Cryo-electron microscopy of tubular arrays of HIV-1Gag resolves structures essential for immaturevirus assemblyTanmay A. M. Bharata,b,c, Luis R. Castillo Menendezb,d,1, Wim J. H. Hagena, Vanda Luxd,2, Sebastien Igonete,f,3,Martin Schorba, Florian K. M. Schura,b, Hans-Georg Kräusslichb,d,4, and John A. G. Briggsa,b,4

aStructural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany; bMolecular Medicine Partnership Unit,European Molecular Biology Laboratory/Universitätsklinikum Heidelberg, Heidelberg, Germany; cStructural Studies Division, MRC Laboratory of MolecularBiology, Cambridge CB2 0QH, United Kingdom; dDepartment of Infectious Diseases, Virology, Universitätsklinikum Heidelberg, 69120 Heidelberg, Germany;eUnité de Virologie Structurale, Departement Virologie, Institut Pasteur, 75724 Paris, France; and fUnité Mixte de Recherche 3569, Centre National de laRecherche Scientifique, 75724 Paris, France

Edited by Robert A. Lamb, Northwestern University, Evanston, IL, and approved April 28, 2014 (received for review January 23, 2014)

The assembly of HIV-1 is mediated by oligomerization of the majorstructural polyprotein, Gag, into a hexameric protein lattice at theplasma membrane of the infected cell. This leads to budding andrelease of progeny immature virus particles. Subsequent pro-teolytic cleavage of Gag triggers rearrangement of the particlesto form mature infectious virions. Obtaining a structural model ofthe assembled lattice of Gag within immature virus particles isnecessary to understand the interactions that mediate assembly ofHIV-1 particles in the infected cell, and to describe the substratethat is subsequently cleaved by the viral protease. An 8-Å resolu-tion structure of an immature virus-like tubular array assembledfrom a Gag-derived protein of the related retrovirus Mason–Pfizermonkey virus (M-PMV) has previously been reported, and a modelfor the arrangement of the HIV-1 capsid (CA) domains has beengenerated based on homology to this structure. Here we haveassembled tubular arrays of a HIV-1 Gag-derived protein with animmature-like arrangement of the C-terminal CA domains andhave solved their structure by using hybrid cryo-EM and tomog-raphy analysis. The structure reveals the arrangement of theC-terminal domain of CA within an immature-like HIV-1 Gag lattice,and provides, to our knowledge, the first high-resolution view ofthe region immediately downstream of CA, which is essential forassembly, and is significantly different from the respective regionin M-PMV. Our results reveal a hollow column of density for thisregion in HIV-1 that is compatible with the presence of a six-helixbundle at this position.

cryo-electron tomography | helical reconstruction | SP1 |electron cryomicroscopy

The major structural component of HIV-1 is the 55-kDa Gagpolyprotein. Gag proteins from all retroviruses contain an

N-terminal membrane-binding matrix (MA) domain; two centraldomains that together comprise capsid (CA); and an RNA-bindingnucleocapsid (NC) domain located toward the C terminus of Gag.Between, and downstream of these domains are regions that differbetween retroviruses. In HIV, a spacer peptide, SP1, is found be-tween CA and NC, whereas downstream of NC are a secondspacer peptide, SP2, and the p6 domain (1).Assembly of an infectious HIV-1 particle proceeds in two stages

(2). In the first stage, Gag oligomerizes into a curved lattice on thecytoplasmic face of the plasma membrane, leading to outwardprotrusion of the membrane and subsequent membrane scission torelease an immature virus particle. Within the immature virus par-ticle, Gag is arranged in a radial manner with the N-terminal MAdomain on the inner surface of the membrane, and the C terminus ofGag pointing toward the center of the particle. In the second stage ofassembly, the viral protease becomes activated and cleaves Gag atfive positions, releasing the component domains, leading to a struc-tural rearrangement within the virus particle. MA remains bound to

the viral membrane, NC and the associated viral genome con-dense in the center of the particle, and CA assembles to forma conical core around the viral genome. This proteolytic matu-ration is required for the particle to become infectious and isa target of antiretroviral drugs (3). The immature virus particletherefore represents a key intermediate in HIV-1 assembly.Obtaining detailed structural information on the arrangement ofGag within immature HIV-1 is essential to reveal the proteininterfaces that mediate assembly of the immature virus, and toreveal the structure of the substrate that must be cleaved by theviral protease to induce maturation to the infectious form.There has been an extensive effort to apply structural biology

methods to understand the structures and interactions that me-diate assembly of HIV-1, and the structural changes associatedwith maturation (4, 5). High-resolution structural data exist for

