The Role And Mechanism Of Autophagy In Viral Protein Expression And Virus Replication During Cytomegalovirus Infection

BACKGROUNDMost of the world's population is infected with human cytomegalovirus(HCMV),among which the sero-prevalance ranged from 40 to>90%.Although most of the infection are asymptomatic in immunocompetent individuals,HCMV retinitis occurs predominantly in those patients who are unable to generate a normal primary T-cell response against the virus or in patients who are carriers of HCMV but have decreased CMV-specific T-cell response due to disease or immuno-suppressive treatment,such as acquired immunodeficiency syndrome(AIDS)and leukemia.Autophagy plays a dual role in virus infection,which not only modulate the primary antiviral response and prevent prolonged and excessive inflammation,but also control viral infections,and finally result in the clearance of viral antigens and other pathogens.It has been demonstrated that cytomegalovirus(CMV)could induce autophagy early upon infection,which might have an impact on virus replication and the survival of host cells,while the virus developed strategies to disrupt autophagy to limit the autophagy-induced immune responses for the virus survival.However,how autophagy was induced by CMV infection and how autophagy influence virus replication is still not well understood,which deserved to be explored further.The major tegument protein p UL83(phosphoprotein pp65)of HCMV is the most abundant protein in the virion,which is involved in virus transcription and replication.The expression of immediately early 1 and 2 gene encoding viral proteins(IE1 and IE2)of HCMV are crucial for the establishment of lytic infection and reactivation from viral latency and play a major role in HCMV pathogenesis and host immunomodulation.While IE1 is conditionally essential,IE2 is indispensable for productive viral replication.Therefore,exploring the mechanisms of how IE2 expression was regulated by autophagy is good point to understand the relationship between autophagy and HCMV replication,which might be a potential therapeutic target to restrict the virus replication.Therefore,in the first part of this study,the human embryonic lung fibroblasts(HELs)and THP-1 derived differentiated macrophages will be used as cell line models in vitro to explore the interaction between viral protein IE2 and the autophagy induced by HCMV infection.Meanwhile,the autophagy inducer rapamycin and the inhibitor 3-MA will be used to regulate autophagy levels before or after HCMV enters the HELs and THP-1 derived macrophages respectively to study the effect of autophagy on the expression of viral proteins IE2 and p UL83.Apoptosis is another important mechanism for the host to clear the virus during lytic infection.However,multiple viruses have developed strategies to suppress regulated cell death through encoding several viral proteis to inhibit apoptotic and necroptotic pathway.The interaction between autophagy and regulated cell death is complicated,which finally play a role in virus replication.In addition,the most common cause of blindness caused by cytomegalovirus retinitis in AIDS and leukemia patients is the necrosis of photoreceptor cells and retinal pigment epithelial cells(RPE).Murine cytomegalovirus(MCMV)has a high homologous with HCMV in geneme and they have similar pathogenic mechanism.Therefore,in vitro and in vivo mouse models infected with MCMV were extensively used to study the pathogenic mechanism of HCMV.MCMV early antigen(EA)is a nucleoprotein that is abundant in viruses,and its expression level can be used as the indicator of virus replication.Therefore,in the second part of the present study,murine cytomegalovirus(MCMV)infected mouse embryonic lung fibroblasts(MEF)and posterior eyecup culture were used in vitro to study the effect of autophagy on MCMV virus replication.In this study,the autophagy inducer rapamycin and autophagy inhibitor 3-MA and chloroquine(CQ)will be used to regulate autophagy levels and to explore further whether