Biochemical Evidence of a Role for Matrix Trimerization in HIV-1

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

Philip R. Tedbury, Mariia Novikova, Sherimay D. Ablan, and Eric O. Freed1

Virus-Cell Interaction Section, HIV Dynamics and Replication Program, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702-1201

Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved November 25, 2015 (received for review August 20, 2015) The matrix (MA) domain of HIV Gag has important functions in such as peripheral blood mononuclear cells, monocyte-derived directing the trafficking of Gag to sites of assembly and mediating macrophages, and most T-cell lines (8, 9). Recent work has the incorporation of the envelope glycoprotein (Env) into assem- shown that the CT mediates an interaction with Rab11 family- bling particles. HIV-1 MA has been shown to form trimers in vitro; interacting protein 1c (FIP1c), which in turn interacts with however, neither the presence nor the role of MA trimers has been Rab14 and appears to direct trafficking of HIV-1 Env to sites of documented in HIV-1 virions. We developed a cross-linking strategy assembly; it is likely that interactions with FIP1c contribute to to reveal MA trimers in virions of replication-competent HIV-1. By the observed requirement for the CT in Env incorporation (10, 11). mutagenesis of trimer interface residues, we demonstrated a corre- It is, however, unclear why lentiviral CTs are so large, because lation between loss of MA trimerization and loss of Env incorpora- far smaller Env CTs also contain essential functional trafficking tion. Additionally, we found that truncating the long cytoplasmic and signaling motifs (12). tail of Env restores incorporation of Env into MA trimer-defective A consequence of the large lentiviral CT is the potential for, or particles, thus rescuing infectivity. We therefore propose a model inevitability of, interactions with the MA domain of Gag during whereby MA trimerization is required to form a lattice capable of particle assembly. The Gag protein forms a hexameric lattice, accommodating the long cytoplasmic tail of HIV-1 Env; in the driven primarily by CA–CA interactions. Whereas the structure absence of MA trimerization, Env is sterically excluded from the of CA in mature particles has been studied extensively, with assembling particle. These findings establish MA trimerization as many high-resolution structures now available (13–16), and recently an obligatory step in the assembly of infectious HIV-1 virions. As progress has been made in determining the structure of the im- MICROBIOLOGY such, the MA trimer interface may represent a novel drug target mature Gag lattice (17), the organization of MA in particles has for the development of antiretrovirals. proven difficult to address directly, with no long-range order dis- cernable for the MA shell. The structure of HIV-1 MA has been HIV | retrovirus | matrix | envelope | trimerization solved in vitro, using both NMR and crystallography approaches (18, 19). The structure of the monomer is very similar using either he assembly and budding of retroviruses involve a series of approach; however, crystallography suggests a trimeric arrangement Tregulated steps, driven primarily by the viral Gag protein for both HIV-1 and simian immunodeficiency virus (SIV) MA (reviewed in refs. 1 and 2). In the case of HIV-1, assembly and proteins (19, 20), whereas NMR reveals only structures for the budding occur predominantly at the plasma membrane. The monomer, with no evidence for higher-order interactions (18). A HIV-1 Gag protein is expressed as a 55-kDa polyprotein, com- third approach visualized 2D lattices of MA or MA-CA, using prising four major domains and two spacer peptides (SPs). The myristylated proteins on a synthetic lipid bilayer with a composition major domains are matrix (MA), capsid (CA), nucleocapsid intended to mimic that of the plasma membrane at sites of assembly (NC), and p6; the spacer peptides are known as SP1 and SP2 and (21). Under these conditions, MA was seen to arrange as hexamers are located between CA-NC and NC-p6, respectively. Assembly of trimers, although the low resolution of this approach precluded and budding from the host cell are driven by the full-length Gag protein; concomitant with, or shortly after budding, viral parti- more-detailed structural analysis. A hexamer-of-trimers arrangement cles undergo maturation, wherein the viral protease (PR) cleaves Gag in an ordered cascade to release the mature proteins (3). Significance In addition to Gag, the other major structural component of retroviral particles is the envelope glycoprotein (Env). HIV-1 The HIV matrix domain of Gag is known to play a key role in Env is synthesized as a 160-kDa precursor that traffics to the plasma the incorporation of the envelope glycoprotein into viral par- membrane via the Golgi apparatus, where it is processed to form ticles. Here we provide evidence that matrix is organized as a the surface glycoprotein gp120 and the transmembrane glycopro- trimer in HIV-1 particles, and that the ability of matrix to form tein gp41 (reviewed in ref. 4). The processed Env glycoproteins trimers is essential for the packaging of the envelope glyco- remain noncovalently associated as a heterodimer; the Env spike protein. The