, supernatants were filtered and virions were pelleted by ultracentrifugation. Virions were resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by the addition of Tris, and then samples were boiled in Laemmli buffer and analyzed by SDS/PAGE and Western blotting for MA. Band volumes were measured and the amount of trimeric Gag was determined as a percentage of the total Gag for that lane and expressed relative to WT. Positions of molecular mass markers are shown. Averages from four independent experiments are shown, ± SEM.

MA Trimer-Defective Mutants Can Be Rescued by Truncation of the Env CT.

We speculated that the loss of Env incorporation in trimer-defective mutants was due to steric hindrance between the long CT of Env and the MA lattice in the immature particle (discussed in more detail in ref. 27). To test this hypothesis, we generated MA trimer-defective virus particles in the context of the gp41 truncation mutant CTdel144. This mutant lacks most of the CT but packages Env and yields infectious particles in cell lines such as HeLa. The CTdel144 mutant is also replication-competent in MT4 and M8166 T cells but not in most T-cell lines or physiologically relevant target cells (8, 9). The trimer-defective mutants were able to incorporate the CT-truncated Env at levels comparable to those in virions produced by WT Gag (Fig. 6A). The MA trimer-defective mutants bearing the CTdel144 Env were also more infectious than those bearing the full-length Env, although not quite to WT levels, reflecting the improved incorporation of CT-truncated Env into trimer-defective mutant particles (Figs. 3B and 6A). Finally, the trimer-defective mutants encoding the CTdel144 Env were replication-competent in MT4 cells, whereas the trimer-defective clones bearing full-length Env displayed a marked delay in replication relative to WT (Fig. 6B).

Fig. 6.

Rescue of MA trimer-defective mutants by truncated Env. (A) HeLa cells were transfected with the HIV-1 mutants indicated. At 48 h posttransfection, virus- and cell-associated samples were collected, separated by SDS/PAGE, and analyzed by Western blotting with anti-gp41 Ab and then HIV Ig to detect Gag. Positions of the Gag precursor Pr55Gag (p55), the Gag processing intermediate Pr41Gag (p41), the mature CA protein, and gp41 (full-length or CTdel144) are indicated. Virus release was calculated as the amount of virion CA relative to total Gag levels, and Env incorporation was expressed as the amount of virion gp41 per virion CA; both were expressed relative to WT. Virus-containing supernatants were used to infect TZM-bl cells; the resulting luciferase signal was normalized to the corresponding RT values to provide a measure of specific infectivity. Averages from four independent experiments are shown, ± SEM. (B) MT4 cells were transfected with the HIV-1 mutants indicated. At 2-d intervals, the cells were split and samples of media were assayed for RT activity.

Discussion

The HIV-1 MA protein has multiple functions during virus assembly and replication (29); MA structure is thus of considerable interest. Although NMR and crystal structures broadly agree on the structure of the MA monomer, these approaches differ in quaternary MA structure and at the trimer interface (30). Whereas NMR structures reveal only a monomer, crystallographic approaches reveal an MA trimer. An MA trimer would be consistent with recently suggested models of CA organization in immature particles; however, no structure is available for MA in the context of either mature or immature virions (17).

Here we developed a cross-linking strategy to demonstrate that MA is trimeric in both mature and immature particles and in a variety of cell types. Notably, this assay makes use of a replication-competent virus, validating the hypothesis that trimeric MA is present in virions. The MA trimer does not appear to be a highly stable structure, and in mature particles is lost when the membrane is disrupted by detergent treatment. These data may explain the discrepancy between NMR and crystallographic approaches. If the MA trimer is maintained by relatively weak interactions, which would be consistent with the small hydrophobic surface area buried at the trimer interface, additional factors may be required for trimer stability. Based on the available data, these additional factors may include an underlying CA lattice and/or an intact membrane, both of which restrict the freedom of movement of MA and increase its effective concentration. This effect may be mimicked in the MA crystal, where the MA protein is at a saturating concentration. By contrast, under the conditions used for solution NMR experiments, the lower MA concentration and absence of physical support may be incompatible with the formation of stable MA trimers.

The CT of HIV-1 Env, like that of most other lentiviruses, is long compared with other retroviruses (reviewed in ref. 4). Although the long CT is essential for Env incorporation into viral particles in physiologically relevant cell types, it also imposes packaging constraints: The Env CT must fit into the area below the viral membrane, which is also occupied by the MA domain of Gag. Various models have been suggested to explain the role of MA in Env incorporation (4, 27, 31); here we present data favoring a model of steric accommodation. Because the central aperture of the hexamer—the region implicated in Env incorporation (Introduction)—is distant from the trimer interface, it is unlikely that any putative MA–Env interaction would be directly affected by mutation of residues at the trimer interface. However, the consequence of a loss of MA trimerization would be a much-reduced aperture at the center of the putative hexamer of MA trimers (Fig. 7 A and B). Although the structure of the CT is currently unknown, this region of gp41 contains three amphipathic helical motifs [referred to as lentivirus lytic peptides (32)] that may lie flat in the plane of the membrane (33). Such a configuration would occupy more space on the surface of the membrane than that of the CT forming a straight rod protruding into the center of the virion, and could explain the sensitivity of Env packaging both to mutations that disrupt MA trimerization and to those that lie around that aperture of the hexamer of trimers. It must be emphasized that this issue remains a matter of speculation and that additional structural information on the gp41 CT would contribute significantly to resolving this issue.

