MINIREVIEW The capsid protein of human immunodeficiency virus: designing inhibitors of capsid assembly Jose ´ L. Neira 1,2 1 Instituto de Biologı ´ a Molecular y Celular, Universidad Miguel Herna ´ ndez, Alicante, Spain 2 Biocomputation and Complex Systems Physics Institute, Zaragoza, Spain Introduction HIV-1, the agent responsible for AIDS, belongs to the retrovirus family. Its genome is formed by two copies of a single-stranded RNA, which are encased within a lipoprotein shell, together with several replicative and accessory proteins. The virus genome is a model of economic packaging because the virus uses complex processing (both in the production and cleavage of mRNAs and in the final viral polypeptides) to generate several proteins that can work in the host cell. Perhaps the most important example of this eco- nomic architecture is the production of Gag and Gag- Pol fusion proteins. The GagPol fusion protein, formed by a ribosomal frame shift event, is cleaved into the Gag and Pol polypeptides by the viral protease. Further cleavage of the Pol chain yields the viral integrase (p31), a new protease (p10), the reverse transcriptase (p50) and RNase H (p15). Conversely, cleavage of the Keywords CTD protein; HIV; NMR; organic molecules; protein–peptide interactions; protein–protein interactions; structure; X-ray crystallography Correspondence J. L. Neira, Instituto de Biologı ´ a Molecular y Celular, Edificio Torregaita ´ n, Universidad Miguel Herna ´ ndez, Avda. del Ferrocarril s ⁄ n, 03202, Elche (Alicante), Spain Fax: +34 9666 58758 Tel: +34 9666 58459 E-mail: jlneira@umh.es Note The author would like to dedicate this paper to Professors Manuel Rico and Alan Fersht for allowing him to learn beside them (Received 9 February 2009, revised 2 July 2009, accepted 24 July 2009) doi:10.1111/j.1742-4658.2009.07314.x The mature capsid of human immunodeficiency virus, HIV-1, is formed by the assembly of copies of a capsid protein (CA). The C-terminal domain of CA, CTD, is able to homodimerize and most of the dimerization interface is formed by a single a-helix from each monomer. Assembly of the HIV-1 capsid critically depends on CA–CA interactions, including CTD interac- tion with itself and with the CA N-terminal domain, NTD. This minireview reports on the search and the design of peptides and small organic com- pounds that are able to interact with the CTD and ⁄ or CA of HIV-1. Such molecules aim to disrupt and ⁄ or alter the oligomerization capability of CTD. The different peptides designed so far interact with CTD mainly via hydrophobic contacts with residues close or belonging to the interface between the dimerization helices. A CTD-binding organic compound also establishes hydrophobic contacts with regions involved in the interface between the NTD and CTD. These results open new venues for the devel- opment of new antiviral drugs that are able to interact with CA and ⁄ or its domains, hampering HIV-1 assembly and infection. Abbreviations CA, capsid protein of HIV-1 (p24); CAC1, a CTD-binding designed peptide; CAI, a CTD-binding phage display peptide; CAP-1, N-(3-chloro-4- methylphenyl)-N¢-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl] ethyl}-urea); CTD, C-terminal domain of CA, comprising residues 146–231 of the intact protein; CTDW184A ⁄ M185A, a double mutant of CTD with Ala substitutions at the positions Trp184 and Met185; HSQC, heteronuclear single quantum coherence; MA, matrix protein of HIV-1; NC, nucleocapsid protein; NTD, N-terminal domain of CA, comprising residues 1–145 of the intact protein; NYAD-1, an improved designed version of CAI. 6110 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS Gag polyprotein, yields the matrix protein (MA), the capsid protein (CA), the nucleocapsid protein (NC) and p6 proteins, as well as two spacer pep- tides. Currently, most of the available drugs in the market against AIDS target the reverse transcriptase and pro- tease enzymes, produced by Pol. In general, the com- pounds involved in the treatment of HIV infection belong to one of the following types: (a) non-nucleo- tide reverse transcriptase inhibitors; (b) nucleotide reverse transcriptase inhibitors; and (c) protease inhibi- tors [1]. Because of the poorly understood side-effects of protease inhibitors in human fat metabolism [2], and the frequent emergence and spread of drug-resis- tant variants of HIV-1 [3], it is necessary to identify new types of drugs that are suitable for long-term usage, which can, at the very least, supplement the cur- rently available drug regimes. Some of those new drug candidates have been reviewed elsewhere [1,4] and their pros and cons for possible anti-HIV chemotheraphy have been discussed; this minireview focuses on CA as a possible target for new anti-HIV-1 drugs. Among its other biophysical features [5], the suitability of CA as a