Molecular determinants for Rous sarcoma virus intasome assemblies involved in retroviral integration.

Integration of retroviral DNA into the host genome involves the formation of integrase (IN)-DNA complexes termed intasomes. Further characterization of these complexes is needed to understand their assembly process. Here, we report the single-particle cryo-EM structure of the Rous sarcoma virus (RSV) strand transfer complex (STC) intasome produced with IN and a preassembled viral/target DNA substrate at 3.36 Å resolution. The conserved intasome core region consisting of IN subunits contributing active sites interacting with viral/target DNA has a resolution of 3 Å. Our structure demonstrated the flexibility of the distal IN subunits relative to the IN subunits in the conserved intasome core, similar to results previously shown with the RSV octameric cleaved synaptic complex intasome produced with IN and viral DNA only. An extensive analysis of higher resolution STC structure helped in the identification of nucleoprotein interactions important for intasome assembly. Using structure-function studies, we determined the mechanisms of several IN-DNA interactions critical for assembly of both RSV intasomes. We determined the role of IN residues R244, Y246, and S124 in cleaved synaptic complex and STC intasome assemblies and their catalytic activities, demonstrating differential effects. Taken together, these studies advance our understanding of different RSV intasome structures and molecular determinants involved in their assembly.

Curvature of the Retroviral Capsid Assembly Is Modulated by a Molecular Switch.

During the maturation step, the retroviral capsid proteins (CAs) assemble into polymorphic capsids. Their acute curvature is largely determined by 12 pentamers inserted into the hexameric lattice. However, how the CA switches its conformation to control assembly curvature remains unclear. We report the high-resolution structural model of the Rous sarcoma virus (RSV) CA T = 1 capsid, established by molecular dynamics simulations combining solid-state NMR and prior cryoelectron tomography restraints. Comparing this with our previous model of the RSV CA tubular assembly, we identify the key residues for dictating the incorporation of acute curvatures. These residues undergo large torsion angle changes, resulting in a 34° rotation of the C-terminal domain relative to its N-terminal domain around the flexible interdomain linker, without substantial changes of either the conformation of individual domains or the assembly contact interfaces. This knowledge provides new insights to help decipher the mechanism of the retroviral capsid assembly.

Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase.

Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last ß-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.

Oligomerization of Retrovirus Integrases.

Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.

A C-terminal "Tail" Region in the Rous Sarcoma Virus Integrase Provides High Plasticity of Functional Integrase Oligomerization during Intasome Assembly.

The retrovirus integrase (IN) inserts the viral cDNA into the host DNA genome. Atomic structures of five different retrovirus INs complexed with their respective viral DNA or branched viral/target DNA substrates have indicated these intasomes are composed of IN subunits ranging from tetramers, to octamers, or to hexadecamers. IN precursors are monomers, dimers, or tetramers in solution. But how intasome assembly is controlled remains unclear. Therefore, we sought to unravel the functional mechanisms in different intasomes. We produced kinetically stabilized Rous sarcoma virus (RSV) intasomes with human immunodeficiency virus type 1 strand transfer inhibitors that interact simultaneously with IN and viral DNA within intasomes. We examined the ability of RSV IN dimers to assemble two viral DNA molecules into intasomes containing IN tetramers in contrast to one possessing IN octamers. We observed that the last 18 residues of the C terminus ("tail" region) of IN (residues 1-286) determined whether an IN tetramer or octamer assembled with viral DNA. A series of truncations of the tail region indicated that these 18 residues are critical for the assembly of an intasome containing IN octamers but not for an intasome containing IN tetramers. The C-terminally truncated IN (residues 1-269) produced an intasome that contained tetramers but failed to produce an intasome with octamers. Both intasomes have similar catalytic activities. The results suggest a high degree of plasticity for functional multimerization and reveal a critical role of the C-terminal tail region of IN in higher order oligomerization of intasomes, potentially informing future strategies to prevent retroviral integration.

Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein.

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 310 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses.

Molecular Architecture of the Retroviral Capsid.

