Rous sarcoma virus Gag protein-oligonucleotide interaction suggests a critical role for protein dimer formation in assembly

The structural protein Gag is the only viral product required for retrovirus assembly. Purified Gag proteins or fragments of Gag are able in vitro to spontaneously form particles resembling immature virions, but this process requires nucleic acid, as well as the nucleocapsid domain of Gag. To examine the role of nucleic acid in the assembly in vitro, we used a purified, slightly truncated version of the Rous sarcoma virus Gag protein, Delta MBD Delta PR, and DNA oligonucleotides composed of the simple repeating sequence GT. Apparent binding constants were determined for oligonucleotides of different lengths, and from these values the binding site size of the protein on the DNA was calculated. The ability of the oligonucleotides to promote assembly in vitro was assessed with a quantitative assay based on electron microscopy. We found that excess zinc or magnesium ion inhibited the formation of virus-like particles without interfering with protein-DNA binding, implying that interaction with nucleic acid is necessary but not sufficient for assembly in vitro. The binding site size of the Delta MBD Delta PR protein, purified in the presence of EDTA to remove zinc ions at the two cysteine-histidine motifs, was estimated to be 11 nucleotides (nt). This value decreased to 8 nt when the protein was purified in the presence of low concentrations of zinc ions. The minimum length of DNA oligonucleotide that promoted efficient assembly in vitro was 22 nt for the zinc-free form of the protein and 16 nt for the zinc-bound form. To account for this striking 1:2 ratio between binding site size and oligonucleotide length requirement, we propose a model in which the role of nucleic acid in assembly is to promote formation of a species of Gag dimer, which itself is a critical intermediate in the polymerizaton of Gag to form the protein shell of the immature virion.     

Nucleic acid-independent retrovirus assembly can be driven by dimerization

The Gag protein of retroviruses alone can polymerize into regular virus-like particles (VLPs) both in vitro and in vivo. In most circumstances the capsid (CA) and nucleocapsid (NC) domains of Gag as well as some form of nucleic acid are required for this process. The mechanism by which NC-nucleic acid interaction promotes assembly has remained obscure. We show here that while deletion of the NC domain of Rous sarcoma virus Gag abolishes formation and budding of VLPs at the plasma membranes of baculovirus-infected insect cells, replacement of NC with a dimer-forming leucine zipper domain restores budding of spherical particles morphologically similar to wild-type VLPs. The positioning of the dimerization domain appears to be critical for proper assembly, as the insertion of a 5-amino-acid flexible linker upstream of the zipper domain leads to budding of tubular rather than spherical particles. Similar tubular particles are formed when the same linker is inserted upstream of NC. The tubes are morphologically distinct from tubes formed when the p10 domain upstream of CA is deleted. The fact that a foreign dimerization domain can functionally mimic NC suggests that the role of nucleic acid in retroviral assembly is not to serve as a scaffold but rather to promote the formation of Gag dimers, which are critical intermediates in the polymerization of the Gag shell.     

A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17.

We report single-point mutations that are located in the matrix protein domain of the gag gene of human immunodeficiency virus type 1 and that prevent Gag particle formation. We show that mutations of p17 that abolish human immunodeficiency virus particle assembly also prevent the dimerization of p17 protein, as measured directly by a protein-protein binding assay. In the three-dimensional structure of p17, mutations that abolish dimerization are located in a single alpha helix that forms part of a fingerlike projection from one side of the molecule. Peptides derived from this region of p17 also reduce the level of p17 dimer when they are added to p17-expressing cells and compete for p17 self-association when present in protein-protein binding assays. We propose that the dimerization of the Gag precursor that occurs by the interdigitation of alpha helices on adjacent matrix molecules is a key stage in virion assembly and that the prevention of such an interaction is the molecular basis of particle misassembly.     