drug target is based on: (a) its own self-assembly properties [6]; and (b) the other protein–protein inter- actions where it is involved [7]. CA in solution is a homodimer with a dissociation constant of 10–18 lm [8]. Homo-oligomerization provides several structural and functional advantages to proteins, such as an improved stability or modular complex formation, whereas the cell minimizes the genome size; indeed, oligomerization is generally employed in nature to build viral capsids, with a minimum of genetic information [9–13]. Fur- thermore, because of the omnipresent nature of pro- tein–protein interactions in key steps during the virus life cycle, it is possible to design antiviral strategies based on the inhibition of those active macromolecular complexes [14]. This minireview focuses on the devel- opment of small molecules (peptides and organic mole- cules) that are potentially able to inhibit HIV-1 infection by impairing the self-assembly of CA. Several peptides and one organic compound have been found to inhibit the C-terminal domain of CA (CTD) dimer- ization or, alternatively, its interactions with the N-ter- minal domain of CA (NTD). This minireview focuses on the structure of the complexes formed by CTD and those molecules, as well as what can be learnt from such studies. The use of drugs such as Bevirimat (Panacos Pharmaceuticals, Watertown, MA, USA) (although it is in Phase II trials) is not discussed because its mechanism of action is not based on inhibi- tion of CA self-assembly. Bevirimat is a novel HIV-1 maturation inhibitor that inhibits specifically the final rate-limiting step in Gag processing; the drug prevents the release of mature capsid protein from its precursor, resulting in the production of immature, non-infectious virus particles [15]. Before describing how those designed molecules work and inhibit dimer formation, it is necessary to describe in some detail the domain architecture of CA of HIV-1. The CA protein is formed by two indepen- dently folded domains, NTD and CTD, separated by a flexible linker [8,16–18]; see also Fig. 1B in Mascare- nhas and Musier-Forsyth [7]. The NTD (residues 1–146 in the numbering of the whole intact protein) is composed of five coiled-coil a-helices (helices 1–5 of CA), with two additional short a-helices (helices 6 and 7) following an extended proline-rich loop [16–18]. The CTD domain (residues 147–231) is a dimer both in solution and in the crystal form [8,19]. Each CTD monomer is composed of a short 3 10 -helix followed by a strand and four a-helices (helices 8–11 of CA): a-helix 8 (residues 160–172), a-helix 9 (residues 178– 191), a-helix 10 (residues 195–202) and a-helix 11 (resi- dues 209–214), which are connected by short loops or turn-like structures (Fig. 1). The dimerization interface of CTD is formed by the mutual docking of a-helix 9 from each monomer, with the side chains of each tryp- tophan (Trp184) deeply buried in the dimer interface [8,19]; this helix has a kink at Thr188. The two addi- tional aromatic residues in each monomer, Tyr164 and Fig. 1. Structure of the CTD dimer. X-ray structure of CTD showing the dimeric structure of the domain. The monomers are depicted in the same colour (blue) and the dimerization helix is highlighted (gold), with the side-chain of the sole trypotophan in each monomer indicated by sticks. The figure was produced using PYMOL (http:// www.pymol.org) [39] using the Protein Data Bank file for CTD (accession no. 1a43) [8]. The different a-helices and the kink at the second one are indicated; the numbering of these helices corre- sponds to helices 8–11 of the whole intact CA. J. L. Neira CA small-molecule interactions FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6111 Tyr169, are located in the hydrophobic core of each monomer, well away from the dimer interface. The dissociation constant of CTD is 10–18 l M [8], similar to that of intact CA (see above). Thus, the CA dimer- ization interface is fully contained within the isolated CTD. This observation has facilitated the initial search for inhibitors of HIV-1 assembly by using simplified experimental setups in vitro; if a small molecule is able to dissociate CTD dimers in solution, then it could be considered a potential inhibitor of CA assembly and HIV-1 infectivity (dependent on that hypothesis always being tested by direct experiments). Furthermore, the same observation validates the view that the structures of CTD small-molecule complexes can be physiologically relevant, and thus they could provide important guide- lines for the rational design of better assembly inhibitors, as well as physiologically acceptable antiviral drugs. Inhibition of CTD self-assembly by peptides Protein assembly processes can be considered as good targets for antivirals because they depend only on recur- ring weak interprotein