Viruses 2022, 14, 318. https://doi.org/10.3390/v14020318 www.mdpi.com/journal/viruses Article Cryo-EM Structure of a Possum Enterovirus Ivy Wang 1 , Sandeep K. Gupta 2 , Guillaume Ems 3,4 , Nadishka Jayawardena 3,5 , Mike Strauss 1, * and Mihnea Bostina 3,6, * 1 Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 0C7, Canada; ivy.wang@mail.mcgill.ca 2 AgResearch, Palmerston North 4442, New Zealand; sandeep.gupta@agresearch.co.nz 3 Department of Microbiology and Immunology, University of Otago, Dunedin 9016, New Zealand; guillaume.ems@student.unamur.be (G.E.); gimshan.jayawardena@oist.jp (N.J.) 4 Faculté des Sciences, Université de Namur, 5000 Namur, Belgium 5 Molecular Cryo-Electron Microscopy Unit, Okinawa Institute of Science and Technology Graduate University, Okinawa 904-0495, Japan 6 Otago Micro and Nano Imaging, University of Otago, Dunedin 9016, New Zealand * Correspondence: mike.strauss@mcgill.ca (M.S.); mihnea.bostina@otago.ac.nz (M.B.) Abstract: Enteroviruses (EVs) represent a substantial concern to global health. Here, we present the cryo-EM structure of a non-human enterovirus, EV-F4, isolated from the Australian brushtail pos- sum to assess the structural diversity of these picornaviruses. The capsid structure, determined to ~3 Å resolution by single particle analysis, exhibits a largely smooth surface, similar to EV-F3 (for- merly BEV-2). Although the cellular receptor is not known, the absence of charged residues on the outer surface of the canyon suggest a different receptor type than for EV-F3. Density for the pocket factor is clear, with the entrance to the pocket being smaller than for other enteroviruses. Keywords: enterovirus; possum; capsid structure; cryo-electron microscopy 1. Introduction Enteroviruses (EVs) form the largest genus within the Picornaviridae family and are one of the most widespread viruses infecting animals. EVs are usually responsible for infections characterized by flu-like symptoms, but can be associated with more severe diseases following the spread of viral replication from the gastro-intestinal or respiratory tract to secondary tissues such as brain, heart, or liver [1]. Currently, there are 15 entero- viruses species that have been identified. EV-A to -D and Rhinoviruses (RV) RV-A to -C primarily infect humans, while EV-E to -L infect livestock and non-human primates [2,3]. Despite efforts, no antivirals for treating enterovirus infections are currently clinically ap- proved [4,5]. With the constant characterisation of new species, EVs remain significant global health burden and increasing threat for further epidemics [6]. The picornavirus single stranded RNA genome encodes a single polyprotein that is cleaved into four structural proteins and seven non-structural proteins. During the viral lifecycle, the capsid precursor domain (P1) is cleaved into VP0, VP1 and VP3 which as- semble into a protomer, the fundamental subunit capable of assembling into an icosahe- dral capsid. Following genome encapsidation, VP0 is further cleaved into VP2 and VP4 forming the mature capsid, with VP1–VP3 responsible for forming the outer surface, while VP4 lines the internal surface of the capsid. A large gallery of structures, solved initially by X-ray crystallography and more recently by cryo-electron microscopy (cryo- EM), have revealed a similar architecture among picornaviruses with distinct structural features [7,8]. A prominent star-shaped ‘mesa’ located at the five-fold axis is surrounded by a depression, named ‘canyon’, formed at the junction between VP1, VP2 and VP3. A pocket within VP1 containing a non-protein factor was identified in the majority of enter- ovirus structures. This “pocket factor” has been implicated in the regulation of viral Citation: Wang, I.; Gupta, S.K.; Ems, G.; Jayawardena, N.; Strauss, M.; Bostina, M. Cryo-EM Structure of a Possum Enterovirus. Viruses 2022, 14, 318. https://doi.org/10.3390/v14020318 Academic Editors: Vernon Ward and John A. Taylor Received: 20 December 2021 Accepted: 1 February 2022 Published: 3 February 2022 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Copyright: © 2022 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (http://crea- tivecommons.org/licenses/by/4.0/).