Significance

HIV-1 undergoes a two-step assembly process. First, an imma-ture noninfectious particle is assembled, which leaves theinfected cell. Second, the structural protein, Gag, is cleaved inthe virus by the viral protease, and this leads to formation ofthe infectious virus. The immature virus particle thereforerepresents the key intermediate in HIV-1 assembly. There iscurrently no high-resolution information available on the ar-rangement of Gag within immature HIV-1. We have assembledpart of HIV-1 Gag in vitro to form immature virus-like tubularprotein arrays, and have solved a subnanometer-resolutionstructure of these arrays by using cryo-EM and tomography.This structure reveals interactions of the C-terminal capsiddomain of Gag that are critical for HIV-1 assembly.

Author contributions: T.A.M.B., L.R.C.M., H.-G.K., and J.A.G.B. designed research; T.A.M.B.,L.R.C.M., W.J.H.H., V.L., S.I., and F.K.M.S. performed research; M.S. contributed newreagents/analytic tools; T.A.M.B., L.R.C.M., and J.A.G.B. analyzed data; and T.A.M.B.,H.-G.K., and J.A.G.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.

Data deposition: The X-ray structures and the fit have been deposited in the Protein DataBank, www.pdb.org (PDB ID codes 4COC, 4COP, and 4D1K) and the cryoEM structure hasbeen deposited in the Electron Microscopy Data Bank (EMDB number EMD-2638).1Present address: Department of Cancer Immunology and AIDS, Dana–Farber Cancer In-stitute, Boston, MA 02215.

2Present address: Faculty of Biology, Center for Medical Biotechnology, University Duis-burg–Essen, 45117 Essen, Germany.

3Present address: Calixar SAS, Lyon, France.4To whom correspondence may be addressed. E-mail: hans-georg.kraeusslich@med.uni-heidelberg.de or john.briggs@embl.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1401455111/-/DCSupplemental.

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all the folded domains of HIV-1 Gag in their isolated forms.Recently, a series of breakthrough studies have revolutionizedour understanding of how the CA domains self-assemble to formthe mature capsid core of the virus (6–8), and a pseudoatomicmodel for the assembled mature core is now available (9). Incontrast, much less structural information is available on theimmature virus. A structure of the HIV-1 Gag lattice withinimmature-like particles assembled in vitro from a Gag-derivedprotein has been resolved to a resolution of ∼17 Å by usingsubtomogram averaging (10). At this resolution, the approximatepositions of the protein domains within the lattice can be de-fined, but their orientation is not clearly determined. We re-cently solved the structure of immature-like tubular assembliesof a Gag-derived protein (ΔProCANC) from another retrovirus,Mason–Pfizer monkey virus (M-PMV), to a resolution of ∼8 Å(11). At this resolution, the arrangement of the CA domainscould be clearly determined, revealing the regions of CA thatinteract within the assembled immature M-PMV Gag lattice.Based on the expected structural homology between the HIV-1and M-PMV CA domains, a model for the arrangement of theCA domains in immature HIV-1 was generated and comparedwith the CA arrangement in mature HIV-1 cores. Strikingly, theinterfaces that stabilize the immature and the mature CA latticesare almost completely distinct in this model.The SP1 peptide plays a crucial role in HIV-1 assembly and

maturation (2). A stretch of ∼25 residues downstream of thefolded C-terminal domain of CA comprising the C-terminalresidues of CA and SP1 is known to be essential for immatureHIV-1 assembly (12, 13). This stretch encompasses the CA-SP1proteolytic cleavage site, mutation of which abolishes formationof mature cores and infectious viruses (14). The bevirimat classof HIV-1 assembly inhibitors blocks HIV-1 replication by spe-cifically targeting processing at this site (15). The structure of thisimportant region of Gag has not yet been resolved with highresolution, however. Early mutational studies and molecularmodeling led to the proposal that this region forms an extendedα-helix (13), and this was supported by subsequent NMR studiesof the isolated sequence (16). The presence of thick columns ofdensity linking the CA and NC layers in low-resolution EMstructures is consistent with an extended six-helix bundle formingin this region (10, 13, 17). M-PMV, which contains no spacerpeptide between CA and NC, diverges structurally from HIV-1in this region (18), and, instead of the thick density columns, sixsplayed densities were observed in the M-PMV ΔProCANCtubes (11).The available structural data therefore do not answer several

