Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.     

Multimerization of human immunodeficiency virus type 1 Gag promotes its localization to barges, raft-like membrane microdomains

The Gag polyprotein of human immunodeficiency virus type 1 (HIV-1) organizes the assembly of nascent virions at the plasma membrane of infected cells. Here we demonstrate that a population of Gag is present in distinct raft-like membrane microdomains that we have termed "barges." Barges have a higher density than standard rafts, most likely due to the presence of oligomeric Gag-Gag assembly complexes. The regions of the Gag protein responsible for barge targeting were mapped by examining the flotation behavior of wild-type and mutant proteins on Optiprep density gradients. N-myristoylation of Gag was necessary for association with barges. Removal of the NC and p6 domains shifted much of the Gag from barges into typical raft fractions. These data are consistent with a model in which multimerization of myristoylated Gag proteins drives association of Gag oligomers into raft-like barges. The functional significance of barge association was revealed by several lines of evidence. First, Gag isolated from virus-like particles was almost entirely localized in barges. Moreover, a comparison of wild-type Gag with Fyn(10)Gag, a chimeric protein containing the N-terminal sequence of Fyn, revealed that Fyn(10)Gag exhibited increased affinity for barges and a two- to fourfold increase in particle production. These results imply that association of Gag with raft-like barge membrane microdomains plays an important role in the HIV-1 assembly process.     

Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly

Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6(Gag) or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.     

Analysis of the initiating events in HIV-1 particle assembly and genome packaging

HIV-1 Gag drives a number of events during the genesis of virions and is the only viral protein required for the assembly of virus-like particles in vitro and in cells. Although a reasonable understanding of the processes that accompany the later stages of HIV-1 assembly has accrued, events that occur at the initiation of assembly are less well defined. In this regard, important uncertainties include where in the cell Gag first multimerizes and interacts with the viral RNA, and whether Gag-RNA interaction requires or induces Gag multimerization in a living cell. To address these questions, we developed assays in which protein crosslinking and RNA/protein co-immunoprecipitation were coupled with membrane flotation analyses in transfected or infected cells. We found that interaction between Gag and viral RNA occurred in the cytoplasm and was independent of the ability of Gag to localize to the plasma membrane. However, Gag:RNA binding was stabilized by the C-terminal domain (CTD) of capsid (CA), which participates in Gag-Gag interactions. We also found that Gag was present as monomers and low-order multimers (e.g. dimers) but did not form higher-order multimers in the cytoplasm. Rather, high-order multimers formed only at the plasma membrane and required the presence of a membrane-binding signal, but not a Gag domain (the CA-CTD) that is essential for complete particle assembly. Finally, sequential RNA-immunoprecipitation assays indicated that at least a fraction of Gag molecules can form multimers on viral genomes in the cytoplasm. Taken together, our results suggest that HIV-1 particle assembly is initiated by the interaction between Gag and viral RNA in the cytoplasm and that this initial Gag-RNA encounter involves Gag monomers or low order multimers. These interactions per se do not induce or require high-order Gag multimerization in the cytoplasm. Instead, membrane interactions are necessary for higher order Gag multimerization and subsequent particle assembly in cells.     

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.     

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.     

Evidence of a role for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery in HIV-1 assembly and release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.     

HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET

The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.     

A Bipartite Membrane-Binding Signal in the Human Immunodeficiency Virus Type 1 Matrix Protein Is Required for the Proteolytic Processing of Gag Precursors in a Cell Type-Dependent Manner

It is unclear whether proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) Gag protein is dependent on virus assembly at the plasma membrane. Mutations that prevent myristylation of HIV-1 Gag proteins have been shown to block virus assembly and release from the plasma membrane of COS cells but do not prevent processing of Gag proteins. In contrast, in HeLa cells similar mutations abolished processing of Gag proteins as well as virus production. We have now addressed this issue with CD4⁺ T cells, which are natural target cells of HIV-1. In these cells, myristylation of Gag proteins was required for proteolytic processing of Gag proteins and production of extracellular viral particles. This result was not due to a lack of expression of the viral protease in the form of a Gag-Pol precursor or a lack of interaction between unmyristylated Gag and Gag-Pol precursors. The processing defect of unmyristylated Gag was partially rescued ex vivo by coexpression with wild-type myristylated Gag proteins in HeLa cells. The cell type-dependent processing of HIV-1 Gag precursors was also observed when another part of the plasma membrane binding signal, a polybasic region in the matrix protein, was mutated. The processing of unmyristylated Gag precursors was inhibited in COS cells by HIV-1 protease inhibitors. Altogether, our findings demonstrate that the processing of HIV-1 Gag precursors in CD4⁺ T cells occurs normally at the plasma membrane during viral morphogenesis. The intracellular environment of COS cells presumably allows activation of the viral protease and proteolytic processing of HIV-1 Gag proteins in the absence of plasma membrane binding.     

Equine infectious anemia virus utilizes a YXXL motif within the late assembly domain of the Gag p9 protein.

