Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

Abstract

Porcine reproductive and respiratory syndrome is one of the most important infectious diseases affecting the global pig industry. Previous studies from our group and other groups showed that cholesterol 25-hydroxylase (CH25H), a multitransmembrane endoplasmic reticulum-associated enzyme, catalyzes the production of 25-hydroxycholesterol (25HC) and inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication. However, PRRSV infection also actively decreases porcine CH25H (pCH25H) expression, through unidentified mechanisms. In this study, we found that the ubiquitin-proteasome pathway plays a major role in pCH25H degradation during PRRSV infection and that the PRRSV-encoded envelope (E) protein interacts with pCH25H. PRRSV E protein degraded pCH25H via ubiquitination, and the ubiquitination site was at pCH25H Lys28. Interestingly, PRRSV E protein appeared to specifically degrade pCH25H but not human CH25H, likely because of a Lys28Arg substitution in the human orthologue. As expected, ubiquitin-mediated degradation by E protein attenuated the antiviral effect of pCH25H by downregulating 25HC production. In addition, we found that knockdown of pCH25H decreased E protein-induced inflammatory cytokine expression and that pCH25H overexpression had the opposite effect. These findings suggested that regulation of pCH25H expression was associated with E protein-induced inflammatory responses. Taken together, our results and those of previous studies of the anti-PRRSV effects of CH25H highlight the complex interplay between PRRSV and pCH25H.IMPORTANCE CH25H has received significant attention due to its broad antiviral activity, which it mediates by catalyzing the production of 25HC. Most studies have focused on the antiviral mechanisms of CH25H; however, whether viruses also actively regulate CH25H expression has not yet been reported. Previous studies demonstrated that pCH25H inhibits PRRSV replication not only via production of 25HC but also by ubiquitination and degradation of viral nonstructural protein 1α. In this study, we expanded on previous work and found that PRRSV actively degrades pCH25H through the ubiquitin-proteasome pathway. PRRSV E protein, a viral structural protein, is involved in this process. This study reveals a novel mechanism of interaction between virus and host during PRRSV infection.

Keywords: CH25H; E protein; PRRSV; cholesterol 25-hydroxylase; porcine reproductive and respiratory syndrome virus; ubiquitin-proteasome pathway.