caspase-3 mediated apoptosis and RIPK1/RIPK3/MLKL-mediated necroptosis are involved in the coordination of autophagy and CMV replication.PURPOSE1.To determine the role of human cytomegalovirus(HCMV)immediate early viral protein 2(IE2)in HCMV infection induced autophagy.2.To explore the effect of different autophagy levels regulation at different time point or different ways on the expression of viral proteins,especially the IE2 with HCMV infection.3.MEF and mice eyecup culture were used to elucidate whether autophagy plays a role in murine cytomegalovirus(MCMV)replication.4.To further investigate the role of caspase-3 dependent apoptosis and RIPK1/RIPK3/MLKL dependent necroptosis in the interaction between MCMV replication and autophagy.5.To further determine the role of autophagy on MCMV virus replication by another autophagy inhibitor CQ,and to explore whether Caspase-3 mediated apoptosis is also involved in this process.METHODS1.The role of autophagy in viral protein expression during HCMV infection1.1 To detect how autophagy levels can be influenced by human HCMV infection and to explore whether viral protein IE2 is participated in the regulation of autophagy.1)Primary human embryonic lung fibroblasts(HELs)and THP-1 derived macrophages were infected with the AD 169 strain of HCMV(MOI1).The total protein was extracted at 6,12,24 and 48 hours post infection(h.p.i.).2)UL122,encoded viral protein IE2,recombinant plasmid transfection was performed to overexpress IE2 in HELs with lipofectamine 3000 reagent.The total protein was extracted at 24 h.p.i..3)Autophagy hallmarks LC3I/II and SQSTM1/p62,autophagy related protein ATG3,viral proteins IE2 and p UL83,were detected by Western blot.4)Image J was used to quantify the target proteins.1.2 Rapamycin or 3-MA were used to regulate the autophagy levels and the effect of autophagy regulation on the viral proteins IE2 and p UL83 expression were investigated.1)Groups:DMEM medium treated mock infection group,DMEM medium treated MCMV infection group,3-MA+DMEM:3-MA treated DMEM medium mock infection group,3-MA+MCMV:3-MA treated MCMV infection group,Rapamycin+DMEM:rapamycin treated DMEM medium mock infection group,Rapamycin+MCMV:rapamycin treated MCMV infection group.2)Autophagy regulation prior to HCMV infection:Autophagy inducer rapamycin(500n M)and inhibitor 3-MA(5m M)were used to stimulate or suppress autophagy 24 hours before HCMV infection of HELs and THP-1 derived macrophages,followed by infecting with HCMV(MOI=1).The total protein was extracted at 24hand 48h post infection.3)Autophagy regulation after HCMV infection:Autophagy inducer rapamycin(500n M)and inhibitor 3-MA(5m M)were used to stimulate or suppress autophagy following HCMV infection of HELs and THP-1 derived macrophages(MOI=1).The total protein was extracted at 24 and 48 h.p.i..4)The expression of LC3I/II,SQSTM1/p62,ATG3,viral protein IE2 and p UL83 were detected by Western blot.5)At 48h after HCMV infection of HELs,immunofluorescence staining was used to detect the distribution and expression of viral protein IE1.6)Image J was used to quantify the target proteins.1.3 The effect of autophagy inhibition by ATG3 gene silencing on viral protein IE2expression.1)ATG3 specific si RNA was synthesized by TSINGKE Biological Technology of Wuhan.2)ATG3 specific si RNA was transiently transformed into HELs with lipofectamine 3000 reagent.At 24 hours post transfection,HELs were infected with HCMV(MOI=1).3)The total protein was extracted at 12 and 24 h.p.i..Expression of LC3I/II,ATG3,and viral protein IE2 were determined by Western blot.4)Image J was used to quantify the target proteins.1.4 The effect of different autophagy levels regulated by rapamycin,3-MA or si ATG3on HCMV-induced apoptotic cell death.1)Groups:same as previously described in 1.2 and 1.3.2)Treatment of autophagy inducer rapamycin(500n M)and inhibitor 3-MA(5m M) before or after HCMV infection of HELs(MOI=1).3)ATG3 specific si RNA or negative blank control si RNA were transiently transfected into HELs and followed by