Reversible binding of recombinant human immunodeficiency virus type 1 gag protein to nucleic acids in virus-like particle assembly in vitro

Recombinant human immunodeficiency virus type 1 (HIV-1) Gag protein can assemble into virus-like particles (VLPs) in suitable buffer conditions with nucleic acid. We have explored the role of nucleic acid in this assembly process. HIV-1 nucleocapsid protein, a domain of Gag, can bind to oligodeoxynucleotides with the sequence d(TG)(n) with more salt resistance than to d(A)(n) oligonucleotides. We found that assembly of VLPs on d(TG)(n) oligonucleotides was more salt resistant than assembly on d(A)(n); thus, the oligonucleotides do not simply neutralize basic residues in Gag but provide a binding surface upon which Gag molecules assemble into VLPs. We also found that Gag molecules could be "trapped" on internal d(TG)(n) sequences within 40-base oligonucleotides, rendering them unable to take part in assembly. Thus, assembly on oligonucleotides requires that Gag proteins bind near the ends of the nucleic acid, and binding of Gag to internal d(TG)(n) sequences is apparently cooperative. Finally, we showed that nucleic acids in VLPs can exchange with nucleic acids in solution; there is a hierarchy of preferences in these exchange reactions. The results are consistent with an equilibrium model of in vitro assembly and may help to explain how Gag molecules in vivo select genomic RNA despite the presence in the cell of a vast excess of cellular mRNA molecules.     

Characterization of Rous sarcoma virus Gag particles assembled in vitro

Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.     

Intermolecular interactions between retroviral Gag proteins in the nucleus

The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these “trapped” Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.     

In Vitro Assembly Properties of Human Immunodeficiency Virus Type 1 Gag Protein Lacking the p6 Domain

Human immunodeficiency virus type 1 (HIV-1) normally assembles into particles of 100 to 120 nm in diameter by budding through the plasma membrane of the cell. The Gag polyprotein is the only viral protein that is required for the formation of these particles. We have used an in vitro assembly system to examine the assembly properties of purified, recombinant HIV-1 Gag protein and of Gag missing the C-terminal p6 domain (Gag Δp6). This system was used previously to show that the CA-NC fragment of HIV-1 Gag assembled into cylindrical particles. We now report that both HIV-1 Gag and Gag Δp6 assemble into small, 25- to 30-nm-diameter spherical particles in vitro. The multimerization of Gag Δp6 into units larger than dimers and the formation of spherical particles required nucleic acid. Removal of the nucleic acid with NaCl or nucleases resulted in the disruption of the multimerized complexes. We conclude from these results that (i) N-terminal extension of HIV-1 CA-NC to include the MA domain results in the formation of spherical, rather than cylindrical, particles; (ii) nucleic acid is required for the assembly and maintenance of HIV-1 Gag Δp6 virus-like particles in vitro and possibly in vivo; (iii) a wide variety of RNAs or even short DNA oligonucleotides will support assembly; (iv) protein-protein interactions within the particle must be relatively weak; and (v) recombinant HIV-1 Gag Δp6 and nucleic acid are not sufficient for the formation of normal-sized particles.     

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.Expression of a single retroviral protein, Gag, in mammalian cells is sufficient for assembly of virus-like particles (VLPs). RNA seems to play an essential role, however, in both the assembly and structure of VLPs. Thus, retrovirus particles always contain RNA; in the absence of genomic RNA, cellular mRNAs replace it in the virus particle (46). RNase treatment of immature murine leukemia virus disrupts the particles (37). Finally, nucleic acid is required for assembly in defined in vitro assembly systems (8, 9).The contribution of nucleic acid to the assembly and structure of retrovirus particles is not yet understood. As one approach to further understanding the role that nucleic acid binding plays in the assembly process, Zhang et al. (59) replaced the principal nucleic acid-binding domain of the HIV-1 Gag protein, nucleocapsid (NC), with a leucine zipper domain. This chimeric protein was able to assemble efficiently in mammalian cells as evidenced through immunoblotting of released VLPs. This observation was extended by Johnson et al. (28), who used Gag-leucine zipper (dimerizing) chimeras of Rous sarcoma virus and studied the morphologies of the resulting particles. The particles assembled from the chimeric proteins were similar, although not identical, to those formed by wild-type (WT) Gag. The fact that NC could be functionally replaced (with respect to particle assembly) with the dimerizing leucine zipper motif led these investigators to propose that the function of nucleic acid in assembly is to promote dimerization. Additional support for this hypothesis comes from the fact that the minimum length of nucleic acid needed to promote assembly is roughly enough to accommodate two molecules of Gag (30, 31).Further studies in which the NC domain of HIV-1 Gag has been replaced by leucine zipper motifs have been presented by Accola et al. (1). Interestingly, they found that a Gag-Zipper (Gag-Z) chimera containing a trimeric zipper motif also assembles efficiently. However, these VLPs, as well as those formed by a chimera containing a dimeric zipper motif, were not characterized morphologically.In the present work, we have extended the analysis of the assembly properties of these HIV-1 Gag-Z chimeras. This study includes the first analysis of recombinant Gag-Z proteins in vitro, as well as detailed characterization of the VLPs formed in mammalian cells. The in vitro assembly results suggest that Gag oligomerization alone is not sufficient to induce particle formation. We raise the possibility here that normal HIV-1 assembly requires neutralization of positive charges in matrix (MA) in addition to nucleic acid-induced oligomerization at the C terminus of the protein.     

