Study Of The Mechanism On Enterovirus Replication Modulated By Neddylation

Background and objective: Human enteroviruses(HEVs),a genus Enterovirus in the family Picornaviridae,comprise polioviruses,coxsackieviruses,echoviruses,and the enterovirus subgroups,which are all belong to four species,Enterovirus A–D.Several enteroviruses,such as EV-A71 and EV-D68,have emerged as serious public health problems in global outbreaks.Neddylation is an important post-translational modification of proteins and plays a key regulatory role in physiological processes such as embryonic development and cell division and proliferation.Similar to the ubiquitination pathway,NEDD8 protein covalently binds to the target protein in a multi-step cascade of E1 activation enzyme(NAE1)E2 ligase(UBE2M and UBE2F)and E3 ligase,and participates in the regulation of protein stability and function.Accumulating evidence suggests that different viruses frequently target Neddylation signaling pathways and modulate the intracellular environment of the host to enhance virus replication inrecently years.Therefore,we speculate that the Neddylation of the modified pathway may be crucial to the replication of EV-A71 and may be a target for antiviral treatment.NAE1 inhibitor MLN4924 has shown significant efficacy as an antineoplastic agent in phase I/II clinical trials in a variety of cancers.Besides,MLN4924 has been found to be an optimal antiviral drug candidate against diverse human viruses.Herein,we set out to investigate the effects of the Neddylation pathway on enterovirus replication and to explore the potential clinical value of MLN4924 as an antiviral drug candidate against enterovirus infection.Methods: 1.The effect of MLN4924 on EV-A71 replication:(1)Human rhabdomyosarcoma RD cells were treated with MLN4924 at various concentrations(0.02,0.05,0.1,0.5,1 and 2 ?M)and challenged with EV-A71(multiplicity of infection,MOI of 0.01).Cytopathic effect(CPE)was observed at 48 h post-infection(hpi),supernatants were collected and viral titers were calculated as TCID50.The protein expression levels of enterovirus VP1 protein were detected using immunoblotting.(2)The cytotoxicity of MLN4924 was determined in mock-infected RD cells.Cells were cultured in media containing a range of MLN4924 concentrations(0–20 ?M),and cell proliferation and cytotoxicity were evaluated using the MTS assay.RD cells were treated with different concentrations of MLN4924 and collected 48 h after EV-A71 infection.Viral RNA levels was detected using q RT-PCR,the curve was drawn,and EC₅₀ was calculated.(3)RD cells were treated with either MLN4924 or DMSO and then incubated with EV-A71 for 2 h at 4 °C or 37 °C to facilitate virus attachment or entry,respectively.q RT-PCR was used to detect viral RNA(v RNA).(4)The concentrations of intracellular and supernatant viral RNA at specific time points post-infection were determined using q RT-PCR,the effect of MLN4924 treatment on EV-A71 VP1 protein expression at specific time points post-infection was detected using immunoblotting.(5)We performed the same experiments in Human colorectal carcinoma cells HT-29 and human lung cancer cells A549.HT-29 and A549 were treated with MLN4924 and challenged with EV-A71.Cytopathic effect(CPE)was observed at 48 h post-infection(hpi),supernatants were collected and viral titers were calculated as TCID50.The protein expression levels of enterovirus VP1 protein were detected using immunoblotting.2.Study on the mechanism of MLN4924 inhibiting EV-A71 replication: We generated stable knockdown RD cell lines using sh RNA targeting endogenous E1(NAE1)and E2(UBE2M or UBE2F)enzymes.The cells were then challenged with EV-A71.CPE was observed in RD cells at 48 hpi.The survival rates of infected cells were determined using trypan blue staining.Viral RNA was determined using q RT-PCR at 48 h post-infection.The expression of VP1 was detected using immunoblotting.Supernatants were collected at 48 hpi,and viral titers were calculated as TCID50.3.The effect of MLN4924 on EV-D68 replication: MLN4924-or DMSO-treated HEK293 T cells and A549 cells were infected with EV-D68.CPE was observed 48 h post-infection,viral RNA(v RNA)was detected using q RT-PCR,EV-D68 VP1 expression were detected using immunoblotting,and viral titer of supernatant was calculated using TCID50 assay.Results: 1.MLN4924 suppresses EV-A71 replication:(1)MLN4924 treatment markedly inhibited EV-A71 CPE and significantly reduced the titers of progeny virus in the supernatant.In addition,EV-A71 VP1 protein expression decreased in a dose-dependent manner in MLN4924-treated cells.MLN4924 inhibited EV-A71 RNA replication in RD cells at an EC₅₀ value of approximately 0.011 ?M.No significant cytotoxicity or decrease in cell proliferation was observed in MLN4924-treated cells,even at concentrations as high as 20 ?M,as determined using the MTS assay.(2)We found that MLN4924 treatment did not significantly affect EV-A71 virus attachment and entry.Time-dose analysis of EV-A71 RNA replication indicated a strong decrease in the accumulation of viral RNA in the cell lysate and in the supernatant of MLN4924-treated RD cells,when compared to that in DMSO-treated cells.We next analyzed VP1 protein expression in RD cells infected with EV-A71 either in the presence of 0.5 ?M MLN4924 or DMSO over a time course.VP1 protein expression was detectable at 8 hpi in both MLN4924-and DMSO-treated cells.At later time points,a dramatic reduction in VP1 protein expression was observed in both the cell-associated and supernatant-associated fractions of MLN4924-treated cells as compared to the DMSO-treated controls.(3)MLN4924 inhibited EV-A71 replication in HT-29 and A549: MLN4924 significantly reduced EV-A71 CPE,RNA replication,VP1 protein expression and progeny virus production.2.EV-A71 replication was inhibited in the stable knockdown RD cell lines sh NAE,sh UBE2 M or sh UBE2M: These results showed that the downregulation of NAE1,UBE2 M,and UBE2 F dramatically reduced enterovirus CPE,the replication of viral RNA,the expression of VP1 protein and the production of infectious virions,as compared with those in control cells.3.MLN4924 inhibits EV-D68 replication: MLN4924 treatment significantly blocked EV-D68 CPE,VP1 protein expression and infectious virion production in HEK293 T.MLN4924 treatment potently inhibited EV-D68 replication in human lung carcinoma A549 cells.Conclusions: 1.MLN424 can inhibit EV-A71 virus replication at low concentrations in different cell lines,suggesting that MLN4924 is a safe and effective inhibitor against enterovirus replication.2.MLN4924 treatment did not significantly affect EV-A71 virus attachment and entry,indicating that MLN4924 suppresses EV-A71 replication at a post-entry step.3.EV-A71 replication was inhibited in the stable knockdown RD cell lines sh NAE,sh UBE2 M or sh UBE2 M,suggesting that Neddylation pathway is a determinant factor for supporting EV-A71 replication and NAE,UBE2 M and UBE2F are required for enterovirus infection.4.MLN4924 treatment potently inhibited EV-D68 replication,which proves that MLN4924 has the potential as a broad-spectrum antiviral drug candidate against enterovirus infection.