requirement for matrix trimerization depends on is a homotrimer of these dimers (5, 6). On the surface of the viral steric constraints between matrix and the cytoplasmic tail of particle, gp120 binds the viral receptor CD4 and the chemokine envelope and matrix. This represents a novel model of enve- coreceptors CXCR4 or CCR5. Binding of gp120 to receptor and lope incorporation in this family of viruses, and suggests that coreceptor triggers structural changes in gp41 that lead to fusion the matrix trimer interface may represent a novel drug target of the viral and target cell membranes. The fusion activity of gp41 to inhibit the production of infectious viral particles and com- is conferred by the ecto- and transmembrane domains of the bat the spread of AIDS. protein (7). A third domain, the cytoplasmic tail (CT), is dispens- Author contributions: P.R.T. and E.O.F. designed research; P.R.T., M.N., and S.D.A. per- able for fusion but plays important roles in Env trafficking and cell formed research; P.R.T., M.N., and E.O.F. analyzed data; and P.R.T. and E.O.F. wrote signaling. Like most lentiviruses, HIV-1 encodes an Env bearing a the paper. very long CT composed of ∼150 amino acids. In contrast, most The authors declare no conflict of interest. other retroviruses encode Env CTs that are ∼25–35 amino acids in This article is a PNAS Direct Submission. length (4). The reasons for the greater length of lentivirus Env CTs 1To whom correspondence should be addressed. Email: [email protected]. are not fully understood. For HIV-1, the CT is required for Env This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. incorporation into particles in physiologically relevant cell types, 1073/pnas.1516618113/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1516618113 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 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 ve- sicular 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.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1516618113 Tedbury et al. Downloaded by guest on September 25, 2021 for MA would be compatible with the most recently suggested model suggesting that MA trimerization probably occurs independent PNAS PLUS for the immature CA lattice, which proposes a hexamer-of-trimers of the cell type used to generate the virions (Fig. 1D). The di- arrangement for the CA amino-terminal domain (CA-NTD), which meric and trimeric MA species were observed to migrate slightly lies immediately below MA (17). more rapidly by SDS/PAGE than their predicted molecular The available structures of MA can be used to place the data weights, probably because the cross-linking sites are centrally acquired through molecular and genetic approaches into context. located in the MA molecule, giving the cross-linked proteins a Mutations have been identified in MA that prevent the in- more compact form than a simple linear polypeptide. To confirm corporation of Env into particles (22–26). The majority of these that the appearance of oligomeric MA species was dependent on mutations map to the tips of the MA trimer (27), a region of the the presence of an intact particle, we treated particles with the protein that lies around the central aperture of the hexamer of nonionic detergent Triton X-100 and the strongly denaturing trimers. These findings implicate this central aperture as the site anionic detergent SDS. In mature particles, in which MA exists of Env incorporation in the particle. This idea is further sup- as a mature protein separate from Gag, both detergents com- ported by the observation that, in cell lines permissive for pletely disrupted the MA trimers (Fig. 1E). In immature particles, packaging of CT-truncated Env, removal of the CT relieves the in which MA is present as a domain of the Gag polyprotein, inhibition of Env incorporation imposed by the MA mutations treatment with SDS but not Triton X-100 disrupted MA oligomers (22, 25, 28). The ability to rescue Env incorporation by removing (Fig. 1E). These data demonstrate the presence of MA trimers in the CT suggests that steric hindrance of Env incorporation may replication-competent HIV-1 particles. be a key mechanism for the loss of Env incorporation imposed by mutations in MA. This view is consistent with our recent data Loss of MA Trimerization Impairs Env Incorporation. Our previous showing that a mutation at the trimer interface was able to res- work revealed that mutation of the highly conserved Thr69 to cue the Env incorporation defects imposed by several MA mu- Arg (69TR) blocked HIV-1 Env incorporation apparently by a tations and a deletion in the Env CT (24). These data suggest mechanism distinct from that of MA mutants previously de- that the MA trimer interface regulates the ability to rescue scribed to block Env incorporation (24). Examination of the MA mutants that are defective for Env incorporation. trimer crystal structure suggested that the 69TR mutation might In this study, we sought to develop a system that would allow sterically hinder MA trimerization (Fig. 2A). We further ex- us to directly determine whether MA forms trimers in virions plored the consequences of introducing mutations at this posi- and, if so, whether MA trimerization plays a role in Env in- tion through vertical scanning mutagenesis, and found that polar

