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The structure of the CA hexamer. (A) Computational slice through a representative tomographic reconstruction of HIV-1 virions. Scale bar, 100 nm. Inset shows a computational slice through the top of the core in the boxed virion, indicating that individual hexamers can be resolved. (B) Cryo-ET reconstruction of the mature CA hexameric lattice (gray isosurface) viewed from outside the core. The final structural model of the hexamer is shown, with the NTD in cyan and the CTD in orange. One monomer is highlighted in blue (NTD) and red (CTD). (C) As in (B), viewed perpendicular to the lattice. See also movie S1. 

The structure of the CA hexamer. (A) Computational slice through a representative tomographic reconstruction of HIV-1 virions. Scale bar, 100 nm. Inset shows a computational slice through the top of the core in the boxed virion, indicating that individual hexamers can be resolved. (B) Cryo-ET reconstruction of the mature CA hexameric lattice (gray isosurface) viewed from outside the core. The final structural model of the hexamer is shown, with the NTD in cyan and the CTD in orange. One monomer is highlighted in blue (NTD) and red (CTD). (C) As in (B), viewed perpendicular to the lattice. See also movie S1. 

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Article
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HIV-1 contains a cone-shaped capsid encasing the viral genome. This capsid is thought to follow fullerene geometry—a curved hexameric lattice of the capsid protein, CA, closed by incorporating 12 CA pentamers. Current models for core structure are based on crystallography of hexameric and cross-linked pentameric CA, electron microscopy of tubular C...

Citations

... The proteolytic cleavages taking place during maturation are well characterized [1,3,7,8]. Indeed, the tridimensional structures of the HIV-1 capsid (CA) in immature [9] and mature [10] virions, as well as in mutants blocked at different stages of maturation have been solved by cryo-electron microscopy [11], revealing the structural switch triggering CA maturation. ...
Article
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Maturation of the HIV-1 viral particles shortly after budding is required for infectivity. During this process, the Pr55Gag precursor undergoes a cascade of proteolytic cleavages, and whilst the structural rearrangements of the viral proteins are well understood, the concomitant maturation of the genomic RNA (gRNA) structure is unexplored, despite evidence that it is required for infectivity. To get insight into this process, we systematically analysed the interactions between Pr55Gag or its maturation products (NCp15, NCp9 and NCp7) and the 5' gRNA region and their structural consequences, in vitro. We show that Pr55Gag and its maturation products mostly bind at different RNA sites and with different contributions of their two zinc knuckle domains. Importantly, these proteins have different transient and permanent effects on the RNA structure, the late NCp9 and NCp7 inducing dramatic structural rearrangements. Altogether, our results reveal the distinct contributions of the different Pr55Gag maturation products on the gRNA structural maturation.
... The retroviral Gag lattice is the most prominent feature in both mature and immature particles, and the complex arrangement of these lattices is dictated by intermolecular protein interactions located primarily within the CA domain (79)(80)(81). While the structural determinants of HIV-1 Gag oligomerization have been extensively studied in authentic particles (114)(115)(116), virus-like particles (117)(118)(119), and in vitro assemblies of purified CA proteins (120), high resolution of the structure of the HTLV-1 has been limited to nuclear magnetic resonance (NMR) studies of the HTLV-1 CA domain (121). Comparative studies probing residues within CA have identified many of the key interaction interfaces required for maintaining replication and morphology of HIV-1 (122, 123), HIV-2 (124), and HTLV-1 (81), including those that dictate the formation of structural features such as the six-helix bundle of CA-SP1 and the two-and three-fold CA interfaces. ...
Article
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Studies of retroviruses have led to many extraordinary discoveries that have advanced our understanding of not only human diseases, but also molecular biology as a whole. The most recognizable human retrovirus, human immunodeficiency virus type 1 (HIV-1), is the causative agent of the global AIDS epidemic and has been extensively studied. Other human retroviruses, such as human immunodeficiency virus type 2 (HIV-2) and human T-cell leukemia virus type 1 (HTLV-1), have received less attention, and many of the assumptions about the replication and biology of these viruses are based on knowledge of HIV-1. Existing comparative studies on human retroviruses, however, have revealed that key differences between these viruses exist that affect evolution, diversification, and potentially pathogenicity. In this review, we examine current insights on disparities in the replication of pathogenic human retroviruses, with a particular focus on the determinants of structural and genetic diversity amongst HIVs and HTLV.
... The vRNP encompasses elements essential to viral genome replication, including two copies of plus-stranded viral RNA (vRNA) and viral NC, reverse transcriptase (RT), and integrase (IN) proteins. The HIV-1 capsid lattice, which is a cone-shaped fullerene shell [4], is assembled from approximately 200 CA hexamer and 12 CA pentamer building blocks [5,6]. Virus replication cycles are broadly divided into early versus late temporal steps, and key early events in the lifecycles of orthoretroviruses include cell entry, reverse transcription, and integration (spumaretroviral infection also relies on these steps, but reverse transcription in this case can occur prior to cell entry [8]). ...
