Studies On Regulatory Mechanisms For Replications Of Zika Virus And Dengue Virus By Host Cell Molecules

Part I: m TOR Signaling Pathway Regulates ZIKV Replication through Cellular Autophagy1.BackgroundZika virus(ZIKV),a mosquito-borne pathogen,causes ZIKV disease,of which the typical symptoms were mostly mild including fever,skin rash and joint pain.Remerging of ZIKV's global outbreak in 2016 spread in America and Southeast Asia.New serious neurological disorders of Guillain-Barré syndrome and microcephaly were observed incidentally,which attracted much more attention worldwide.ZIKV is a single-stranded positive sense RNA virus that belongs to the Flaviviridae family.ZIKV genomic RNA encodes a long polyprotein,which is cleaved and processed into 3 structural proteins and 7 nonstructural proteins.ZIKV can replicate and establish infections in several types of cells.To date,the limited knowledge of molecular mechanisms underlying the early stages of ZIKV infection and its interaction with host cells troubles effectively preventing viral epidemic and treating ZIKV disease.Autophagy is a highly conserved catabolic process in which long-lived cytoplasmic components or damaged organelles are sequestered and degraded.Autophagy is adopted by host cells to remove damaged organelles and ensure cell survival so that it resists virus infection,like herpes simplex virus.Meanwhile,some viruses evade the monitoring and protective functions of cellular autophagy and utilize it to benefit their own replication,such as dengue virus and hepatitis C virus.m TOR signaling pathway plays an essential part in autophagy regulation.As the target of autophagy inducer rapamycin,m TOR forms 2 complexes,known as m TORC1 and m TORC2.Inhibition of m TORC1 can activate ULK1 and Beclin-1,initiating autophagy through promoting the production and prolongation of precursor membrane structure.Besides,m TORC1 promotes protein synthesis mainly through the phosphorylation of two key effectors,S6 K and 4E-BP1.The most important role of m TORC2 is to phosphorylate AKT and activate PI3K/AKT signaling pathway.TSC2 can be regulated by several upstream factors,including AKT,AMPK,MAPK.Phosphorylation of TSC2 inhibits m TORC1 activity.However,AMPK and Beclin-1 can also directly regulate autophagy in a m TORC1-independent manner.At autophagy initiation,microtubuleassociated protein 1 light chain(LC3-I)esterified to LC3-II,which is considered as an early marker of autophagy.Previous studies have found that the NS4 A and NS4 B proteins of ZIKV can induce autophagy through m TOR signaling pathway at infection of fetal neural stem cells,which impairs the nervous system.Therefore,further studies are needed to explore the exact mechanism of ZIKV-induced autophagy and its functional roles in viral life cycle.Since ZIKV can be transmitted to humans via mosquito-transmission,systemic viral infection requires ZIKV to infiltrate blood vessels and spread through the circulatory system.Besides,ZIKV can cross the blood-brain barrier to impair the nerve system.This work aimed to study the autophagy induction of human umbilical vein endothelial cells(HUVEC)after ZIKV infection and explore its functional role in viral replication by using pharmacological drugs or siRNAs.In order to further determine the interaction between m TOR and autophagy during ZIKV infection,the regulation of m TOR signaling on autophagy and viral replication were examined by interfering the key molecules of m TOR signaling pathway.2.MethodsHUVEC was infected with lentivirus expressing m Tag RFP-m Wasabi-LC3,followed by ZIKV infection for another 24 hours.Intracellular LC3 fluorescent protein were observed by confocal microscopy to examine the induction of autophagy.Different titers of ZIKV infected HUVEC at different time points to detect the conversion of LC3-I to LC3-II and P62 expression by immunoblotting.The amount of fluorescent LC3 puncta was calculated by confocal microscopy to analyze the autophagic flux in ZIKV-infected HUVEC.Cells