HCMV(MOI=1)infection or DMEM mock medium 24 hours after transfection.4)Cell suspension were collected at 24h and 48h after HCMV infection,and PE Annexin V 7-AAD staining was used to detect the levels of cell apoptosis by flow cytometry.2.The role of autophagy in murine cytomegalovirus(MCMV)replication and its mechanism2.1 Effect of MCMV infection on autophagy levels and programmed cell death1)Tissue and cell infection model:Eyecups were isolated from adult C57BL/6J mice and infected with K181 strain of MCMV(5×10~3PFU)or DMEM mock medium.MEF were infected with K181 strain of MCMV(MOI=0.1)or DMEM mock medium.2)Total protein was extracted at 24,48,72 and 96 h.p.i.from MEF,or at 4,7,10,and 14 days post infection(d.p.i.)from eyecup.3)Autophagy hallmarks LC3I/II and SQSTM1/p62,phospho-p70 S6kinase(T389),and cleaved caspase-3 were detected using Western blot.Image J was used to quantify the target proteins.4)Double staining of TUNEL and cleaved caspase-3 or phosphor-MLKL at 48 h.p.i.of MEF to localize the TUNEL positive cells and cleaved caspase-3 or phosphor-MLKL.2.2 The effect of autophagy regulation by rapamycin or 3-MA on MCMV replication1)Groups:DMEM medium mock infection group,MCMV infection group,3-MA+MCMV:3-MA treated MCMV infection group,Rapamycin+MCMV:rapamycin treated MCMV infection group.2)MEF were infected with K181 strain of MCMV(MOI=0.1).Eyecups were isolated from adult C57BL/6J mice and infected with K181 strain of MCMV(5×10~3 PFU).3)Rapamycin(500n M),3-MA(5m M)or DMEM medium were added to different groups 2 hours after MCMV attached the MEF or eyecup.4)Culture supernatants were collected at 24,48,72 and 96 h.p.i.in MEF,and at 4,7,10,and 14 d.p.i.in eyecup,which were treated with medium mocks or autophagy inhibitor 3-MA,or autophagy inducer rapamycin.These culture supernatants were used for plaque assay to detect the virus titer.5)MEF were fixed with 4%paraformaldehyde at 48 h.p.i.,and then double immunofluorescence staining of TUNEL and virus early antigen(EA)was performed to detect the expression of EA with treatment of autophagy inhibitor 3-MA,or autophagy inducer rapamycin.6)Immunofluorescence staining was performed to detect EA positive cells in the slides section and whole flat mount of eyecup treated with autophagy inhibitor 3-MA,or autophagy inducer rapamycin at 4,7,10,and 14 d.p.i..7)Image J was used to quantify the expression of EA in MEF and eyecup culture.2.3 Caspase-3 dependent apoptosis was involved in the MCMV replication suppression caused by 3-MA.1)Groups and MCMV infection:DMEM medium mock infection group,MCMV infection group,3-MA+DMEM:3-MA treated DMEM medium mock infection group,3-MA+MCMV:3-MA treated MCMV infection group,Rapamycin+DMEM:rapamycin treated DMEM medium mock infection group,Rapamycin+MCMV:rapamycin treated MCMV infection group.MEF and eyecup were infected with K181 strain of MCMV as described in 2.2.2)Rapamycin(500n M),3-MA(5m M)or DMEM medium were added to different groups 2 hours after MCMV attachment and penetration.3)The cytotoxicity test of autophagy inducer rapamycin and inhibitor 3-MA:TUNEL assay was performed in MEF treated with 3-MA(5m M)or DMEM for 48 hours and in eyecup treated with rapamycin(500n M),3-MA(5m M)or DMEM for 7 days to detect cell death numbers caused by chemicals.4)Double staining of TUNEL and retina pigment cells(RPE)at 4,7,and 14 d.p.i.of eyecup,or double staining of TUNEL and EA at 48 h.p.i.of MEF,were performed to test the apoptotic cells distribution and EA expression.5)The total protein was extracted at 4,7,10,14 d.p.i.of eyecup and 24,48 h.p.i.of MEF.Western blot was used to detect the related protein expression including autophagy marker proteins LC3I/II and SQSTM1/p62,and phospho-p70 S6 kinase (T389),m TOR,phospho-m TOR(pm TOR),Cleaved caspase-3 and cleaved caspase-8 and RIPK1/RIPK3/MLKL pathway.6)Image J was used to quantify the TUNEL and EA positive cells and the relative expression of target proteins.2.4 The effect of another autophagy inhibitor chloroquine(CQ)on MCMV-induced apoptosis and