A Two-Pronged Structural Analysis of Retroviral Maturation Indicates that Core Formation Proceeds by a Disassembly-Reassembly Pathway Rather than a Displacive Transition

Retrovirus maturation involves sequential cleavages of the Gag polyprotein, initially arrayed in a spherical shell, leading to formation of capsids with polyhedral or conical morphology. Evidence suggests that capsids assemble de novo inside maturing virions from dissociated capsid (CA) protein, but the possibility persists of a displacive pathway in which the CA shell remains assembled but is remodeled. Inhibition of the final cleavage between CA and spacer peptide SP1/SP blocks the production of mature capsids. We investigated whether retention of SP might render CA assembly incompetent by testing the ability of Rous sarcoma virus (RSV) CA-SP to assemble in vitro into icosahedral capsids. Capsids were indeed assembled and were indistinguishable from those formed by CA alone, indicating that SP was disordered. We also used cryo-electron tomography to characterize HIV-1 particles produced in the presence of maturation inhibitor PF-46396 or with the cleavage-blocking CA5 mutation. Inhibitor-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. Some CA protein is generated but usually not enough for a mature core to assemble. We propose that inhibitors like PF-46396 bind to the Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. CA5 particles, which exhibit no cleavage at the CA-SP1 site, have spheroidal shells with relatively thin walls. It appears that this lattice progresses displacively toward a mature-like state but produces neither conical cores nor infectious virions. These observations support the disassembly-reassembly pathway for core formation.     

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.     

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.     

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.     

Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket

Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for in vitro. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.     

Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in?vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.     

Residual HIV-1 DNA Flap-independent nuclear import of cPPT/CTS double mutant viruses does not support spreading infection

Background

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1.

Results

In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly.

Conclusions

This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.     

The TY3 Gag3 spacer controls intracellular condensation and uncoating

Cells expressing the yeast retrotransposon Ty3 form concentrated foci of Ty3 proteins and RNA within which virus-like particle (VLP) assembly occurs. Gag3, the major structural protein of the Ty3 retrotransposon, is composed of capsid (CA), spacer (SP), and nucleocapsid (NC) domains analogous to retroviral domains. Unlike the known SP domains of retroviruses, Ty3 SP is highly acidic. The current studies investigated the role of this domain. Although deletion of Ty3 SP dramatically reduced retrotransposition, significant Gag3 processing and cDNA synthesis occurred. Mutations that interfered with cleavage at the SP-NC junction disrupted CA-SP processing, cDNA synthesis, and electron-dense core formation. Mutations that interfered with cleavage of CA-SP allowed cleavage of the SP-NC junction, production of electron-dense cores, and cDNA synthesis but blocked retrotransposition. A mutant in which acidic residues of SP were replaced with alanine failed to form both Gag3 foci and VLPs. We propose a speculative "spring" model for Gag3 during assembly. In the first phase during concentration of Gag3 into foci, intramolecular interactions between negatively charged SP and positively charged NC domains of Gag3 limit multimerization. In the second phase, the NC domain binds RNA, and the bound form is stabilized by intermolecular interactions with the SP domain. These interactions promote CA domain multimerization. In the third phase, a negatively charged SP domain destabilizes the remaining CA-SP shell for cDNA release.     

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we have used the single particle cryo-electron microscopy method to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 Å resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag lattice structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10–12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.     

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.     

Phosphorylation and proteolytic cleavage of gag proteins in budded simian immunodeficiency virus

