Structure of the Fanconi anaemia monoubiquitin ligase complex.

The Fanconi anaemia (FA) pathway repairs DNA damage caused by endogenous and chemotherapy-induced DNA crosslinks, and responds to replication stress1,2. Genetic inactivation of this pathway by mutation of genes encoding FA complementation group (FANC) proteins impairs development, prevents blood production and promotes cancer1,3. The key molecular step in the FA pathway is the monoubiquitination of a pseudosymmetric heterodimer of FANCD2-FANCI4,5 by the FA core complex-a megadalton multiprotein E3 ubiquitin ligase6,7. Monoubiquitinated FANCD2 then recruits additional protein factors to remove the DNA crosslink or to stabilize the stalled replication fork. A molecular structure of the FA core complex would explain how it acts to maintain genome stability. Here we reconstituted an active, recombinant FA core complex, and used cryo-electron microscopy and mass spectrometry to determine its structure. The FA core complex comprises two central dimers of the FANCB and FA-associated protein of 100 kDa (FAAP100) subunits, flanked by two copies of the RING finger subunit, FANCL. These two heterotrimers act as a scaffold to assemble the remaining five subunits, resulting in an extended asymmetric structure. Destabilization of the scaffold would disrupt the entire complex, resulting in a non-functional FA pathway. Thus, the structure provides a mechanistic basis for the low numbers of patients with mutations in FANCB, FANCL and FAAP100. Despite a lack of sequence homology, FANCB and FAAP100 adopt similar structures. The two FANCL subunits are in different conformations at opposite ends of the complex, suggesting that each FANCL has a distinct role. This structural and functional asymmetry of dimeric RING finger domains may be a general feature of E3 ligases. The cryo-electron microscopy structure of the FA core complex provides a foundation for a detailed understanding of its E3 ubiquitin ligase activity and DNA interstrand crosslink repair.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.

UNLABELLED: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Combined approaches to flexible fitting and assessment in virus capsids undergoing conformational change.

Fitting of atomic components into electron cryo-microscopy (cryoEM) density maps is routinely used to understand the structure and function of macromolecular machines. Many fitting methods have been developed, but a standard protocol for successful fitting and assessment of fitted models has yet to be agreed upon among the experts in the field. Here, we created and tested a protocol that highlights important issues related to homology modelling, density map segmentation, rigid and flexible fitting, as well as the assessment of fits. As part of it, we use two different flexible fitting methods (Flex-EM and iMODfit) and demonstrate how combining the analysis of multiple fits and model assessment could result in an improved model. The protocol is applied to the case of the mature and empty capsids of Coxsackievirus A7 (CAV7) by flexibly fitting homology models into the corresponding cryoEM density maps at 8.2 and 6.1Å resolution. As a result, and due to the improved homology models (derived from recently solved crystal structures of a close homolog - EV71 capsid - in mature and empty forms), the final models present an improvement over previously published models. In close agreement with the capsid expansion observed in the EV71 structures, the new CAV7 models reveal that the expansion is accompanied by ∼5° counterclockwise rotation of the asymmetric unit, predominantly contributed by the capsid protein VP1. The protocol could be applied not only to viral capsids but also to many other complexes characterised by a combination of atomic structure modelling and cryoEM density fitting.

Structural and functional analysis of coxsackievirus A9 integrin αvß6 binding and uncoating.

Coxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin αv family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for αvß6, which recognizes the arginine-glycine-aspartic acid (RGD) loop present on the C terminus of viral capsid protein, VP1. As the atomic model of CVA9 lacks the RGD loop, we used surface plasmon resonance, electron cryo-microscopy, and image reconstruction to characterize the capsid-integrin interactions and the conformational changes on genome release. We show that the integrin binds to the capsid with nanomolar affinity and that the binding of integrin to the virion does not induce uncoating, thereby implying that further steps are required for release of the genome. Electron cryo-tomography and single-particle image reconstruction revealed variation in the number and conformation of the integrins bound to the capsid, with the integrin footprint mapping close to the predicted site for the exposed RGD loop on VP1. Comparison of empty and RNA-filled capsid reconstructions showed that the capsid undergoes conformational changes when the genome is released, so that the RNA-capsid interactions in the N termini of VP1 and VP4 are lost, VP4 is removed, and the capsid becomes more porous, as has been reported for poliovirus 1, human rhinovirus 2, enterovirus 71, and coxsackievirus A7. These results are important for understanding the structural basis of integrin binding to CVA9 and the molecular events leading to CVA9 cell entry and uncoating.

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating.

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.

The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination.

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.

Structure of Nora virus at 2.7 Å resolution and implications for receptor binding, capsid stability and taxonomy.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein ß-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.

Deep learning enables the atomic structure determination of the Fanconi Anemia core complex from cryoEM.