interactions, and the disruption of only a few of these interactions may be sufficient to suppress infectivity. In principle, destabilization of the CA dimeric species and, subsequently, HIV capsid assembly can be carried out by using isolated CTD as a competitive inhibitor [20,21]. Alternatively, virion assembly can be hampered by the use of small peptides or organic molecules which bind to: (a) CTD (or CA) monomers and occupy the dimerization interface or (b) nearby sites to the dimerization interface, thus weak- ening the binding to the other monomer. Below, the structural implications of peptides that are able to bind to CTD are reviewed, as well as how they work based on the reported structural studies. There was an early attempt to design peptides that could hamper dimerization of the whole CA in solu- tion [22]. The peptide with the highest affinity for the whole CA has the sequence IPVGEIYKRW, which corresponds to the a-helix 7 of NTD. This region is important for the formation of CA hexamers [6]; there- fore, this peptide could inhibit the formation of the hexamers, and assembly of the HIV-1 capsid, but no evidence is yet available. A peptide-mimicking the dimerization-helix of CTD as an inhibitor of CA dimerization Both structural [8] and thermodynamic [23] analyses indicate that most of the dimerization interface in CA (Fig. 1) is contained within a single helical segment (helix 9). Thus, it was reasonable to assume, as a first approach, that a synthetic peptide mimicking the sequence of helix 9 could constitute a good inhibitor of CTD dimerization. Accordingly, our group designed a peptide comprising the dimerization helix of CTD, plus three residues on each site to avoid fraying effects: the sequence of this peptide, CAC1, is: Ac-EQ- ASQEVKNWMTETLLVQNA-CONH 2 [24]. We first tested whether CAC1 was bound to CTD in solution by using several biophysical techniques. Thermal dena- turations followed by CD, NMR and size-exclusion chromatography provided evidence for the interaction between CAC1 and CTD. Gel filtration analysis also provided evidence for dissociation of the CTD dimer mediated by the peptide because the protein eluted at larger volumes in the presence of equimolar peptide concentrations. A quantitative value of the affinity constant of the CAC1–CTD complex was determined by fluorescence (using an anthraniloyl-labeled peptide), affinity chro- matography and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant of the complex, in the order of 50 lm, which is only three- to five-fold higher than the dissociation constant of dimeric CTD (10–18 lm) [8]. Because CAC1 is random-coil when isolated in solu- tion, as shown by 1D-NMR experiments, it is impor- tant to note that the above determined affinity constant must take into account the entropy penalty to fold the peptide. In view of this fact, and the previous observation that not every residue energetically involved in the CTD interface was contained within CAC1 [23], the comparable affinity of the peptide– CTD complex and the CTD–CTD dimer was unex- pected. However, later studies have provided a possible explanation for this observation. As in the CAC1 pep- tide, a-helix 9 becomes only permanently structured when the CTD monomers dimerize [25,26], and then a substantial entropy penalty must be paid on the forma- tion of both the peptide–CTD and the CTD–CTD complexes [6]. As expected, CAC1 is able to efficienty inhibit the in vitro assembly of the HIV-1 capsid (R. Bocanegra and M. G. Mateu, unpublished results). To map the region of CTD where CAC1 actually binds, 2D-NMR titration experiments were attempted but, because of the low solubility of the peptide, we were unable to describe the interacting residues. We have started the design of modified peptides of CAC1 with improved solubilities (in collaboration with C. Cavasotto, Houston, TX, USA), and have been able to map the region of CTD where a modified CAC1 peptide is bound, by using 1 H- 15 N- heteronuclear single quantum coherence (HSQC) NMR experiments. The CA small-molecule interactions J. L. Neira 6112 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS residues of dimeric CTD that experienced the largest changes in chemical shifts, and ⁄ or their intensities were significantly decreased in the presence of the peptide (in a 2 : 1 peptide ⁄ protein molar concentration), were Asp152, Arg154, Val165, Leu190, Gly206 and Thr210 (J. L. Neira, unpublished results). All of these residues are involved in the dimerization interface of nonmutat- ed CTD, or are located proximal to the interface (as shown by the X-ray structure of CTD) [8]. Although it has not been possible to ascertain the structure of CAC1 upon binding to CTD by transfer NOE experi- ments, it may be reasonable to assume (based on other structural studies, see below) that it acquires an a-helix-like