Hepatitis E Virus Genome Structure and Replication Strategy Scott P. Kenney1 and Xiang-Jin Meng2 1Food Animal Health Research Program, The Ohio State University, Wooster, Ohio 44691 2Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Correspondence: [email protected]; [email protected] Hepatitis E virus (HEV) possesses many of the features of other positive-stranded RNA viruses but also adds HEV-specific nuances, making its virus–host interactions unique. Slow virus replication kinetics and fastidious growth conditions, coupled with the historical lack of an efficient cell culture system to propagate the virus, have left many gaps in our understanding of its structure and replication cycle. Recent advances in culturing selected strains of HEV and resolving the 3D structure of the viral capsid are filling in knowledge gaps, but HEV remains an extremely understudied pathogen. Many steps in the HEV life cycle and many aspects of HEV pathogenesis remain unknown, such as the host and viral factors that determine cross- species infection, the HEV-specific receptor(s) on host cells, what determines HEV chronicity and the ability to replicate in extrahepatic sites, and what regulates processing of the open reading frame 1 (ORF1) nonstructural polyprotein. epatitis E virus (HEV) is one of the leading incidence of HEV infection within the United Hcauses of acute viral hepatitis worldwide. In States is unknown owing to the lack of Food and developing countries with poor sanitation con- Drug Administration (FDA)–approved diag- ditions, an estimated 20 million infections occur nostics for testing patients. Further complicat- annually with approximately 3.3 million acute ing diagnosis, the incubation period of hepatitis HEV cases, resulting in about 56,600 deaths per E following exposure to the virus ranges from 2 year (Lozano et al. 2012; Rein et al. 2012). In to 10 weeks. The symptoms of acute hepatitis E www.perspectivesinmedicine.org industrialized countries, clinical cases of HEV are indistinguishable from other types of acute tend to be more sporadic, with zoonotic trans- viral hepatitis, which include mild fever, anorex- mission via direct contact with infected animals ia, nausea, and vomiting lasting for days. Less or consumption of contaminated animal meats common symptoms include abdominal pain, being the most likely routes of infection. Domes- skin rash, or joint pain. In addition, patients tic animals, including pigs, deer, and rabbits, can develop jaundice with dark urine and hepa- harbor the zoonotic genotype (gt)3 HEV, which tomegaly. In rare instances, fulminant hepatitis can contaminate commercial meat products may also occur (Goel and Aggarwal 2016). The bound for human consumption (Feagins et al. rate of fulminant hepatitis greatly increases in 2007; Cossaboom et al. 2011, 2016). The true HEV-infected women who are in their second Editors: Stanley M. Lemon and Christopher Walker Additional Perspectives on Enteric Hepatitis Viruses available at www.perspectivesinmedicine.org Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031724 1 S.P. Kenney and X.-J. Meng or third trimester of pregnancy, resulting in mink, but also is not thought to infect humans. acute liver failure, fetal loss, and mortality rates The Orthohepevirus D species consists of iso- as high as 25%. More recently, chronic HEV lates found in bats (Smith et al. 2015). The infections have become a significant clinical taxonomy of the Hepeviridae family will con- problem in immunosuppressed populations, es- tinue to evolve as more genetically divergent pecially in solid organ transplant recipients (Ka- strains are identified from an expanding host mar et al. 2011, 2013; Kamar and Izopet 2014; of animal species. Fang and Han 2017), which could allow the vi- rus to acquire mutations that could potentially PROPERTIES OF VIRIONS AND VIRION enhance pathogenicity and transmissibility (van STRUCTURE Tong et al. 2016). The HEV virion is a small (320–340 Å) particle (Balayan et al. 1983) with a T = 3 icosohedral CLASSIFICATION capsid lattice comprised of 180 copies of the HEV is a single-stranded positive-sense RNA capsid protein (Xing et al. 1999, 2010; Guu virus classified within the Hepeviridae family, et al. 2009). Crystal structures revealed three of which there are two genera based on sequence functional domains named S (shell), M (mid- divergence: Orthohepevirus and Piscihepevirus. dle), and P (protruding). The S domain is the The genus Piscihepevirus contains a single mem- most conserved region among HEV genotypes ber known as the cutthroat trout virus (Batts (Zhai et al. 2006) and forms the icosahedral shell et al. 2011). The genus Orthohepevirus includes serving as the base for the M and P domains. avian and mammalian HEVs and are subdivided The S domain crystal structure reveals a jelly-roll into four distinct species, Orthohepevirus A–D. fold common to many small