Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex.

Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.

Oligomeric assembly of the C-terminal and transmembrane region of SARS-CoV-2 nsp3.

As for all single-stranded, positive-sense RNA (+RNA) viruses, intracellular RNA synthesis relies on extensive remodeling of host cell membranes that leads to the formation of specialized structures. In the case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus causing COVID-19, endoplasmic reticulum membranes are modified, resulting in the formation of double-membrane vesicles (DMVs), which contain the viral dsRNA intermediate and constitute membrane-bound replication organelles. The non-structural and transmembrane protein nsp3 is a key player in the biogenesis of DMVs and, therefore, represents an interesting antiviral target. However, as an integral transmembrane protein, it is challenging to express for structural biology. The C-terminus of nsp3 encompasses all the membrane-spanning, -interacting, and -remodeling elements. By using a cell-free expression system, we successfully produced the C-terminal region of nsp3 (nsp3C) and reconstituted purified nsp3C into phospholipid nanodiscs, opening the way for structural studies. Negative-stain transmission electron microscopy revealed the presence of nsp3C oligomers very similar to the region abutting and spanning the membrane on the cytosolic side of DMVs in a recent subtomogram average of the SARS-CoV-2 nsp3-4 pore (1). AlphaFold-predicted structural models fit particularly well with our experimental data and support a pore-forming hexameric assembly. Altogether, our data give unprecedented clues to understand the structural organization of nsp3, the principal component that shapes the molecular pore that spans the DMVs and is required for the export of RNA in vivo. IMPORTANCE: Membrane remodeling is at the heart of intracellular replication for single-stranded, positive-sense RNA viruses. In the case of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this leads to the formation of a network of double-membrane vesicles (DMVs). Targeting DMV biogenesis offers promising prospects for antiviral therapies. This requires a better understanding of the molecular mechanisms and proteins involved. Three non-structural proteins (nsp3, nsp4, and nsp6) direct the intracellular membrane rearrangements upon SARS-CoV-2 infection. All of them contain transmembrane helices. The nsp3 component, the largest and multi-functional protein of the virus, plays an essential role in this process. Aiming to understand its structural organization, we used a cell-free protein synthesis assay to produce and reconstitute the C-terminal part of nsp3 (nsp3C) including transmembrane domains into phospholipid nanodiscs. Our work reveals the oligomeric organization of one key player in the biogenesis of SARS-CoV-2 DMVs, providing basis for the design of future antiviral strategies.

Energetics and Kinetic Assembly Pathways of Hepatitis B Virus Capsids in the Presence of Antivirals.

Capsid assembly modulators (CAMs) are antiviral molecules that disturb the formation of icosahedral viral capsids, in particular, those of the Hepatitis B virus (HBV). We report an integrated, physics-driven study elucidating quantitatively the effects of two classes of CAMs on the HBV capsid assembly. Time-resolved small-angle X-ray scattering measurements revealed accelerated self-assembly processes that implied the increase of subunit binding energy from 9- up to 18-fold the thermal energy due to CAMs. Cryotransmission electron microscopy images showed that both classes induce various changes in capsid morphology: from a slight elongation, unrecognized in previous work, to a strong deformation with a capsid size more than twice as large. The observed capsid morphologies were closely reproduced in coarse-grained simulations by varying the Föppl-von-Kármán number, thus pointing out the role of CAMs in altering the capsid elastic energy. Our results illuminate the mechanisms of action of CAMs on HBV capsid assembly at high spatiotemporal resolution and may bring perspectives on virus-derived nanocapsules with tunable morphologies.

De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1's N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis.

The endocytic recycling compartment serves as a viral factory for hepatitis E virus.

Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Systematic Method for the Exploration, Representation, and Classification of the Diphenylalanine Solvatomorphic Space.

An understanding of the conditions that govern the self-assembly process of peptides is a fundamental step toward the design of new nanostructures that possess interesting properties. In this work, we first synthesize and explore extensively diphenylalanine (FF) self-assembling crystals formed in different solvents (i.e., solvatomorphs) using polarized optical microscopy and transmission electron microscopy. Then, we develop a numerical method that allows an unambiguous classification of the solvatomorphs through a K-means automatic clustering method. In addition, we generate a two-dimensional (2D) representation of the solvatomorphic space together with the clustering results via a principal component analysis (PCA). The classification is based on structural similarities of solvatomorphs as revealed by the analysis of their respective infrared spectra. Among the 20 samples considered, 4 clear clusters are extracted within which the compounds show very similar crystalline structures. The information extracted allows us to assign many of the peaks that appear in the complex IR spectra of the samples considered. The implementation of the overall procedure we propose, i.e., "GAULOIS" and "REFRACT-R", is transferable to other types of spectra and paves the way for a systematic, fast, and accurate classification method applicable to various types of experimental spectroscopic data.

Relationships between RNA topology and nucleocapsid structure in a model icosahedral virus.

The process of genome packaging in most of viruses is poorly understood, notably the role of the genome itself in the nucleocapsid structure. For simple icosahedral single-stranded RNA viruses, the branched topology due to the RNA secondary structure is thought to lower the free energy required to complete a virion. We investigate the structure of nucleocapsids packaging RNA segments with various degrees of compactness by small-angle x-ray scattering and cryotransmission electron microscopy. The structural differences are mild even though compact RNA segments lead on average to better-ordered and more uniform particles across the sample. Numerical calculations confirm that the free energy is lowered for the RNA segments displaying the larger number of branch points. The effect is, however, opposite with synthetic polyelectrolytes, in which a star topology gives rise to more disorder in the capsids than a linear topology. If RNA compactness and size account in part for the proper assembly of the nucleocapsid and the genome selectivity, other factors most likely related to the host cell environment during viral assembly must come into play as well.