conformation. We are currently developing and designing new variants of CAC1 not only with an improved solubility, but also with a larger helicity to assess whether the latter improves the affinity for CTD. A phage display peptide as an inhibitor of CA assembly A CTD-binding, 12-mer peptide, CAI, was identified by Stitch et al. [27] using phage display techniques. The sequence of CAI is: ITFEDLLDYYGP, which does not bear any resemblance to the sequence of the dimerization helix of CTD (see above). CAI is the first reported peptide able to inhibit the assembly of the mature HIV-1 capsid in vitro. However, because of the lack of permeability of cells to CAI, the peptide was unable to inhibit HIV-1 infection ex vivo. The CAI peptide isolated in solution is random-coil, but it adopts an a-helical conformation upon binding to CTD, as shown by the X-ray structure of the nonmu- tated-CTD–CAI complex [28] (Fig. 2A, main panel). The aromatic moiety of Phe3, together with Leu6 and Ile1, are the residues of CAI with the largest number of protein contacts (Fig. 2A, inset); these data suggest that the CTD–CAI interaction occurs mainly through hydrophobic contacts. The X-ray structure of the CAI–CTD complex is a five helix bundle [28] (Fig. 2A, main panel). In this bundle, the dimerization helix of CTD (a-helix 9) reorients to maximize interactions with CAI; this movement results in a change of the buried surface at the dimeric interface when compared with the buried interface in the dimeric CTD. The affinity constant of CAI for CTD was measured by mapping the changes in 15 N-HSQC NMR spectra upon peptide addition [27]; the dissociation constant is 15 ± 7 lm, which is comparable to that of the CTD–CTD dimer, and higher than that of CAC1. Similar to the results obtained with CAC1 (see above), this value can be explained because an entropy penalty must be paid during folding of both the CAI peptide and a-helix 9 of CTD during formation of the peptide– protein and protein–protein complexes. These NMR experiments also allowed the determina- tion of the CTD binding site. However, instead of using the dimeric wild-type CTD species, Stitch et al. [27] employed the monomeric CTD mutant (CTDW184A ⁄ M185A) in the titration experiments. Thus, they were able to observe the chemical shifts of the whole dimer- ization helix of CTD [27] (otherwise, those chemical shifts are too broad to be observed because of the presence of interconverting conformers) [29]. The bind- ing site of CTD encompasses residues Tyr169 to Val191, which agrees with the X-ray structure of the nonmutated-CTD-CAI complex [28]; thus, the peptide binds to a groove created by a-helices 8, 9 (the dimerization helix) and 11 of CTD (Fig. 2A, main panel) and, in addition, the kink at Thr188 in CTD is less pronounced than in each of the monomers of the dimeric CTD. Finally, it is important to note that some of the residues of CTD bound to CAI are also involved in binding to CAC1 and vice versa (see above); these data suggest that the modes of binding of both peptides are quite similar, if not identical. Bartonova et al. [30] have recently shown that resi- dues Tyr169, Asn183, Glu187 and Leu211, all of which form the binding pocket site of CAI, are necessary for the quaternary structure integrity of CTD [30]. When residues Tyr169, Asn183, Glu187 and Leu 211 were replaced with alanine, the resulting mutants were competent for immature capsid-like particle assembly in vitro and budding of immature-like capsids. The results obtained by Bartonova et al. [30] pinpoint an indirect (allosteric) effect of CAI binding on the assem- bly of the immature capsid lattice, but a direct binding effect in the conserved CTD binding pocket during mature assembly. On the other hand, mutations at Tyr169 and Leu211 do not yield mature-like particles and give rise to non-infectious virions with nonregular mature cores. The X-ray structures of these two mutants differ from that of wild-type, and those of the assembly-competent mutants E187A and N183A: the conformations of the two assembly-incompetent mutants (Y169A and L211A) in the absence of CAI are the same as the structures of any mutant when complexed to the peptide. Interestingly, Glu187 (together with Ser178, Glu180 and Gln192) is one of the residues that decreases the association constant of CTD, as previously demonstrated in an alanine scanning of the dimerization interface [23]. Barklis et al. [31] have shown that CAI does not block Gag assembly [27], but dismantles HIV-1 CA J. L. Neira CA small-molecule interactions FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6113 tubes [31]. The authors suggest two possible explana- tions: (a) CAI modifies the CTD dimer interface (as suggested based on the X-ray structure of the different complexes [28,30]) and (b) CAI is able to replace the a-helix 4 of NTD, which