were pre-treated with autophagy inhibitors(Wortmannin and chloroquine),or Beclin-1 specific sh RNA to explore the function of autophagy inhibition on viral production by immunoblotting and QRT-PCR.Autophagy inducer rapamycin was also used to explore the function of autophagy induction on ZIKV production by immunoblotting and QRT-PCR.Next,study focused on m TOR in ZIKV-infected HUVEC,the target of rapamycin.HUVEC was infected with ZIKV at 12 and 18 hours post infection(hpi)to detect the expression of m TOR signaling pathway molecules.Rapamycin and its derivatives(everolimus,temsirolimus),or sim TOR were used to disturb m TOR itself;si Raptor and si Rictor were used to disturb m TORC1 and m TORC2,respectively;si TSC2,si AMPK,or AKT activator SC79 were used to disturb m TORC1 upstream factors: Immunoblotting,QRT-PCR and plaque assay were utilized to explore the function of m TOR signaling pathway molecules on viral production.HUVEC was infected with m Tag RFP-m Wasabi-LC3 lentivirus followed by ZIKV infection and observed by confocal microscope to visually verify the effects of disturbing m TOR signaling pathway molecules on ZIKV-induced autophagy.3.ResultsZIKV infection and autophagy inducer rapamycin significantly increased the amount of intracellular fluorescent LC3 puncta or positive cells expressing LC3 fluorescent protein.The expression of LC3-II in ZIKV-infected cells increased significantly at 18 and 24 hpi and maintained until 36 hpi.P62 expression was decreased starting at 12 hpi and significant declined at 48 hpi.A slight shift from yellow to partially red fluorescence was observed in ZIKV-infected cells expressing m Tag RFP-m Wasabi-LC3 from 18 to 24 hpi by confocal microscope.Autophagy inhibitor Wortmannin reduced the expression of LC3-II and reduced extracellular viral progeny content.Chloroquine increased the expression of LC3-II,but reduced viral protein NS3 expression and extracellular viral progeny content.Knockdown of Beclin-1 also reduced viral protein NS3 expression and extracellular viral progeny content.Rapamycin increased the expression of LC3-II,but did not affect viral protein NS3 expression and extracellular viral progeny content.ZIKV infection decreased m TOR phosphorylation and promoted the phosphorylation of upstream factors TSC2 and AMPK and the dephosphorylation of AKT.Downstream factors S6 K and 4E-BP1 dephosphorylated,indicating the blocking of protein translation,while activation of ULK1,expression of Beclin-1 and LC3 II increased,indicating enhanced autophagy.Inhibiting of m TOR molecule directly by rapamycin and its derivatives or sim TOR increased intracellular viral RNA merely but viral protein NS1 expression and extracellular viral progeny content were not affected.Knockdown of Raptor or Rictor promoted viral protein NS1 expression,intracellular viral RNA and extracellular viral progeny content.Interfering with upstream molecules of m TORC1 by si TSC2 or SC79 reduced viral protein NS1 expression,intracellular viral RNA and extracellular viral progeny content.The amount of cellular LC3 fluorescent puncta did not change in the cells transfected with si AMPK,decreased in those with si TSC2 or activation of AKT,and increased in those with rapamycin or siRNAs targeting to Rictor,Raptor or m TOR.It was consistent with the previous results of ZIKVinduced autophagy.4.ConclusionZIKV infection induces autophagy in HUVEC and autophagic flux is promoted.Autophagy inhibition can reduce viral protein expression and progeny release.However,autophagy inducer rapamycin does not affect viral protein expression and progeny release.ZIKV infection inhibits the AKT/TSC2/m TORC1 signaling pathway in HUVEC,upregulating the activation of ULK1 and expression of Beclin-1 and LC3 II enhance autophagy and dephosphorylating of S6 K,4E-BP1 to hinder protein translation.Autophagy can promote ZIKV production but viral protein expression is regulated by m TOR signaling pathway.This study clarifies the mechanism of autophagy in the ZIKV life