... Transmission electron microscopy (TEM) of negatively stained samples aided the initial characterization of HIV-1 as a retrovirus [174]. Higher resolution cryogenic electron microscopy techniques, such as cryogenic electron tomography [3,[5][6][7], have further informed HIV-1 ultrastructure analyses. ...
Article
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Integrase is the retroviral protein responsible for integrating reverse transcripts into cellular genomes. Co-packaged with viral RNA and reverse transcriptase into capsid-encased viral cores, human immunodeficiency virus 1 (HIV-1) integrase has long been implicated in reverse transcription and virion maturation. However, the underlying mechanisms of integrase in these non-catalytic-related viral replication steps have remained elusive. Recent results have shown that integrase binds genomic RNA in virions, and that mutational or pharmacological disruption of integrase-RNA binding yields eccentric virion particles with ribonucleoprotein complexes situated outside of the capsid shell. Such viruses are defective for reverse transcription due to preferential loss of integrase and viral RNA from infected target cells. Parallel research has revealed defective integrase-RNA binding and eccentric particle formation as common features of class II integrase mutant viruses, a phenotypic grouping of viruses that display defects at steps beyond integration. In light of these new findings, we propose three new subclasses of class II mutant viruses (a, b, and c), all of which are defective for integrase-RNA binding and particle morphogenesis, but differ based on distinct underlying mechanisms exhibited by the associated integrase mutant proteins. We also assess how these findings inform the role of integrase in HIV-1 particle maturation.
... Interestingly, AFM analysis reveals that disassembly is observed to begin always near the narrow end of the core, where the local density of capsid pentamers is highest [21,62,63] ( Figure 2C). However, recent studies using EM show that the virus capsid disassembles by losing patches of the capsid lattice [64]. ...
Article
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Obtaining an understanding of the mechanism underlying the interrelations between the structure and function of HIV-1 is of pivotal importance. In previous decades, this mechanism was addressed extensively in a variety of studies using conventional approaches. More recently, atomic force microscopy, which is a relatively new technique with unique capabilities, has been utilized to study HIV-1 biology. Atomic force microscopy can generate high-resolution images at the nanometer-scale and analyze the mechanical properties of individual HIV-1 virions, virus components (e.g., capsids), and infected live cells under near-physiological environments. This review describes the working principles and various imaging and analysis modes of atomic force microscopy, and elaborates on its distinctive contributions to HIV-1 research in areas such as mechanobiology and the physics of infection.
... In the mature core, six CA NTD monomers form an outer hexameric ring with a central 18-helix bundle that provides intra-hexamer stability, while the CA CTD portions reside below the hexameric ring and contribute inter-hexamer interactions. Intrahexameric NTD-CTD interactions between adjacent CA molecules add further stability to the capsid [101][102][103]. The mature capsid core is somewhat pleomorphic in shape, modelled as a fullerene structure in which the hexameric lattice is closed through the incorporation of 12 CA pentameric vertices [104][105][106][107]. ...
Article
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The assembly of HIV-1 particles is a concerted and dynamic process that takes place on the plasma membrane of infected cells. An abundance of recent discoveries has advanced our understanding of the complex sequence of events leading to HIV-1 particle assembly, budding, and release. Structural studies have illuminated key features of assembly and maturation, including the dramatic structural transition that occurs between the immature Gag lattice and the formation of the mature viral capsid core. The critical role of inositol hexakisphosphate (IP6) in the assembly of both the immature and mature Gag lattice has been elucidated. The structural basis for selective packaging of genomic RNA into virions has been revealed. This review will provide an overview of the HIV-1 assembly process, with a focus on recent advances in the field, and will point out areas where questions remain that can benefit from future investigation.
... Curvature leading to the assembly of a closed structure is accomplished by insertion of 12 pentamers (coloured in blue); seven pentamers towards the broad end and five pentamers toward the narrow end. In a more recent landmark study, Mattei et al. [35] showed the full structural power of cryo-ET and STA by revealing the precise molecular architecture of the natively assembled mature HIV-1 capsid core at 7 Å resolution. Some of the technicalities necessary for achieving the reported resolution included a dose-symmetric tilt acquisition scheme that optimally preserves high-resolution information, 3D CTF (contrast transfer function) correction and dose-filtering [36,37]. ...
Article
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Viruses can be enveloped or non-enveloped, and require a host cell to replicate and package their genomes into new virions to infect new cells. To accomplish this task, viruses hijack the host-cell machinery to facilitate their replication by subverting and manipulating normal host cell function. Enveloped viruses can have severe consequences for human health, causing various diseases such as acquired immunodeficiency syndrome (AIDS), seasonal influenza, COVID-19, and Ebola virus disease. The complex arrangement and pleomorphic architecture of many enveloped viruses pose a challenge for the more widely used structural biology techniques, such as X-ray crystallography. Cryo-electron tomography (cryo-ET), however, is a particularly well-suited tool for overcoming the limitations associated with visualizing the irregular shapes and morphology enveloped viruses possess at macromolecular resolution. The purpose of this review is to explore the latest structural insights that cryo-ET has revealed about enveloped viruses, with particular attention given to their architectures, mechanisms of entry, replication, assembly, maturation and egress during infection. Cryo-ET is unique in its ability to visualize cellular landscapes at 3–5 nanometer resolution. Therefore, it is the most suited technique to study asymmetric elements and structural rearrangements of enveloped viruses during infection in their native cellular context.