Solution structure of a double mutant of the carboxy-terminal dimerization domain of the HIV-1 capsid protein

As in other retroviruses, the HIV-1 capsid (CA) protein is composed of two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), joined by a flexible linker. The dimerization of the CTD is thought to be a critical step in the assembly of the immature and mature viral capsids. The precise nature of the functional form of CTD dimerization interface has been a subject of considerable interest. Previously, the CTD dimer was thought to involve a face-to-face dimerization observed in the early crystallographic studies. Recently, the crystallographic structure for a domain-swapped CTD dimer has been determined. This dimer, with an entirely different interface that includes the major homology region (MHR) has been suggested as the functional form during the Gag assembly. The structure determination of the monomeric wt CTD of HIV-1 has not been possible because of the monomer-dimer equilibrium in solution. We report the NMR structure of the [W184A/M185A]-CTD mutant in its monomeric form. These mutations interfere with dimerization without abrogating the assembly activity of Gag and CA. The NMR structure shows some important differences compared to the CTD structure in the face-to-face dimer. Notably, the helix-2 is much shorter, and the kink seen in the crystal structure of the wt CTD in the face-to-face dimer is absent. These NMR studies suggest that dimerization-induced conformational changes may be present in the two crystal structures of the CTD dimers and also suggest a mechanism that can simultaneously accommodate both of the distinctly different dimer models playing functional roles during the Gag assembly of the immature capsids.     

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.     

Structural and functional studies of an intermediate on the pathway to operator binding by Escherichia coli MetJ

We present the results of in vitro DNA-binding assays for a mutant protein (Q44K) of the E. coli methionine repressor, MetJ, as well as the crystal structure at 2.2 A resolution of the apo-mutant bound to a 10-mer oligonucleotide encompassing an 8 bp met-box sequence. The wild-type protein binds natural operators co-operatively with respect to protein concentration forming at least a dimer of repressor dimers along operator DNAs. The minimum operator length is thus 16 bp, each MetJ dimer interacting with a single met-box site. In contrast, the Q44K mutant protein can also bind stably as a single dimer to 8 bp target sites, in part due to additional contacts made to the phosphodiester backbone outside the 8 bp target via the K44 side-chains. Protein-protein co-operativity in the mutant is reduced relative to the wild-type allowing the properties of an intermediate on the pathway to operator site saturation to be examined for the first time. The crystal structure of the decamer complex shows a unique conformation for the protein bound to the single met-box site, possibly explaining the reduced protein-protein co-operativity. In both the extended and minimal DNA complexes formed, the mutant protein makes slightly different contacts to the edges of DNA base-pairs than the wild-type, even though the site of amino acid substitution is distal from the DNA-binding motif. Quantitative binding assays suggest that this is not due to artefacts caused by the crystallisation conditions but is most likely due to the relatively small contribution of such direct contacts to the overall binding energy of DNA-protein complex formation, which is dominated by sequence-dependent distortions of the DNA duplex and by the protein-protein contact between dimers.     

