Cholesterol 25-Hydroxylase Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication through Enzyme Activity-Dependent and -Independent Mechanisms

Abstract

Cholesterol 25-hydroxylase (CH25H) has recently been identified as a host restriction factor that exerts antiviral effects by catalyzing the production of 25-hydroxycholesterol (25HC). CH25H can be rapidly induced upon infection with some viruses. Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, has ranked among the most important swine pathogens since it was discovered in the late 1980s. In this study, we found that PRRSV infection significantly downregulated the expression of CH25H in cells by a so-far unknown mechanism, suggesting that CH25H exerts antiviral activity against PRRSV. Indeed, overexpression of CH25H inhibited PRRSV replication, whereas knockdown of CH25H by short interfering RNA (siRNA) promoted PRRSV infection. The anti-PRRSV effect of 25HC operates via inhibition of viral penetration. Interestingly, a CH25H mutant (CH25H-M) lacking hydroxylase activity still inhibited PRRSV infection. Screening using a yeast two-hybrid system followed by coimmunoprecipitation and immunofluorescence colocalization analyses confirmed that both CH25H and CH25H-M interact with the nonstructural protein 1 alpha (nsp1α) of PRRSV. Unexpectedly, the expression of nsp1α decreased following coexpression with CH25H or CH25H-M. Detailed analyses demonstrated that CH25H/CH25H-M could degrade nsp1α through the ubiquitin-proteasome pathway and that site K169 in the nsp1α protein is the key site of ubiquitination. Taken together, our findings demonstrate that CH25H restricts PRRSV replication by targeting viral penetration as well as degrading nsp1α, revealing a novel antiviral mechanism used by CH25H. PRRSV has been a continuous threat to the global swine industry, and current vaccines are insufficient to provide sustainable control. CH25H has been found to exert a broad antiviral effect; thus, it is an attractive target for the development of anti-PRRSV drugs. Here, we demonstrate that CH25H is an interferon-stimulated gene that is highly expressed in porcine alveolar macrophages. CH25H exerts its anti-PRRSV effect not only via the production of 25HC to inhibit viral penetration but also by degrading viral protein through the ubiquitin-proteasome pathway, suggesting that CH25H is a candidate for the development of antiviral therapeutics. However, PRRSV infection appears to actively decrease CH25H expression to promote viral replication, highlighting the complex game between PRRSV and its host.

Cholesterol 25-hydroxylase negatively regulates porcine intestinal coronavirus replication by the production of 25-hydroxycholesterol

“…Previous studies have indicated that CH25H has an antiviral effect against some viruses and generates antiviral activities through the production of 25HC. For example, CH25H restricts HCV replication through the obstruction of membranous web formation (Anggakusuma et al, 2015); CH25H inhibits PRV by blocking viral attachment and internalization ; 25HC reduces PRRSV infection by inhibiting viral penetration but does not affect adsorption, replication, or release of PRRSV (Ke et al, 2017); and 25HC suppresses Lassa virus infection through aberrant GP1 glycosylation (Shrivastava-Ranjan et al, 2016). In the present study, the overexpression of CH25H or 25HC inhibited PEDV infection by impeding virion entry, which is similar to that observed in PRRSV.…”

S100A9 regulates porcine reproductive and respiratory syndrome virus replication by interacting with the viral nucleocapsid protein

“…PRRs include host restriction factors, many of which are simulated by interferon, which directly or indirectly target multiple stages of the viral life cycle. For instance, the membrane-associated enzyme, cholesterol 25-hydroxylase, inhibits viral entry and degrades viral proteins (Evans et al, 2017;Ke et al, 2017). Moreover, viperin (an IFN-stimulated gene) suppresses HCMV infection by inhibiting viral structural protein synthesis, assembly, and maturation (Chin and Cresswell, 2001).…”

Nuclear localization signal in TRIM22 is essential for inhibition of type 2 porcine reproductive and respiratory syndrome virus replication in MARC-145 cells

“…Primers (Table 2) were designed using the Primer Express software (version 3.0; Applied Biosystems, Carlsbad, CA). Absolute quantitative mRNA levels were calculated using standard curves as previously described [50].…”

“…To investigate the role of endogenous TRIM22 on PRRSV replication, MARC-145 cells were treated with control-siRNA or si-TRIM22 followed by PRRSV infection, then viral titers were measured by TCID 50 , and viral total RNA were quantified by qPCR at 36 hpi. Compared to control cells, we observed that there was a statistically significant increase of the virus yields in the supernatants of si-TRIM22-transfected cells (Fig.…”

“…c The position and sequence of the NLS in TRIM22. MARC-145 cells transfected with Flag-TRIM22 (WT or NLS lacking mutants) were infected with 0.5 MOI of PRRSV for 48 h. d Production of progeny virus were determined using TCID50 assay. e The total cellular RNA was extracted and the mRNA levels of PRRSV N gene were determined by qPCR (Colorfigure online)…”