Fig. 7.

Schematic illustrating the role of MA trimerization in Env incorporation. Adapted from Tedbury and Freed (27). (A) Combining the models proposed by Alfadhli et al. (21) and Hill et al. (19) reveals a hexamer-of-trimers arrangement of MA, with a large (∼45-nm) central aperture ringed by residues where mutations are known to be able to block Env incorporation (indicated in blue). Hypothetical positions of the gp41 CT are depicted in green. (B) An earlier model by Alfadhli et al. (51) showed a hexameric configuration for MA, without trimers; in this model, the MA lattice has a much smaller central aperture (∼30 nm). The narrowed central aperture could cause the steric exclusion of the HIV-1 Env CT. (C) The truncation of the Env CT rescues Env incorporation into MA trimer-defective particles, possibly by relieving the steric clash between MA and the Env CT.

If the importance of MA trimerization is the steric accommodation of the large gp41 CT, it would be anticipated that mutations that block MA trimerization could be rescued by pseudotyping with CT-truncated Env (Fig. 7C), and indeed this is what we observed. Because the particles defective for MA trimerization displayed efficient particle assembly, our data suggest that Env incorporation is the primary step in the late phase of HIV-1 replication that is dependent on MA trimerization. However, we cannot definitively rule out the possibility that interactions between MA trimers and the gp41 CT could impact postbudding events, for example membrane fusion. It is notable that the trimer-defective mutants seemed to incorporate more truncated Env than the WT yet were less infectious by a small but statistically significant margin. This result suggests the possibility of an additional role for MA trimerization in mediating particle infectivity, or could reflect a trimerization-independent function of MA.

The hypothesis that HIV-1 MA forms trimers to accommodate the long gp41 CT has implications for the structures of related viruses. Because the CT-truncated Env was efficiently packaged into HIV-1 particles that lacked a trimeric MA, all other genera of orthoretroviruses might be expected to accommodate their short-tailed Envs irrespective of the lattice organization of MA. Indeed, whereas the CA-NTD of immature HIV-1 appears to be organized as a hexamer of trimers (17), strongly reminiscent of the proposed arrangement of MA (21), the CA-NTD of Mason-Pfizer monkey virus (M-PMV) appears to adopt a predominantly dimeric organization (34); these data illustrate differences in the fundamental Gag lattice structures of diverse retroviruses. HIV-1 and M-PMV also differ in the organization of their Gag proteins: HIV-1 CA lies immediately C-terminal of MA, whereas in M-PMV there are two additional protein domains (pp24/16 and p12) between MA and CA (35, 36). Consequently, there is more uncertainty in attempting to infer MA arrangement for the CA arrangement in M-PMV. Furthermore, because M-PMV has a short-tailed Env, it may not require the additional space made available by MA trimerization. Given the technical challenges of imaging MA directly in viral particles, in the short term it may prove easier to infer MA arrangement from the CA-NTD configuration (at least for those viruses in which the CA domain is located immediately C-terminal to MA).

Evolutionarily, it is unclear how and why the lentiviruses evolved long-tailed Envs and, presumably, trimeric MA. The potential uniqueness of the HIV-1 MA trimer configuration could suggest that the CT is extremely important functionally, and drove the rearrangement of the Gag lattice to accommodate a long CT. Or, alternatively, the hexamer-of-trimers configuration of CA arose first, driven by functional requirements for virus particle assembly, and the evolution of a long CT and an accommodating MA followed through minor modifications of MA to promote trimerization. It is worth noting that the structure of the MA monomer is well-conserved among diverse retroviruses (37, 38). In vitro evidence for MA trimerization has been observed for several lentiviruses—HIV-1 (19), SIV (20), and equine infectious anemia virus (EIAV) (39)—whereas feline immunodeficiency virus (FIV) MA crystalized as a dimer (40). It may be relevant in this regard that FIV has the shortest Env CT among the lentiviruses (4, 41). By contrast, when considering retroviruses outside the lentiviruses, none of the MA structures available for human T-cell leukemia virus (HTLV) II (42), bovine leukemia virus (BLV) (43), Moloney murine leukemia virus (Mo-MLV) (38), Rous sarcoma virus (RSV) (44), or M-PMV (45) revealed an MA trimer, although, in every case except RSV, the authors felt that a trimeric MA was possible, based primarily on their close structural conservation with HIV-1 and SIV MA. It has been suggested that the formation of MA trimers is a precursor to Gag assembly in BLV and M-PMV (43, 45); however, our data suggest that in the case of HIV-1, loss of MA trimerization does not significantly impact virus-like particle assembly and therefore does not play a major role in Gag lattice formation. It would be of considerable interest to determine whether MA trimerization occurs in retroviruses with short Env CTs, and, if so, what function it performs.

