Adaptation of a Genotype 3 Hepatitis E Virus to Efficient Growth in Cell Culture Depends on an Inserted Human Gene Segment Acquired by Recombination

Abstract

An infectious cDNA clone of a genotype 3 strain of hepatitis E virus adapted to growth in HepG2/C3A human hepatoma cells was constructed. This virus was unusual in that the hypervariable region of the adapted virus contained a 171-nucleotide insertion that encoded 58 amino acids of human S17 ribosomal protein. Analyses of virus from six serial passages indicated that genomes with this insert, although initially rare, were selected during the first passage, suggesting it conferred a significant growth advantage. RNA transcripts from this cDNA and the viruses encoded by them were infectious for cells of both human and swine origin, the major host species for this zoonotic virus. Mutagenesis studies demonstrated that the S17 insert was a major factor in cell culture adaptation. Introduction of 54 synonymous mutations into the insert had no detectable effect, thus implicating protein, rather than RNA, as the important component. Truncation of the insert by 50% decreased the levels of successful transfection by ϳ3-fold. Substitution of the S17 sequence by a different ribosomal protein sequence or by GTPase-activating protein sequence resulted in a partial enhancement of transfection levels, whereas substitution with 58 amino acids of green fluorescent protein had no effect. Therefore, both the sequence length and the amino acid composition of the insert were important. The S17 sequence did not affect transfection of human hepatoma cells when inserted into the hypervariable region of a genotype 1 strain, but this chimeric genome acquired a dramatic ability to replicate in hamster cells.

Hepatitis E virus replication and interferon responses in human placental cells

“…The reporter viruses harbor a Gaussia luciferase, which replaces parts of the ORF2 gene 23. The human hepatocellular carcinoma cell line HepG2 was tested as a positive control in parallel, and RBV was used as an HEV RNA replication inhibitor.…”

“…AF444002, with insertion of an S17 sequence in the hypervariable region) and one the Kernow‐C1 p6, were used to generate HEV in vitro transcripts as described 22, 23, 24. Capping was performed using Ribom7G Cap Analog (Promega, Madison, WI).…”

The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

“…JQ679013). The Kernow-C1 P1 strain expressed the HEV ORF2 protein in less than 2% of Huh7 S10-3 liver cells transfected with viral RNA, whereas the Kernow-C1 P6 HEV strain expressed the HEV ORF2 protein in 10 to 45% of transfected Huh7 S10-3 liver cells (24). Having two infectious cDNA clones derived from the same patient fecal sample, with one lacking elements required for enhanced replication, gave us a unique opportunity to assess the molecular determinants allowing the Kernow-C1 P6 strain to replicate better than the Kernow-C1 P1 strain.…”

“…The HVR has been proposed to play a role in HEV persistence in immunocompromised individuals (22). Additionally, insertions and rearrangements within the ORF1 protein have given an enhanced ability to replicate in cell culture (23,24).…”

“…Recently, unique strains of HEV containing insertions of 171 nucleotides in the HVR of the HEV genome, derived from the human ribosomal proteins S17 and S19 (RPS17 and RPS19), were identified from an English patient coinfected with human immunodeficiency virus and an American liver transplant patient chronically infected with HEV, respectively (24,25). The RPS17 insertion mutant was further selected during serial passages of HEV-containing fecal materials in HepG2C3A cells and exhibited an expanded host range, infecting cells from pig, deer, chicken, cat, dog, mouse, and hamster in vitro (26).…”