Nucleic acid-induced dimerization of HIV-1 Gag protein.

HIV-1 Gag is a highly flexible multidomain protein that forms the protein lattice of the immature HIV-1 virion. In vitro, it reversibly dimerizes, but in the presence of nucleic acids (NAs), it spontaneously assembles into virus-like particles (VLPs). High-resolution structures have revealed intricate details of the interactions of the capsid (CA) domain of Gag and the flanking spacer peptide SP1 that stabilize VLPs, but much less is known about the assembly pathway and the interactions of the highly flexible NA-binding nucleocapsid (NC) domain. Here, using a novel hybrid fluorescence proximity/sedimentation velocity method in combination with calorimetric analyses, we studied initial binding events by monitoring the sizes and conformations of complexes of Gag with very short oligonucleotides. We observed that high-affinity binding of oligonucleotides induces conformational changes in Gag accompanied by the formation of complexes with a 2:1 Gag/NA stoichiometry. This NA-liganded dimerization mode is distinct from the widely studied dimer interface in the CA domain and from protein interactions arising in the SP1 region and may be mediated by protein-protein interactions localized in the NC domain. The formation of the liganded dimer is strongly enthalpically driven, resulting in higher dimerization affinity than the CA-domain dimer. Both detailed energetic and conformational analyses of different Gag constructs revealed modulatory contributions to NA-induced dimerization from both matrix and CA domains. We hypothesize that allosterically controlled self-association represents the first step of VLP assembly and, in concert with scaffolding along the NA, can seed the formation of two-dimensional arrays near the NA.

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation.

UNLABELLED: By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals--electrostatic, hydrophobic, and lipid-specific-to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE: Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane-bounded protein lattice that recruits genomic RNA into the virus and forms the shell of a budding immature viral capsid. In binding studies of HIV-1 Gag MA to model membranes with well-controlled lipid composition, we dissect the multiple interactions of the MA domain with its target membrane. This results in a detailed understanding of the thermodynamic aspects that determine membrane association, preferential lipid recruitment to the viral shell, and those aspects of Gag assembly into the membrane-bound protein lattice that are determined by MA.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

UNLABELLED: HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE: Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation.

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus.

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein.

UNLABELLED: Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactions in vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particles in vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interaction in vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. IMPORTANCE: Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.

Diverse interactions of retroviral Gag proteins with RNAs.

Retrovirus particles are constructed from a single virus-encoded protein, termed Gag. Given that assembly is an essential step in the viral replication cycle, it is a potential target for antiviral therapy. However, such an approach has not yet been exploited because of the lack of fundamental knowledge concerning the structures and interactions responsible for assembly. Assembling an infectious particle entails a remarkably diverse array of interactions, both specific and nonspecific, between Gag proteins and RNAs. These interactions are essential for the construction of the particle, for packaging of the viral RNA into the particle, and for placement of the primer for viral DNA synthesis. Recent results have provided some new insights into each of these interactions. In the case of HIV-1 Gag, it is clear that more than one domain of the protein contributes to Gag-RNA interaction.

A conformational transition observed in single HIV-1 Gag molecules during in vitro assembly of virus-like particles.

UNLABELLED: The conformational changes within single HIV-1 Gag molecules that occur during assembly into immature viruses are poorly understood. Using an in vitro assembly assay, it has been proposed that HIV-1 Gag undergoes a conformational transition from a compact conformation in solution to an extended rod-like conformation in virus-like particles (VLPs). Here we used single-molecule Förster resonance energy transfer (smFRET) to test this model by directly probing the conformation of single HIV-1 Gag molecules. We demonstrate that monomeric HIV-1 Gag lacking the p6 domain and the N-terminal myristoyl moiety is found in solution predominantly in a compact conformation. Gag in this conformation, and in the presence of nucleic acid, assembles into 30-nm-diameter particles. However, with the addition of inositol hexakisphosphate, Gag adopts a linear conformation and assembles into full-sized ∼100-to-150-nm-diameter VLPs. Parallel fluorescence correlation spectroscopy measurements show that this conformational transition occurs early in the assembly process when Gag oligomers are small, perhaps as early as upon dimerization. Thus, smFRET measurements confirm that HIV-1 Gag transitions from a compact to a linear conformation during the formation of VLPs. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid. IMPORTANCE: The establishment of single-molecule fluorescence techniques reveals the conformational state of individual HIV-1 Gag molecules prior to and during in vitro assembly into virus-like particles. The data demonstrate that Gag in distinct conformations forms particles with different morphologies. In the compact conformation, in the presence of nucleic acid, Gag forms spherical particles of a diameter of approximately 30 nm. In the extended conformation, Gag forms spherical virus-like particles of approximately 100-nm diameter. The adoption of the extended conformation required the presence of inositol hexakisphosphate in addition to nucleic acid. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.

