Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells

The major structural elements of retroviruses are contained in a single polyprotein, Gag, which in human immunodeficiency virus type 1 (HIV-1) comprises the MA, CA, spacer peptide 1 (SP1), NC, SP2, and p6 polypeptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the N-terminal MA domain at the membrane and C-terminal NC-SP2-p6 region nearest to the center. Here, we report the three-dimensional structures of individual immature HIV-1 virions, as obtained by electron cryotomography. The concentric shells of the Gag polyprotein are clearly visible, and radial projections of the different Gag layers reveal patches of hexagonal order within the CA and SP1 shells. Averaging well-ordered unit cells leads to a model in which each CA hexamer is stabilized by a bundle of six SP1 helices. This model suggests why the SP1 spacer is essential for assembly of the Gag lattice and how cleavage between SP1 and CA acts as a structural switch controlling maturation.     

Role of the SP2 Domain and Its Proteolytic Cleavage in HIV-1 Structural Maturation and Infectivity

HIV-1 buds as an immature, noninfectious virion. Proteolysis of its main structural component, Gag, is required for morphological maturation and infectivity and leads to release of four functional domains and the spacer peptides SP1 and SP2. The N-terminal cleavages of Gag and the separation of SP1 from CA are all essential for viral infectivity, while the roles of the two C-terminal cleavages and the role of SP2, separating the NC and p6 domains, are less well defined. We have analyzed HIV-1 variants with defective cleavage at either or both sites flanking SP2, or largely lacking SP2, regarding virus production, infectivity, and structural maturation. Neither the presence nor the proteolytic processing of SP2 was required for particle release. Viral infectivity was almost abolished when both cleavage sites were defective and severely reduced when the fast cleavage site between SP2 and p6 was defective. This correlated with an increased proportion of irregular core structures observed by cryo-electron tomography, although processing of CA was unaffected. Mutation of the slow cleavage site between NC and SP2 or deletion of most of SP2 had only a minor effect on infectivity and did not induce major alterations in mature core morphology. We speculate that not only separation of NC and p6 but also the processing kinetics in this region are essential for successful maturation, while SP2 itself is dispensable.     

Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation

The human immunodeficiency virus type 1 (HIV-1) protease (PR) has recently been shown to be inhibited by its propeptide p6* in vitro. As p6* itself is a PR substrate, the primary goal of this study was to determine the importance of p6* cleavage for HIV-1 maturation and infectivity. For that purpose, short peptide variants mimicking proposed cleavage sites within and flanking p6* were designed and analyzed for qualitative and quantitative hydrolysis in vitro. Proviral clones comprising the selected cleavage site mutations were established and analyzed for Gag and Pol processing, virus maturation, and infectivity in cultured cells. Amino-terminal cleavage site mutation caused aberrant processing of nucleocapsid proteins and delayed replication kinetics. Blocking the internal cleavage site resulted in the utilization of a flanking site at a significantly decreased hydrolysis rate in vitro, which however did not affect Gag-Pol processing and viral replication. Although mutations blocking cleavage at the p6* carboxyl terminus yielded noninfectious virions exhibiting severe Gag processing defects, mutations retarding hydrolysis of this cleavage site neither seemed to impact viral infectivity and propagation in cultured cells nor seemed to interfere with overall maturation of released viruses. Interestingly, these mutants were shown to be clearly disadvantaged when challenged with wild-type virus in a dual competition assay. In sum, we conclude that p6* cleavage is absolutely essential to allow complete activation of the PR and subsequent processing of the viral precursors.     

Sequential Steps in Human Immunodeficiency Virus Particle Maturation Revealed by Alterations of Individual Gag Polyprotein Cleavage Sites

Retroviruses are produced as immature particles containing structural polyproteins, which are subsequently cleaved by the viral proteinase (PR). Extracellular maturation leads to condensation of the spherical core to a capsid shell formed by the capsid (CA) protein, which encases the genomic RNA complexed with nucleocapsid (NC) proteins. CA and NC are separated by a short spacer peptide (spacer peptide 1 [SP1]) on the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein and released by sequential PR-mediated cleavages. To assess the role of individual cleavages in maturation, we constructed point mutations abolishing cleavage at these sites, either alone or in combination. When all three sites between CA and NC were mutated, immature particles containing stable CA-NC were observed, with no apparent effect on other cleavages. Delayed maturation with irregular morphology of the ribonucleoprotein core was observed when cleavage of SP1 from NC was prevented. Blocking the release of SP1 from CA, on the other hand, yielded normal condensation of the ribonucleoprotein core but prevented capsid condensation. A thin, electron-dense layer near the viral membrane was observed in this case, and mutant capsids were significantly less stable against detergent treatment than wild-type HIV-1. We suggest that HIV maturation is a sequential process controlled by the rate of cleavage at individual sites. Initial rapid cleavage at the C terminus of SP1 releases the RNA-binding NC protein and leads to condensation of the ribonucleoprotein core. Subsequently, CA is separated from the membrane by cleavage between the matrix protein and CA, and late release of SP1 from CA is required for capsid condensation.     

