A peptide inhibitor of HIV-1 assembly in vitro

Formation of infectious HIV-1 involves assembly of Gag polyproteins into immature particles and subsequent assembly of mature capsids after proteolytic disassembly of the Gag shell. We report a 12-mer peptide, capsid assembly inhibitor (CAI), that binds the capsid (CA) domain of Gag and inhibits assembly of immature- and mature-like capsid particles in vitro. CAI was identified by phage display screening among a group of peptides with similar sequences that bind to a single reactive site in CA. Its binding site was mapped to CA residues 169-191, with an additional contribution from the last helix of CA. This result was confirmed by a separate X-ray structure analysis showing that CAI inserts into a conserved hydrophobic groove and alters the CA dimer interface. The CAI binding site is a new target for antiviral development, and CAI is the first known inhibitor directed against assembly of immature HIV-1.     

Larger helical populations in peptides derived from the dimerization helix of the capsid protein of HIV-1 results in peptide binding toward regions other than the "hotspot" interface

The C-terminal domain (CTD) of the capsid protein (CA) of HIV-1 participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization to form the viral capsid. Intact CA and the CTD are able to homodimerize with similar affinity (~15 μM); CTD homodimerization involves mainly an α-helical region. We have designed peptides derived from that helix with predicted higher helical propensities than the wild-type sequence while keeping residues important for dimerization. These peptides showed a higher helicity than that of the wild-type peptide, although not as high as theoretically predicted, and proved to be able to self-associate with apparent affinities similar to that of the whole CTD. However, binding to CTD mainly occurs at the last helical region of the protein. Accordingly, most of those peptides are unable to inhibit CA polymerization in vitro. Therefore, there is a subtle tuning between monomer-monomer interactions important for CTD dimerization and the maximal helical content achieved by the wild-type sequence of the interface.     

Discovery of dual inhibitors targeting both HIV-1 capsid and human cyclophilin A to inhibit the assembly and uncoating of the viral capsid

HIV-1 assembly and disassembly (uncoating) processes are critical for the HIV-1 replication. HIV-1 capsid (CA) and human cyclophilin A (CypA) play essential roles in these processes. We designed and synthesized a series of thiourea compounds as HIV-1 assembly and disassembly dual inhibitors targeting both HIV-1 CA protein and human CypA. The SIV-induced syncytium antiviral evaluation indicated that all of the inhibitors displayed antiviral activities in SIV-infected CEM cells at the concentration of 0.6–15.8 μM for 50% of maximum effective rate. Their abilities to bind CA and CypA were determined by ultraviolet spectroscopic analysis, fluorescence binding affinity and PPIase inhibition assay. Assembly studies in vitro demonstrated that the compounds could potently disrupt CA assembly with a dose-dependent manner. All of these molecules could bind CypA with binding affinities (Kd values) of 51.0–512.8 μM. Fifteen of the CypA binding compounds showed potent PPIase inhibitory activities (IC₅₀ values < 1 μM) while they could not bind either to HIV-1 Protease or to HIV-1 Integrase in the enzyme assays. These results suggested that 15 compounds could block HIV-1 replication by inhibiting the PPIase activity of CypA to interfere with capsid disassembly and disrupting CA assembly.     

Design of antiviral stapled peptides containing a biphenyl cross-linker

Here we report the design and synthesis of a panel of stapled peptides containing a distance-matching biphenyl cross-linker based upon a peptide capsid assembly inhibitor reported previously. Compared with the linear peptide, the biphenyl-stapled peptides exhibited significantly enhanced cell penetration and potent antiviral activity in the cell-based infection assays. Isothermal titration calorimetry and surface plasmon resonance experiments revealed that the most active stapled CAI peptide binds to the C-terminal domain of HIV capsid protein as well as envelop glycoprotein gp120 with low micromolar binding affinities, and as a result, inhibits both the HIV-1 virus entry and the virus assembly.     

The dimerization domain of the HIV-1 capsid protein binds a capsid protein-derived peptide: a biophysical characterization

The type 1 HIV presents a conical capsid formed by approximately 1500 units of the capsid protein, CA. Homodimerization of CA via its C-terminal domain, CA-C, constitutes a key step in virion assembly. CA-C dimerization is largely mediated by reciprocal interactions between residues of its second alpha-helix. Here, we show that an N-terminal-acetylated and C-terminal-amidated peptide, CAC1, comprising the sequence of the CA-C dimerization helix plus three flanking residues at each side, is able to form a complex with the entire CA-C domain. Thermal denaturation measurements followed by circular dichroism (CD), NMR, and size-exclusion chromatography provided evidence of the interaction between CAC1 and CA-C. The apparent dissociation constant of the heterocomplex formed by CA-C and CAC1 was determined by several biophysical techniques, namely, fluorescence (using an anthraniloyl-labeled peptide), affinity chromatography, and isothermal titration calorimetry. The three techniques yielded similar values for the apparent dissociation constant, in the order of 50 microM. This apparent dissociation constant was only five times higher than was the dissociation constant of both CA-C and the intact capsid protein homodimers (10 microM).     

Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein

The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal domain and dimerization of the C-terminal domain of CA (CAC), and both domains constitute potential targets for anti-HIV therapy. CAC homodimerization occurs mainly through its second helix, and it is abolished when its sole tryptophan is mutated to alanine. This mutant, CACW40A, resembles a transient monomeric intermediate formed during dimerization. Its tertiary structure is similar to that of the subunits in the dimeric, non-mutated CAC, but the segment corresponding to the second helix samples different conformations. The present study comprises a comprehensive examination of the CACW40A internal dynamics. The results obtained, with movements sampling a wide time regime (from pico- to milliseconds), demonstrate the high flexibility of the whole monomeric protein. The conformational exchange phenomena on the micro-to-millisecond time scale suggest a role for internal motions in the monomer-monomer interactions and, thus, flexibility of the polypeptide chain is likely to contribute to the ability of the protein to adopt different conformational states, depending on the biological environment.     

A trimer of dimers is the basic building block for human immunodeficiency virus-1 capsid assembly

Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 μM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small molecule CA binding agents may disturb this essential step in the HIV-1 life cycle.     

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.     

A cell-penetrating helical peptide as a potential HIV-1 inhibitor

The capsid domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is a critical determinant of virus assembly, and is therefore a potential target for developing drugs for AIDS therapy. Recently, a 12-mer α-helical peptide (CAI) was reported to disrupt immature- and mature-like capsid particle assembly in vitro; however, it failed to inhibit HIV-1 in cell culture due to its inability to penetrate cells. The same group reported the X-ray crystal structure of CAI in complex with the C-terminal domain of capsid (C-CA) at a resolution of 1.7 Å. Using this structural information, we have utilized a structure-based rational design approach to stabilize the α-helical structure of CAI and convert it to a cell-penetrating peptide (CPP). The modified peptide (NYAD-1) showed enhanced α-helicity. Experiments with laser scanning confocal microscopy indicated that NYAD-1 penetrated cells and colocalized with the Gag polyprotein during its trafficking to the plasma membrane where virus assembly takes place. NYAD-1 disrupted the assembly of both immature- and mature-like virus particles in cell-free and cell-based in vitro systems. NMR chemical shift perturbation analysis mapped the binding site of NYAD-1 to residues 169-191 of the C-terminal domain of HIV-1 capsid encompassing the hydrophobic cavity and the critical dimerization domain with an improved binding affinity over CAI. Furthermore, experimental data indicate that NYAD-1 most likely targets capsid at a post-entry stage. Most significantly, NYAD-1 inhibited a large panel of HIV-1 isolates in cell culture at low micromolar potency. Our study demonstrates how a structure-based rational design strategy can be used to convert a cell-impermeable peptide to a cell-permeable peptide that displays activity in cell-based assays without compromising its mechanism of action. This proof-of-concept cell-penetrating peptide may aid validation of capsid as an anti-HIV-1 drug target and may help in designing peptidomimetics and small molecule drugs targeted to this protein.     

The Maturational Refolding of the β-Hairpin Motif of Equine Infectious Anemia Virus Capsid Protein Extends Its Helix α1 at Capsid Assembly Locus

A retroviral capsid (CA) protein consists of two helical domains, CA^N and CA^C, which drive hexamer and dimer formations, respectively, to form a capsid lattice. The N-terminal 13 residues of CA refold to a β-hairpin motif upon processing from its precursor polyprotein Gag. The β-hairpin is essential for correct CA assembly but unexpectedly it is not within any CA oligomeric interfaces. To understand the β-hairpin function we studied the full-length CA protein from equine infectious anemia virus (EIAV), a lentivirus sharing the same cone-shaped capsid core as HIV-1. Solution NMR spectroscopy is perfectly suited to study EIAV-CA that dimerizes weaker than HIV-1-CA. Comparison between the wild-type (wt) EIAV-CA and a variant lacking the β-hairpin structure demonstrated that folding of the β-hairpin specifically extended the N terminus of helix α1 from Tyr²⁰ to Pro¹⁷. This coil to helix transition involves the conserved sequence of Thr¹⁶-Pro¹⁷-Arg¹⁸ (Ser¹⁶-Pro¹⁷-Arg¹⁸ in HIV-1-CA). The extended region of helix α1 constituted an expanded EIAV-CA^N oligomeric interface and overlapped with the HIV-1-CA hexamer-core residue Arg¹⁸, helical in structure and pivotal in assembly. Therefore we propose the function of the maturational refolding of the β-hairpin in CA assembly is to extend helix α1 at the N terminus to enhance the CA^N oligomerization along the capsid assembly core interface. In addition, NMR resonance line broadening indicated the presence of micro-millisecond exchange kinetics due to the EIAV-CA^N domain oligomerization, independent to the faster EIAV-CA^C domain dimerization.     