corporation. By using a combination of biochemical, genetic, and and hydrophobic residues (69TA, 69TN, and 69TV) were well- MICROBIOLOGY virological approaches, we demonstrate the presence of MA tolerated, whereas introduction of charged residues (69TD, 69TK, trimers in replication-competent HIV-1 particles, and show a and 69TR) resulted in a loss of viral replication (Fig. 2B). The strong correlation between loss of MA trimerization and impaired 69TW mutant possessed an intermediate phenotype, suggesting Env incorporation. These MA trimerization-defective mutants that a large side chain impaired function, but was less inhibitory could be rescued by removal of the long Env CT, demonstrating than the charged side chains. Further examination of these mu- that the requirement for MA trimerization in Env incorporation tants showed that virus assembly and release were efficient in all is linked to the presence of the long gp41 CT. This report both cases, but Env incorporation and infectivity were severely im- demonstrates the existence of MA trimers in infectious HIV-1 paired in those mutants bearing charged residues at position 69 particles and establishes the importance of this structure for (Fig. 2C). MA trimerization was then examined in a subset of Env incorporation. mutant viruses that were either competent (69TA and 69TW) or impaired (69TD and 69TR) for Env incorporation (Fig. 2D). The Results mutants that were able to incorporate Env contained trimeric Detection of MA Trimers in Replication-Competent HIV-1 Particles. In MA, whereas those unable to incorporate Env lacked MA tri- our previous study, we proposed that residues Gln62 and Ser66 merization in this assay. are positioned in the putative MA trimer such that Gln62 of Similar data were generated with an additional set of mutants chain A is less than 5 Å from Ser66 of chain C [Protein Data in which cysteine instead of lysine was introduced at positions Bank (PDB) ID code 1HIW]. We described genetic evidence for Gln62 and Ser66, permitting spontaneous disulfide-bridge for- interaction between these positions, and found that these resi- mation between MA molecules in particles. However, although dues could be mutated to arginine, either singly or together, these mutants were replication-competent, this approach yielded without causing major defects in viral replication (24). The tol- only dimers, which were lost in the presence of the MA trimer- erance for arginine suggested that the introduction of physico- defective mutation 69TR (Fig. S2). We suspect this failure to cross- chemically similar lysine residues at positions 62 and 66 should link trimers may be due to the shorter side chain of cysteine, likewise be tolerated; furthermore, the presence of primary relative to lysine, and the absence of the glutaraldehyde, limiting amines in the lysine side chain could potentially allow for the the chances of simultaneously cross-linking three MA mono- formation of intratrimer cross-links following treatment with mers; consequently, we did not pursue this approach further. glutaraldehyde (Fig. 1A). Having generated a 62QK/66SK dou- We expanded the analysis of MA trimer formation through a ble mutant, we confirmed its replication competence in Jurkat T series of conservative and nonconservative mutations at, or near, cells, where only a small delay relative to WT replication was the trimer interface predicted by the crystal structure (Fig. 3). observed (Fig. 1B). We then performed a cross-linking assay. Although several polar residues were sufficiently close together Viral particles were harvested and treated with very low (0.0001– to potentially form hydrogen bonds at the trimer interface (Asn46, 0.0064%) concentrations of glutaraldehyde, which, as a bi- Gln58, and Gln68), we found no evidence that such interactions functional cross-linker, is able to react with two primary amine contributed to trimer formation (Fig. 3A). In contrast, non- groups. We were able to detect both dimers and trimers of MA conservative mutations at positions 44, 71, and 74 inhibited MA in particles, but only in the 62QK/66SK mutant (Fig. 1C); at trimerization. Again, mutants that were deficient for MA trimeri- these concentrations of glutaraldehyde, no cross-linking of WT zation were also impaired for Env incorporation and infectivity, MA was observed. Likewise, no MA dimers or trimers were whereas the conservative 74LV mutation retained both MA tri- observed when the cross-linking assay was performed using an merization and Env incorporation (Fig. 3B). These results suggest MA mutant with only one residue substituted for lysine (62QK) that a small cluster of hydrophobic interactions appears to be re- (Fig. S1). We repeated the trimerization assay using the T-cell quired for the formation of the MA trimer, whereas the peripheral lines Jurkat and MT4 and observed MA trimerization in both, hydrophilic residues are largely dispensable (Fig. 3 C and D).

