@article{b916c75429954f1f95903fb036adf0d6,
title = "Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication",
abstract = "Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annu-ally. HEV{\textquoteright}s open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the {\textquoteleft}pPCP{\textquoteright} domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.",
author = "Robert Ledesma and Brigitte Heller and Abhishek Biswas and Stephanie Maya and Stefania Gili and John Higgins and Alexander Ploss",
note = "Funding Information: We kindly thank Susan Emerson and Patricia Farci (NIAID) for providing us with the pSK SAR55, pBSK(+) Kc1 ORF1 WT GLuc plasmid, XJ Meng (Virginia-Maryland College of Veterinary Medicine) for providing us the plasmids pGEM-9zf-pSHEV3 and pGEM-7Zf(-)-TW6196E encoding the infectious pSHEV3 (gt 3) and TW6196 (gt 4) clone, respectively. HEV ORF1 RNA dependent RNA Polymerase structural data was kindly provided by J{\'e}r{\^o}me Gouttenoire. We would also like to thank Christina DeCoste and Katherine Rittenbach in the Molecular Biology flow cytometry core facility and Dr. Gary Laevsky and the Molecular Biology Confocal Microscopy Facility which is a Nikon Center of Excellence for their excellent technical support. We further thank Dr. Frederick Hughson for indispensable advice and expertise, as well as Jaden Shirkey and Kevin DAmico of the Hughson lab, and all members of the Ploss lab for critical discussions and comments throughout experimentation and preparation of the manuscript. Work in the lab is supported by grants from the National Institutes of Health (R01 AI138797, R01 AI107301, R01 AI146917, R01 AI153236 to AP), a Burroughs Wellcome Fund Award for Investigators in Pathogenesis (#101539 to AP) and funding from Princeton University. RL and SM were supported by the National Institute of General Medicine Sciences of the National Institutes of Health under Award Number T32GM007388. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. (DGE-2039656) awarded to RL. The Molecular Biology Flow Cytometry Resource Facility is partially supported by the Cancer Institute of New Jersey Cancer Center Support grant (P30CA072720). Publisher Copyright: {\textcopyright} LeDesma et al.",
year = "2023",
doi = "10.7554/eLife.80529",
language = "English (US)",
volume = "12",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}