critical questions: Most importantly, (i) does the arrangement ofthe CA domains in immature HIV correspond to the modelgenerated based on structural homology from the M-PMV–

derived tube structure; and (ii) what is the structure of the regionimmediately downstream of CA that is critical for immatureassembly and the regulation of maturation?The key step in obtaining detailed structural data for the im-

mature M-PMV lattice was to generate immature-like tubulararrays of a Gag construct. The helical symmetry of the M-PMVΔProCANC tubes allowed application of a hybrid cryo-electrontomography (ET) and cryo-EM image processing approach forstructure determination (11). HIV-1 Gag derivatives that as-semble tubular arrays adopt a mature-like arrangement of CA(19, 20), and have indeed been important tools in determiningthe structure of the mature CA core (9, 21). In contrast, HIV-1Gag derivatives known to assemble into immature-like arraysform approximately spherical structures (22, 23) that areunsuitable for application of high-resolution helical recon-struction methods.Here we identify an HIV Gag-derived protein competent to

assemble partially immature-like tubular arrays, and determinetheir structure to a resolution of 9.4 Å. The structure reveals the

immature-virus-like arrangement of the C-terminal domain ofHIV CA (CA-CTD) and the CA-SP1 linker region.

Results and DiscussionImmature-Like Tubular Arrays. We previously assembled imma-ture-like tubular arrays of M-PMV Gag from a protein com-prising CA, NC, and the intervening residues, but lacking theN-terminal proline residue of CA (ΔProCANC) (11). SimilarHIV-1 Gag-derived proteins assemble narrow tubular arrays(Fig. 1A) (19, 20, 24), which have a mature-like arrangement ofCA. Prior studies had indicated that certain HIV-1 Gag-derivedproteins can assemble tubular or spherical particles dependingon the conditions (22) and that certain mutations in the CA-SP1region converted the assembly from spherical to tubular or ab-errant sheet-like structures (12, 25). Furthermore, mutations inthe region targeted by the capsid assembly inhibitor CAI do notalter assembly of immature particles in vitro or in tissue culture,whereas they completely disrupt assembly of the mature-likelattice (26). We therefore purified CANC variants and otherGag-derived proteins with these mutations, and tested them forassembly of wide tubular particles. We found that CANC pro-teins with the mutation Y169L or Y169S yielded tubes witha wider diameter upon negative stain EM, whereas all other

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Fig. 1. Arrangement of the Gag lattice is immature-like in the HIV-1 CANCY169L tubes. (A) WT HIV-1 CANC protein was assembled along with DNA. Ona slice through a tomogram of the assembled tubes, thin mature-like tubeswere observed. (B) A single amino acid mutation Y169S/L converts thinmature-like tubes to thick immature-like tubes (Fig. S1). (C) Comparison ofradial density profiles of CANC Y169L tubes with immature HIV-1 virusparticles and mature-like WT CANC tubes. (D) A schematic representation ofthe HIV-1 Gag polyprotein aligned with the radial density profile shown in C(green). Positions of the NC, SP1, CA-CTD, CA-NTD, and MA domains alongwith the viral membrane are indicated.

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constructs tested did not lead to this phenotype or abolished invitro assembly. Other constructs tested included Y169S/Lmutations within constructs beginning after the N-terminalproline residue of CA or carrying mutations in predictedN-terminal interfaces, constructs with deletion or partial deletionof the N-terminal β-hairpin sequences, and hybrid constructsmade by fusing the N-terminal domain of CA (CA-NTD) fromM-PMV with the CA-CTD and the downstream sequence of Gagfrom HIV-1. Accordingly, CANC variants Y169L and Y169Swere selected for further study.We assembled CANC Y169L or Y169S in vitro in the pres-

