Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture

Abstract

Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line

“…It is only recently that optimised cell models have been developed and high HEV viral loads detected in HepG2/C3A, A549, PLC/PRF/5 and Huh-7-Lunet BLR cells [24][25][26]. Increased viral replication has also been reported in hepatoma cells expressing HEV-1 ORF4 [27]. Our group previously developed an in vitro system of HEV infection in human HepaRG cells embedded in 3D Matrigel [28].…”

Mechanism of Cross-Species Transmission, Adaptive Evolution and Pathogenesis of Hepatitis E Virus

“…ORF4 protein is reported to enhance the replication of HEV-1 by promoting the viral RNA-dependent RNA polymerase (RdRp) activity of ORF1 [ 35 ]. A recent report demonstrated that ORF4 provided in trans enhances the viral replication of genotype 3 HEV (HEV-3) although it does not naturally encode ORF4 in its genome [ 36 ].…”

Hepatitis E and Pregnancy: An Unholy Alliance Unmasked from Kashmir, India

“…An additional ORF4, spanning nt2835-3308 and overlapping with ORF1, is present in HEV-gt1 alone and its protein expression is regulated via an IRES-like RNA element (nt2701-2787). ORF4 appears to cause endoplasmic reticulum (ER) stress which may be involved in promoting viral replication [ 38 , 39 ] ( Figure 2 ). As C-terminal 19 amino acids are absent in around half of the genomes, only the N-terminal 124 amino acids of pORF4 can interact with other viral and host proteins.…”

“…Other hepatitis viruses (HAV, HBV and HCV) and other HEV genotypes (HEV-gt2 and HEV-gt3) do not cause higher deaths in pregnant women [ 150 ]. Among the HEV family, HEV-gt1 alone has ORF4 which encodes a protein, pORF4 of 124 aa [ 38 , 39 ]. The encoded pORF4 by the HEV-gt1 genome interact with other viral and host proteins, enhance viral polymerase activity and promote viral replication [ 123 ].…”