Retroviral capsid cores are proteinaceous containers that self-assemble to encase the viral genome and a handful of proteins that promote infection. Their function is to protect and aid in the delivery of viral genes to the nucleus of the host, and, in many cases, infection pathways are influenced by capsid-cellular interactions. From a mathematical perspective, capsid cores are polyhedral cages and, as such, follow well-defined geometric rules. However, marked morphological differences in shapes exist, depending on virus type. Given the specific roles of capsid in the viral life cycle, the availability of detailed molecular structures, particularly at assembly interfaces, opens novel avenues for targeted drug development against these pathogens. Here, we summarize recent advances in the structure and understanding of retroviral capsid, with particular emphasis on assemblies and the capsid cores.

Contributions of Charged Residues in Structurally Dynamic Capsid Surface Loops to Rous Sarcoma Virus Assembly.

UNLABELLED: Extensive studies of orthoretroviral capsids have shown that many regions of the CA protein play unique roles at different points in the virus life cycle. The N-terminal domain (NTD) flexible-loop (FL) region is one such example: exposed on the outer capsid surface, it has been implicated in Gag-mediated particle assembly, capsid maturation, and early replication events. We have now defined the contributions of charged residues in the FL region of the Rous sarcoma virus (RSV) CA to particle assembly. Effects of mutations on assembly were assessed in vivo and in vitro and analyzed in light of new RSV Gag lattice models. Virus replication was strongly dependent on the preservation of charge at a few critical positions in Gag-Gag interfaces. In particular, a cluster of charges at the beginning of FL contributes to an extensive electrostatic network that is important for robust Gag assembly and subsequent capsid maturation. Second-site suppressor analysis suggests that one of these charged residues, D87, has distal influence on interhexamer interactions involving helix α7. Overall, the tolerance of FL to most mutations is consistent with current models of Gag lattice structures. However, the results support the interpretation that virus evolution has achieved a charge distribution across the capsid surface that (i) permits the packing of NTD domains in the outer layer of the Gag shell, (ii) directs the maturational rearrangements of the NTDs that yield a functional core structure, and (iii) supports capsid function during the early stages of virus infection. IMPORTANCE: The production of infectious retrovirus particles is a complex process, a choreography of protein and nucleic acid that occurs in two distinct stages: formation and release from the cell of an immature particle followed by an extracellular maturation phase during which the virion proteins and nucleic acids undergo major rearrangements that activate the infectious potential of the virion. This study examines the contributions of charged amino acids on the surface of the Rous sarcoma virus capsid protein in the assembly of appropriately formed immature particles and the maturational transitions that create a functional virion. The results provide important biological evidence in support of recent structural models of the RSV immature virions and further suggest that immature particle assembly and virion maturation are controlled by an extensive network of electrostatic interactions and long-range communication across the capsid surface.

Crystal structure of the Rous sarcoma virus intasome.

Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses. Retrovirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroviruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous sarcoma virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy virus integrase. Our work highlights the diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroviruses.

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

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

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly.

UNLABELLED: The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ∼8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCE: Retroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.

Atomic Modeling of an Immature Retroviral Lattice Using Molecular Dynamics and Mutagenesis.

Defining the molecular interaction between Gag proteins in an assembled hexagonal lattice of immature retrovirus particles is crucial for elucidating the mechanisms of virus assembly and maturation. Recent advances in cryo-electron microscopy have yielded subnanometer structural information on the morphology of immature Gag lattices, making computational modeling and simulations feasible for investigating the Gag-Gag interactions at the atomic level. We have examined the structure of Rous sarcoma virus (RSV) using all-atom molecular dynamics simulations and in vitro assembly, to create the first all-atom model of an immature retroviral lattice. Microseconds-long replica exchange molecular dynamics simulation of the spacer peptide (SP)-nucleocapsid (NC) subdomains results in a six-helix bundle with amphipathic properties. The resulting model of the RSV Gag lattice shows features and dynamics of the capsid protein with implications for the maturation process, and confirms the stabilizing role of the upstream and downstream regions of Gag, namely p10 and SP-NC.

Rous sarcoma virus synaptic complex capable of concerted integration is kinetically trapped by human immunodeficiency virus integrase strand transfer inhibitors.

