Biology subjects

Domanska, A.

Publications and source records attributed to Domanska, A..

4 recordsLinked to original sources

bioRxiv2023

The structure of immature tick-borne encephalitis virus

Tick-borne encephalitis virus (TBEV) is a medically important flavivirus that poses a significant health threat in Europe and Asia. However, the structure of the immature form of TBEV remains unknown. Here, we employed state-of-the-art cryogenic electron microscopy (cryoEM) to determine the structure of the immature TBEV particle. The immature TBEV particle has a diameter of 56 nm and its surface glycoproteins are organised into spikes characteristic of immature flaviviruses. The cryoEM reconstructions of the whole virus and of the individual spike enabled us to build atomic models of the major viral components, the E and prM proteins. The insights obtained from our study provide a foundation for understanding the early stages of TBEV assembly and maturation. The pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in metastable conformation. Destabilisation of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM cleavage, the collapse of E protein ectodomains onto the virion surface concurrent with significant movement of the membrane domains of both E and M, and release of the pr fragment from the particle render the virus mature and infectious. This knowledge contributes to our understanding of the flavivirus life cycle.

microbiology↗

bioRxiv2022

Structural studies reveal that endosomal cations promote formation of infectious CVA9 A particles, facilitating RNA and VP4 release.

Coxsackievirus A9, an enterovirus, is a common cause of paediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N-termini and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here we show that fatty-acid free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results show that CVA9 treatment with albumin or endosomal ions generates a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-EM and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control non-expanded virion to 3.5, 3.3 and 2.9 [A] resolutions, respectively. Where albumin promotes stabile expanded virions, the endosomal ionic concentrations induce unstable CVA9 virions which easily disintegrate losing their genome. Loss of most of the VP4 molecules and exposure of negatively-charged amino acid residues in the capsids interior after expansion, create a repulsive viral RNA-capsid interface, aiding genome release. ImportanceCoxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH is required. Here, we show by biophysical separation, cryogenic electron microscopy and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalised and the genome is repelled by the negatively-charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

microbiology↗

bioRxiv2020

Identification of a conserved virion-stabilizing network inside the interprotomer pocket of enteroviruses

Major efforts have been underway to develop broad-spectrum high potency capsid binders that inhibit the life cycle of enteroviruses, a large group (family Picornaviridae) whose members include poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses, and rhinoviruses. These diverse viruses cause a wide variety of illnesses, ranging from the mild common cold to hand-foot-and-mouth disease, myocarditis, pancreatitis, aseptic meningitis, and encephalitis. So-called classical capsid binders target a surface exposed hydrophobic pocket in one of the viral coat proteins (VP1) to prevent the genome uncoating process. However, efficacy, toxicity, emergence of drug-resistant viruses, and existence of certain enteroviral species that lack the VP1 pocket limit their clinical benefit. Recently, we identified a new druggable site at a conserved interface formed by multiple capsid proteins, the VP1-VP3 interprotomer pocket. To further study the properties that confer druggability at this site, we have determined high-resolution cryo-electron microscopy structures of two enteroviruses, coxsackieviruses B3 and B4, complexed with interprotomer-targeting compounds, CP17 and CP48 respectively. Until now, there has been no structure available for Coxsackievirus B4 despite the fact that the virus has long been implicated in the development of insulin-dependent diabetes mellitus. At better than 3 Å resolution, we could identify the detailed interactions that facilitate ligand binding. Both compounds target the same three conserved residues, each from a different polypeptide chain, to form a virion-stabilizing network inside the pocket. We measured the in silico binding energy for both inhibitors when anchored to the network and found a global stabilizing effect on the order of thousands of kcal/mol under saturating conditions (60 total sites per virion). Intriguingly, a recent X-ray structure has revealed that glutathione targets the same network within the interprotomer site of bovine enterovirus F3, where it is thought to facilitate virus assembly. In summary, our findings provide the structural basis for how a newly designed class of capsid binders target and stabilize enteroviruses. Future efforts to chemically optimize drugs for enhanced targeting to the interprotomer pocket is a promising endeavor in the fight against enteroviruses, especially given the possibility of synergistic effects when used in combination with classical VP1 binders like pleconaril.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2018

2.8 A resolution cryo-EM structure of human parechovirus 3 in complex with Fab from a neutralizing antibody

Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and lack of a vaccine or specific antiviral therapy. Towards the latter, we present a 2.8 [A]-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody AT12-015 using cryo-EM and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally at high-resolution, it was possible to model a stretch of RNA inside the virion and from this identify the key features that drive and stabilize protein-RNA association during assembly.\n\nImportance\n\nHPeV3 is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, which despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Towards this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high-resolution it was possible to precisely define the epitope that when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interaction, viral pathogenesis mechanisms, and for finding potential cures for infection and disease.

microbiology↗