The lentiviral Gag polyprotein (Pr55(Gag)) is cleaved by the viral protease during the late stages of the virus life cycle. Proteolytic cleavage of Pr55(Gag) is necessary for virion maturation, a structural rearrangement required for infectivity that occurs in budded virions. In this study, we investigate the relationship between phosphorylation of capsid (CA) domains in Pr55(Gag) and its cleavage intermediates and their cleavage by the viral protease in simian immunodeficiency virus (SIV). First, we demonstrate that phosphorylated forms of Pr55(Gag), several CA-containing cleavage intermediates of Pr55(Gag), and the free CA protein are detectable in SIV virions but not in virus-producing cells, indicating that phosphorylation of these CA-containing Gag proteins may require an environment that is unique to the virion. Second, we show that the CA domain of Pr55(Gag) can be phosphorylated in budded virus and that this phosphorylation does not require the presence of an active viral protease. Further, we provide evidence that CA domains (i.e., incompletely cleaved CA) are phosphorylated to a greater extent than free (completely cleaved) CA and that CA-containing Gag proteins can be cleaved by the viral protease in SIV virions. Finally, we demonstrate that Pr55(Gag) and several of its intermediates, but not free CA, are actively phosphorylated in budded virus. Taken together, these data indicate that, in SIV virions, phosphorylation of CA domains in Pr55(Gag) and several of its cleavage intermediates likely precedes the cleavage of these domains by the viral protease.     

Evidence for a stable interaction of gp41 with Pr55(Gag) in immature human immunodeficiency virus type 1 particles

Assembly of infectious human immunodeficiency virus type 1 (HIV-1) virions requires incorporation of the viral envelope glycoproteins gp41 and gp120. Several lines of evidence have suggested that the cytoplasmic tail of the transmembrane glycoprotein, gp41, associates with Pr55(Gag) in infected cells to facilitate the incorporation of HIV-1 envelope proteins into budding virions. However, direct evidence for an interaction between gp41 and Pr55(Gag) in HIV-1 particles has not been reported. To determine whether gp41 is associated with Pr55(Gag) in HIV-1 particles, viral cores were isolated from immature HIV-1 virions by sedimentation through detergent. The cores contained a major fraction of the gp41 that was present on untreated virions. Association of gp41 with cores required the presence of the gp41 cytoplasmic tail. In HIV-1 particles containing a functional protease, a mutation that prevents cleavage of Pr55(Gag) at the matrix-capsid junction was sufficient for the detergent-resistant association of gp41 with the isolated cores. In addition to gp41, a major fraction of virion-associated gp120 was also detected on immature HIV-1 cores. Isolation of cores under conditions known to disrupt lipid rafts resulted in the removal of a raft-associated protein incorporated into virions but not the HIV-1 envelope proteins. These results provide biochemical evidence for a stable interaction between Pr55(Gag) and the cytoplasmic tail of gp41 in immature HIV-1 particles. Moreover, findings in this study suggest that the interaction of Pr55(Gag) with gp41 may regulate the function of the envelope proteins during HIV-1 maturation.     

Genetic Determinants of Rous Sarcoma Virus Particle Size

The Gag proteins of retroviruses are the only viral products required for the release of membrane-enclosed particles by budding from the host cell. Particles released when these proteins are expressed alone are identical to authentic virions in their rates of budding, proteolytic processing, and core morphology, as well as density and size. We have previously mapped three very small, modular regions of the Rous sarcoma virus (RSV) Gag protein that are necessary for budding. These assembly domains constitute only 20% of RSV Gag, and alterations within them block or severely impair particle formation. Regions outside of these domains can be deleted without any effect on the density of the particles that are released. However, since density and size are independent parameters for retroviral particles, we employed rate-zonal gradients and electron microscopy in an exhaustive study of mutants lacking the various dispensable segments of Gag to determine which regions would be required to constrain or define the particle dimensions. The only sequence found to be absolutely critical for determining particle size was that of the initial capsid cleavage product, CA-SP, which contains all of the CA sequence plus the spacer peptides located between CA and NC. Some regions of CA-SP appear to be more important than others. In particular, the major homology region does not contribute to defining particle size. Further evidence for interactions among CA-SP domains was obtained from genetic complementation experiments using mutant ΔNC, which lacks the RNA interaction domains in the NC sequence but retains a complete CA-SP sequence. This mutant produces low-density particles heterogeneous in size. It was rescued into particles of normal size and density, but only when the complementing Gag molecules contained the complete CA-SP sequence. We conclude that CA-SP functions during budding in a manner that is independent of the other assembly domains.     

