Structural aspects of hepatitis E virus

Abstract

Hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide. Hepatitis E is an enterically transmitted zoonotic disease that causes large waterborne epidemic outbreaks in developing countries and has become an increasing public-health concern in industrialized countries. In this setting, the infection is usually acute and self-limiting in immunocompetent individuals, although chronic cases in immunocompromised patients have been reported, frequently associated with several extrahepatic manifestations. Moreover, extrahepatic manifestations have also been reported in immunocompetent individuals with acute HEV infection. HEV belongs to the alphavirus-like supergroup III of single-stranded positive-sense RNA viruses, and its genome contains three partially overlapping open reading frames (ORFs). ORF1 encodes a nonstructural protein with eight domains, most of which have not been extensively characterized: methyltransferase, Y domain, papain-like cysteine protease, hypervariable region, proline-rich region, X domain, Hel domain, and RNA-dependent RNA polymerase. ORF2 and ORF3 encode the capsid protein and a multifunctional protein believed to be involved in virion release, respectively. The novel ORF4 is only expressed in HEV genotype 1 under endoplasmic reticulum stress conditions, and its exact function has not yet been elucidated. Despite important advances in recent years, the biological and molecular processes underlying HEV replication remain poorly understood, primarily due to a lack of detailed information about the functions of the viral proteins and the mechanisms involved in host-pathogen interactions. This review summarizes the current knowledge concerning HEV proteins and their biological properties, providing updated detailed data describing their function and focusing in detail on their structural characteristics. Furthermore, we review some unclear aspects of the four proteins encoded by the ORFs, highlighting the current key information gaps and discussing potential novel experimental strategies for shedding light on those issues.

Keywords: Hepatitis E virus; Review; Structural biology.

Fig. 1

Schematic representation of ORF1 of HEV. The HEV genome is approximately 7.2 kb in length, with a methyl guanosine cap (Cap) at the 5’ end and a polyA at the 3’ end, containing two untranslated regions (NCRs) at the 5’ and 3’ ends. HEV contains three partially overlapping open reading frames (ORFs). ORF1 includes eight putative domains: Y domain (Y), papain-like cysteine protease (PCP), hypervariable region (HVR), proline-rich region (PRO), X domain (X), helicase (HEL), and RNA-dependent RNA polymerase (RdRp). Four cis-reactive elements (CRE) with a stem-loop structure (SL) are indicated, the second of which is located in the junction region (JR). The black lightning bolt symbol represents the cleavage site for the serine protease cellular factor Xa, and the red lightning bolt symbols represent cleavage sites for the serine protease thrombin in ORF1. “MB” indicates the membrane-binding site in the MT-Y iceberg region. The nucleotide and amino acid positions are according to HEV strain Sar55 (GenBank accession number AF444002).

Fig. 2

Summary of ORF2 structure and characteristics. (A) ORF2 contains a signal peptide (SP) and three domains: the shell domain (S), middle domain (M), and protruding domain (P). The black triangles indicate the glycosylation sites in ORF2 (N137, N310, and N562), and the black star indicates the proline-rich hinge between the M and P domains. The secondary structure of the HEV3 capsid protein (PDB ID 2ZTN) is displayed. S, M, and P domains are shown in blue, green, and black, respectively. α-Helices, β-sheets, and loops are represented by yellow rectangles, pink arrows, and grey thick lines. Red dashed lines indicate the disordered regions. The nucleotide and amino acid positions are according to HEV strain Sar55 (GenBank accession number AF444002). (B) 3D structure of the capsid protein (HEV3 PDB ID 2ZTN). N-linked glycosylation sites (N137 and N310 in the S domain and N562 in the P domain) are indicated in red.

Fig. 3

Major features and motifs of ORF3. The two hydrophobic domains (D1 and D2) and the two proline-rich domains (P1 and P2) are shown.

Fig. 4

Novel ORF4, present only in HEV1. The IRES-like element (nt 2701–2787) and ORF4 protein (overlapping ORF1), with its putative ubiquitination site, are shown.