ence of nucleic acid and imaged the assembly products by cryo-ET. Both proteins assembled into tubular arrays with an overallaverage diameter of 744 ± 23 Å (Fig. 1B and Fig. S1) and wereindistinguishable in cryo-ET images. Radial density profiles werecalculated for CANC Y169L tubular assemblies from the cryo-ET data. The results were compared with those obtained for WTCANC tubes (diameter, 300–400 Å), and for immature HIV-1particles produced from transfected cells in the presence ofa protease inhibitor (Fig. 1 C and D). CANC Y169L tubesshowed a similar radial density profile as immature HIV-1: bothdisplayed three separate peaks corresponding (from the inside)to the ribonucleoprotein complex (NC layer), the CA-CTD, andthe CA-NTD, whereas immature HIV-1 exhibited two additionalpeaks corresponding to the MA-bound lipid bilayer. Theseobservations suggest that CANC Y169L and CANC Y169S tubesassemble immature-like protein lattices.CA residue Y169 is located in a tight interface formed be-

tween the CA-NTD and CA-CTD within the assembled matureHIV-1 core (7, 27). It strongly coevolves with its interactionpartners within this interface (28). Mutation of Y169 abolishesproper mature core formation and thereby infectivity, but is fullycompatible with immature assembly in vitro and with release ofvirus particles with regular immature morphology (26). It seemslikely that, for Y169L and Y169S mutants, the mature lattice isdestabilized by the mutation, making assembly of an immature-like arrangement of CA the most energetically favorable state.

Solving the Structure of the Tubular Arrays. To obtain a 3D struc-ture of the tubes, we applied the same hybrid tomography/helicalreconstruction methodology that we had previously used to de-termine the structure of immature-like M-PMV ΔProCANCtubular arrays (11) (Fig. 2, Table S1, and SI Materials andMethods). Data from both mutants was pooled and two in-dependent reconstructions were generated and found to beequivalent to a resolution of 9.4 Å (Fig. S2A). They were aver-aged together to generate the final structure (Fig. 2 C–F).As previously described for in vitro assembled HIV Gag

spheres (10), the tubes show three layers of density: outermost isthe CA-NTD layer, below this the CA-CTD layer, and innermostis the NC layer (Fig. 2B). The CA-CTD and NC layers are joinedby column-like densities. Although the NC layer appears disor-dered, the two CA layers are structured (Fig. 2 B and C). Theresolution of the final reconstruction varied through the struc-ture from an average resolution of 10.0 Å in CA-NTD, 9.4 Å inCA-CTD, and 10.8 Å at SP1 (Fig. S2B), likely reflecting differingdegrees of order at different radii. In both CA layers, the elec-tron density contains three independent copies of the mono-mer (Fig. S3), which are not related to one another by thetwofold symmetry or the helical symmetry implicit in thereconstruction protocol.We wished to compare the resulting density with high-reso-

lution crystal structures of the individual protein domains.Crystal structures are available for the NTD and CTD of CA (5).To ensure that the mutations at position 169 of CA do not causesubstantial changes in the structure of the CA-CTD, weexpressed, purified, and crystallized both Y169L and Y169S CA-CTD according to established protocols (SI Materials andMethods), and solved their structures by X-ray crystallography

(Table S2). We found them to be similar to the previouslyresolved structures of the HIV CA-CTD Y169A protein (26)(rmsd, 0.50 Å and 0.74 Å for CA-CTD Y169S and Y169Lproteins, respectively).We carried out rigid body fitting of the Y169S CA-CTD X-ray

structure into the central density layer of the cryo-EM structure.We also carried out rigid body fitting of an available high-reso-lution atomic structure of the CA-NTD [Protein Data Bank(PDB) ID code 2JPR] (29) into the outer density layer of thecryo-EM structure and manually joined the domains. We thenapplied established molecular dynamics-based flexible fitting(MDFF) (30) methods to the joined structure to generate animproved model of the CA layer (Figs. 2 E and F and 3, Fig. S3,and Movie S1). For the CA-NTD and CA-CTD domains, a goodrigid body fit was observed into each of the three independentcopies of the monomer, and only minimal movements of helicestook place during flexible fitting (Fig. S4). This indicates that thestructures of the domains within the tubes are similar to thoseresolved in the input atomic structures.Finally, to allow direct comparison of the protein arrange-

ments in the HIV-1 CANC Y169S/L tubes with those previouslydescribed for M-PMV ΔProCANC tubes, we also revisited theM-PMV data and applied the same MDFF-based flexible fittingto generate a revised structural model of the M-PMV–derivedtubes (Fig. S3F).

