A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody.

Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (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.IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for 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. Toward 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 interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

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.

The Structure of Human Parechovirus 1 Reveals an Association of the RNA Genome with the Capsid.

UNLABELLED: Parechoviruses are human pathogens that cause diseases ranging from gastrointestinal disorders to encephalitis. Unlike those of most picornaviruses, parechovirus capsids are composed of only three subunits: VP0, VP1, and VP3. Here, we present the structure of a human parechovirus 1 (HPeV-1) virion determined to a resolution of 3.1 Å. We found that interactions among pentamers in the HPeV-1 capsid are mediated by the N termini of VP0s, which correspond to the capsid protein VP4 and the N-terminal part of the capsid protein VP2 of other picornaviruses. In order to facilitate delivery of the virus genome into the cytoplasm, the N termini of VP0s have to be released from contacts between pentamers and exposed at the particle surface, resulting in capsid disruption. A hydrophobic pocket, which can be targeted by capsid-binding antiviral compounds in many other picornaviruses, is not present in HPeV-1. However, we found that interactions between the HPeV-1 single-stranded RNA genome and subunits VP1 and VP3 in the virion impose a partial icosahedral ordering on the genome. The residues involved in RNA binding are conserved among all parechoviruses, suggesting a putative role of the genome in virion stability or assembly. Therefore, putative small molecules that could disrupt HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors. IMPORTANCE: Human parechoviruses (HPeVs) are pathogens that cause diseases ranging from respiratory and gastrointestinal disorders to encephalitis. Recently, there have been outbreaks of HPeV infections in Western Europe and North America. We present the first atomic structure of parechovirus HPeV-1 determined by X-ray crystallography. The structure explains why HPeVs cannot be targeted by antiviral compounds that are effective against other picornaviruses. Furthermore, we found that the interactions of the HPeV-1 genome with the capsid resulted in a partial icosahedral ordering of the genome. The residues involved in RNA binding are conserved among all parechoviruses, suggesting an evolutionarily fixed role of the genome in virion assembly. Therefore, putative small molecules disrupting HPeV RNA-capsid protein interactions could be developed into antiviral inhibitors.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.