Measuring the Binding Stoichiometry of HIV-1 Gag to Very-Low-Density Oligonucleotide Surfaces Using Surface Plasmon Resonance Spectroscopy

The interaction of the HIV Gag polyprotein with nucleic acid is a critical step in the assembly of viral particles. The Gag polyprotein is composed of the matrix (MA), capsid (CA), and nucleocapsid (NC) domains. The NC domain is required for nucleic acid interactions, and the CA domain is required for Gag-Gag interactions. Previously, we have investigated the binding of the NC protein to d(TG)(n) oligonucleotides using surface plasmon resonance (SPR) spectroscopy. We found a single NC protein is able to bind to more than one immobilized oligonucleotide, provided that the oligonucleotides are close enough together. As NC is believed to be the nucleic acid binding domain of Gag, we might expect Gag to show the same complex behavior. We wished to analyze the stoichiometry of Gag binding to oligonucleotides without this complication due to tertiary complex formation. We have therefore analyzed Gag binding to extremely low oligonucleotide density on SPR chips. Such low densities of oligonucleotides are difficult to accurately quantitate. We have determined by Fourier transform ion cyclotron (FTICR) mass spectrometry that four molecules of NC bind to d(TG)(10) (a 20-base oligonucleotide). We developed a method of calibrating low-density surfaces using NC calibration injections. Knowing the maximal response and the stoichiometry of binding, we can precisely determine the amount of oligonucleotide immobilized at these very-low-density surfaces (<1 Response Unit). Using this approach, we have measured the binding of Gag to d(TG)(10). Gag binds to a 20-mer with a stoichiometry of greater than 4. This suggests that once Gag is bound to the immobilized oligonucleotide, additional Gag molecules can bind to this complex.     

Placement of leucine zipper motifs at the carboxyl terminus of HIV-1 protease significantly reduces virion production

Natural HIV-1 protease (PR) is homodimeric. Some researchers believe that interactions between HIV-1 Gag-Pol molecules trigger the activation of embedded PR (which mediates Gag and Gag-Pol cleavage), and that Gag-Pol assembly domains outside of PR may contribute to PR activation by influencing PR dimer interaction in a Gag-Pol context. To determine if the enhancement of PR dimer interaction facilitates PR activation, we placed single or tandem repeat leucine zippers (LZ) at the PR C-terminus, and looked for a correlation between enhanced Gag processing efficiency and increased Gag-PR-LZ multimerization capacity. We found significant reductions in virus-like particles (VLPs) produced by HIV-1 mutants, with LZ fused to the end of PR as a result of enhanced Gag cleavage efficiency. Since VLP production can be restored to wt levels following PR activity inhibition, this assembly defect is considered PR activity-dependent. We also found a correlation between the LZ enhancement effect on Gag cleavage and enhanced Gag-PR multimerization. The results suggest that PR dimer interactions facilitated by forced Gag-PR multimerization lead to premature Gag cleavage, likely a result of premature PR activation. Our conclusion is that placement of a heterologous dimerization domain downstream of PR enhances PR-mediated Gag cleavage efficiency, implying that structural conformation, rather than the primary sequence outside of PR, is a major determinant of HIV-1 PR activation.     

Analysis of human immunodeficiency virus type 1 Gag dimerization-induced assembly

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.     

Interactions of HIV-1 Gag with assembly cofactors

HIV-1 Gag is the only protein required for retroviral particle assembly. There is evidence suggesting that phosphatidylinositol phosphate and nucleic acid are essential for viruslike particle assembly. To elucidate structural foundations of interactions of HIV-1 Gag with the assembly cofactors PI(4,5)P2 and RNA, we employed mass spectrometric protein footprinting. In particular, the NHS-biotin modification approach was used to identify the lysine residues that are exposed to the solvent in free Gag and are protected from biotinylation by direct protein-ligand or protein-protein contacts in Gag complexes with PI(4,5)P2 and/or RNA. Of 21 surface lysines readily modified in free Gag, only K30 and K32, located in the matrix domain, were strongly protected in the Gag-PI(4,5)P2 complex. Nucleic acid also protected these lysines, but only at significantly higher concentrations. In contrast, nucleic acids and not PI(4,5)P2 exhibited strong protection of two nucleocapsid domain residues: K391 and K424. In addition, K314, located in the capsid domain, was specifically protected only in the presence of both PI(4,5)P2 and nucleic acid. We suggest that concerted binding of PI(4,5)P2 and nucleic acid to the matrix and nucleocapsid domains, respectively, promotes protein-protein interactions involving capsid domains. These protein-protein interactions must be involved in virus particle assembly.     

Functional Redundancy in HIV-1 Viral Particle Assembly

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.     