We determined conditions to produce milligram quantities of the soluble Rous sarcoma virus (RSV) synaptic complex that is kinetically trapped by HIV strand transfer inhibitors (STIs). Concerted integration catalyzed by RSV integrase (IN) is effectively inhibited by HIV STIs. Optimized assembly of the RSV synaptic complex required IN, a gain-of-function 3'-OH-recessed U3 oligonucleotide, and an STI under specific conditions to maintain solubility of the trapped synaptic complex at 4 °C. A C-terminal truncated IN (1-269 residues) produced a homogeneous population of trapped synaptic complex that eluted at ∼ 151,000 Da upon Superdex 200 size-exclusion chromatography (SEC). Approximately 90% of input IN and DNA are incorporated into the trapped synaptic complex using either the C-terminally truncated IN or wild type IN (1-286 residues). No STI is present in the SEC running buffer suggesting the STI-trapped synaptic complex is kinetically stabilized. The yield of the trapped synaptic complex correlates with the dissociative half-life of the STI observed with HIV IN-DNA complexes. Dolutegravir, MK-2048, and MK-0536 are equally effective, whereas raltegravir is ∼ 70% as effective. Without an STI present in the assembly mixture, no trapped synaptic complex was observed. Fluorescence and mass spectroscopy analyses demonstrated that the STI remains associated with the trapped complex. SEC-multiangle light scattering analyses demonstrated that wild type IN and the C-terminal IN truncation are dimers that acted as precursors to the tetramer. The purified STI-trapped synaptic complex contained a tetramer as shown by cross-linking studies. Structural studies of this three-domain RSV IN in complex with viral DNA may be feasible.

Effect of multimerization on membrane association of Rous sarcoma virus and HIV-1 matrix domain proteins.

In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.

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.

NC-mediated nucleolar localization of retroviral gag proteins.

The assembly and release of retrovirus particles from the cell membrane is directed by the Gag polyprotein. The Gag protein of Rous sarcoma virus (RSV) traffics through the nucleus prior to plasma membrane localization. We previously reported that nuclear localization of RSV Gag is linked to efficient packaging of viral genomic RNA, however the intranuclear activities of RSV Gag are not well understood. To gain insight into the properties of the RSV Gag protein within the nucleus, we examined the subnuclear localization and dynamic trafficking of RSV Gag. Restriction of RSV Gag to the nucleus by mutating its nuclear export signal (NES) in the p10 domain or interfering with CRM1-mediated nuclear export of Gag by leptomycin B (LMB) treatment led to the accumulation of Gag in nucleoli and discrete nucleoplasmic foci. Retention of RSV Gag in nucleoli was reduced with cis-expression of the 5' untranslated RU5 region of the viral RNA genome, suggesting the psi (Ψ) packaging signal may alter the subnuclear localization of Gag. Fluorescence recovery after photobleaching (FRAP) demonstrated that the nucleolar fraction of Gag was highly mobile, indicating that there was rapid exchange with Gag proteins in the nucleoplasm. RSV Gag is targeted to nucleoli by a nucleolar localization signal (NoLS) in the NC domain, and similarly, the human immunodeficiency virus type 1 (HIV-1) NC protein also contains an NoLS consisting of basic residues. Interestingly, co-expression of HIV-1 NC or Rev with HIV-1 Gag resulted in accumulation of Gag in nucleoli. Moreover, a subpopulation of HIV-1 Gag was detected in the nucleoli of HeLa cells stably expressing the entire HIV-1 genome in a Rev-dependent fashion. These findings suggest that the RSV and HIV-1 Gag proteins undergo nucleolar trafficking in the setting of viral infection.

A structural model for the generation of continuous curvature on the surface of a retroviral capsid.