RNAviruses (Ross- These species are classified based on host range mann and Johnson 1989). The P domain is hy- and sequence identities (Fig. 1). The species Or- pothesized to be the binding site for the cellular thohepevirus A includes at least seven HEV ge- receptor and is recognized by neutralizing anti- notypes (HEV-1 to HEV-7), which collectively bodies (He et al. 2008). The P domain is further infect humans and a number of other animal divided into the P1 region, which has threefold species, including pigs, boars, deer, mongooses, protrusions, and the P2 region, which has two- rabbits, and camels (Smith et al. 2016). gt1 to 4 fold spikes. Both P regions possess β-barrel folds infect humans, with gt1 and gt2 being restricted and potential polysaccharide-binding sites that to humans and gt3 and gt4 being zoonotic, in- are postulated to help in cell–receptor binding fecting both humans and several other animal and/or capsid disassembly (Guu et al. 2009). www.perspectivesinmedicine.org species. HEV-5 and HEV-6 have only been iden- The M domain interacts strongly with both the tified in wild boars thus far (Smith et al. 2014). S and P domains, and likely contributes to virion HEV-7 from camelids has recently been report- particle stability (Xing et al. 2010). The amino- ed to infect humans and has caused chronic dis- terminal region of the capsid protein appears to ease in a liver transplant patient (Lee et al. 2016). be critical for forming the T = 3 icosahedral Orthohepevirus B species was originally known structure, as capsid protein expression products as the avian HEV and primarily infects chickens, lacking the amino-terminal 111 amino acids as- causing hepatitis-splenomegaly syndrome with semble into virus-like particles (VLPs) with a decreased egg production (Haqshenas et al. T = 1 symmetry that are smaller than the native 2001). More recently, members of the Orthohep- virions. In addition, the carboxy-terminal re- evirus B species have also been detected in wild gion of the capsid, although not involved in birds (Reuter et al. 2016; Zhang et al. 2017). virion morphology, is required for virus replica- Avian HEV does not appear to be able to tion. Virus particles lacking the 52 carboxy- infect mammals (Yugo et al. 2014). The Ortho- terminal amino acids showed defective RNA en- hepevirus C species consists of isolates from rat, capsidation and had less stable virions (Shiota greater bandicoot, Asian musk shrew, ferret, and et al. 2013). 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031724 HEV Genome Structure and Replication Strategy Orthohepevirus A AB290312 AY115488 AB369689 13794 Genotype 3 AF082843 AP003430 59 JQ0 053 JQ953664 FJ7 FJ998008 84 0977 Orthohepevirus C AF4557 AB248521AB290313 EU36 GU345042 KF511797 KJ873911 NC_023425JN597006 AY5350042 AB369687 Orthohepevirus B AY535004 EU723513 Orthohepevirus D JQ001749 FJ906895 HQ731075 JQ013791 Piscihepevirus KJ013415 AB197673 HPECG AY723745 X9 8292 DQ279091 HPESV HP JF4437 AY230 P J272108 ENSSP A 8 AY204877 KJ4 21 KJ496144 202 Genotype 1 96143 AB36968 37 AB602441 AB1085 AB220974 DQ450072 AB573435 Genotype 2 AB856243 Genotype 4 GU119961 Genotype 7 AB074915 Genotype 5 Genotype 6 Orthohepevirus A Figure 1. A phylogenetic tree encompassing all of the newly International Committee for Taxonomy of Viruses (ICTV)–recognized reference strains of the family Hepeviridae. All recently proposed reference strains for www.perspectivesinmedicine.org hepatitis E virus (HEV) (Smith et al. 2014, 2016) were aligned and placed into a circular phylogenetic tree using Geneious software v10.2.2 by Biomatters (see geneious.com). Sequence alignment parameters included global alignment with free end gaps with a cost matrix of 65% similarity. The tree was generated using the Tamura-Nei genetic distance model with a neighbor joining tree build method. The more divergent strains of HEV are located toward the center (Piscihepevirus) and the more conserved strains are located around the circumference (Or- thohepevirus A strains). The Orthohepevirus A strains are color-coded to match their respective genotype. Cartoons depict which host species the viruses are known to infect. Orthohepevirus C are also able to infect ferrets, mink, Asian musk shrew, and greater bandicoot (Smith et al. 2014). Orthohepevirus B are reported to infect turkeys, little egrets (Reuter et al. 2016), and wild birds (Zhang et al. 2017). GENOME STRUCTURE ∼6650 nucleotides long (Huang et al. 2004). HEV genome RNA possesses a 50 7-methylgua- Genome Organization 0 nosine cap structure followed by a short 5 un- The mammalian HEV genome is a single- translated region (UTR) of 26 nucleotides, three stranded, positive-sense RNA ∼7200 nucleotides major open reading frames ([ORFs]: ORF1, in length, whereas the avian HEV genome is ORF2, and ORF3), and a 30UTR (Fig. 2) (Zhang Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a031724 3 S.P.