Apolipoprotein E polymorphisms and their protective effect on hepatitis E virus replication

Potential conflict of interest: Nothing to report. TO THE EDITOR: With interest, we read the report by Zhang et al. published in November 2015 entitled “Apolipoprotein E and protection against hepatitis E viral infection in American non‐Hispanic blacks.”1 In a large candidate gene study of selected data from the Third National Health and Nutrition Examination Survey, 1991‐1994, Zhang et al. suggested a possible correlation between certain apolipoprotein E (ApoE) single‐nucleotide polymorphisms (SNPs) and protection against hepatitis E virus (HEV) infection. Of 6,272 participants from three different ethnic/racial populations in the United States, they selected 497 SNPs from 190 genes potentially associated with influencing the cellular lipid metabolism and immune response upon HEV infection. In the literature, three allelic isoforms of the ApoE gene are described (ϵ2, ϵ3, and ϵ4).2 Comparing the allele frequencies of the ApoE variants and the prevalence of anti‐HEV immunoglobulin Gs in the cohort study, the researchers provide evidence for a strong correlation of ApoEϵ3 and ApoEϵ4 with protection against HEV infection in non‐Hispanic blacks, but not in non‐Hispanic whites or Mexican Americans. To characterize the underlying mechanism of this observation, we investigated the role of ApoE SNPs on HEV replication in tissue culture. Huh‐7.5 cells silenced in endogenous ApoE expression3 were rescued to ectopically express the three ApoE allelic isoforms as determined by an ApoE‐specific enzyme‐linked immunosorbent assay (ELISA; Fig. 1A). HEV RNA replication was analyzed by transfection of in vitro transcribed genotype 3 reporter replicon encoding a Gaussia luciferase.4 HEV replication levels were not influenced by any of the ApoE isoforms (Fig. 1B). Next, we challenged the same cell lines with genotype 3 full‐length HEV RNA and determined viral replication with ribavirin (RBV) as control by the detection of intracellular RNA copy numbers (Fig. 1C). Moreover, the efficiency of virus assembly and release was determined by quantification of the accumulation of open reading frame 2 (ORF2) protein in culture fluid of transfected cells by using an ORF2‐specific ELISA5 (Fig. 1D). Importantly, HEV full‐length RNA replication and virus assembly were not affected irrespective of which ApoE isoform was expressed, whereas RBV treatment decreased HEV RNA replication and virus particle release (Fig. 1C,D). In summary, these results show that at least in the Huh‐7.5 model, HEV RNA replication and virus production are not affected by ApoE polymorphisms, unlike the epidemiological findings reported by Zhang et al.1Figure 1: HEV replication is not influenced by expression of ApoE isoforms. Huh‐7.5 3′sh ApoE KD cells were silenced of ApoE expression with a short hairpin RNA targeting the 3′‐untranslated region of the human ApoE gene and rescued by ectopic expression of the three common ApoE isoforms. (A) ApoE‐specific secretion was determined by commercially available ApoE‐ELISA (n = 3). (B) Cell lines expressing different ApoE variants were transfected with HEVp6 subgenomic replicon encoding for a Gaussia luciferase reporter, and replication was determined at 4, 24, 48, and 72 hours postelectroporation (h p.e.) by monitoring Gaussia activity in the supernatants. Data are depicted as relative light units (RLU) normalized to 4 hours (n = 3). (C,D) HEVp6 full‐length in vitro transcripts were transfected into cell lines expressing different ApoE variants, and intracellular HEV RNA (n = 3) (C) as well as secreted HEV ORF2 (n = 2) (D) was conducted as described and normalized to 4‐hour values. For each assay the median and range of the biological replicates are depicted.