virus replication.1)Groups:DMEM medium mock infection group,MCMV infection group,3-MA+DMEM:3-MA treated DMEM medium mock infection group,3-MA+MCMV:3-MA treated MCMV infection group,CQ+DMEM:CQ treated DMEM medium mock infection group,CQ+MCMV:CQ treated MCMV infection group.2)MEF were infected with K181 strain of MCMV(MOI=0.1).Eyecups were isolated from adult C57BL/6J mice and infected with K181 strain of MCMV(5×10~3PFU).3)Rapamycin(500n M),3-MA(5m M),CQ(100?M)or DMEM medium were added to different groups 2 hours after MCMV attachment and penetration.Culture supernatants were collected at 24 and 48 h.p.i.in MEF,and at 7 d.p.i.in eyecup.Plaque assay was performed to detect the virus titer in MEF and eyecup treated with autophagy inhibitor 3-MA,or CQ.4)The morphology of MEF was observed and pictures were taken at 24 and 48 h.p.i.with the inverted microscope.Double staining of MCMV early antigen EA and TUNEL was carried out to detect the viral replication levels and apoptotic cells caused by CQ at 48 h.p.i..5)The total protein was extracted at 24 and 48 h.p.i.and Western blot was used to detect the related protein expression including autophagy marker proteins LC3I/II and SQSTM1/p62,and phospho-p70 S6 kinase(T389),and cleaved caspase-3.6)Double staining of MCMV EA or retinal pigment epithelial cells(RPE)and TUNEL in eyecup was performed to detect the viral replication levels,morphological changes of RPE and apoptotic cells caused by CQ at 7 d.p.i..7)Image J was used to quantify the EA,TUNEL positive cells and the relative expression of target proteins.RESULTS1.Autophagy inhibition by 3-MA and si ATG3 can suppress HCMV IE2 expression in HELs.1)The early stage of HCMV infection or IE2 overexpression can induce autophagy in HELs:the LC3I/II ratio was lower than the control group at 6 h.p.i.(p<0.05);at 12 and 24 h.p.i.,the LC3I/II ratio showed the same trend as that of 6 h.p.i.,while the LC3I/II ratio was higher than the control group at 48 h.p.i.(p<0.05).The expression of SQSTM1/P62 was higher than the control group at 24 h.p.i.(p<0.05),which indicated that infection of HCMV promotes the lipidation of LC3 before 24 h.p.i.,which was accompanied by the increased expression of viral protein IE2.When only IE2 was overexpressed via UL122 recombinant plasmid transfection without HCMV infection,the autophagy related proteins LC3II and ATG3 were upregulated(P<0.05),which indicated that not only HCMV infection,but also IE2 overexpression could upregulate autophagy.2)HCMV infection of THP-1 derived macrophage can activate autophagy:the autophagy-related protein LC3II was higher than the control group at 6 and 48 h.p.i.,(p<0.05),and the expression of SQSTM1/P62 was lower than the control group at 12 and 24 h.p.i.(p<0.05),indicating that autophagy was activated early at 6 h.p.i..HCMV IE2 protein expression was gradually increased.3)Pretreatment of 3-MA or rapamycin before HCMV attachment can inhibit viral proteins expression:?Treatment with autophagy inducer rapamycin:In HELs,although the expression of LC3II in both the mock infection and HCMV infection groups were decreased at 24 h.p.i.(P<0.05),the expression of SQSTM1/p62 also decreased(P<0.05).The expression of LC3II was increased in HCMV infected HELs at 48 h.p.i.(P<0.05),and the expression of SQSTM1/p62 was decreased(P<0.05),indicating that rapamycin pretreatment can induce autophagy.Meanwhile the immediate early protein IE2 and the tegument protein p UL83 were significantly reduced at 24 and48 h.p.i.(P<0.05).In THP-1 derived macrophage,rapamycin-induced autophagy is mainly manifested as decreased expression of autophagy substrate SQSTM1/p62(p<0.05).Meanwhile,the viral protein IE2 expression was decreased at 24 h.p.i.,but increased at 48 h.p.i.,while the expression of tegument protein p UL83 was increased at 24 h.p.i.compared with the control group(p<0.05).?Treatment with autophagy inhibitor 3-MA:In HELs,the conversion of LC3I to LC3II was inhibited,while SQSTM1/p62 expression was increased at 24 and 48 h.p.i.,indicating that autophagy was inhibited.Meanwhile,the viral protein IE2 expression was significantly reduced at 24 and 48 h.p.i.