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Fig. 2. Structure of the HIV-1 CANC Y169S/L tubes at 9.4-Å resolution. (A)Structure of a single CANC Y169L tube solved by using real-space helicalreconstruction. One hexamer of Gag is colored red to illustrate the ar-rangement of the lattice. Isosurface threshold is 1.5σ away from the mean.(B) The same structure as in A rotated 90° and magnified to show the dif-ferent layers in the lattice (annotated). (C) On averaging the asymmetric unitextracted from 41 different tubes, a 9.4-Å resolution structure was obtained(Fig. S2 and Movie S1). Isosurface threshold is 1.0σ away from the mean in C–E.One pseudohexameric asymmetric unit of Gag CA has been colored red. Thedotted line shows the position of the clipping plane used to produce E and F.(D) The same reconstruction as C, in an orthogonal orientation. (E) Six copies ofHIV-1 CA-NTD molecules (blue ribbon) were flexibly fitted into the outermostlayer of the pseudohexameric asymmetric unit. (F) Six corresponding HIV-1 CA-CTD molecules (orange ribbon) were similarly fitted into the inner layer of thehexamer (SI Materials and Methods and Fig. S3). Isosurface threshold is 1.5σaway from the mean.

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Arrangement of the CA Domains. Unexpectedly, the CA-NTDsin the HIV-1 CANC Y169S/L tubes are not assembled toform a hexameric lattice, as had been observed in M-PMVΔProCANC tubes and in previous studies on immature HIV-1Gag assemblies (10, 17), but instead pack together to forma lattice that is “p2” in crystallographic terms, containing fourtwofold positions (Figs. 2E and 3B and Fig. S5 A and C). Al-though a lattice with this symmetry can form a tubular array, it isdifficult to adapt it to form spherical structures as would benecessary in the virus. Within the lattice, clear additional den-sities outside the positions of the fitted first seven CA α-helicesare observed, corresponding to the N-terminal β-hairpin of theCA-NTD (Fig. 3E). This feature cannot be present in the im-mature virus, because the β-hairpin only forms upon proteolyticcleavage of the N terminus of CA from the preceding MA do-main. The β-hairpin appears to mediate interactions betweenCA-NTDs in the HIV-1 CANC Y169S/L tubes. Taken together,

these observations suggest that the arrangement of the CA-NTDwithin the HIV-1 CANC Y169S/L tubes is unlikely to representthe arrangement of the CA-NTD within the immature virus andthat it is most probably specific to the in vitro assembly of thismutant protein. We have therefore not interpreted it further.However, we note the remarkable capacity of the CA-NTDto oligomerize in at least three different ways to form curvedlattices—first as seen in the mature HIV-1 capsid, second as seenin the M-PMV ΔProCANC tubes, and third as seen in the HIV-1CANC Y169S/L tubes described here.The CA-CTD monomers are well resolved (Figs. 2F and 3C

and Fig. S2 E and F). They form an approximately hexamericlattice, “p6” in crystallographic terms (Fig. 3C and Fig. S5 B andD). The lattice spacing derived from the helical parameters was72.5 Å at the CA-CTD:SP1 interface, corresponding to 81 Å atthe CA-NTD. This is in good agreement with the interhexamericdistance of ∼80 Å at the CA-NTD measured in the immatureHIV-1 Gag lattice by previous cryo-EM studies (17, 31), andsignificantly smaller than the ∼95 Å interhexameric spacing ob-served in the mature lattice (6). In the structure, there is a spacebetween the CA-NTD and CA-CTD domains (Fig. 3D). Thespace between the domains is larger than that previously ob-served in the M-PMV ΔProCANC tubes. No direct protein–protein contacts were observed between the two domains, butthe polypeptide chain linking the domains (the 7–8 linker) isresolved (Fig. 3F and Fig. S3 C and D). Because of the mismatchbetween the p2 symmetry of the CA-NTD and p6 symmetry ofthe CA-CTD lattices, there are three different relative positionsof the CA-CTD and CA-NTD. All these positions differ from therelative positions of the CA-CTD and CA-NTD in the M-PMVΔProCANC tubes and in the mature HIV-1 CA lattice (Fig. S6).To link the domains, there are therefore three different ori-entations of the 7–8 linker (Figs. S3 C and D and S6). Together,these data indicate that the arrangement of the CA-CTD withinthe immature-like lattice can be formed independent of the ar-rangement of the CA-NTD, and that the linker between them ishighly flexible.The CA-CTD layer is made of compact α-helical domains