A pocket-factor-triggered conformational switch in the hepatitis B virus capsid.

Viral hepatitis is growing into an epidemic illness, and it is urgent to neutralize the main culprit, hepatitis B virus (HBV), a small-enveloped retrotranscribing DNA virus. An intriguing observation in HB virion morphogenesis is that capsids with immature genomes are rarely enveloped and secreted. This prompted, in 1982, the postulate that a regulated conformation switch in the capsid triggers envelopment. Using solid-state NMR, we identified a stable alternative conformation of the capsid. The structural variations focus on the hydrophobic pocket of the core protein, a hot spot in capsid-envelope interactions. This structural switch is triggered by specific, high-affinity binding of a pocket factor. The conformational change induced by the binding is reminiscent of a maturation signal. This leads us to formulate the "synergistic double interaction" hypothesis, which explains the regulation of capsid envelopment and indicates a concept for therapeutic interference with HBV envelopment.

Recapitulating Trafficking of Nucleosides Into the Active Site of Polymerases of RNA Viruses: The Challenge and the Prize.

Nucleoside analogs are very effective antiviral agents with currently over 25 compounds approved for the therapy of viral infections. Still, their successful use against RNA viruses is very recent, despite RNA viruses comprising some of the most damaging human pathogens (e.g., Coronaviruses, Influenza viruses, or Flaviviridae such as dengue, Zika and hepatitis C viruses). The breakthrough came in 2013-2014, when the nucleoside analog Sofosbuvir became one of the cornerstones of current curative treatments for hepatitis C virus (HCV). An analog designed on the same principles, Remdesivir, has been the first approved compound against SARS-CoV-2, the coronavirus that causes the current COVID-19 pandemic. Both of these nucleoside analogs target the RNA-dependent RNA polymerase (RdRp) (NS5B for HCV, nsp12 for SARS-CoV-2). RdRps of RNA viruses display a peculiar elaboration of the classical polymerase architecture that leads to their active site being caged. Thus, triphosphate nucleosides and their analogs must access this active site in several steps along a narrow and dynamic tunnel. This makes straightforward computational approaches such as docking unsuitable for getting atomic-level details of this process. Here we give an account of ribose-modified nucleoside analogs as inhibitors of viral RdRps and of why taking into account the dynamics of these polymerases is necessary to understand nucleotide selection by RdRps. As a case study we use a computational protocol we recently described to examine the approach of the NTP tunnel of HCV NS5B by cellular metabolites of Sofosbuvir. We find major differences with natural nucleotides even at this early stage of nucleotide entry.

Nonsymmetrical Dynamics of the HBV Capsid Assembly and Disassembly Evidenced by Their Transient Species.

As with many protein multimers studied in biophysics, the assembly and disassembly dynamical pathways of hepatitis B virus (HBV) capsid proteins are not symmetrical. Using time-resolved small-angle X-ray scattering and singular value decomposition analysis, we have investigated these processes in vitro by a rapid change of salinity or chaotropicity. Along the assembly pathway, the classical nucleation-growth mechanism is followed by a slow relaxation phase during which capsid-like transient species self-organize in accordance with the theoretical prediction that the capture of the few last subunits is slow. By contrast, the disassembly proceeds through unexpected, fractal-branched clusters of subunits that eventually vanish over a much longer time scale. On the one hand, our findings confirm and extend previous views as to the hysteresis phenomena observed and theorized in capsid formation and dissociation. On the other hand, they uncover specifics that may directly relate to the functions of HBV subunits in the viral cycle.

Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity.

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.

In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes.

Single-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic maturation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions.

The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif.

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modeling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that at least some of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.

Septin 9 has Two Polybasic Domains Critical to Septin Filament Assembly and Golgi Integrity.

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins interact with PIs and then assemble and shape membranes is poorly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similar to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association is not dependent on these PB domains, but on putative PB-adjacent amphipathic helices. The presence of PB domains guarantees protein enrichment in PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings offer further insight into the role of septins in organelle morphology.

TTClust: A Versatile Molecular Simulation Trajectory Clustering Program with Graphical Summaries.

It is extremely helpful to be able to partition the thousands of frames produced in molecular dynamics simulations into a limited number of most dissimilar conformations. While robust clustering algorithms are already available to do so, there is a distinct need for an easy-to-use clustering program with complete user control, taking as input a trajectory from any molecular dynamics (MD) package and outputting an intuitive display of results with plots allowing at-a-glance analysis. We present TTClust (for Trusty Trajectory Clustering), a python program that uses the MDTraj package to fill this need.

Nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging genome or polyelectrolyte.

The survival of viruses partly relies on their ability to self-assemble inside host cells. Although coarse-grained simulations have identified different pathways leading to assembled virions from their components, experimental evidence is severely lacking. Here, we use time-resolved small-angle X-ray scattering to uncover the nonequilibrium self-assembly dynamics of icosahedral viral capsids packaging their full RNA genome. We reveal the formation of amorphous complexes via an en masse pathway and their relaxation into virions via a synchronous pathway. The binding energy of capsid subunits on the genome is moderate (~7kBT0, with kB the Boltzmann constant and T0 = 298 K, the room temperature), while the energy barrier separating the complexes and the virions is high (~ 20kBT0). A synthetic polyelectrolyte can lower this barrier so that filled capsids are formed in conditions where virions cannot build up. We propose a representation of the dynamics on a free energy landscape.

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of "dynamic" domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.