PRRSV Regulates Cholesterol Synthesis And Its Interaction With Cholesterol 25 Hydroxylase

Since PRRS's discovery in the late 1980s,the disease has spread throughout the world major countries,causing huge economic strike to the worldwide pig industry.Due to the highly variable,super immune suppression and immune escape ability characteristics of PRRSV,existing vaccines are difficult to provide complete protection,and there is no specific anti-PRRSV drug,which makes the prevention and control of PRRS very difficult.Therefore,in-depth study of the interaction between PRRSV and the host,the screening of antiviral host factors,and the discovery of potential drug targets are of great significance for the effective prevention and control of PRRS.Cellular cholesterol plays a vital role in the life cycle of enveloped viruses,and a large number of studies have confirmed that cholesterol and its metabolites participate in antiviral natural immunity.At present,it is not clear how PRRSV regulates intracellular cholesterol synthesis and the relationship between cholesterol synthesis and host antiviral natural immunity.This study analyzed the mechanism of PRRSV regulating intracellular cholesterol synthesis,and the anti-PRRSV effect of cholesterol catalyzed product 25-hydroxycholesterol(25HC)and the mechanism of PRRSV antagonizing the antiviral activity of cholesterol 25-hydroxylase(CH25H)were studied.The specific research content is as follows:1.PRRSV nsp4 up-regulates intracellular cholesterol levels through the PP2A-HMGCR pathwayThe dynamic changes of intracellular cholesterol content at different time points in porcine alveolar macrophages(PAMs)and PK-15^CD163 cells infected with PRRSV(WUH-3 strain)were analyzed,and found that PRRSV infection significantly increased intracellular cholesterol levels and the activity of the HMGCR.We further analyzed whether AMPK and PP2A that regulate HMGCR activity,are involved in PRRSV up-regulating HMGCR,and it was found that PRRSV up-regulates HMGCR activity by regulating PP2A phosphorylation.In order to screen which protein encoded by PRRSV regulates HMGCR activity,the effects of all viral proteins encoded by PRRSV on HMGCR phosphorylation were analyzed,and it was found that PRRSV mainly up-regulates HMGCR activity through nsp4;overexpression experiments further confirmed that nsp4 up-regulated intracellular cholesterol levels and independs on its3C-like protease activity.By constructing truncation of different functional domains of nsp4,it was confirmed that domain I(amino acids 1-80)of PRRSV nsp4 interacts with the structural subunit PR65?of PP2A and the catalytic subunit PP2Ac of PP2A.Overexpressing domain I of nsp4 up-regulates the intracellular cholesterol content and inhibits the production of IFN-?induced by Sendai virus,and this inhibitory effect can be eliminated by the HMGCR inhibitor Lovastatin.Lovastatin pretreated cells significantly inhibited the production of cholesterol induced by PRRSV,and at the same time weakened the inhibitory effect of PRRSV on IFN-?.Therefore,Lovastatin significantly inhibits the proliferation of PRRSV.The above results indicate that PRRSV mainly up-regulates intracellular cholesterol content through the non-structural protein nsp4,and that nsp4 up-regulates intracellular cholesterol level is one of the strategies of PRRSV to antagonize the host's innate immunity.2.Cholesterol 25-hydroxylase(CH25H)inhibits PRRSV replication in two waysCH25H is an enzyme related to the endoplasmic reticulum,which can oxidize cholesterol to produce 25-hydroxy cholesterol(25HC),reducing the accumulation of cholesterol in the cell,and 25HC negative feedback regulates cholesterol synthesis.Analysis of the role of swine CH25H(pCH25H)in PRRSV infection revealed that overexpression of pCH25H inhibits PRRSV replication,and interfering the expression of pCH25H with siRNA promotes PRRSV infection,suggesting that pCH25H is a host limiting factor.The effect of 25HC on PRRSV infection and its mechanism were further analyzed.It is found that 25HC mainly inhibits PRRSV replication by inhibiting virus invasion.When analyzing the anti-PRRSV effect of pCH25H,it was also unexpectedly found that pCH25H-M still has the ability to inhibit PRRSV infection.Through yeast two-hybrid screening,immunoprecipitation and immunofluorescence colocalization experiments,it was confirmed that both pCH25H and pCH25H-M interact with the PRRSV non-structural protein 1?,and can degrade nsp1?through the ubiquitin proteasome pathway.Through the construction of nsp1?potential ubiquitination site mutants,it was confirmed that the 169th lysine(K169)is the key amino acid site for the degradation of nsp1?by pCH25H/pCH25H-M.The above results indicate that pCH25H not only inhibits the invasion of PRRSV through 25HC,but also degrades nsp1?through the ubiquitin proteasome pathway to inhibit virus replication.3.PRRSV E protein degrades porcine CH25H through the ubiquitin proteasome pathwayFurther analyze the expression level of pCH25H at different times in PAMs infected with PRRSV.The results show that PRRSV infection down-regulated the protein expression of pCH25H in cells,especially in the middle and late stages of infection,suggesting that PRRSV may adopt a certain strategy to antagonize the antiviral effect of pCH25H.Further analysis found that in addition to the interaction with nsp1?,pCH25H also interacts with PRRSV E protein.When E protein and pCH25H are co-expressed,the significant degradation of pCH25H can be observed.Analyzing the possible pathways for E protein to degrade pCH25H,the results confirm that E protein mainly degrades pCH25H through the ubiquitin proteasome pathway.By constructing pCH25H potential ubiquitination site mutants,it was confirmed that the28th lysine(K28)of pCH25H was the ubiquitination site.Significantly,this site(K28)is not conserved in human,monkey and murine CH25H(all are arginine),suggesting that the degradation of CH25H by PRRSV E protein is specific,and experiments have also confirmed that PRRSV E protein cannot degrade human CH25H.In view of the specific degradation of pCH25H by PRRSV E protein,the 25HC content in cells with overexpression E protein and PRRSV infected were further analyzed,and it was found that the 25HC content in cells with overexpression E protein and in the middle and late stages of PRRSV infection significantly decreased.On the one hand,overexpression E protein reduced the inhibitory effect of pCH25H on PRRSV;on the other hand,it weakened the inhibitory effect of 25HC on the inflammatory response.The above results indicate that PRRSV E protein can degrade pCH25H through the ubiquitin proteasome pathway,which is a strategy for PRRSV to antagonize the host antiviral effect and induce late inflammatory response.