(Fig. 2F and Fig. S2 E and F). The overall arrangement ofthe CA-CTD monomers resembles that seen in the M-PMVΔProCANC tubes (11) and in one of the previously derivedmodels of the immature HIV-1 CA-CTD lattice (10). The CA-CTD monomers in the HIV-1 CANC Y169S/L tubes show a kinkin helix 9 of CA, a feature also observed in the X-ray atomicstructure, but not observed in the M-PMV ΔProCANC tubes.The dimer interface and the overall arrangement of the HIV-1CA-CTD dimers around the hexameric axis are similar to thatobtained for M-PMV (Fig. S3 E and F). In HIV-1 and M-PMVtubes, CA-CTD dimerization is mediated by a hydrophobic in-terface between helix 9 in each monomer (Figs. S3 E and F andS7). In HIV-1 CA, residues W184 and M185 are close to thisinterface, whereas, in M-PMV CA, residue Y180 (in the se-quence VDYV) is close to this interface. The relative orientationof the CA-CTD monomers within a CA-CTD:CA-CTD dimer issimilar to that in the M-PMV ΔProCANC tubes (only 1° dif-ference in the crossing angle of the two helix 9s), and to that inthe crystal structure of CA Y169A (4° difference in crossingangles). In contrast, the relative orientation differs strongly from“mature-like” dimeric forms such as mature-like CA arrays (138°difference) or HIV-1 CA crystals (118° difference; Fig. S7).Despite the highly similar structures of the dimers, the sizes ofthe hexamers formed by these CA-CTD dimers are different:72.5 Å for HIV-1 and 86 Å for M-PMV (measured at the CA–

SP1 interface). To accommodate the smaller hexamer size, ad-jacent CA-CTDs around the sixfold axis are closer together inthe HIV-1 CANC Y169S/L tubes (18.9 Å center to center) thanin the M-PMV ΔProCANC tubes (22.6 Å; Fig. S3 E and F). Theinterface between adjacent CA-CTDs around the sixfold is cor-respondingly more extensive in the HIV-1 CANC Y169S/L tubes

A

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Fig. 3. Arrangement of CA domains in HIV-1 CANC Y169S/L tubes. (A) Thearrangement of the CA lattice on the HIV-1 CANC Y169S/L tubes viewedfrom outside the surface of the tube. The CA-NTD domains are colored blueand the CA-CTD domains are colored orange. A gray hexagon is super-imposed on the image to show the pseudohexameric asymmetric unit on thesurface of the tubes. (B) Arrangement of the CA-NTD lattice. (C) Arrange-ment of the CA-CTD lattice. Fig. S5 A and B show CA-NTD and CA-CTDsymmetry axes. (D) The CA lattice viewed from the side of the tube. A gap isobserved between the CA-NTD and CA-CTD layers. (E) Additional densitiesare observed in the final reconstruction, outside the fitted first sevenα-helices of CA, which are consistent with the folded N-terminal β-hairpin.Isosurface threshold is 1.0σ away from the mean. (F) The CA-CTD and CA-NTD layers are connected by the 7–8 linker (arrowheads), which is flexibleand forms three different arrangements (Fig. S6). Isosurface threshold is0.75σ away from the mean.

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(including CA residues R154-P157, E212, and M215-T216) thanin the M-PMV ΔProCANC tubes [including CA residues K149,P152 (in the sequence VKQGPD), R211, and S214 (in the se-quence IRLCSD)]. The major homology region, conservedacross retroviruses, covers the base of helix 8, and the adjacentpart of the 7–8 linker. This region includes residues likely tofunction in positioning the 7–8 linker and stabilizing the CA-CTD fold, but also the residues R154–P157 that contribute tothe interactions around the hexamer.

