Open Reading Frame 4 - A Multifunctional Protein of Hepatitis E Virus

Hepatitis E virus (HEV) is a small virus belonging to the family Hepeviridae. HEV is the chief cause of acute hepatitis worldwide, causing approximately 20 million infections annually, which results in 60,000 deaths. The genome of HEV is a single-stranded, positive-sense RNA of about 15 kb in length and is categorized into three open reading frames (ORFs): ORF1, ORF2 and ORF3. HEV GT 1 is known to encode an additional fourth reading frame ORF4, which is expressed only during endoplasmic reticulum (ER) stress. The ORF4 is believed to play a critical functional role in the viral replication of HEV GT 1 isolates. Although the disease was discovered in the late 1980s, the precise molecular mechanisms underlying the higher pregnancy mortality associated with HEV remain elusive. The absence of an efficient culture system and a GT 1 animal pregnancy model is a significant barrier to understanding the mechanisms behind HEV-induced pregnancy mortality. This introductory chapter focuses on the morphology, and genomic organization, along with structural and nonstructural proteins of HEV. The information provided in this review on the HEV proteins is likely to assist in understanding the deeper functioning of structural as well as non-structural biology of HEV proteins.

Hepatitis E virus (HEV), a member of the family Hepeviridae, is the major cause of hepatitis E infections across the globe. Though the open reading frame 4 (ORF4) indispensability in the replication of HEV has been demonstrated, its physicochemical properties remain to be explored. Therefore, a thorough investigation of the physical and chemical properties of the ORF4 proteins, obtained from various HEV isolates, was carried out to examine their peculiar characteristics. The physicochemical properties considered for the present study included a number of charged amino acid residues, instability indices, aliphatic indices and grand average of hydropathy (GRAVY) scores. The comparative analyses predicted the basic, unstable, thermostable and hydrophobic nature of the ORF4 proteins. Delineation of various physicochemical components of ORF4 will assist in revealing specific patterns of these proteins, further which will increase our knowledge of HEV biology.

Hepatitis E virus (HEV) is a small RNA virus. The unique component open reading frame 4 (ORF4) has been demonstrated to perform crucial functions in the HEV Genotype I (GT I). The amino acid pattern of HEV-ORF4 was systematically examined by computer predictors to explicate its role in viral pathogenesis. Amino acid distribution showed ORF4 was enriched with disorder-promoting residues (Ala, Arg, Pro, Ser, Gly) and a few order-promoting residues (Leu), in combination with structure-breaking resides (Gly and Pro). The ORF4 showed a deficiency in order-promoting residues, like Asn, Phe, Tyr, and Trp. This initial examination revealed a preponderance of disordered regions interpreting ORF4 as proteins consisting of disordered regions, i.e., proteins consisting of disordered regions with structured globular domains). The IDRs (intrinsically disordered regions) as IDPs (intrinsically disordered proteins)/IDPRs (intrinsically disordered protein regions) play a critical role in various regulatory functions of viruses, thus were examined that revealed ORF4 exhibited the characteristics of ORDP (ordered protein), IDPR and IDP. Further, the identified disorder-based protein binding sites revealed the involvement of ORF4 in diverse crucial biological functions, substantiating them as targets of regulation. As ORF4 functions are yet to be completely explored, thus, our data could help in elucidating its functions. Collectively, data from this comprehensive investigation suggest the ORF4 protein’s role in the regulation and pathogenesis of HEV.

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation worldwide. The open reading frame 4 (ORF4) has been demonstrated to enhance the replication in HEV Genotype 1 (G1) isolates through regulation of an internal ribosome entry site (IRES)-like RNA element. HEV accounts for up to 30% mortality rate in pregnant women with the highest incidences reported for HEV G1. The HEV physiopathogenesis and adaptation to new hosts are attributed to its large genomic variability and constant evolution. Although previous investigations have reported extensive genetic diversity in HEV strains, data on the prevalence of genomic variability in ORF4 protein remains unexplored. The present study attempted a detailed characterization of the factors contributing to genetic variability in HEV ORF4 which included mutational, entropy and selection pressure analyses. The sequences of ORF4 genomes were retrieved and a total of three different datasets were built, representative of each host Human, Rat and Ferret, and comparative analyses were performed. Resulting alignments of ORF4 protein genes inspected for different datasets revealed the prevalence of both synonymous and non-synonymous mutations. Datasets I, II and III were observed with 1, 63 and 23 entropy sites, respectively, with Dataset I possessing the least variation and Dataset II with the largest variation. Several non-synonymous mutations in conjunction with higher entropy values were observed in the II (Rat) and III (Ferret) datasets, however, limited variation was observed in the I dataset (Human). Selection pressure analysis revealed ORF4 protein genes under both purifying (I) and positive (II and III) selection. A higher transition to transversion ratio was observed signifying bias towards transition in ORF4 genomes. This chapter aims to collect information and discuss new advances in the role of ORF4 heterogeneity in HEV variability. These findings are likely to augment information about the molecular evolution, adaptation and biology of this emerging viral protein.

Hepatitis E virus (HEV) is the causative agent of hepatitis E worldwide. The indispensability of the open reading frame 4 (ORF4) region in the replication of HEV Genotype 1 (G1) has been demonstrated. Intrinsically disordered protein (IDP) offers enormous potential as a druggable target. Intrinsically disordered protein regions (IDPRs)/intrinsically disordered proteins (IDPs) are characterized by a lack of defined tertiary structure under physiological conditions. Given their prevalence in various diseases, IDPs are attractive therapeutic targets. The ORF4 has shown its involvement in the regulation of HEV due to the prevalence of intrinsically disordered regions (IDRs) as IDPR or IDP. In this context, the current study explores the ORF4 protein as a drug target due to its intrinsic disorder (ID) protein characteristic. Utilizing the homology modelling algorithm, the 3-dimensional (3D) structures of the ORF4 target protein were designed and further assessed through PROCHECK for the presence of clefts, tunnels and pores. Subsequently, the ORF4 was examined for the overall fraction of intrinsic disorder content. In this chapter, an overview of ORF4 has been provided in terms of its structure-function relationship and contribution to several biological processes through PPIs. The results propose that ORF4 protein could act as a potential drug molecule, henceforth, accelerating the process of drug designing strategies against HEV.