(p<0.05),and tegument protein p UL83 was increased at 24 h.p.i.but was inhibited at 48 h.p.i.(p<0.05).In THP-1 derived macrophages,the viral protein IE2 expression showed a decreasing trend as autophagy was inhibited(p<0.05),but the viral protein p UL83 expression increased significantly at 48 h.p.i.(p<0.05).4)Treatment of 3-MA or rapamycin after HCMV attachment can affect viral proteins expression:?Treatment with autophagy inducer rapamycin:In HELs,the viral protein IE2 and p UL83 expression were increased at 48 h.p.i.(p<0.05),but there was no significant effect on the expression of viral protein IE1(p>0.05).?Treatment with autophagy inhibitor 3-MA:In HELs,the expression of viral proteins IE2,IE1 and p UL83 were all significantly inhibited at 24 and 48 h.p.i.(p<0.05).?In THP-1 derived macrophages,the expression of viral proteins IE2 and p UL83 expression was not stable and inconsistent enough with the treatment of rapamycin and 3-MA.5)Autophagy inhibition by si ATG3 can inhibit the expression of viral protein IE2:ATG3 si RNA transient transfection significantly reduced ATG3 expression(p<0.001),as well as the expression of LC3II(p<0.01),indicating that the autophagy was inhibited.Meanwhile,the viral protein IE2 expression was also suppressed at 12 and 24 h.p.i.of HCMV(p<0.01).6)The role of autophagy in programmed cell death induced by HCMV infection:At 24 h.p.i.of HCMV in HELs,autophagy regulation by rapamycin or 3-MA before HCMV attachment and penetration had no significant effect on the number of apoptotic cell death(p>0.05),while 3-MA treatment after HCMV infection can,to some extent,inhibit apoptotic cell death of HELs,though it is of no significant difference(p>0.05).At 48 h.p.i.of HCMV in HELs,3-MA and rapamycin pretreatments have the tendency to promote the apoptosis of HELs induced by HCMV infection,although there was no statistical significance(p>0.05).In addition,autophagy inhibition by si ATG3 can somehow inhibit apoptotic cell death but it is of no significant difference (p>0.05).2.Autophagy inhibition by 3-MA can inhibit MCMV replication in MEF and eyecup culture.1)MCMV infection can induce autophagy early but inhibit autophagy during late stage:LC3I/II ratio was significantly decreased(LC3I decreased but LC3II increased relatively)at 24 h.p.i.of MCMV in MEF,while LC3I/II ratio was increased at 48 h.p.i.(P<0.05).These results indicated that autophagy was activated at 24 h.p.i.but inhibited at 48 h.p.i.of MCMV in MEF.In the cultured eyecup isolated from C57BL/6J mice,MCMV infection also increased LC3II expression at 4 and 7 d.p.i.(P<0.05),but the ratio of LC3I/II was increased at 10 d.p.i.(P<0.05)and there was no significant difference at 14 d.p.i.(P>0.05)between MCMV infected and mocks treated eyecups,which means that autophagy was activated at 4 and 7 d.p.i.but inhibited at 10 d.p.i.following MCMV infection in eyecups.2)Autophagy inhibitor 3-MA can restrict MCMV replication in MEF and eyecup culture: Once treated with 3-MA,the virus titer from MEF was significantly reduced at 48,72,and 96 h.p.i.of MCMV(p<0.05),while there was no significant change in virus replication with treatment of rapamycin(p>0.05).In eyecup culture,the virus titer decreased significantly at 4,7,10,and 14 d.p.i.with treatment of 3-MA(p<0.05).With rapamycin treatment,the virus titer was also reduced at 4 and 7 d.p.i.(p<0.05) but didn't change a lot at 10 and 14 d.p.i.of MCMV(p>0.05).Meanwhile,the EA expression in eyecup was decreased at 4,7,10 and 14 d.p.i.with 3-MA treatment (p<0.05).In rapamycin treated eyecup,EA expression did not change a lot at 4 and 10 d.p.i.(p>0.05),although at 7 and 14 d.p.i..EA expression was increased(p<0.05).3.Caspase-3 mediated apoptosis promoted by autophagy inhibitor 3-MA and CQ was involved in the mechanism of its inhibition role in MCMV replication.1)MCMV infection can induce caspase-3 dependent apoptosis:MCMV infection significantly increased cleaved caspase-3 levels at 48 h.p.i.