Tedbury et al. PNAS Early Edition | 3of9 Downloaded by guest on September 25, 2021 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.

We additionally confirmed that the trimer interface mutants that MA mutations in the context of 62QK/66SK into a PR(−)back- were deficient for MA trimerization, Env incorporation, and in- bone. This allowed us to repeat the trimerization assay in immature fectivity were also deficient for replication in Jurkat cells (Fig. 4). As particles; again, the introduction of the trimer-defective mutations previously observed with the Thr69 mutants, trimer-deficient mu- 74LE and 74LG inhibited MA trimerization (Fig. 5). These data tants (44AE, 71SR, 71SW, 74LE, and 74LG) were unable to rep- demonstrate a strong and consistent correlation between Env in- licate in Jurkat cells (Fig. 4). Finally, we introduced trimer-defective corporation and formation of WT MA trimers. At this point, we

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1516618113 Tedbury et al. Downloaded by guest on September 25, 2021 PNAS PLUS MICROBIOLOGY

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, in- dicating in red positions at which mutations inhibited trimerization (labeled) and in green positions at which mutations did not affect MA trimerization.

Tedbury et al. PNAS Early Edition | 5of9 Downloaded by guest on September 25, 2021 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.

cannot exclude the possibility that the failure to detect trimers could immature particles; however, no structure is available for MA in be due to a more subtle change in MA structure that is refractory the context of either mature or immature virions (17). to both glutaraldehyde cross-linking and Env incorporation rather Here we developed a cross-linking strategy to demonstrate than a complete loss of MA trimerization. that MA is trimeric in both mature and immature particles and in a variety of cell types. Notably, this assay makes use of a repli- MA Trimer-Defective Mutants Can Be Rescued by Truncation of the cation-competent virus, validating the hypothesis that trimeric Env CT. We speculated that the loss of Env incorporation in MA is present in virions. The MA trimer does not appear to be a trimer-defective mutants was due to steric hindrance between highly stable structure, and in mature particles is lost when the 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 membrane is disrupted by detergent treatment. These data may 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). Discussion The HIV-1 MA protein has multiple functions during virus as- Fig. 5. MA trimer-defective mutants block MA trimerization in immature particles. sembly and replication (29); MA structure is thus of considerable 293T cells were transfected with the HIV-1 mutants indicated. After 48 h, super- interest. Although NMR and crystal structures broadly agree on natants were filtered and virions were pelleted by ultracentrifugation. Virions were the structure of the MA monomer, these approaches differ in resuspended in PBS and treated with glutaraldehyde. Cross-linking was stopped by theadditionofTris,andthensampleswereboiledinLaemmlibufferandanalyzed quaternary MA structure and at the trimer interface (30). Whereas by SDS/PAGE and Western blotting for MA. Band volumes were measured and the NMR structures reveal only a monomer, crystallographic amount of trimeric Gag was determined as a percentage of the total Gag for approaches reveal an MA trimer. An MA trimer would be con- that lane and expressed relative to WT. Positions of molecular mass markers sistent with recently suggested models of CA organization in are shown. Averages from four independent experiments are shown, ± SEM.

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1516618113 Tedbury et al. Downloaded by guest on September 25, 2021 PNAS PLUS MICROBIOLOGY

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.

explain the discrepancy between NMR and crystallographic ap- amphipathic helical motifs [referred to as lentivirus lytic peptides proaches. If the MA trimer is maintained by relatively weak in- (32)] that may lie flat in the plane of the membrane (33). Such a teractions, which would be consistent with the small hydrophobic configuration would occupy more space on the surface of the surface area buried at the trimer interface, additional factors membrane than that of the CT forming a straight rod protruding may be required for trimer stability. Based on the available data, into the center of the virion, and could explain the sensitivity of these additional factors may include an underlying CA lattice Env packaging both to mutations that disrupt MA trimerization and/or an intact membrane, both of which restrict the freedom of and to those that lie around that aperture of the hexamer of movement of MA and increase its effective concentration. This trimers. It must be emphasized that this issue remains a matter of effect may be mimicked in the MA crystal, where the MA protein speculation and that additional structural information on the is at a saturating concentration. By contrast, under the conditions gp41 CT would contribute significantly to resolving this issue. used for solution NMR experiments, the lower MA concentra- If the importance of MA trimerization is the steric accommo- tion and absence of physical support may be incompatible with dation of the large gp41 CT, it would be anticipated that mutations the formation of stable MA trimers. that block MA trimerization could be rescued by pseudotyping with The CT of HIV-1 Env, like that of most other lentiviruses, is CT-truncated Env (Fig. 7C), and indeed this is what we observed. long compared with other retroviruses (reviewed in ref. 4). Al- Because the particles defective for MA trimerization displayed ef- though the long CT is essential for Env incorporation into viral ficient particle assembly, our data suggest that Env incorporation is particles in physiologically relevant cell types, it also imposes the primary step in the late phase of HIV-1 replication that is de- packaging constraints: The Env CT must fit into the area below pendent on MA trimerization. However, we cannot definitively rule the viral membrane, which is also occupied by the MA domain of out the possibility that interactions between MA trimers and the Gag. Various models have been suggested to explain the role of gp41 CT could impact postbudding events, for example membrane MA in Env incorporation (4, 27, 31); here we present data fa- fusion. It is notable that the trimer-defective mutants seemed to voring a model of steric accommodation. Because the central incorporate more truncated Env than the WT yet were less in- aperture of the hexamer—the region implicated in Env infectious by a small but statistically significant margin. This result corporation (Introduction)—is distant from the trimer interface, suggests the possibility of an additional role for MA trimerization in it is unlikely that any putative MA–Env interaction would be mediating particle infectivity, or could reflect a trimerization- directly affected by mutation of residues at the trimer interface. independent function of MA. However, the consequence of a loss of MA trimerization would The hypothesis that HIV-1 MA forms trimers to accommo- be a much-reduced aperture at the center of the putative hex- date the long gp41 CT has implications for the structures of amer of MA trimers (Fig. 7 A and B). Although the structure of related viruses. Because the CT-truncated Env was efficiently the CT is currently unknown, this region of gp41 contains three packaged into HIV-1 particles that lacked a trimeric MA, all