HIV Gag-leucine zipper chimeras form ABCE1-containing intermediates and RNase-resistant immature capsids similar to those formed by wild-type HIV-1 Gag

During HIV-1 assembly, Gag polypeptides multimerize to form an immature capsid and also package HIV-1 genomic RNA. Assembling Gag forms immature capsids by progressing through a stepwise pathway of assembly intermediates containing the cellular ATPase ABCE1, which facilitates capsid formation. The NC domain of Gag is required for ABCE1 binding, acting either directly or indirectly. NC is also critical for Gag multimerization and RNA binding. Previous studies of GagZip chimeric proteins in which NC was replaced with a heterologous leucine zipper that promotes protein dimerization but not RNA binding established that the RNA binding properties of NC are dispensable for capsid formation per se. Here we utilized GagZip proteins to address the question of whether the RNA binding properties of NC are required for ABCE1 binding and for the formation of ABCE1-containing capsid assembly intermediates. We found that assembly-competent HIV-1 GagZip proteins formed ABCE1-containing intermediates, while assembly-incompetent HIV-1 GagZip proteins harboring mutations in residues critical for leucine zipper dimerization did not. Thus, these data suggest that ABCE1 does not bind to NC directly or through an RNA bridge, and they support a model in which dimerization of Gag, mediated by NC or a zipper, results in exposure of an ABCE1-binding domain located elsewhere in Gag, outside NC. Additionally, we demonstrated that immature capsids formed by GagZip proteins are insensitive to RNase A, as expected. However, unexpectedly, immature HIV-1 capsids were almost as insensitive to RNase A as GagZip capsids, suggesting that RNA is not a structural element holding together immature wild-type HIV-1 capsids.     

Mutation of dileucine-like motifs in the human immunodeficiency virus type 1 capsid disrupts virus assembly, gag-gag interactions, gag-membrane binding, and virion maturation

The human immunodeficiency virus type 1 (HIV-1) Gag precursor protein Pr55(Gag) drives the assembly and release of virus-like particles in the infected cell. The capsid (CA) domain of Gag plays an important role in these processes by promoting Gag-Gag interactions during assembly. The C-terminal domain (CTD) of CA contains two dileucine-like motifs (L189/L190 and I201/L202) implicated in regulating the localization of Gag to multivesicular bodies (MVBs). These dileucine-like motifs are located in the vicinity of the CTD dimer interface, a region of CA critical for Gag-Gag interactions during virus assembly and CA-CA interactions during core formation. To study the importance of the CA dileucine-like motifs in various aspects of HIV-1 replication, we introduced a series of mutations into these motifs in the context of a full-length, infectious HIV-1 molecular clone. CA mutants LL189,190AA and IL201,202AA were both severely impaired in virus particle production because of a variety of defects in the binding of Gag to membrane, Gag multimerization, and CA folding. In contrast to the model suggesting that the CA dileucine-like motifs regulate MVB targeting, the IL201,202AA mutation did not alter Gag localization to the MVB in either HeLa cells or macrophages. Revertants of single-amino-acid substitution mutants were obtained that no longer contained dileucine-like motifs but were nevertheless fully replication competent. The varied phenotypes of the mutants reported here provide novel insights into the interplay among Gag multimerization, membrane binding, virus assembly, CA dimerization, particle maturation, and virion infectivity.     

New roles for key residues in helices H1 and H2 of the Escherichia coli H-NS N-terminal domain: H-NS dimer stabilization and Hha binding

Bacterial nucleoid-associated proteins H-NS and Hha modulate gene expression in response to environmental factors. The N-terminal domain of H-NS is involved in homomeric and heteromeric protein-protein interactions. Homomeric interaction leads to the formation of dimers and higher oligomers. Heteromeric interactions with Hha-like proteins modify the modulatory properties of H-NS. In this study, we have used NMR and mutagenesis of the N-terminal domain of H-NS to identify the Hha-binding region around helices H1 and H2 of H-NS. Two conserved arginine residues, R12 and R15, located in the same side and in adjacent turns of helix H2 are shown to be involved in two different protein-protein interactions: R12 is essential for Hha binding and does not affect H-NS dimer formation, and R15 does not affect Hha binding but is essential for the proper folding of H-NS dimers. Our results demonstrate a close structural connection between Hha-H-NS interactions and H-NS dimerization that may be involved in a possible mechanism for the modulation of the H-NS regulatory activity by Hha.