The Mechanism Of PRRSV Regulating Cell Translation And Autophagy

Viruses are strict intracellular parasites and in order to utilize cellular resources for efficient replication,different viruses have evolved different strategies to regulate the cellular metabolism,such as translation and autophagy.Porcine reproductive and respiratory syndrome is a viral epidemic threatening the pig industry and has caused huge economic losses.The causative agent PRRSV has outbroken in most countries since its discovery in the late 1980 s.PRRSV was well known by its high variation and inhibiting the host immunity,thus the previous studies mainly focused on the variation pattern and the mechanism of immunosuppression,while the cellular metabolism changes were less studied.Whether PRRSV can directly regulate host protein translation has not been reported,and the mechanism of PRRSV induced autophagy remains unclear,too.Our study proved that PRRSV infection inhibits the cellular translation and nsp2 as well as nsp5 plays a key role in this process.Then the exact mechanisms of PRRSV nsp2 induced translation shutoff,as well as nsp5 induced autophagy were studied.The main research contents are as follows: 1.PRRSV infection inhibits the cellular translationPuromycin can indicate translation level and is used in the indirect immunofluorescence assay to verify that PRRSV infection can inhibit the translation level of MARC-145 cells.Western blot results also showed that PRRSV can significantly inhibit the translation of MARC-145 cells at 24 h post infection.The inhibition became more and more obvious with time.PRRSV infection could also dose-dependently inhibit the translation of MARC-145 cells and PAMs cells,but UV-inactivated PRRSV treatment showed limited effect on the cellular translation,suggesting that PRRSV induced translation shutoff is dependent on viral replication.To screen non-structural proteins of PRRSV WUH3 strain that inhibit translation,Hela cells were transfected with the eukaryotic expression plasmids of these non-structural proteins.The indirect immunofluorescence assay results showed that both nsp2 and nsp5 can significantly inhibit translation.At the same time,we also tested the effects of viral non-structural proteins on the translation level of HEK293 T cells by western blot and obtained the same conclusions.We selected nsp2 and nsp5 for subsequent research,respectively.2.The mechanism of PRRSV nsp2 induced host shutoffTo investigate the mechanism of PRRSV nsp2 induced host shutoff,we constructed three truncated mutants of nsp2(N-terminal papain-like protease domain(PL2),intermediate hypervariable region(HV)and C-terminal transmembrane region(TM)).The plasmids were transfected into HEK293 T cells and the results showed that nsp2 TMdomain can inhibit translation,which was dependent on its location on mitochondria.What's more,nsp2 and its TM domain did not affect the EGFP m RNA level,while thefluorescence of EGFP was significantly decreased,indicating that the translation processwas inhibited specifically.eIF2? phosphorylation is a key modification in the process of cell translationregulation,but nsp2 and TM domain did not affect eIF2? phosphorylation,indicatingthat nsp2 and TM domain induced cellular translation shutoff is not dependent on eIF2?phosphorylation,which also suggests that PRRSV induced translation shutoff does notrely on eIF2? phosphorylation only.To check for this possibility,we applied a drugnamed ISRIB,which can eliminate the host translation shutoff effect of eIF2?phosphorylation.ISRIB treatment only partially recovered the low translation leveldecreased by PRRSV infection,suggesting that PRRSV can inhibit translation in aneIF2? phosphorylation independent manner.We investigated whether PRRSV can induce host translation shutoff by inhibitingmTOR signaling pathway.The western blot results showed that PRRSV significantlyinhibited the phosphorylation of mTOR,4E-BP1,S6 K and e IF4 E.The agonist of mTORsignaling pathway can restore the cellular translation level suppressed by PRRSVinfection,indicating that PRRSV can induce translation shutoff by inhibiting the mTORsignaling pathway.What's more,we proved that overexpression of PRRSV nsp2 andTM domain also inhibited mTOR signaling pathway.In summary of the effects of PRRSV infection and nsp2 overexpression ontranslation,PRRSV inhibits translation by at least two mechanisms: inducing eIF2? phosphorylation and inhibiting the mTOR signaling pathway by encoding nsp2.3.The mechanism of PRRSV nsp5 induced incomplete autophagyWe have found that PRRSV nsp5 inhibits the cellular translation level,while a previous study proved that the fusion protein of PRRSV nsp5,nsp6 and nsp7(nsp567)can induce autophagy.We proposed that PRRSV nsp5 may inhibit translation by inducing autophagy.To check for this possibility,we constructed eukaryotic expression plasmids encoding PRRSV nsp5,nsp56,nsp67,nsp7 or nsp567 and the plasmids were transfected into HEK293 T cells.The results showed that nsp5,nsp56 and nsp567,but not nsp67 and nsp7,increased the level of LC3-II and the aggregation of GFP-LC3,indicating that nsp5 can induce autophagy.The accumulation of autophagosomes may caused by the increased production or the decreased degradation.To investigate the mechanism of PRRSV nsp5 induced autophagy,cells were treated with chloroquine to block the autophagosomes degradation pathway and the western blot results showed that nsp5 did not up-regulate LC3-II anymore,indicating that nsp5 does not induce autophagosomes production.In addition,we found that autophagosomes and lysosomes did not co-localize in nsp5 overexpressing cells,which further confirmed that nsp5 inhibits the degradation of autophagosomes.What's more,we also proved that PRRSV infection induced accumulation of autophagosomes was caused by the blockage of degradation,too.Since the interactions of SNAP29 and STX17 or VAMP8 directly mediate the fusion of autophagosomes and lysosomes,we first tested whether nsp5 affects the interaction between these SNARE proteins.The Co-IP and indirect immunofluorescence assay results showed that overexpression of nsp5 could inhibit the interaction between STX17 and SNAP29,but the interaction between VAMP8 and SNAP29 showed no significant change.The further research showed that nsp5 interacted with the N-terminal domain and SNARE domain of STX17.Since the SNARE domain mediates the interactions between SNARE proteins,the interaction of PRRSV nsp5 with the SNARE domain of STX17 may inhibit the interaction between STX17 and SNAP29,which may contribute to PRRSV induced incomplete autophagy.In summary,this study proved that PRRSV inhibits cellular translation by encoding nsp2 to inhibit the mTOR pathway,and PRRSV inhibits the fusion of autophagosomes and lysosomes by encoding nsp5 to inhibit the interaction of STX17 and SNAP29.This study provides a reference for us to better understand how PRRSV regulates host cellular metabolism and the biological significances of the regulation.