Tedbury et al. PNAS Early Edition | 7of9 Downloaded by guest on September 25, 2021 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 rep- Fig. 7. Schematic illustrating the role of MA trimerization in Env in- resent a target for the development of anti-HIV therapeutics. corporation. Adapted from Tedbury and Freed (27). (A) Combining the models proposed by Alfadhli et al. (21) and Hill et al. (19) reveals a hexamer- Materials and Methods of-trimers arrangement of MA, with a large (∼45-nm) central aperture Cell Lines, Antibodies, and Plasmids. HeLa and TZM-bl cells were cultured in ringed by residues where mutations are known to be able to block Env in- Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% (vol/vol) corporation (indicated in blue). Hypothetical positions of the gp41 CT are FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine depicted in green. (B) An earlier model by Alfadhli et al. (51) showed a (Gibco). TZM-bl is a HeLa-derived indicator cell line that expresses luciferase hexameric configuration for MA, without trimers; in this model, the MA following infection by HIV (46). 293T cells were cultured in DMEM supple- ∼ lattice has a much smaller central aperture ( 30 nm). The narrowed central mented with 10% (vol/vol) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, + aperture could cause the steric exclusion of the HIV-1 Env CT. (C) The trun- and 2 mM L-glutamine. Jurkat CD4 T cells were cultured in Roswell Park cation of the Env CT rescues Env incorporation into MA trimer-defective Memorial Institute (RPMI) 1640 medium supplemented with 10% (vol/vol) particles, possibly by relieving the steric clash between MA and the Env CT. 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). other genera of orthoretroviruses might be expected to accom- MA was detected with mouse monoclonal antiserum 2D11 (ZeptoMetrix). modate their short-tailed Envs irrespective of the lattice organization of MA. Indeed, whereas the CA-NTD of immature Plasmids. HIV-1 particles were generated using the full-length proviral clone HIV-1 appears to be organized as a hexamer of trimers (17), pNL4-3 (48). Point mutations were introduced by first subcloning the strongly reminiscent of the proposed arrangement of MA (21), HindIII-SpeI fragment from pNL4-3 into pBluescript (Stratagene). Muta- the CA-NTD of Mason-Pfizer monkey virus (M-PMV) appears tions were introduced using the QuikChange method (Stratagene) fol- to adopt a predominantly dimeric organization (34); these data lowing the manufacturer’s instructions, and the mutant BssHII-SphI illustrate differences in the fundamental Gag lattice structures of fragment was recloned into pNL4-3. All mutations were confirmed by DNA sequencing (Macrogen). For experiments requiring immature particles, a diverse retroviruses. HIV-1 and M-PMV also differ in the orga- clone bearing a PR active-site mutation was used. CTdel144 has been nization of their Gag proteins: HIV-1 CA lies immediately described previously (25); MA mutations were introduced into pNL4-3/ C-terminal of MA, whereas in M-PMV there are two additional CTdel144 using the subcloning strategy described above. protein domains (pp24/16 and p12) between MA and CA (35, 36). Consequently, there is more uncertainty in attempting to infer MA Virus Replication, Release, and Infectivity. HIV-1 replication was assayed by the arrangement for the CA arrangement in M-PMV. Furthermore, rate of spreading infection in Jurkat cells as reported previously (49). Virus because M-PMV has a short-tailed Env, it may not require the replication was monitored by measuring reverse transcriptase (RT) activity as additional space made available by MA trimerization. Given the described (50). The efficiency of virus particle assembly and release was technical challenges of imaging MA directly in viral particles, assayed by Western blot of cell- and virion-associated Gag. The percentage in the short term it may prove easier to infer MA arrangement of the total expressed Gag that is released from the cell as virion-associated from the CA-NTD configuration (at least for those viruses in material indicates the efficiency of particle release. For infectivity assays, virus-containing supernatants were generated by transfecting HeLa cells in which the CA domain is located immediately C-terminal to MA). 