cycle,as well as the dual functions of m TOR signaling pathway in protein synthesis and autophagy induction during ZIKV infection.It also provides theoretical support for developing highly effective anti-ZIKV drugs based on autophagy-related molecules of mTOR signaling pathway.Part II: PTEN Regulates DENV Replication through Lipid Metabolism1.BackgroundDengue virus(DENV)is an arthropod-borne viral pathogen worldwide with 390 million people at risk annually from tropic to subtropical regions.DENV infections can be asymptomatic,mild clinical features,even life-threatening dengue hemorrhagic fever or dengue shock syndrome.As a member of Flaviviridae family,the virion is an enveloped particle containing a positive single stranded RNA,encoding a single polyprotein that is cleaved into 3 structural proteins and 7 non-structural proteins.No potent vaccines or antiviral drugs are available for DENV infection,in part due to the limited understanding of DENV-host interactions.Phosphatase and tensin homolog deleted on chromosome 10(PTEN)is an important tumor suppressor,encoding a dual-specificity phosphatase with both lipid and protein phosphatase activity that antagonizes PI3 K to block AKT signaling pathway to regulate lipid metabolism.DENV can hijack cellular lipid metabolism and utilize to lipid drops(LDs)as viral assembly platform to promote viral replication and deplete LDs for cellular ?-oxidation to provide energy for viral production.This study aimed to explore the regulation of PTEN phosphatase activity on lipid metabolism and autophagy during DENV infection in Huh7 cells,as well as the effect on viral production.2.MethodsHuh7 cells were infected with DENV-2 for 48 hours.Immunoblotting and QRT-PCR were used to examine PTEN expression.Cells were infected with lentiviruses overexpressing PTEN(wt PTEN)and PTEN sh RNA followed by DENV-2 infection to detect the effect on virus production by immunoblotting,QRT-PCR and plaque assay.Cells were infected with 3 forms of PTEN mutants(G129E-PTEN lipid phosphatase inactive form,Y138L-PTEN protein phosphatase inactive form,C124S-PTEN both the lipid and protein phosphatase inactive form)lentiviruses followed by DENV-2 infection to detect the effect of PTEN phosphatase activity on viral production by immunoblotting,QRT-PCR and plaque assay.DENV NS3 antibody and Oil Red O were used to stain intracellular viral protein and LDs respectively to detect the effect of PTEN phosphatase activity on lipid metabolism in DENV-infected Huh7 cells by confocal microscope.Immunoblotting was used to detect the expression of AKT signaling pathway molecules,lipid synthase,and autophagy-related protein LC3.3.ResultsDENV infection reduced PTEN protein expression by immunoblotting,but did not influence PTEN m RNA level by QRT-PCR.PTEN overexpression increased viral protein NS1 expression,intracellular viral RNA and extracellular viral progeny content,while knockdown of PTEN did the opposite effect.Wt PTEN and Y138 L promoted viral protein NS1 expression,intracellular viral RNA and extracellular viral progeny content,while G129 E and C124 S did not affect.DENV infection reduced the area and number of cellular LDs by confocal microscope.Wt PTEN and Y138 L decreased the area and number of cellular LDs compared with DENV-infected cells instead of G129 E and C124 S.Wt PTEN and Y138 L decreased AKT phosphorylation,promoted the phosphorylation of Fox O1 and Maf1,reduced the expression of lipid synthase and increased LC3-II protein expression.However,G129 E and C124 S did not affect and sh PTEN did the opposite effect.4.ConclusionDENV infection downregulates PTEN expression through post-transcriptional regulation.The lipid phosphatase activity of PTEN enhances DENV production through AKT/Fox O1/Maf1 signaling pathway and autophagy,resulting in lipid reduction and LDs consumption.It helps to clarify the interaction mechanism between PTEN and lipid metabolism in the process of DENV infection and replication,providing new clues for antiDENV therapy.