The genome of a retrovirus is surrounded by a convex protein shell, or capsid, that helps facilitate infection. The major part of the capsid surface is formed by interlocking capsid protein (CA) hexamers. We report electron and X-ray crystallographic analysis of a variety of specimens assembled in vitro from Rous sarcoma virus (RSV) CA. These specimens all contain CA hexamers arranged in planar layers, modeling the authentic capsid surface. The specimens differ only in the number of layers incorporated and in the disposition of each layer with respect to its neighbor. The body of each hexamer, formed by the N-terminal domain of CA, is connected to neighboring hexamers through C-terminal domain dimerization. The resulting layer structure is very malleable due to inter-domain flexibility. A helix-capping hydrogen bond between the two domains of RSV CA creates a pivot point, which is central to controlling their relative movement. A similar mechanism for the governance of inter-domain motion was recently described for the human immunodeficiency virus type 1 (HIV-1) capsid, although there is negligible sequence identity between RSV and HIV-1 CA in the region of contact, and the amino acids involved in creating the pivot are not conserved. Our observations allow development of a physically realistic model for the way neighboring hexamers can tilt out of plane, deforming the hexamer layer and generating the continuously curved surfaces that are a feature of all retroviral capsids.

Conserved and variable features of Gag structure and arrangement in immature retrovirus particles.

The assembly of retroviruses is driven by oligomerization of the Gag polyprotein. We have used cryo-electron tomography together with subtomogram averaging to describe the three-dimensional structure of in vitro-assembled Gag particles from human immunodeficiency virus, Mason-Pfizer monkey virus, and Rous sarcoma virus. These represent three different retroviral genera: the lentiviruses, betaretroviruses and alpharetroviruses. Comparison of the three structures reveals the features of the supramolecular organization of Gag that are conserved between genera and therefore reflect general principles of Gag-Gag interactions and the features that are specific to certain genera. All three Gag proteins assemble to form approximately spherical hexameric lattices with irregular defects. In all three genera, the N-terminal domain of CA is arranged in hexameric rings around large holes. Where the rings meet, 2-fold densities, assigned to the C-terminal domain of CA, extend between adjacent rings, and link together at the 6-fold symmetry axis with a density, which extends toward the center of the particle into the nucleic acid layer. Although this general arrangement is conserved, differences can be seen throughout the CA and spacer peptide regions. These differences can be related to sequence differences among the genera. We conclude that the arrangement of the structural domains of CA is well conserved across genera, whereas the relationship between CA, the spacer peptide region, and the nucleic acid is more specific to each genus.

Proton-driven assembly of the Rous Sarcoma virus capsid protein results in the formation of icosahedral particles.

In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic resolution structural analysis. CA hexamers and pentamers are the fundamental building blocks of the capsid, however the pentameric state, in particular, remains poorly characterized. We have developed an efficient in vitro protocol for studying the assembly of Rous sarcoma virus (RSV) CA that involves mild acidification and produces structures modeling the authentic viral capsid. These structures include regular spherical particles with T = 1 icosahedral symmetry, built from CA pentamers alone. These particles were subject to cryoelectron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM-derived three-dimensional density map. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned underneath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra are the only detectable species and that these exist in reversible equilibrium at pH 5. These same acidic conditions are known to promote formation of a RSV CA CTD dimer, present within the icosahedral particle, which facilitates capsid assembly. The results are consistent with a model in which RSV CA assembly is a nucleation-limited process driven by very weak protein-protein interactions.

Visualization of a missing link in retrovirus capsid assembly.

For a retrovirus such as HIV to be infectious, a properly formed capsid is needed; however, unusually among viruses, retrovirus capsids are highly variable in structure. According to the fullerene conjecture, they are composed of hexamers and pentamers of capsid protein (CA), with the shape of a capsid varying according to how the twelve pentamers are distributed and its size depending on the number of hexamers. Hexamers have been studied in planar and tubular arrays, but the predicted pentamers have not been observed. Here we report cryo-electron microscopic analyses of two in-vitro-assembled capsids of Rous sarcoma virus. Both are icosahedrally symmetric: one is composed of 12 pentamers, and the other of 12 pentamers and 20 hexamers. Fitting of atomic models of the two CA domains into the reconstructions shows three distinct inter-subunit interactions. These observations substantiate the fullerene conjecture, show how pentamers are accommodated at vertices, support the inference that nucleation is a crucial morphologic determinant, and imply that electrostatic interactions govern the differential assembly of pentamers and hexamers.