The Translated Amino Acid Sequence of an Insertion in the Hepatitis E Virus Strain 47832c Genome, But Not the RNA Sequence, Is Essential for Efficient Cell Culture Replication

Abstract

The hepatitis E virus (HEV) can cause hepatitis E in humans. Recently, the occurrence of HEV strains carrying insertions in their hypervariable genome region has been described in chronically infected patients. The insertions originate from human genes or from the HEV genome itself. Although their distinct functions are largely unknown, an involvement in efficient cell culture replication was shown for some strains. The HEV strain 47832c, originally isolated from a chronically infected transplant patient, carries a bipartite insertion composed of HEV genome duplications. Here, several mutants with deletions and substitutions of the insertion were generated and tested in cell culture. Complete deletion of the insertion abolished virus replication and even a single glycine to arginine substitution led to reduced cell culture growth. A mutant encoding a frameshift of the inserted sequence was not infectious, whereas a mutant carrying synonymous codons in this region replicated similar like the wild type. Substitution of the insertion with the S17 insertion from HEV strain Kernow C1-p6 did not result in viable virus, which might indicate strain- or cell type-specificity of the insertions. Generally, the translated amino acid sequence of the insertion, but not the RNA sequence, seems to be responsible for the observed effect.

Isolation of 15 hepatitis E virus strains lacking ORF1 rearrangements from wild boar and pig organ samples and efficient replication in cell culture

“…The HVR is known to be an intrinsically disordered region, to which functions such as the regulation of replication are attributed to (Purdy et al., 2012). In this context, effects of insertions, mutations and deletions within the HVR on the efficiency of HEV RNA replication and viral growth were proven (Pudupakam et al., 2011; Scholz et al., 2021). Especially insertions originating from human genes have been assumed to lead to a replicative advantage in vitro (Lhomme et al., 2014; Nguyen et al., 2012; Shukla et al., 2011).…”

A Modular Hepatitis E Virus Replicon System for Studies on the Role of ORF1-Encoded Polyprotein Domains

“…This is consistent with the similarity of the expression kinetics of p6LucA26 and 47832mcLucA26. The VXH.47832mc fragment is derived from a cell culture adapted strain and also contains an insertion in the HVR, which is critical for growth in cell culture [ 20 ]. The influence of these insertions on virus replication is still not fully understood, though some evidence exists that they contain nuclear localization signals [ 20 , 37 ].…”

“…The VXH.47832mc fragment is derived from a cell culture adapted strain and also contains an insertion in the HVR, which is critical for growth in cell culture [ 20 ]. The influence of these insertions on virus replication is still not fully understood, though some evidence exists that they contain nuclear localization signals [ 20 , 37 ]. However, nuclear localization of the polyprotein by itself does not appear to improve replication in cell culture [ 37 ].…”

“…However, nuclear localization of the polyprotein by itself does not appear to improve replication in cell culture [ 37 ]. Notably, Scholz et al demonstrated that exchanging the insertion in 47832mc for the insertion of p6 attenuates the virus beyond recovery [ 20 ]. The sequence length of VXH fragment exchanged in this work is larger, which may account for the comparably lower difference between the replicons.…”

“…While some progress has been made in recent years, particularly with the development of cell culture models based on HEV-3 strains Kernow-C1 [ 17 ] and 47832c [ 18 ], the efficient cultivation of HEV in vitro is still limited to specific strains and cell lines. Both HEV strains were isolated from persistently infected patients and exhibit specific insertions within the HVR of the nonstructural protein encoding ORF1, which are critical for maintenance in cell culture [ 19 , 20 ]. In addition, optimized and well-adapted protocols allowed replication of HEV strains without particular insertions and production of high titers in cell culture [ 21 ].…”