Essential functions of inositol hexakisphosphate (IP6) in murine leukemia virus replication.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.IMPORTANCEInositol hexakisphosphate (IP6) is crucial for the assembly and replication of HIV-1. IP6 is packaged in HIV-1 particles and stabilizes the viral core enabling it to synthesize viral DNA early in viral infection. While its importance for HIV-1 is well established, its significance for other retroviruses is unknown. Here we report the role of IP6 in the gammaretrovirus, murine leukemia virus (MLV). We found that like HIV-1, MLV packages IP6, and as in HIV-1, IP6 stabilizes the MLV core thus promoting reverse transcription. Interestingly, we discovered a key difference in the role of IP6 in MLV versus HIV-1: while HIV-1 is not dependent upon IP6 levels in target cells, MLV replication is significantly reduced in IP6-deficient cell lines. We suggest that this difference in IP6 requirements reflects key differences between HIV-1 and MLV replication.

Essential functions of Inositol hexakisphosphate (IP6) in Murine Leukemia Virus replication.

We have investigated the function of inositol hexakisphosphate (IP6) and inositol pentakisphosphate (IP5) in the replication of murine leukemia virus (MLV). While IP6 is known to be critical for the life cycle of HIV-1, its significance in MLV remains unexplored. We find that IP6 is indeed important for MLV replication. It significantly enhances endogenous reverse transcription (ERT) in MLV. Additionally, a pelleting-based assay reveals that IP6 can stabilize MLV cores, thereby facilitating ERT. We find that IP5 and IP6 are packaged in MLV particles. However, unlike HIV-1, MLV depends upon the presence of IP6 and IP5 in target cells for successful infection. This IP6/5 requirement for infection is reflected in impaired reverse transcription observed in IP6/5-deficient cell lines. In summary, our findings demonstrate the importance of capsid stabilization by IP6/5 in the replication of diverse retroviruses; we suggest possible reasons for the differences from HIV-1 that we observed in MLV.

Human Immunodeficiency Virus Type 1 Gag Polyprotein Modulates Membrane Physical Properties like a Surfactant: Potential Implications for Virus Assembly.

Human immunodeficiency virus (HIV) assembly at an infected cell's plasma membrane requires membrane deformation to organize the near-spherical shape of an immature virus. While the cellular expression of HIV Gag is sufficient to initiate budding of virus-like particles, how Gag generates membrane curvature is not fully understood. Using highly curved lipid nanotubes, we have investigated the physicochemical basis of the membrane activity of recombinant nonmyristoylated Gag-Δp6. Gag protein, upon adsorption onto the membrane, resulted in the shape changes of both charged and uncharged nanotubes. This shape change was more pronounced in the presence of charged lipids, especially phosphatidylinositol bisphosphate (PI(4,5)P2). We found that Gag modified the interfacial tension of phospholipid bilayer membranes, as judged by comparison with the effects of amphipathic peptides and nonionic detergent. Bioinformatic analysis demonstrated that a region of the capsid and SP1 domains junction of Gag is structurally similar to the amphipathic peptide magainin-1. This region accounts for integral changes in the physical properties of the membrane upon Gag adsorption, as we showed with the synthetic CA-SP1 junction peptide. Phenomenologically, membrane-adsorbed Gag could diminish the energetic cost of increasing the membrane area in a way similar to foam formation. We propose that Gag acts as a surface-active substance at the HIV budding site that softens the membrane at the place of Gag adsorption, lowering the energy for membrane bending. Finally, our experimental data and theoretical considerations give a lipid-centric view and common mechanism by which proteins could bend membranes, despite not having intrinsic curvature in their molecular surfaces or assemblies.

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome.

All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs--only four nucleotides per genomic RNA--reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.

RNA aptamers directed to human immunodeficiency virus type 1 Gag polyprotein bind to the matrix and nucleocapsid domains and inhibit virus production.