12-well plates. One microgram of DNA was transfected using PolyJet (SignaGen) Evolutionarily, it is unclear how and why the lentiviruses according to the manufacturer’s instructions. Supernatants were harvested 48 h evolved long-tailed Envs and, presumably, trimeric MA. The posttransfection and assayed for RT activity as described (50). TZM-bl cells potential uniqueness of the HIV-1 MA trimer configuration were infected with the supernatants, and the luciferase signal was measured could suggest that the CT is extremely important functionally, 24 h postinfection using Britelite Plus (PerkinElmer). Infectivity was defined and drove the rearrangement of the Gag lattice to accommodate as the level of luciferase expressed by TZM-bl cells divided by the total a long CT. Or, alternatively, the hexamer-of-trimers configura- amount of virus (RT) with which they were infected. tion of CA arose first, driven by functional requirements for virus particle assembly, and the evolution of a long CT and an ac- Env Incorporation into Virions. Virions were harvested 48 h posttransfection μ commodating MA followed through minor modifications of MA by filtering supernatant through a 0.45- m membrane and then pelleted by centrifugation at 76,000 × g for 1 h at 4 °C. Virions were resuspended in 2× to promote trimerization. It is worth noting that the structure of Laemmli buffer [120 mM Tris·Cl, pH 6.8, 4% (vol/vol) SDS, 20% (vol/vol) the MA monomer is well-conserved among diverse retroviruses glycerol, 10% (vol/vol) β-mercaptoethanol, 0.02% bromophenol blue] and (37, 38). In vitro evidence for MA trimerization has been ob- analyzed by Western blotting. Protein samples were separated by SDS/PAGE served for several lentiviruses—HIV-1 (19), SIV (20), and equine and transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon; infectious anemia virus (EIAV) (39)—whereas feline immuno- Millipore). Membranes were probed with primary antibody overnight at deficiency virus (FIV) MA crystalized as a dimer (40). It may be 4 °C, washed, and then incubated for 1 h with species-specific horseradish per- relevant in this regard that FIV has the shortest Env CT among oxidase-conjugated secondary antibody. After the final washes, bands were the lentiviruses (4, 41). By contrast, when considering retroviruses revealed by chemiluminescence and membranes were exposed to a charge-cou- outside the lentiviruses, none of the MA structures available for pled device in a Universal Hood II (Bio-Rad). Quantification was performed using human T-cell leukemia virus (HTLV) II (42), bovine leukemia virus ImageLab software (Bio-Rad). To calculate Env incorporation, membranes were probed first for gp41 and then reprobed for CA. Band volumes (average pixel (BLV) (43), Moloney murine leukemia virus (Mo-MLV) (38), intensity multiplied by the area covered by the band) were determined for Rous sarcoma virus (RSV) (44), or M-PMV (45) revealed an MA each gp41 band and divided by the volume for the corresponding CA band. trimer, although, in every case except RSV, the authors felt that a trimeric MA was possible, based primarily on their close structural MA Trimerization. Cross-linkable MA was generated by introducing the mu- conservation with HIV-1 and SIV MA. It has been suggested that tations 62QK/66SK as described above. Molecular clones were transfected the formation of MA trimers is a precursor to Gag assembly in BLV into HeLa cells using PolyJet, and virions were harvested 48 h posttransfection