The isolated major homology region of the HIV capsid protein is mainly unfolded in solution and binds to the intact protein

Assembly of the mature human immunodeficiency virus type 1 (HIV-1) capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, the CA protein undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. The most conserved region of CA, the major homology region (MHR), is located in the C-terminal domain of CA (CTD). The MHR is involved in both immature and mature virus assembly; however, its exact function during both assembly stages is unknown. To test its conformational preferences and to provide clues on its role during CA assembly, we have used a minimalist approach by designing a peptide comprising the whole MHR (MHRpep, residues Asp152 to Ala174). Isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CTD with an affinity of ~30μM (as shown by fluorescence and ITC); the CTD binding region comprises residues belonging to α-helices 10 and 11. In the immature virus capsid, the MHR and α-helix 11 regions of two CTD dimers also interact [Briggs JAG, Riches JD, Glass B, Baratonova V, Zanetti G and Kr?usslich H-G (2009) Proc. Natl. Acad. Sci. USA 106, 11090-11095]. These results can be considered a proof-of-concept that the conformational preferences and binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.     

Structure of a monomeric mutant of the HIV-1 capsid protein

The capsid protein (CA) of HIV-1 plays a significant role in the assembly of the immature virion and is the critical building block of its mature capsid. Thus, there has been significant interest in the CA protein as a target in the design of inhibitors of early and late stage events in the HIV-1 replication cycle. However, because of its inherent flexibility from the interdomain linker and the monomer-dimer equilibrium in solution, the HIV-1 wild-type CA monomer has defied structural determinations by X-ray crystallography and nuclear magnetic resonance spectroscopy. Here we report the detailed solution structure of full-length HIV-1 CA using a monomeric mutant that, though noninfective, preserves many of the critical properties of the wild-type protein. The structure shows independently folded N-terminal (NTD) and C-terminal domains (CTD) joined by a flexible linker. The CTD shows some differences from that of the dimeric wild-type CTD structures. This study provides insights into the molecular mechanism of the wild-type CA dimerization critical for capsid assembly. The monomeric mutant allows investigation of interactions of CA with human cellular proteins exploited by HIV-1, directly in solution without the complications associated with the monomer-dimer equilibrium of the wild-type protein. This structure also permits the design of inhibitors directed at a novel target, viz., interdomain flexibility, as well as inhibitors that target multiple interdomain interactions critical for assembly and interactions of CA with host cellular proteins that play significant roles within the replication cycle of HIV-1.     

Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro capsid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemical series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analyses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electron microscopic (EM) studies showed that the BD compounds prevented virion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.     

Synthesis and antiviral activities of novel acylhydrazone derivatives targeting HIV-1 capsid protein

HIV-1 capsid protein (CA) plays important roles in the viral replication cycle. A number of acylhydrazone derivatives that act as inhibitors of HIV-1 CA assembly, were designed and synthesized. The synthesized compounds were tested for their antiviral activities and cytotoxicities using CEM cells. Some derivatives also were assayed for their ability to inhibit HIV-1 CA assembly in vitro. Among them, compounds 14f and 14i display the most promising potency with EC₅₀ values of 0.21 and 0.17 μΜ, respectively.     

Residues in the HIV-1 capsid assembly inhibitor binding site are essential for maintaining the assembly-competent quaternary structure of the capsid protein

Morphogenesis of infectious HIV-1 involves budding of immature virions followed by proteolytic disassembly of the Gag protein shell and subsequent assembly of processed capsid proteins (CA) into the mature HIV-1 core. The dimeric interface between C-terminal domains of CA (C-CA) has been shown to be important for both immature and mature assemblies. We previously reported a CA-binding peptide (CAI) that blocks both assembly steps in vitro. The three-dimensional structure of the C-CA/CAI complex revealed an allosteric effect of CAI that alters the C-CA dimer interface. Based on this structure, we now investigated the phenotypes of mutations in the binding pocket. CA variants carrying mutations Y169A, L211A, or L211S had a reduced affinity for CAI and were unable to form mature-like particles in vitro. These mutations also blocked morphological conversion to mature virions in tissue culture and abolished infectivity. X-ray crystallographic analyses of the variant C-CA domains revealed that these alterations induced the same allosteric change at the dimer interface observed in the C-CA/CAI complex. These results point to a role of key interactions between conserved amino acids in the CAI binding pocket of C-CA in maintaining the correct conformation necessary for mature core assembly.     

How to assemble a capsid

Retroviral capsids are composed of hexagonal arrays of the viral CA protein. In this issue of Cell, Ganser-Pornillos et al. (2007) provide a molecular model of the hexagonal HIV-1 CA lattice obtained from a new electron cryomicroscopic reconstruction. This study reveals the three principal stabilizing interfaces in the capsid lattice and explains how two different classes of inhibitors can block capsid assembly.     

Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.