In conclusion, we have demonstrated the presence of MA trimers in HIV-1 particles, and shown that disruption of the MA trimer leads to the loss of Env incorporation and viral infectivity. The functional importance of the MA trimer suggests that this structure may represent a target for the development of anti-HIV therapeutics.

Materials and Methods

Cell Lines, Antibodies, and Plasmids.

HeLa and TZM-bl cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% (vol/vol) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine (Gibco). TZM-bl is a HeLa-derived indicator cell line that expresses luciferase following infection by HIV (46). 293T cells were cultured in DMEM supplemented with 10% (vol/vol) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. Jurkat CD4⁺ T cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% (vol/vol) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. Anti–HIV-1 IgG is pooled patient serum obtained from the NIH AIDS Reagent Program. HIV-1 gp41 was detected with the 2F5 monoclonal antibody (47). MA was detected with mouse monoclonal antiserum 2D11 (ZeptoMetrix).

Plasmids.

HIV-1 particles were generated using the full-length proviral clone pNL4-3 (48). Point mutations were introduced by first subcloning the HindIII-SpeI fragment from pNL4-3 into pBluescript (Stratagene). Mutations were introduced using the QuikChange method (Stratagene) following the manufacturer’s instructions, and the mutant BssHII-SphI fragment was recloned into pNL4-3. All mutations were confirmed by DNA sequencing (Macrogen). For experiments requiring immature particles, a clone bearing a PR active-site mutation was used. CTdel144 has been described previously (25); MA mutations were introduced into pNL4-3/CTdel144 using the subcloning strategy described above.

Virus Replication, Release, and Infectivity.

HIV-1 replication was assayed by the rate of spreading infection in Jurkat cells as reported previously (49). Virus replication was monitored by measuring reverse transcriptase (RT) activity as described (50). The efficiency of virus particle assembly and release was assayed by Western blot of cell- and virion-associated Gag. The percentage of the total expressed Gag that is released from the cell as virion-associated material indicates the efficiency of particle release. For infectivity assays, virus-containing supernatants were generated by transfecting HeLa cells in 12-well plates. One microgram of DNA was transfected using PolyJet (SignaGen) according to the manufacturer’s instructions. Supernatants were harvested 48 h posttransfection and assayed for RT activity as described (50). TZM-bl cells were infected with the supernatants, and the luciferase signal was measured 24 h postinfection using Britelite Plus (PerkinElmer). Infectivity was defined as the level of luciferase expressed by TZM-bl cells divided by the total amount of virus (RT) with which they were infected.

Env Incorporation into Virions.

Virions were harvested 48 h posttransfection by filtering supernatant through a 0.45-μm membrane and then pelleted by centrifugation at 76,000 × g for 1 h at 4 °C. Virions were resuspended in 2× Laemmli buffer [120 mM Tris⋅Cl, pH 6.8, 4% (vol/vol) SDS, 20% (vol/vol) glycerol, 10% (vol/vol) β-mercaptoethanol, 0.02% bromophenol blue] and analyzed by Western blotting. Protein samples were separated by SDS/PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon; Millipore). Membranes were probed with primary antibody overnight at 4 °C, washed, and then incubated for 1 h with species-specific horseradish peroxidase-conjugated secondary antibody. After the final washes, bands were revealed by chemiluminescence and membranes were exposed to a charge-coupled device in a Universal Hood II (Bio-Rad). Quantification was performed using ImageLab software (Bio-Rad). To calculate Env incorporation, membranes were probed first for gp41 and then reprobed for CA. Band volumes (average pixel intensity multiplied by the area covered by the band) were determined for each gp41 band and divided by the volume for the corresponding CA band.

MA Trimerization.

Cross-linkable MA was generated by introducing the mutations 62QK/66SK as described above. Molecular clones were transfected into HeLa cells using PolyJet, and virions were harvested 48 h posttransfection by filtering supernatant through a 0.45-μm membrane and then pelleting by centrifugation at 76,000 × g for 1 h at 4 °C. Particles were resuspended in a small volume of PBS (e.g., virus from 1.5 mL supernatant from a six-well plate was resuspended in 20 μL). Cross-linking was induced by the addition of glutaraldehyde at a final concentration of 0.001% (or at the range of concentrations indicated) and allowed to proceed for 10 min at room temperature. Cross-linking was stopped by the addition of 100 mM (final concentration) Tris (pH 7.5). Samples were lysed in Laemmli buffer and analyzed by Western blotting for MA.

Structural Modeling.

Models of MA and mutants were generated in MacPyMOL (DeLano Scientific) using PDB ID code 1HIW (19).