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1516618113 Tedbury et al. Downloaded by guest on September 25, 2021 by filtering supernatant through a 0.45-μm membrane and then pelleting by Structural Modeling. Models of MA and mutants were generated in MacPyMOL PNAS PLUS centrifugation at 76,000 × g for 1 h at 4 °C. Particles were resuspended in a (DeLano Scientific) using PDB ID code 1HIW (19). 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 ACKNOWLEDGMENTS. We thank members of the E.O.F. laboratory and of glutaraldehyde at a final concentration of 0.001% (or at the range of con- S. Welbourn for helpful discussion and critical review of the manuscript. The HIV-Ig was obtained through the NIH AIDS Research and Reference centrations indicated) and allowed to proceed for 10 min at room temperature. Reagent Program. Work in the E.O.F. laboratory is supported by the Cross-linking was stopped by the addition of 100 mM (final concentration) Tris Intramural Research Program of the Center for Cancer Research, National (pH 7.5). Samples were lysed in Laemmli buffer and analyzed by Western blot- Cancer Institute, NIH, and by the Intramural AIDS Targeted Antiviral ting for MA. Program.

1. Sundquist WI, Kräusslich H-G (2012) HIV-1 assembly, budding, and maturation. Cold fective for Gag membrane targeting and envelope glycoprotein incorporation. J Mol Spring Harb Perspect Med 2(7):a006924. Biol 427(6 Pt B):1413–1427. 2. Freed EO (2015) HIV-1 assembly, release and maturation. Nat Rev Microbiol 13(8): 29. Freed EO (1998) HIV-1 Gag proteins: Diverse functions in the virus life cycle. Virology 484–496. 251(1):1–15. 3. Konvalinka J, Kräusslich H-G, Müller B (2015) Retroviral proteases and their roles in 30. Massiah MA, et al. (1996) Comparison of the NMR and X-ray structures of the HIV-1 virion maturation. Virology 479-480:403–417. matrix protein: Evidence for conformational changes during viral assembly. Protein 4. Checkley MA, Luttge BG, Freed EO (2011) HIV-1 envelope glycoprotein biosynthesis, Sci 5(12):2391–2398. trafficking, and incorporation. J Mol Biol 410(4):582–608. 31. Johnson MC (2011) Mechanisms for Env glycoprotein acquisition by retroviruses. AIDS 5. Lyumkis D, et al. (2013) Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 Res Hum Retroviruses 27(3):239–247. – envelope trimer. Science 342(6165):1484 1490. 32. Miller MA, Garry RF, Jaynes JM, Montelaro RC (1991) A structural correlation between 6. Julien J-P, et al. (2013) Crystal structure of a soluble cleaved HIV-1 envelope trimer. lentivirus transmembrane proteins and natural cytolytic peptides. AIDS Res Hum – Science 342(6165):1477 1483. Retroviruses 7(6):511–519. 7. Doms RW, Moore JP (2000) HIV-1 membrane fusion: Targets of opportunity. J Cell Biol 33. Viard M, et al. (2008) Photoinduced reactivity of the HIV-1 envelope glycoprotein 151(2):F9–F14. with a membrane-embedded probe reveals insertion of portions of the HIV-1 Gp41 8. Murakami T, Freed EO (2000) The long cytoplasmic tail of gp41 is required in a cell cytoplasmic tail into the viral membrane. Biochemistry 47(7):1977–1983. type-dependent manner for HIV-1 envelope glycoprotein incorporation into virions. 34. Bharat TAM, et al. (2012) Structure of the immature retroviral capsid at 8 Å resolution Proc Natl Acad Sci USA 97(1):343–348. by cryo-electron microscopy. Nature 487(7407):385–389. 9. Akari H, Fukumori T, Adachi A (2000) Cell-dependent requirement of human immu- 35. Bradac JA, Hunter E (1986) Polypeptides of Mason-Pfizer monkey virus. III. Trans- nodeficiency virus type 1 gp41 cytoplasmic tail for Env incorporation into virions. lational order of proteins on the gag and env gene specified precursor polypeptides. J Virol 74(10):4891–4893. Virology 150(2):503–508. 10. Qi M, et al. (2013) Rab11-FIP1C and Rab14 direct plasma membrane sorting and 36. Sonigo P, Barker C, Hunter E, Wain-Hobson S (1986) Nucleotide sequence of Mason- particle incorporation of the HIV-1 envelope glycoprotein complex. PLoS Pathog 9(4): Pfizer monkey virus: An immunosuppressive D-type retrovirus. Cell 45(3):375–385. MICROBIOLOGY e1003278. 37. Hamard-Peron E, Muriaux D (2011) Retroviral matrix and lipids, the intimate in- 11. Qi M, et al. (2015) A tyrosine-based motif in the HIV-1 envelope glycoprotein tail mediates cell-type- and Rab11-FIP1C-dependent incorporation into virions. Proc Natl teraction. Retrovirology 8(1):15. Acad Sci USA 112(24):7575–7580. 38. Riffel N, et al. (2002) Atomic resolution structure of Moloney murine leukemia virus 12. Tedbury PR, Freed EO (2015) The cytoplasmic tail of retroviral envelope glycoproteins. matrix protein and its relationship to other retroviral matrix proteins. Structure – Prog Mol Biol Transl Sci 129:253–284. 10(12):1627 1636. 13. Briggs JAG, Wilk T, Welker R, Kräusslich H-G, Fuller SD (2003) Structural organization 39. Chen K, et al. (2008) Solution NMR characterizations of oligomerization and dynamics of authentic, mature HIV-1 virions and cores. EMBO J 22(7):1707–1715. of equine infectious anemia virus matrix protein and its interaction with PIP2. 