Arrangement of the CA-SP1 Region. Below the CA-CTD layer arecolumns of ordered density that connect the CA-CTD to the NCalong the sixfold symmetry axes of the lattice (Fig. 4 A and B andFig. S2G). They are formed by six extended structures sur-rounding a central cavity. The arrangement is approximatelysixfold symmetric, but deviates from this symmetry toward theNC. These columns of density are in the position correspondingto the C-terminal residues of CA and N-terminal part of SP1,a region of the protein that is essential for assembly. Theseresidues can adopt a helical conformation in solution (16), butthere is currently no high-resolution structural informationavailable for this region in an oligomeric state. The resolution ofour cryo-EM structure is not high enough to permit ab initiomodeling of this region, but the density we observe is consistentwith the presence of the predicted six α-helices arranged arounda central hole (Fig. S2G). To test whether the density has thedimensions expected for six helices packed together, we com-pared it with the X-ray structure of the six-helix bundle fromcucumber mosaic virus coat protein (32). The dimensions of thehelical bundles are almost identical (Fig. 4A). Together, theseobservations provide strong support for the presence of a hollowsix-helix bundle in this region.The hollow columns of density seen in the HIV-1 CANC

Y169S/L tubes is in contrast to the six splayed densities that

point away from the sixfold symmetry axis in the M-PMVΔProCANC tubes (Fig. 4 C and D). This contrast indicatessubstantial structural divergence in this region in the two viruses.The point at which the two electron densities strongly divergecorresponds approximately to the position of HIV-1 CA residueL231 (the last residue of CA) based on a provisional model ofthe region downstream of CA (SI Materials and Methods and Fig.S8 B and D). The observed structural divergence is consistent,with structural differences observed in lower-resolution struc-tures of spherical in vitro assembled HIV-1 and MPMV imma-ture virus-like particles (18) and also with sequence divergence:the region downstream of CA in HIV-1 shows a strong hydro-phobic helical moment compatible with the presence of a helicalbundle (13), whereas no strong helical-hydrophobic moment isseen in this region of the M-PMV sequence (18, 33). Never-theless, it is not obvious how to reconcile the observed structuraldivergence of this region with the apparent functional equiva-lence: mutagenesis data indicates that M-PMV containsa “spacer-like” sequence that, as in HIV-1, functions as a keydeterminant of assembly (34). Higher-resolution structural anal-ysis will be required to understand whether the differentstructures of this sequence in the two viruses could conceal re-lated mechanisms for stabilizing the assembling Gag lattice.

Implications for Assembly and Its Inhibition. It has long been knownfrom structural studies of isolated domains that CA-CTD andCA-NTD can fold independently of one another. Here we foundthat the CA-CTD can assemble an immature-like lattice in-dependent of the arrangement of the CA-NTD. This “mis-matched” lattice is accommodated by the flexibility of the linkerbetween the domains. These observations raise the question:what is the role of the CA-NTD during assembly in vivo? Indeed,previous observations have shown that proteins consisting onlyof the CA-CTD and the adjacent SP1 peptide, replacing up-stream regions with a myristoylation site and downstream regionswith a leucine zipper, are competent to assemble VLPs in cells,albeit at lower efficiency, despite the absence of CA-NTD (35).Conceivably, the CA-NTD may modulate assembly, while beingmore important for its structural role within the mature capsid,or its role in the early stages of infection, mediated via inter-actions with cellular factors such as CypA, CPSF-6, or nuclearporins. Consistent with this suggestion, although assemblyinhibitors with binding sites in the CA-NTD in some cases leadto a reduction in virus production, binding of other molecules tothe CA-NTD instead inhibit virus maturation (36) or act duringearly infection (37).The independence of the two CA domains may also be func-

tionally important in maturation. Cleavage upstream or down-stream of CA is not sufficient to induce disassembly ofa preassembled immature Gag lattice, but cleavage on both sidesof CA is required (38). The data presented here indicate thatcleavage between CA and MA could even permit β-hairpinformation—and potentially rearrangement of the NTD—with-out destabilizing the immature CA-CTD arrangement. Low-resolution structural studies of inhibited viruses suggest that thebevirimat class of inhibitors, which inhibit proteolytic cleavage atthe CA–SP1 boundary, stabilize an immature conformation ofthe lattice (39). We speculate that bevirimat could also arrest thevirus in such a hybrid maturation state, in which partial matu-ration of the CA-NTD has already occurred.Essentially all stages of the viral lifecycle have been proposed

as potential targets for antiretroviral drugs (15), including as-sembly and cleavage of the immature virus. In the absence ofstructural data on the immature Gag lattice, the binding sites ofassembly inhibitors relative to the protein–protein interfacesremain unknown. Indeed for inhibitors such as bevirimat, thebinding site exists only in the assembled immature lattice. Forthe CA-CTD, the structure presented here provides the beststructural model to date for the arrangement of the CA-CTD