Gag orchestrates the assembly and release of human immunodeficiency virus type 1 (HIV-1) particles. We explored here the potential of anti-Gag RNA aptamers to inhibit HIV-1 replication. In vitro, RNA aptamers raised against an HIV-1 Gag protein, lacking the N-terminal myristate and the C-terminal p6 (DP6-Gag), could bind to matrix protein (MA), nucleocapsid protein (NC), or entire DP6-Gag protein. Upon cotransfection with pNL4-3.Luc molecular clone into 293T cells, six of the aptamers caused mild inhibition (2- to 3-fold) in the extracellular capsid levels, and one aptamer displayed 20-fold inhibition. The reduction was not due to a release defect but reflected Gag mRNA levels. We hypothesized that the aptamers influence genomic RNA levels via perturbation of specific Gag-genomic RNA interactions. Binding studies revealed that the "NC-binders" specifically compete with the packaging signal (ψ) of HIV-1 for binding to DP6-Gag. Therefore, we tested the ability of two NC-binders to inhibit viruses containing ψ-region deletions (ΔSL1 or ΔSL3) and found that the NC-binders were no longer able to inhibit Gag synthesis. The inability of these aptamers to inhibit ψ-deleted viruses correlated with the absence of competition with the corresponding ψ transcripts lacking SL1 or SL3 for binding DP6-Gag in vitro. These results indicate that the NC-binding aptamers disrupt Gag-genomic RNA interaction and negatively affect genomic RNA transcription, processing, or stability. Our results reveal an essential interaction between HIV-1 Gag and the ψ-region that may be distinct from that which occurs during the encapsidation of genomic RNA. Thus, anti-Gag aptamers can be an effective tool to perturb Gag-genomic RNA interactions.

Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding.

Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA(3)(Lys) serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA(3)(Lys) placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA(3)(Lys) annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing.

Solution properties of murine leukemia virus gag protein: differences from HIV-1 gag.

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.

On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Electrostatic interactions and binding orientation of HIV-1 matrix studied by neutron reflectivity.

The N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions.

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein.

The HIV-2 genomic RNA serves both as a messenger for protein synthesis and as a genome for viral assembly and particle production. Our previous work has shown that the HIV-2 genomic RNA encodes two additional Gag proteins that are N-terminal truncated isoforms of the p57 Gag polyprotein. In this study, by the use of mono- and bicistronic RNAs we show that translation at the three AUGs is driven by three distinct and independent internal ribosome entry segments both in vitro and ex vivo. Furthermore we used the recombinant Gag and HIV-2 protease to show that, in vitro, translation is tightly regulated by these two viral proteins. This regulation is exerted both at the level of protein production and also on the selection of the AUG initiation site which changes the ratio at which the three different Gag isoforms are produced.

Assembly properties of human immunodeficiency virus type 1 Gag-leucine zipper chimeras: implications for retrovirus assembly.

Expression of the retroviral Gag protein leads to formation of virus-like particles in mammalian cells. In vitro and in vivo experiments show that nucleic acid is also required for particle assembly. However, several studies have demonstrated that chimeric proteins in which the nucleocapsid domain of Gag is replaced by a leucine zipper motif can also assemble efficiently in mammalian cells. We have now analyzed assembly by chimeric proteins in which nucleocapsid of human immunodeficiency virus type 1 (HIV-1) Gag is replaced by either a dimerizing or a trimerizing zipper. Both proteins assemble well in human 293T cells; the released particles lack detectable RNA. The proteins can coassemble into particles together with full-length, wild-type Gag. We purified these proteins from bacterial lysates. These recombinant "Gag-Zipper" proteins are oligomeric in solution and do not assemble unless cofactors are added; either nucleic acid or inositol phosphates (IPs) can promote particle assembly. When mixed with one equivalent of IPs (which do not support assembly of wild-type Gag), the "dimerizing" Gag-Zipper protein misassembles into very small particles, while the "trimerizing" protein assembles correctly. However, addition of both IPs and nucleic acid leads to correct assembly of all three proteins; the "dimerizing" Gag-Zipper protein also assembles correctly if inositol hexakisphosphate is supplemented with other polyanions. We suggest that correct assembly requires both oligomeric association at the C terminus of Gag and neutralization of positive charges near its N terminus.

Distinct Contributions of Different Domains within the HIV-1 Gag Polyprotein to Specific and Nonspecific Interactions with RNA.

Viral genomic RNA is packaged into virions with high specificity and selectivity. However, in vitro the Gag specificity towards viral RNA is obscured when measured in buffers containing physiological salt. Interestingly, when the binding is challenged by increased salt concentration, the addition of competing RNAs, or introducing mutations to Gag protein, the specificity towards viral RNA becomes detectable. The objective of this work was to examine the contributions of the individual HIV-1 Gag polyprotein domains to nonspecific and specific RNA binding and stability of the initial protein-RNA complexes. Using a panel of Gag proteins with mutations disabling different Gag-Gag or Gag-RNA interfaces, we investigated the distinct contributions of individual domains which distinguish the binding to viral and nonviral RNA by measuring the binding of the proteins to RNAs. We measured the binding affinity in near-physiological salt concentration, and then challenged the binding by increasing the ionic strength to suppress the electrostatic interactions and reveal the contribution of specific Gag-RNA and Gag-Gag interactions. Surprisingly, we observed that Gag dimerization and the highly basic region in the matrix domain contribute significantly to the specificity of viral RNA binding.