14. Pornillos O, Ganser-Pornillos BK, Yeager M (2011) Atomic-level modelling of the HIV Biochemistry 47(7):1928–1937. capsid. Nature 469(7330):424–427. 40. Serrière J, Robert X, Perez M, Gouet P, Guillon C (2013) Biophysical characterization 15. Zhao G, et al. (2013) Mature HIV-1 capsid structure by cryo-electron microscopy and and crystal structure of the feline immunodeficiency virus p15 matrix protein. all-atom molecular dynamics. Nature 497(7451):643–646. Retrovirology 10(1):64. 16. Gres AT, et al. (2015) X-ray crystal structures of native HIV-1 capsid protein reveal 41. Talbott RL, et al. (1989) Nucleotide sequence and genomic organization of feline conformational variability. Science 349(6243):99–103. immunodeficiency virus. Proc Natl Acad Sci USA 86(15):5743–5747. 17. Schur FKM, et al. (2015) Structure of the immature HIV-1 capsid in intact virus particles 42. Christensen AM, Massiah MA, Turner BG, Sundquist WI, Summers MF (1996) Three- at 8.8 Å resolution. Nature 517(7535):505–508. dimensional structure of the HTLV-II matrix protein and comparative analysis of 18. Massiah MA, et al. (1994) Three-dimensional structure of the human immunodefi- matrix proteins from the different classes of pathogenic human retroviruses. J Mol ciency virus type 1 matrix protein. J Mol Biol 244(2):198–223. Biol 264(5):1117–1131. 19. Hill CP, Worthylake D, Bancroft DP, Christensen AM, Sundquist WI (1996) Crystal 43. Matthews S, Mikhailov M, Burny A, Roy P (1996) The solution structure of the bovine structures of the trimeric human immunodeficiency virus type 1 matrix protein: Im- leukaemia virus matrix protein and similarity with lentiviral matrix proteins. EMBO J plications for membrane association and assembly. Proc Natl Acad Sci USA 93(7): 15(13):3267–3274. – 3099 3104. 44. McDonnell JM, et al. (1998) Solution structure and dynamics of the bioactive retro- 20. Rao Z, et al. (1995) Crystal structure of SIV matrix antigen and implications for virus viral M domain from Rous sarcoma virus. J Mol Biol 279(4):921–928. – assembly. Nature 378(6558):743 747. 45. Conte MR, Klikova M, Hunter E, Ruml T, Matthews S (1997) The three-dimensional 21. Alfadhli A, Barklis RL, Barklis E (2009) HIV-1 matrix organizes as a hexamer of trimers solution structure of the matrix protein from the type D retrovirus, the Mason-Pfizer on membranes containing phosphatidylinositol-(4,5)-bisphosphate. Virology 387(2): monkey virus, and implications for the morphology of retroviral assembly. EMBO J 466–472. 16(19):5819–5826. 22. Mammano F, Kondo E, Sodroski J, Bukovsky A, Göttlinger HG (1995) Rescue of human 46. Platt EJ, Wehrly K, Kuhmann SE, Chesebro B, Kabat D (1998) Effects of CCR5 and CD4 immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins cell surface concentrations on infections by macrophagetropic isolates of human with short cytoplasmic domains. J Virol 69(6):3824–3830. immunodeficiency virus type 1. J Virol 72(4):2855–2864. 23. Ono A, Huang M, Freed EO (1997) Characterization of human immunodeficiency virus 47. Purtscher M, et al. (1994) A broadly neutralizing human monoclonal antibody against type 1 matrix revertants: Effects on virus assembly, Gag processing, and Env in- gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 10(12): corporation into virions. J Virol 71(6):4409–4418. 1651–1658. 24. Tedbury PR, Ablan SD, Freed EO (2013) Global rescue of defects in HIV-1 envelope 48. Adachi A, et al. (1986) Production of acquired immunodeficiency syndrome-associ- glycoprotein incorporation: Implications for matrix structure. PLoS Pathog 9(11): e1003739. ated retrovirus in human and nonhuman cells transfected with an infectious molec- – 25. Freed EO, Martin MA (1995) Virion incorporation of envelope glycoproteins with long ular clone. J Virol 59(2):284 291. but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in 49. Freed EO, Orenstein JM, Buckler-White AJ, Martin MA (1994) Single amino acid the human immunodeficiency virus type 1 matrix. J Virol 69(3):1984–1989. changes in the human immunodeficiency virus type 1 matrix protein block virus 26. Brandano L, Stevenson M (2012) A highly conserved residue in the C-terminal helix of particle production. J Virol 68(8):5311–5320. HIV-1 matrix is required for envelope incorporation into virus particles. J Virol 86(4): 50. Willey RL, et al. (1988) In vitro mutagenesis identifies a region within the envelope 2347–2359. gene of the human immunodeficiency virus that is critical for infectivity. J Virol 62(1): 27. Tedbury PR, Freed EO (2014) The role of matrix in HIV-1 envelope glycoprotein in- 139–147. corporation. Trends Microbiol 22(7):372–378. 51. Alfadhli A, Huseby D, Kapit E, Colman D, Barklis E (2007) Human immunodeficiency 28. Tedbury PR, Mercredi PY, Gaines CR, Summers MF, Freed EO (2015) Elucidating the virus type 1 matrix protein assembles on membranes as a hexamer. J Virol 81(3): mechanism by which compensatory mutations rescue an HIV-1 matrix mutant de- 1472–1478.

Tedbury et al. PNAS Early Edition | 9of9 Downloaded by guest on September 25, 2021