Cryo-electron microscopy of protein complexes often leads to moderate resolution maps (4-8 Å), with visible secondary-structure elements but poorly resolved loops, making model building challenging. In the absence of high-resolution structures of homologues, only coarse-grained structural features are typically inferred from these maps, and it is often impossible to assign specific regions of density to individual protein subunits. This paper describes a new method for overcoming these difficulties that integrates predicted residue distance distributions from a deep-learned convolutional neural network, computational protein folding using Rosetta, and automated EM-map-guided complex assembly. We apply this method to a 4.6 Šresolution cryoEM map of Fanconi Anemia core complex (FAcc), an E3 ubiquitin ligase required for DNA interstrand crosslink repair, which was previously challenging to interpret as it comprises 6557 residues, only 1897 of which are covered by homology models. In the published model built from this map, only 387 residues could be assigned to the specific subunits with confidence. By building and placing into density 42 deep-learning-guided models containing 4795 residues not included in the previously published structure, we are able to determine an almost-complete atomic model of FAcc, in which 5182 of the 6557 residues were placed. The resulting model is consistent with previously published biochemical data, and facilitates interpretation of disease-related mutational data. We anticipate that our approach will be broadly useful for cryoEM structure determination of large complexes containing many subunits for which there are no homologues of known structure.

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair.

Vertebrate DNA crosslink repair excises toxic replication-blocking DNA crosslinks. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause Fanconi anemia (FA). A key step in crosslink repair is monoubiquitination of the FANCD2-FANCI heterodimer, which then recruits nucleases to remove the DNA lesion. Here, we use cryo-EM to determine the structures of recombinant chicken FANCD2 and FANCI complexes. FANCD2-FANCI adopts a closed conformation when the FANCD2 subunit is monoubiquitinated, creating a channel that encloses double-stranded DNA (dsDNA). Ubiquitin is positioned at the interface of FANCD2 and FANCI, where it acts as a covalent molecular pin to trap the complex on DNA. In contrast, isolated FANCD2 is a homodimer that is unable to bind DNA, suggestive of an autoinhibitory mechanism that prevents premature activation. Together, our work suggests that FANCD2-FANCI is a clamp that is locked onto DNA by ubiquitin, with distinct interfaces that may recruit other DNA repair factors.

Interpretation of medium resolution cryoEM maps of multi-protein complexes.

Electron cryo-microscopy (cryoEM) is used to determine structures of biological molecules, including multi-protein complexes. Maps at better than 3.0Å resolution are relatively straightforward to interpret since atomic models of proteins and nucleic acids can be built directly. Still, these resolutions are often difficult to achieve, and map quality frequently varies within a structure. This results in data that are challenging to interpret, especially when crystal structures or suitable homology models are not available. Recent advances in mass spectrometry techniques, computational methods and model building tools facilitate subunit/domain fitting into maps, elucidation of protein contacts, and de novo generation of atomic models. Here, we review techniques for map interpretation and provide examples from recent studies of multi-protein complexes.

Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA.

Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantly-expressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0.

Erratum: Genomic RNA folding mediates assembly of human parechovirus.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Genomic RNA folding mediates assembly of human parechovirus.

Assembly of the major viral pathogens of the Picornaviridae family is poorly understood. Human parechovirus 1 is an example of such viruses that contains 60 short regions of ordered RNA density making identical contacts with the protein shell. We show here via a combination of RNA-based systematic evolution of ligands by exponential enrichment, bioinformatics analysis and reverse genetics that these RNA segments are bound to the coat proteins in a sequence-specific manner. Disruption of either the RNA coat protein recognition motif or its contact amino acid residues is deleterious for viral assembly. The data are consistent with RNA packaging signals playing essential roles in virion assembly. Their binding sites on the coat proteins are evolutionarily conserved across the Parechovirus genus, suggesting that they represent potential broad-spectrum anti-viral targets.The mechanism underlying packaging of genomic RNA into viral particles is not well understood for human parechoviruses. Here the authors identify short RNA motifs in the parechovirus genome that bind capsid proteins, providing approximately 60 specific interactions for virion assembly.

Strain-dependent neutralization reveals antigenic variation of human parechovirus 3.

Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.

Multiple capsid-stabilizing interactions revealed in a high-resolution structure of an emerging picornavirus causing neonatal sepsis.

The poorly studied picornavirus, human parechovirus 3 (HPeV3) causes neonatal sepsis with no therapies available. Our 4.3-Å resolution structure of HPeV3 on its own and at 15 Å resolution in complex with human monoclonal antibody Fabs demonstrates the expected picornavirus capsid structure with three distinct features. First, 25% of the HPeV3 RNA genome in 60 sites is highly ordered as confirmed by asymmetric reconstruction, and interacts with conserved regions of the capsid proteins VP1 and VP3. Second, the VP0 N terminus stabilizes the capsid inner surface, in contrast to other picornaviruses where on expulsion as VP4, it forms an RNA translocation channel. Last, VP1's hydrophobic pocket, the binding site for the antipicornaviral drug, pleconaril, is blocked and thus inappropriate for antiviral development. Together, these results suggest a direction for development of neutralizing antibodies, antiviral drugs based on targeting the RNA-protein interactions and dissection of virus assembly on the basis of RNA nucleation.