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.     

Effects of Gag mutation and processing on retroviral dimeric RNA maturation

After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.     

Inhibition of HIV-1 maturation via drug association with the viral Gag protein in immature HIV-1 particles

The small molecule 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently inhibits human immunodeficiency virus, type 1 (HIV-1) replication by interfering with proteolytic cleavage of the viral Gag protein at a specific site. Here we have demonstrated that the antiviral mechanism involves the association of DSB with Gag at a 1:1 stoichiometry within immature HIV-1 particles. The binding was specific, as mutations in Gag that confer resistance to DSB inhibited the association, which could be competed by DSB but not by the inactive compound betulinic acid. The addition of DSB to purified immature viral cores inhibited the cleavage of Gag at the CA-SP1 junction in vitro, thus reproducing the effect of the drug when present during maturation of HIV-1 particles. Based on these findings, we propose a model in which a trimer of DSB associates with the CA-SP1 junction of adjacent subunits within the Gag polymer. The model may explain the ability of highly similar compounds to specifically target the seemingly unrelated steps of HIV-1 maturation and virus entry.     

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.     

Human immunodeficiency virus type 1 resistance to the small molecule maturation inhibitor 3-O-(3',3'-dimethylsuccinyl)-betulinic acid is conferred by a variety of single amino acid substitutions at the CA-SP1 cleavage site in Gag

The compound 3-O-(3',3'-dimethylsuccinyl)-betulinic acid (DSB) potently and specifically inhibits human immunodeficiency virus type 1 (HIV-1) replication by delaying the cleavage of the CA-SP1 junction in Gag, leading to impaired maturation of the viral core. In this study, we investigated HIV-1 resistance to DSB by analyzing HIV-1 mutants encoding a variety of individual amino acid substitutions in the CA-SP1 cleavage site. Three of the substitutions were lethal to HIV-1 replication owing to a deleterious effect on particle assembly. The remaining mutants exhibited a range of replication efficiencies; however, each mutant was capable of replicating in the presence of concentrations of DSB that effectively inhibited wild-type HIV-1. Mutations conferring resistance to DSB also led to impaired binding of the compound to immature HIV-1 virions and loss of DSB-mediated inhibition of cleavage of Gag. Surprisingly, two of the DSB-resistant mutants retained an intermediate ability to bind the compound, suggesting that binding of DSB to immature HIV-1 particles may not be sufficient for antiviral activity. Overall, our results indicate that Gag amino acids L363 and A364 are critical for inhibition of HIV-1 replication by DSB and suggest that these residues form key contacts with the drug in the context of the assembling HIV-1 particle. These results have implications for the design of and screening for novel inhibitors of HIV-1 maturation.     

Effects of blocking individual maturation cleavages in murine leukemia virus gag

A single protein, termed Gag, is responsible for retrovirus particle assembly. After the assembled virion is released from the cell, Gag is cleaved at several sites by the viral protease (PR). The cleavages catalyzed by PR bring about a wide variety of physical changes in the particle, collectively termed maturation, and convert the particle into an infectious virion. In murine leukemia virus (MLV) maturation, Gag is cleaved at three sites, resulting in formation of the matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. We introduced mutations into MLV that inhibited cleavage at individual sites in Gag. All mutants had lost the intensely staining ring characteristic of immature particles; thus, no single cleavage event is required for this feature of maturation. Mutant virions in which MA was not cleaved from p12 were still infectious, with a specific infectivity only approximately 10-fold below that of the wild type. Particles in which p12 and CA could not be separated from each other were noninfectious and lacked a well-delineated core despite the presence of dense material in their interiors. In both of these mutants, the dimeric viral RNA had undergone the stabilization normally associated with maturation, suggesting that this change may depend upon the separation of CA from NC. Alteration of the C-terminal end of CA blocked CA-NC cleavage but also reduced the efficiency of particle formation and, in some cases, severely disrupted the ability of Gag to assemble into regular structures. This observation highlights the critical role of this region of Gag in assembly.