(P<0.05)but not at 24 h.p.i.(P>0.05)in MEF.Meanwhile,the cleaved caspase-3 was also increased at 4,7,10 and 14 d.p.i.of MCMV in cultured eyecups,especially at 7 and 10 d.p.i.(P<0.05).There were some TUNEL positive cells stained with cleaved caspase-3 at the same time,although not all apoptotic cells overlapped with cleaved caspase-3.The staining of TUNEL cells did not overlap with phospho-MLKL staining,but phospho-MLKL always stand by TUNEL positive cells.2)Autophagy inhibitor 3-MA can promote MCMV infection induced caspase-3 dependent apoptosis:?Firstly,double staining results of TUNEL with EA or RPE showed that 3-MA increased the number of TUNEL positive cells but decreased EA expression in MEF(P<0.05).Meanwhile,in eyecup,the number of TUNEL positive cells was also increased at 7 and 14 d.p.i.with 3-MA treatment(P<0.05)but not rapamycin treatment.?Secondly,Western blot results showed that 3-MA treatment significantly increased caspase-3 cleavage in MCMV infected MEF at 48 h.p.i.(P<0.05)compared with MCMV infected MEF without 3-MA treatment,but at 24 h.p.i.,it seemed that MCMV infection can convert 3-MA induced increasing of caspase-3 cleavage in MEF.In MEF,it seemed that 3-MA has no significant and consistent effects on RIPK1/RIPK3/MLKL mediated necroptosis pathway.However,in MCMV infected eyecups,results showed that the cleaved caspase-3 and caspase-8 were increased at 4,7,10 and 14 d.p.i.(P<0.05),indicating apoptosis was activated. But the expression of active RIPK1,phospho-RIPK3,and MLKL were reduced in MCMV infected eyecups treated with 3-MA compared with MCMV infected eyecups at 4,7 and 10 d.p.i.(P<0.05),indicating the necroptosis was somehow inhibited.3)Another autophagy inhibitor CQ also can inhibit MCMV infection induced caspase-3 dependent apoptosis:?Like 3-MA,CQ not only could inhibit virus replication in MCMV infected MEF at 24 and 48 h.p.i.(P<0.05),but also could inhibit MCMV replication in eyecups at 7 d.p.i.(P<0.05).CQ even had stronger effect on limiting MCMV replication at 48 h.p.i.in MEF(P<0.05),as well as at 7 d.p.i.in eyecup,though it is of no significant difference(p>0.05).?Double staining of TUNEL and EA results showed that at 48 h.p.i.in MEF,CQ treatment increased TUNEL positive cells and decreased EA expression(p<0.05),also in eyecups,the number of TUNEL positive cells increased while the EA expression reduced at 7 d.p.i.(p<0.05).?Meanwhile,the Western blot results showed that CQ can also significantly promote the caspase-3 cleavage(P<0.05),which even stronger than 3-MA at 48h.p.i.in MEF.CONCLUSIONS1.IE2 was involved in human cytomegalovirus(HCMV)infection induced autophagy.2.Autophagy levels could interfere with virus entry during HCMV infecting HELs in vitro.3.Autophagy inhibition by 3-MA or silencing ATG3 expression could significantly suppress HCMV viral protein IE2 expression in HELs.4.Although autophagy can be induced by HCMV infection in THP-1 derived macrophage,the role of autophagy levels regulated by rapamycin and 3-MA on the expression of viral proteins IE2 and p UL83 was irregular and inconsistent,which might by caused by the primary cell resource and its related complex immune responses.5.Autophagy was activated at the early stage following MCMV infection both in MEF and eyecup culture,while it was inhibited at late stage.Autophagy inhibitor 3-MA and CQ can significantly reduce MCMV EA expression and restrict the production and release of virus progeny in MEF and eyecup culture.6.Meanwhile,MCMV infection later can induce caspase-3 dependent apoptosis in MEF and eyecup.Furthermore,autophagy inhibitor,3-MA and CQ both can promote caspase-3 dependent apoptosis caused by MCMV infection,which indicated that during the late infection stage,3-MA and CQ restrict MCMV replication indirectly via promoting caspase-3 dependent apoptosis but not RIPK1/RIPK3/MLKL mediated necroptosis.However,during the early stage of MCMV infection,inhibited autophagy plays the leading role in limiting MCMV replication caused by 3-MA and CQ.