The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles.

The vpr gene of human immunodeficiency virus type 1 (HIV-1) encodes a virion-associated regulatory protein. Mutagenesis has shown that the virion association of Vpr requires sequences near the C terminus of the HIV-1 Gag polyprotein Pr55gag. To investigate whether Vpr incorporation is mediated by a specific domain of Pr55gag, we examined the ability of chimeric HIV-1/Moloney murine leukemia virus (MLV) Gag polyproteins to direct the incorporation of Vpr. Vpr expressed in trans did not associate with particles formed by the authentic MLV Gag polyprotein or with particles formed by chimeric Gag polyproteins that had the matrix (MA) or capsid (CA) domain of MLV precisely replaced by the corresponding domain of HIV-1HXB2. By contrast, Vpr was efficiently incorporated upon replacement of the C-terminal nucleocapsid (NC) domain of the MLV Gag polyprotein with HIV-1 p15 sequences. Vpr was also efficiently incorporated into particles formed by a MLV Gag polyprotein that had the HIV-1 p6 domain fused to its C terminus. Furthermore, a deletion analysis revealed that a conserved region near the C terminus of the p6 domain is essential for Vpr incorporation, whereas sequences downstream of the conserved region are dispensable. These results show that a virion association motif for Vpr is located within residues 1 to 46 of p6.     

Charged amino acid residues of human immunodeficiency virus type 1 nucleocapsid p7 protein involved in RNA packaging and infectivity.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Gag precursor polyprotein (Pr55Gag) with the viral genomic RNA is required for retroviral replication. Mutations that reduce RNA packaging efficiency have been localized to the highly basic nucleocapsid (NC) p7 domain of Pr55Gag, but the importance of the basic amino acid residues in specific viral RNA encapsidation and infectivity has not been thoroughly investigated in vivo. We have systematically substituted the positively charged residues of the NC domain of Pr55Gag in an HIV-1 viral clone by using alanine scanning mutagenesis and have assayed the effects of these mutations on virus replication, particle formation, and RNA packaging in vivo. Analysis of viral clones with single substitutions revealed that certain charged amino acid residues are more critical for RNA packaging efficiency and infectivity than others. Analysis of viral clones with multiple substitutions indicates that the presence of positive charge in each of three independent domains--the zinc-binding domains, the basic region that links them, and the residues that Hank the two zinc-binding domains--is necessary for efficient HIV-1 RNA packaging. Finally, we note that some mutations affect virus replication more drastically than RNA incorporation, providing in vivo evidence for the hypothesis that NC p7 may be involved in aspects of the HIV life cycle in addition to RNA packaging.     

HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice

Maturation of nascent virions, a key step in retroviral replication, involves cleavage of the Gag polyprotein by the viral protease into its matrix (MA), capsid (CA), and nucleocapsid (NC) components and their subsequent reorganization. Bevirimat (BVM) defines a new class of antiviral drugs termed maturation inhibitors. BVM acts by blocking the final cleavage event in Gag processing, the separation of CA from its C-terminal spacer peptide 1 (SP1). Prior evidence suggests that BVM binds to Gag assembled in immature virions, preventing the protease from accessing the CA-SP1 cleavage site. To investigate this hypothesis, we used cryo-electron tomography to examine the structures of (noninfectious) HIV-1 viral particles isolated from BVM-treated cells. We find that these particles contain an incomplete shell of density underlying the viral envelope, with a hexagonal honeycomb structure similar to the Gag lattice of immature HIV but lacking the innermost, NC-related, layer. We conclude that the shell represents a remnant of the immature Gag lattice that has been processed, except at the CA-SP1 sites, but has remained largely intact. We also compared BVM-treated particles with virions formed by the mutant CA5, in which cleavage between CA and SP1 is also blocked. Here, we find a thinner CA-related shell with no visible evidence of honeycomb organization, indicative of an altered conformation and further suggesting that binding of BVM stabilizes the immature lattice. In both cases, the observed failure to assemble mature capsids correlates with the loss of infectivity.     