binds to a CTD groove, and which helps align NTDs and CTDs around the CA hexamers [6]. Thus, binding of CAI to CTD would weaken the hexamers, impair assembly and destabilize the assembled cores. In that way, CAI would act dur- ing assembly and uncoating of the tubes. In summary, CAI is the first peptide that has been shown to efficiently inhibit CA assembly. Variants of CAI peptide Armed with this structural information on CAI, Zhang et al. [32] used a structure-based rational design approach to stabilize the a-helical structure of CAI and also to convert it into a cell-penetrating peptide. The resulting peptide, NYAD-1, contains a covalent bridge between two amino acids separated four resi- dues away; this bridge (the so-called ‘hydrocarbon sta- ple’) acts by restricting the conformational freedom of CAI. The sequence of NYAD-1 is: ITFXDLLXYY GKKK, where X is the nonstandard amino acid (S)-2- A B C Fig. 2. The complexes of CTD with different peptides and organic molecules. (A) Main panel: structure of CTD (green) in complex with the peptide CAI (purple) (accession no. 2buo) [28,30]; the helices are named after the elements of structure in the wild-type CTD in Fig. 1. Inset: the interface region is shown with residues Phe3 (CAI) and Glu180 and Asn183 (CTD) indicated (the labels are in different colours: blue for protein residues and black for the peptide amino acids); for the sake of clarity, only these two residues of the CTD are shown. (B) Main panel: structure of CTD (green) in complex with the peptide NYAD-1 (cyan) (accession no. 2k1c) [33]; the helices are named after the elements of structure in the wild-type CTD in Fig. 1. Inset: the interface region is shown with residues Phe3 (CAI) and Asn183 (CTD) indicated (the labels are in dif- ferent colours); for clarity, only Asn183 is shown. (C) The structure of NTD in complex with the small organic molecule CAP-1 (accession no. 2pxr) [36]; the helices are in red, the b-sheets in yellow and the loops in green. The circle at the bottom of the figure indicates the region where the binding of CAP-1 occurs. The figures were produced using PYMOL (http://www.pymol.org) [39]. CA small-molecule interactions J. L. Neira 6114 FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS (2¢-pentenyl) alanine, which works as the ‘staple’ between both residues of the peptide; that staple makes CAI more helical. The NYAD-1 peptide is able to penetrate cells and disrupts the assembly of both immature- and mature- like virus particles in cell-free and cell-based systems in vitro [32]. The NYAD-1 peptide demonstrates enhanced helicity when isolated in solution, which appears to suggest that the higher the helicity, the higher the inhibition of assembly. The affinity of NAYD-1 for CTD is the largest reported to date, with values close to 1 lm. The difference in affinity from the original wild-type CAI (approximately 15 lm; see above) and with the CTD domain itself can be explained by the higher helicity of the isolated peptide (i.e. the NYAD-1 peptide, unlike CAC1, CAI and even the CTD monomer, does not have to pay such a large entropy penalty during binding to CTD because it is already helically structured). Zhang et al. [32] have also shown by using 15 N- HSQC-NMR that the binding site of NAYD-1 to the protein encompasses residues Phe169 to Val191, desta- bilizing rather than dissociating the nonmutated dimeric CTD (i.e. NYAD-1 does not disrupt the dimeric structure of the wild-type CTD). The residues observed in the binding region are the same as in CAI (see above). Furthermore, Zhang et al. [32] have solved the solution structure of the monomeric double mutant CTDW184A ⁄ M185A in complex with NYAD-1 (Fig. 2B, main panel). In the structure, the a-helix 9 is fully formed, with the kink present at the Thr188 [33], in contrast to what happens with the structure of the isolated monomeric double mutant [34]; thus, NYAD- 1 makes the dimerization helix of CTD adopt a more native-like structure (if we assume native-like structure to comprise that shown by each of the monomers in the wild-type dimeric CTD). The peptide binds to a hydrophobic pocket formed by residues of the four helices, and it adopts a helical conformation (as CAI) where its hydrophobic side chains (especially Phe3) make extensive contacts with specific hydrophobic patches of the double monomeric mutant (Fig. 2B, inset). When a comparison of the NMR structure of the complex NYAD-1-CTDW184A ⁄ M185A with the X-ray structure of CAI-CTDW184A ⁄ M185A is carried out, the differences are mostly observed in the movement of a-helix 9 (the dimerization helix in the wild-type nonmutated CTD); this helix is pushed away much further from its original wild-type position in the design of Stitch et al. [27] (6 A ˚ ) than in that of Zhang et al. [32] (3 A ˚ ). It is tempting to suggest that this