A CHIV M-PMV

B D

Fig. 4. Arrangement of the region immediately downstream of CA. (A)View of a hexamer of HIV Gag from inside the tube. The region downstreamof helix 11 of CA forms columns of density that connect the CA-CTD to theNC along the pseudo–six-fold symmetry axes of the lattice (a slice throughthe density is presented in Fig. S2G). This arrangement is consistent witha six-helix bundle model. The six-helix bundle found in cucumber mosaicvirus (green) has been placed in the density for comparison. (B) A view fromthe side, orthogonal to A. Six extended densities are observed linking the CAand NC layers. Isosurface threshold is 1.0σ away from the mean. (C) A view ofthe previously solved M-PMV ΔProCANC tubes in the same orientation as A.No density consistent with a six-helix bundle is seen. (D) A view of theM-PMV ΔProCANC tubes from the side. Isosurface threshold is 1.5σ awayfrom the mean.

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and downstream regions in immature HIV-1, and, together withthe M-PMV structure presented previously (11), provides aframework for understanding how inhibitor-binding sites relateto the positions of interfaces important in virus assembly. Mu-tagenesis analysis has shown the CA-CTD regions to be mostcritical in mediating virus assembly, and the interfaces they formduring assembly include the most conserved residues across ret-roviruses. The observed similarity in CA-CTD:CA-CTD inter-actions between M-PMV and HIV makes it reasonable to assumethat these interactions will be conserved across retroviruses.

Materials and MethodsProtein Purification and in Vitro Assembly. HIV-1 CA-CTD and CANC proteinswere expressed and purified from Escherichia coli as described previously (22,26). Assembly of purified proteins into tubes was induced as describedpreviously (22). Immature HIV-1 particles (for radial-density profiles) wereproduced by transfection of 293T cells with the HIV-1 proviral plasmidpNLC4-3 in the presence of 5μM amprenavir followed by purification asdescribed previously (40). (SI Materials and Methods).

X-Ray Structure Determination of Y169S and Y169L CA-CTD Proteins. Crystal-lization, data collection, structure refinement, and model building of CA-CTDY169S and CA-CTD Y169L proteins were performed as described previously(26) (SI Materials and Methods). PDB accession codes are 4COC for Y169L and4COP for Y169S.

Cryo-EM and Image Processing. Cryo-EM sample preparation and data col-lection was performed as described previously (11). The helical parameters ofeach tube were extracted using cryo-ET and subtomogram averaging (10, 11,41). Real-space helical reconstruction was conducted by using extractedhelical parameters (42). The pseudohexameric asymmetric units from allreconstructed tubes were averaged to obtain a final reconstruction of theHIV-1 CANC Y169S/L tubes at 9.4 Å (SI Materials and Methods). The structureand fit are deposited under EMDB accession number EMD-2638, and PDBaccession code 4D1K.

Atomic Structure Fitting and Analysis. Atomic structures of retroviral CAdomains were fitted into the cryo-EM densities by using MDFF (30). Analysisof final obtained PDB files was done by using the UCSF Chimera package(43). (SI Materials and Methods).

ACKNOWLEDGMENTS. The authors thank Leonardo Trabuco for help withrunning MDFF, Maria Anders for preparing amprenavir-inhibited virus,Marie-Christine Vaney for help with X-ray data processing and structurerefinement, Ahmed Haouz and Patrick Weber (robotized crystallizationfacility Proteopole, Institut Pasteur) for help in crystal screening, and theEuropean Molecular Biology Laboratory (EMBL) Information TechnologyServices Unit and Frank Thommen for technical support. This study wassupported by Deutsche Forschungsgemeinschaft Grants BR 3635/2-1 (toJ.A.G.B.) and KR 906/7-1 (to H.-G.K.) and a Federation of EuropeanBiochemical Societies long-term fellowship (to T.A.M.B.). The laboratoryof J.A.G.B. acknowledges financial support from EMBL and the Chica undHeinz Schaller Stiftung.

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