Structural analysis of HIV-1 maturation using cryo-electron tomography

HIV-1 buds form infected cells in an immature, non-infectious form. Maturation into an infectious virion requires proteolytic cleavage of the Gag polyprotein at five positions, leading to a dramatic change in virus morphology. Immature virions contain an incomplete spherical shell where Gag is arranged with the N-terminal MA domain adjacent to the membrane, the CA domain adopting a hexameric lattice below the membrane, and beneath this, the NC domain and viral RNA forming a disordered layer. After maturation, NC and RNA are condensed within the particle surrounded by a conical CA core. Little is known about the sequence of structural changes that take place during maturation, however. Here we have used cryo-electron tomography and subtomogram averaging to resolve the structure of the Gag lattice in a panel of viruses containing point mutations abolishing cleavage at individual or multiple Gag cleavage sites. These studies describe the structural intermediates correlating with the ordered processing events that occur during the HIV-1 maturation process. After the first cleavage between SP1 and NC, the condensed NC-RNA may retain a link to the remaining Gag lattice. Initiation of disassembly of the immature Gag lattice requires cleavage to occur on both sides of CA-SP1, while assembly of the mature core also requires cleavage of SP1 from CA.     

Proteolytic processing of the p2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.     

Localization of the Vpx packaging signal within the C terminus of the human immunodeficiency virus type 2 Gag precursor protein.

Viral protein X (Vpx) is a human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus accessory protein that is packaged into virions in molar amounts equivalent to Gag proteins. To delineate the processes of virus assembly that mediate Vpx packaging, we used a recombinant vaccinia virus-T7 RNA polymerase system to facilitate Gag protein expression, particle assembly, and extracellular release. HIV genes were placed under control of the bacteriophage T7 promoter and transfected into HeLa cells expressing T7 RNA polymerase. Western immunoblot analysis detected p55gag and its cleavage products p39 and p27 in purified particles derived by expression of gag and gag-pol, respectively. In trans expression of vpx with either HIV-2 gag or gag-pol gave rise to virus-like particles that contained Vpx in amounts similar to that detected in HIV-2 virus produced from productively infected T cells. Using C-terminal deletion and truncation mutants of HIV-2 Gag, we mapped the p15 coding sequence for determinants of Vpx packaging. This analysis revealed a region (residues 439 to 497) downstream of the nucleocapsid protein (NC) required for incorporation of Vpx into virions. HIV-1/HIV-2 gag chimeras were constructed to further characterize the requirements for incorporation of Vpx into virions. Chimeric HIV-1/HIV-2 Gag particles consisting of HIV-1 p17 and p24 fused in frame at the C terminus with HIV-2 p15 effectively incorporate Vpx, while chimeric HIV-2/HIV-1 Gag particles consisting of HIV-2 p17 and p27 fused in frame at the C terminus with HIV-1 p15 do not. Expression of a 68-amino-acid sequence of HIV-2 containing residues 439 to 497 fused to the coding regions of HIV-1 p17 and p24 also produced virus-like particles capable of packaging Vpx in amounts similar to that of full-length HIV-2 Gag. Sucrose gradient analysis confirmed particle association of Vpx and Gag proteins. These results demonstrate that the HIV-2 Gag precursor (p55) regulates incorporation of Vpx into virions and indicates that the packaging signal is located within residues 439 to 497.     

Flexibility in the P2 domain of the HIV-1 Gag polyprotein

The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA(CTD)) through the NC domain of Gag (CA(CTD)-p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA(CTD) and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA(CTD) and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an alpha-helical conformation. The presence of a transient coil-to-helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA(CTD)-p2-NC forms dimers and self-associates with an equilibrium constant (Kd = 1.78 +/- 0.5 microM) similar to that observed for the intact capsid protein (Kd = 2.94 +/- 0.8 microM), suggesting that Gag self-association is not significantly influence by the P2 domain.     

Insulin-like growth factor II mRNA binding protein 1 associates with Gag protein of human immunodeficiency virus type 1, and its overexpression affects virus assembly

The assembly of human immunodeficiency virus type 1 (HIV-1) particles is driven by viral Gag protein. This function of Gag not only benefits from its self-multimerization property but also depends on its interaction with a number of cellular factors such as TSG101 and ALIX/AIP1 that promote virus budding and release from cell surfaces. However, interaction with Gag also allows some cellular factors such as APOBEC3G and Trim5alpha to access viral replication machinery and block viral replication. In this study, we report a new HIV-1 Gag-binding factor named insulin-like growth factor II mRNA binding protein 1 (IMP1). Gag-IMP1 interaction requires the second zinc finger of the nucleocapsid (NC) domain of Gag and the KH3 and KH4 domains of IMP1. A fourfold reduction of HIV-1 infectivity was seen with overexpression of the wild-type IMP1 and its mutant that is able to interact with Gag but not with overexpression of IMP1 mutants exhibiting Gag-binding deficiency. The decreased viral infectivity was further shown as a result of diminished viral RNA packaging, abrogated Gag processing on the cellular membranes, and impeded maturation of virus particles. Together, these results demonstrate that IMP1 interacts with HIV-1 Gag protein and is able to block the formation of infectious HIV-1 particles.     