... Nonetheless, several studies have yielded high-resolution density maps resolving secondary structural elements, including coat protein complex I (ref. 6 ), nuclear pore complex 4,7 , polysomes 8 , chemotaxis signaling arrays 9 , retroviruses assembly [10][11][12][13][14] , bacteria surface layer 15 and ribosomes 16 . ...
... Users define the boundary of subregion(s) in the tomogram for later reconstruction (Steps 5-7). The particles are then picked using template matching (Steps [8][9][10][11][12]. emClarity manages the subtomogram-associated metadata in a MATLAB database and updates the metadata after each processing step throughout the pipeline (Step 13). ...
Article
Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA.
... Additionally, since the crosscorrelation cleaning approach was not applicable when using Relion, we attempted to increase the homogeneity of the subvolumes used for refinement by a more stringent exclusion of subvolumes that deviate from ideal lattice geometry. In order to retain only subvolumes containing the structurally most similar local CA hexamers we employed a cleaning strategy based on local geometry, similar to what has been previously used for mature HIV-1 and RSV CA assemblies (Mattei et al., 2016;Obr et al., 2021) (see also Figure S4 and Materials & Methods for more details). ...
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The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo- EM) image processing has been well documented for single particle analysis (SPA). Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Angstrom resolution range. In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Angstrom. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
... Substantial developments in protein engineering, structural biology, and molecular modeling have led to an improved understanding of the molecular architecture and interaction surfaces of various CA assembly forms (12,38,40,(52)(53)(54)(55)(56). Indeed, several critical binding interactions between CA and host factors have been defined. ...
Article
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The HIV-1 capsid core participates in several replica-tion processes. The mature capsid core is a lattice composed of capsid (CA) monomers thought to assemble first into CA dimers, then into ∼250 CA hex-amers and 12 CA pentamers. CA assembly requires conformational flexibility of each unit, resulting in the presence of unique, solvent-accessible surfaces. Significant advances have improved our understanding of the roles of the capsid core in replication; however, the contributions of individual CA assembly forms remain unclear and there are limited tools available to evaluate these forms in vivo. Here, we have selected aptamers that bind CA lattice tubes. We describe aptamer CA15-2, which selectively binds CA lattice, but not CA monomer or CA hexamer, suggesting that it targets an interface present and accessible only on CA lattice. CA15-2 does not compete with PF74 for binding, indicating that it likely binds a non-overlapping site. Furthermore, CA15-2 inhibits HIV-1 replication when expressed in virus producer cells, but not target cells, suggesting that it binds a biologically-relevant site during virus production that is either not accessible during post-entry replication steps or is accessible but unaltered by aptamer binding. Importantly, CA15-2 represents the first aptamer that specifically recognizes the HIV-1 CA lattice.
... (f) Tilt (left) and twist (right) angles between hexamers within a single core. Insets show a schematic illustration of tilt and twist angles [76]. The tilt/twist angle is indicated by the colour of the connecting lines between hexamer positions, from blue (less tilt along the long axis of the core) to red (more tilt along the circumference). ...
... The pentameric structure recently determined from native capsid by cryoET and subtomogram averaging is different from the previous crystal structure of the cross-linked pentamer [72,76]. Its NTDs are rotated by approximately 19 degrees compared to its hexameric counterpart. ...
... In doing so, it excludes helix 3 from the interface and forms a 10-helix bundle with its neighbouring hexamer instead. Moreover, the binding site for host factors and small molecules such as PF74 is more open at the pentamer NTD-CTD interface in comparison to the hexameric one [76]. The five arginine residues (R18) at the center of the pentamer in HIV CA (or the corresponding residue K17 in RSV CA) have been proposed to regulate the transition between hexamer and pentamer by balancing its electrostatic destabilization with stabilizing the lattice [38,79]. ...
Article
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Retroviruses have a very complex and tightly controlled life cycle which has been studied intensely for decades. After a virus enters the cell, it reverse-transcribes its genome, which is then integrated into the host genome, and subsequently all structural and regulatory proteins are transcribed and translated. The proteins, along with the viral genome, assemble into a new virion, which buds off the host cell and matures into a newly infectious virion. If any one of these steps are faulty, the virus cannot produce infectious viral progeny. Recent advances in structural and molecular techniques have made it possible to better understand this class of viruses, including details about how they regulate and coordinate the different steps of the virus life cycle. In this review we summarize the molecular analysis of the assembly and maturation steps of the life cycle by providing an overview on structural and biochemical studies to understand these processes. We also outline the differences between various retrovirus families with regards to these processes.