We have previously demonstrated that the Gag p9 protein of equine infectious anemia virus (EIAV) is functionally homologous with Rous sarcoma virus (RSV) p2b and human immunodeficiency virus type 1 (HIV-1) p6 in providing a critical late assembly function in RSV Gag-mediated budding from transfected COS-1 cells (L. J. Parent et al., J. Virol. 69:5455-5460, 1995). In light of the absence of amino acid sequence homology between EIAV p9 and the functional homologs of RSV and HIV-1, we have now designed an EIAV Gag-mediated budding assay to define the late assembly (L) domain peptide sequences contained in the EIAV p9 protein. The results of these particle budding assays revealed that expression of EIAV Gag polyprotein in COS-1 cells yielded extracellular Gag particles with a characteristic density of 1.18 g/ml, while expression of EIAV Gag polyprotein lacking p9 resulted in a severe reduction in the release of extracellular Gag particles. The defect in EIAV Gag polyprotein particle assembly could be corrected by substituting either the RSV p2b or HIV-1 p6 protein for EIAV p9. These observations demonstrated that the L domains of EIAV, HIV-1, and RSV were interchangeable in mediating assembly of EIAV Gag particles in the COS-1 cell budding assay. To localize the L domain of EIAV p9, we next assayed the effects of deletions and site-specific mutations in the p9 protein on its ability to mediate budding of EIAV Gag particles. Analyses of EIAV Gag constructs with progressive N-terminal or C-terminal deletions of the p9 protein identified a minimum sequence of 11 amino acids (Q20N21L22Y23P24D25L26S27E28I29K30) capable of providing the late assembly function. Alanine scanning studies of this L-domain sequence demonstrated that mutations of residues Y23, P24, and L26 abrogated the p9 late budding function; mutations of other residues in the p9 L domain did not substantially affect the level of EIAV Gag particle assembly. These data indicate that the L domain in EIAV p9 utilizes a YXXL motif which we hypothesize may interact with cellular proteins to facilitate virus particle budding from infected cells.     

An assembly domain of the Rous sarcoma virus Gag protein required late in budding.

The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding.     

HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.

Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.     

Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.     

The effect of HIV-1 Gag myristoylation on membrane binding

During the viral life cycle, an HIV protein, Gag, assembles at the host membrane, specifically at lipid raft regions, at very high concentrations leading to viral particle budding. Gag is post-translationally modified with an N-terminal myristate group which is thought to target Gag to lipid rafts thus aiding in assembly. Here we have analyzed the membrane binding of myristoylated HIV-1 Gag and a non-myristoylated form of HIV-1 Gag to various membrane models. After assessing the extent of myristoylation by HPLC and radiometric assays, we compared membrane binding using fluorescence methods. We found that myristoylated Gag shows a greater than twofold increase in binding affinity to model rafts. A structural model to explain these results is presented.     

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.     

Specificity of plasma membrane targeting by the rous sarcoma virus gag protein

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.     

Role of HIV-1 Gag domains in viral assembly

After entry of the human immunodeficiency virus type 1 (HIV-1) into T cells and the subsequent synthesis of viral products, viral proteins and RNA must somehow find each other in the host cells and assemble on the plasma membrane to form the budding viral particle. In this general review of HIV-1 assembly, we present a brief overview of the HIV life cycle and then discuss assembly of the HIV Gag polyprotein on RNA and membrane substrates from a biochemical perspective. The role of the domains of Gag in targeting to the plasma membrane and the role of the cellular host protein cyclophilin are also reviewed.     

p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease.

The human immunodeficiency virus type 1 (HIV-1) Gag protein precursor, Pr55Gag, contains at its C-terminal end a proline-rich, 6-kDa domain designated p6. Two functions have been proposed for p6: incorporation of the HIV-1 accessory protein Vpr into virus particles and virus particle production. To characterize the role of p6 in the HIV-1 life cycle and to map functional domains within p6, we introduced a number of nonsense and single and multiple amino acid substitution mutations into p6. Following the introduction of the mutations into the full-length HIV-1 molecular clone pNL4-3, the effects on Gag protein expression and processing, virus particle production, and virus infectivity were analyzed. The production of mutant virus particles was also examined by transmission electron microscopy. The results indicate that (i) p6 is required for efficient virus particle production from a full-length HIV-1 molecular clone; (ii) a Pro-Thr-Ala-Pro sequence, located between residues 7 and 10 of p6, is critical for virus particle production; (iii) mutations outside the Pro-Thr-Ala-Pro motif have little or no effect on virus assembly and release; (iv) the p6 defect is manifested at a late stage in the budding process; and (v) mutations in p6 that severely reduce virion production in HeLa cells also block or significantly delay the establishment of a productive infection in the CEM (12D-7) T-cell line. We further demonstrate that mutational inactivation of the viral protease reverses the p6 defect, suggesting a functional linkage between p6 and the proteolytic processing of the Gag precursor protein during the budding of progeny virions.