The Effect of Inositol Hexakisphosphate on HIV-1 Particle Production and Infectivity can be Modulated by Mutations that Affect the Stability of the Immature Gag Lattice

The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.     

The relationship between HIV-1 genome RNA dimerization, virion maturation and infectivity

The relationship between virion protein maturation and genomic RNA dimerization of human immunodeficiency virus type 1 (HIV-1) remains incompletely understood. We have constructed HIV-1 Gag cleavage site mutants to enable the steady state observation of virion maturation steps, and precisely study Gag processing, RNA dimerization, virion morphology and infectivity. Within the virion maturation process, the RNA dimer stabilization begins during the primary cleavage (p2-NC) of Pr55 Gag. However, the primary cleavage alone is not sufficient, and the ensuing cleavages are required for the completion of dimerization. From our observations, the increase of cleavage products may not put a threshold on the transition from fragile to stable dimeric RNA. Most of the RNA dimerization process did not require viral core formation, and particle morphology dynamics during viral maturation did not completely synchronize with the transition of dimeric RNA status. Although the endogenous virion RT activity was fully acquired at the initial step of maturation, the following process was necessary for viral DNA production in infected cell, suggesting the maturation of viral RNA/protein plays critical role for viral infectivity other than RT process.     

Replacement of the P1 amino acid of human immunodeficiency virus type 1 Gag processing sites can inhibit or enhance the rate of cleavage by the viral protease

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag precursor is highly regulated, with differential rates of cleavage at the five major processing sites to give characteristic processing intermediates. We examined the role of the P1 amino acid in determining the rate of cleavage at each of these five sites by using libraries of mutants generated by site-directed mutagenesis. Between 12 and 17 substitution mutants were tested at each P1 position in Gag, using recombinant HIV-1 protease (PR) in an in vitro processing reaction of radiolabeled Gag substrate. There were three sites in Gag (MA/CA, CA/p2, NC/p1) where one or more substitutions mediated enhanced rates of cleavage, with an enhancement greater than 60-fold in the case of NC/p1. For the other two sites (p2/NC, p1/p6), the wild-type amino acid conferred optimal cleavage. The order of the relative rates of cleavage with the P1 amino acids Tyr, Met, and Leu suggests that processing sites can be placed into two groups and that the two groups are defined by the size of the P1' amino acid. These results point to a trans effect between the P1 and P1' amino acids that is likely to be a major determinant of the rate of cleavage at the individual sites and therefore also a determinant of the ordered cleavage of the Gag precursor.     

In vitro resistance to the human immunodeficiency virus type 1 maturation inhibitor PA-457 (Bevirimat)

3-O-(3',3'-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.     

Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.     

The structural biology of HIV assembly

HIV assembly and replication proceed through the formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in the assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting the formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.     

Cumulative mutations of ubiquitin acceptor sites in human immunodeficiency virus type 1 gag cause a late budding defect

The p6 domain of human immunodeficiency virus type 1 (HIV-1) Gag has long been known to be monoubiquitinated. We have previously shown that the MA, CA, and NC domains are also monoubiquitinated at low levels (E. Gottwein and H. G. Krausslich, J. Virol. 79:9134-9144, 2005). While several lines of evidence support a role for ubiquitin in virus release, the relevance of Gag ubiquitination is unclear. To directly address the function of Gag ubiquitination, we constructed Gag variants in which lysine residues in the NC, SP2, and p6 domains were mutated to arginine either in individual domains or in combination. Using these mutants, we showed that in addition to MA, CA, NC, and p6, SP2 is also mono- or di-ubiquitinated at levels comparable to those of the other domains. Replacement of all lysine residues in only one of the domains had minor effects on virus release, while cumulative mutations in NC and SP2 or in NC and p6 resulted in an accumulation of late budding structures, as observed by electron microscopy analysis. Strikingly, replacement of all lysine residues downstream of CA led to a significant reduction in virus release kinetics and a fivefold accumulation of late viral budding structures compared to wild-type levels. These results indicate that ubiquitination of lysine residues in Gag in the vicinity of the viral late domain is important for HIV-1 budding, while no specific lysine residue may be needed and individual domains can functionally substitute. This is consistent with Gag ubiquitination being functionally involved in a transient protein interaction network at the virus budding site.