Hepatitis E Virus Genome Structure and Replication Strategy

Abstract

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.

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 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 (Orthohepevirus 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).

Figure 2.

Genomic organization of hepatitis E virus (HEV). The genome of HEV is ∼7.2 kb in length and is composed of a single-stranded positive-sense RNA molecule. The genome contains a 7-methylguanosine RNA cap at the 5′ end and is polyadenylated at the 3′ terminus. There are three conserved open reading frames (ORFs) found in all known HEV strains: ORF1, ORF2, and ORF3. ORF1 encodes the nonstructural polyproteins with putative functional domains including methyltransferase (Met), Y domain, papain-like cysteine protease (PCP), hypervariable region (HVR), helicase (Hel), and RNA-dependent RNA polymerase (RdRp). ORF2 encodes the capsid structural protein. ORF3 encodes a multifunctional phosphoprotein (also known as VP13). ORF2 and ORF3 proteins are translated from a bicistronic subgenomic RNA 2.2 kb in length. In addition to these three ORFs, the genotype (gt)1 HEV encodes an ORF4, which generates a protein from an internal ribosome entry site (IRES)-like element in response to endoplasmic reticulum (ER) stress. This ORF4 protein is a viral replication enhancer. A number of RNA structural elements within the HEV genome contribute to RNA packaging (5′ RNA stem loops), translation of ORF2, ORF3 (junction region CREs), and ORF4 (IRES-like element), and in binding of the genomic RNA to the RdRp (3′ CRE). Positions and lengths of ORFs and indicated features are based on the prototypical gt1 HEV (Sar55 strain) sequence (GenBank accession number AF444002.1).

Figure 3.

Life cycle of hepatitis E virus (HEV). (1) Nonenveloped HEV virions bind to cellular receptors. Heparin sulfate proteoglycans (HSPGs) and heat shock cognate protein 70 (HSC70) are thought to be the attachment receptors for HEV but more research is required to determine the entry receptors for HEV. Once bound to the cell, virions are taken into the cell through a dynamin-2, clathrin, and cholesterol-dependent process. (2) Quasi-enveloped HEV virions are capable of entering the cell via a clathrin-mediated pathway. This entry pathway appears to be dependent on Rab 5 and Rab7 GTPases and, unlike the unenveloped HEV virions, also requires lysosome function before uncoating within the host cell. (3) Heat shock proteins including HSP90 and Grp78 have been implicated in transporting virions to sites of genome release. (4) Once released into the cytoplasm, the positive-strand genomic RNA serves as messenger RNA (mRNA) for the translation of the open reading frame (ORF)1 nonstructural polyprotein. (5) Whether or not processing occurs for the ORF1 polyprotein remains debatable, but the viral nonstructural proteins within ORF1 transcribe the positive-sense RNA genome into a negative-strand intermediate. This step occurs at either the rough endoplasmic reticulum (RER) or RER-derived membrane vesicles and involves host proteins, including elongation initiation factors, and viral proteins, including ORF4 for genotype (gt)1 HEV replication. The subgenomic bicistronic mRNA is generated from the negative-stranded RNA and serves as messenger for translation of ORF2 and ORF3 proteins. (6) Host ribosomes translate the newly synthesized RNAs into more nonstructural proteins and the ORF2 and ORF3 proteins. (7) There are a number of unknown steps to virus assembly. ORF2 binds to viral genomic RNA and begins to multimerize. ORF3 also binds to ORF2 during transition to the plasma membrane. (8) Particles become membrane-associated likely by budding into intracellular vesicles via interaction of the ORF3 protein with host vacuolar sorting proteins such as tumor suppressor gene 101. These enveloped particles retain the trans-Golgi network protein 2 protein and CD63 on their lipid surface until particle release. (9) The plasma membrane serves as the final site for release of enveloped virions via the exosomal release pathway and is sensitive to disruption of Rab27a and Hrs. (10) At some point during transit through the host digestive system, the viral envelope is lost. HEV released into the bloodstream appears to retain its quasi-envelope, which is thought to mask the capsid shell from the host immune system.