Hepatitis E virus ORF3 protein hijacking thioredoxin domain-containing protein 5 (TXNDC5) for its stability to promote viral particle release

Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Among the three open reading frames (ORFs) of the HEV genome, the ORF3 protein is involved in virus release. However, the host proteins involved in HEV release need to be clarified. In this study, a host protein, thioredoxin domain-containing protein 5 (TXNDC5), interacted with the non-palmitoylated ORF3 protein by co-immunoprecipitation analysis. We determined that the overexpression or knockdown of TXNDC5 positively regulated HEV release from the host cells. The 17FCL19 mutation of the ORF3 protein lost the ability to interact with TXNDC5. The releasing amounts of HEV with the ORF3 mutation (FCL17-19SSP) were decreased compared with wild-type HEV. The overexpression of TXNDC5 can stabilize and increase ORF3 protein amounts, but not the TXNDC5 mutant with amino acids 1-88 deletion. Meanwhile, we determined that the function of TXNDC5 on the stabilization of ORF3 protein is independent of the Trx-like domains. Knockdown of TXNDC5 could lead to the degradation of ORF3 protein by the endoplasmic reticulum (ER)-associated protein degradation-proteasome system. However, the ORF3 protein cannot be degraded in the knockout-TXNDC5 stable cells, suggesting that it may hijack other proteins for its stabilization. Subsequently, we found that the other members of protein disulfide isomerase (PDI), including PDIA1, PDIA3, PDIA4, and PDIA6, can increase ORF3 protein amounts, and PDIA3 and PDIA6 interact with ORF3 protein. Collectively, our study suggested that HEV ORF3 protein can utilize TXNDC5 for its stability in ER to facilitate viral release.IMPORTANCE Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design. Hepatitis E virus (HEV) infection is the leading cause of acute viral hepatitis worldwide. After the synthesis and modification in the cells, the mature ORF3 protein is essential for HEV release. However, the host protein involved in this process has yet to be determined. Here, we reported a novel host protein, thioredoxin domain-containing protein 5 (TXNDC5), as a chaperone, contributing to HEV release by facilitating ORF3 protein stability in the endoplasmic reticulum through interacting with non-palmitoylated ORF3 protein. However, we also found that in the knockout-TXNDC5 stable cell lines, the HEV ORF3 protein may hijack other proteins for its stabilization. For the first time, our study demonstrated the involvement of TXNDC5 in viral particle release. These findings provide some new insights into the process of the HEV life cycle, the interaction between HEV and host factors, and a new direction for antiviral design.