Human picornaviruses : uncoating, assembly and interaction with cellular receptors

Pathogenic human picornaviruses are known to cause a wide variety of diseases ranging from mild colds to severe paralysis. In addition to their importance in causing disease, they also serve as models for understanding the basic mechanisms of host-pathogen interactions, virus entry, viral genome release, viral synthesis and viral assembly. In picornaviruses, the majority of the structural and host-cell interaction studies have been conducted on polioviruses and human rhinoviruses. Picornaviruses like coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1 have not been so well studied because of difficulties in culturing them. Recently, the number of cases reported for infection by these viruses has increased dramatically due to better detection methods, thus making structural studies of these viruses and their interactions with their host cells important in order to understand their mode of infection so that better therapeutics can be designed against them. I have studied coxsackievirus A 7, coxsackievirus A 9 and human parechovirus 1, which are all pathogenic picornaviruses, in order to understand the mechanism of pathogenesis, tropism, viral entry and assembly for these viruses in particular and for picornaviruses in general. Two studies dealt with determining the structure of coxsackievirus A 7, a Human Enterovirus A species for which there was no structural information available at the time when this study was conducted. The genome-filled and empty structure of coxsackievirus A 7 were determined using cryo electron microscopy to sub-nanometer resolution which helped in building pseudo-atomic models for them using homology modelling and flexible fitting. With the help of these models, the majority of the strain variations in the capsid proteins were identified on the surface of VP1. Such variations are the likely cause of differences in pathogenesis and tropism between strains. Furthermore, superimposition of these models showed that the capsid underwent a conformational change on RNA release. In the process, generalised methods for optimising and comparing results from flexible fitting were developed. The next structural study elucidated the interaction of coxsackievirus A 9, a Human Enterovirus B species, with a cellular receptor. Integrins were found to bind sub-stoichiometrically to the capsid using electron cryo-tomography (cryo-ET). Asymmetric reconstruction indicated that this was probably due to steric hindrance. The affinity of this interaction was calculated to be 1nM using surface plasmon resonance. Additionally, the conformational changes which occur on its RNA release were quantified. The fourth study explained the importance of viral RNA in picornavirus assembly. Pentameric intermediates of human parechovirus 1 were isolated and used to identify packaging signals in the viral RNA required for capsid assembly using aptamer library screening and next generation sequencing analysis. Poly-U was identified as the common motif for these packaging signals present on the stem or the loop of the RNA secondary structure. Overall, this thesis gives an insight into many important aspects of host-virus interactions especially the events occurring on viral RNA exit and during its encapsidation. The work in this thesis could be utilized to identify potential targets for antiviral synthesis and also to define general virus assembly principles.N/

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

Peer reviewe

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.Peer reviewe

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

Structural and Functional Analysis of Coxsackievirus A9 Integrin alpha(v)beta(6) Binding and Uncoating

Coxsackievirus A9 (CVA9) is an important pathogen of the Picornaviridae family. It utilizes cellular receptors from the integrin v family for binding to its host cells prior to entry and genome release. Among the integrins tested, it has the highest affinity for v6, 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.Peer reviewe

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.Peer reviewe

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

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.Peer reviewe