short movement is also responsible for the larger affinity constant of NYAD-1. Although the ability of NYAD-1 to dismantle HIV-1 CA tubes has not been tested, by analogy to the CAI peptide [31], it is likely to have similar activity. In summary, NYAD-1, a variant of CAI, is the first peptide able to interfere with CA interactions during HIV-1 assembly (both mature- and immature-like virus particles), penetrate cells and inhibit the infectivity of HIV-1. Small organic molecules as inhibitors of CTD self-assembly The first breakthrough in identifying small-molecule inhibitors of HIV-1 assembly was reported by Tang et al. [35], with molecules not designed specifically against CTD, but rather the whole CA. The first mole- cule, N-(3-chloro-4-methylphenyl)-N¢-{2-[({5-[(dimeth- ylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) (Scheme 1), has dose-dependent inhibition in viral infectivity assays, and its affinity for the NTD of CA is approximately 800 lm. Both X-ray and NMR show that CAP-1 binds to the full-length CA, in a pocket formed at the point where helices 1, 2, 4 and 7 of the NTD interact [36] (interestingly, this region is spatially close to the groove created by a-helices 8 and 9 of CTD, where CAI binds, see above) (Fig. 2C). The protein undergoes a remarkable conformational change as CAP-1 binds with the aromatic residue of Phe32, which is moved away from its buried position in the protein. This movement creates a large cavity where the aromatic ring of CAP-1 inserts; the urea and dimethyla- monium groups of CAP-1 also make contacts with the side chains and ⁄ or backbones of several residues in the protein. Thus, most of the contacts of CAP-1 with CTD are hydrophobic, similar to the peptides (see above). Scheme 1. The CAP-1 and CAP-2 comp- unds designed by Tang et al. [35] and Kelly et al. [36]. J. L. Neira CA small-molecule interactions FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6115 Helices 4 and 7 of NTD form the interface required to create a stable hexagonal CA lattice of the virion capsid [37,38], and these helices pack against a-helices 8, 9 (the dimerization helix) and 11 of the CTD domain. Thus, CAP-1 does not inhibit capsid assem- bly through direct binding to CTD but rather because it hampers the proper docking of both NTD and CTD. A second organic compound, CAP-2 (Scheme 1), was shown also to bind the NTD of CA, but it is cytotoxic and it was not investigated further [35]. Conclusions In summary, it appears that even closely designed pep- tides (such as CAI and NYAD-1) display subtle, but key structural differences that account for the different inhibition properties exhibited in vitro and in vivo.Itis also clear that there are multiple ways to disrupt (or better destabilize) the dimeric CTD because all the peptide designs suggest slightly different modes of binding. These modes of binding appear to be related to: (a) the high flexibility of the a-helix-8- a-helix-9 interface (Fig. 1) and (b) a high number of hydropho- bic contacts through the aromatic moieties of the pep- tides (the hydrophobic contribution also appears to be important in binding of CAP-1). Rather than targeting dimerization of the CTD directly, studies with this molecule demonstrate that another effective strategy for disrupting the assembly of the virion capsid involves targeting the whole CA protein and regions close to the CTD. Thus, these peptides and small organic molecules, together with the structures of the complexes that they form with CA, provide an impor- tant basis for the design of new anti-HIV-1 agents aimed at inhibiting capsid assembly in vivo. Acknowledgements Both referees are thanked for their helpful suggestions and discussion. This work was supported by grants from Ministerio de Sanidad y Consumo (FIS 01⁄ 0004-02), Ministerio Ciencia e Innovacio ´ n (SAF 2008-05742-C02-01; CSD2008-00005), FIPSE private Foundation (Exp: 36557⁄ 06) and Generalitat Valenci- ana (ACOMP ⁄ 2009⁄ 185). Drs Mauricio G. Mateu and Francisco N. Barrera are thanked for critically reading the manuscript and for their collaboration over all these years. Dr Anjali P. 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Cell 131, 17–19. 38 Ganser-Pornillos BK, Cheng A & Yeager M (2007) Structure of a full-length HIV-1 CA: a model for the mature capsid lattice. Cell 131, 70–79. 39 DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA. J. L. Neira CA small-molecule interactions FEBS Journal 276 (2009) 6110–6117 ª 2009 The Author Journal compilation ª 2009 FEBS 6117 . 2009) doi:10.1111/j.1742-4658.2009.07314.x The mature capsid of human immunodeficiency virus, HIV-1, is formed by the assembly of copies of a capsid protein (CA). The C-terminal domain of CA,. MINIREVIEW The capsid protein of human immunodeficiency virus: designing inhibitors of capsid assembly Jose ´ L. Neira 1,2 1 Instituto