# Hepatitis E ORF2 Blocks Trophoblast Autophagy to Induce Miscarriage via LC3B Binding Rather than PI3K/Akt/mTOR Pathway Suppression

## TL;DR

This study shows how the Hepatitis E virus protein ORF2 blocks cell cleanup processes in pregnant mice, leading to miscarriage.

## Contribution

The study identifies ORF2 as the key HEV protein that inhibits autophagy by directly binding to LC3B, not through the PI3K/Akt/mTOR pathway.

## Key findings

- ORF2, not ORF3, causes autophagy suppression in trophoblast cells by accumulating p62 and reducing LC3B.
- ORF2 binds directly to LC3B, blocking autophagosome formation, confirmed by co-immunoprecipitation.
- ORF2 suppresses PI3K/Akt/mTOR while activating AMPK and TFEB, indicating complex autophagy regulation.

## Abstract

Hepatitis E virus (HEV) is a zoonotic pathogen that can infect pregnant women and cause adverse pregnancy outcomes, including miscarriage and preterm delivery. The previous study demonstrated that HEV genotype 3 (HEV-3) inhibits complete autophagic flux in both mouse placental tissue and human trophoblast cells (JEG-3), evidenced by reduced expression of ATG proteins (including LC3, Beclin1, ATG4B, ATG5, and ATG9A) and accumulation of p62. However, the specific regulatory pathway involved remains unclear. Thus, eukaryotic expression vectors for HEV open reading frames (ORFs) were constructed, and ORF2 and ORF3 proteins were transiently overexpressed in JEG-3 cells via liposome transfection. While both ORF2 and ORF3 significantly reduced LC3B protein levels (p < 0.01), only ORF2 induced p62 accumulation (p < 0.01), indicative of autophagic inhibition, which indicates that ORF2 was the key viral protein mediating autophagy suppression in JEG-3. The results of WB and RT-qPCR showed that ORF2 suppressed the PI3K/Akt/mTOR pathway while enhancing nuclear translocation of TFEB (p < 0.01) and AMPK phosphorylation (p < 0.01), suggesting paradoxical activation of upstream autophagy regulators. Through co-transfection of mCherry-LC3 with ORF2, co-localization studies, and AlphaFold 3-based intermolecular interaction predictions, we propose that ORF2 directly binds LC3B to block autophagosome formation. Finally, co-immunoprecipitation confirmed physical interaction between HEV ORF2 and LC3B, elucidating the molecular mechanism of HEV-induced autophagy suppression in trophoblasts. These findings reveal the molecular mechanism by which HEV inhibits autophagy leading to miscarriage in mice, providing new insights into HEV-induced reproductive damage.