Human Enterovirus 68 Interferes With The Host Cell Cycle To Facilitate Viral Production

Background:Human enterovirus 68(EV-D68)is an emerging pathogen that can cause severe respiratory disease and is associated with cases of acute flaccid paralysis and cranial nerve dysfunction,especially among children.It was first isolated from samples obtained in California in 1962 from four children with pneumonia and bronchiolitis.Over the past 10 years,EV-D68 infection outbreaks have been reported in Italy,the United States,Germany,China,and several other countries.And in 2014,the United States and Canada had the largest and most widespread EV-D68 outbreaks,and these were associated with severe clinical manifestations.Unfortunately,no vaccines for prevention or medicines for treatment are currently available for future outbreaks,mainly due to the fact that information on host factors required for EV-D68 replication is scarce.As a feature of their pathogenic mechanism,many viruses facilitate their own replication by interacting with host factors that regulate cell cycle progression.In a previous study,we found that human EV-A71 and Coxsackievirus A16,manipulate the host cell cycle at S phase in order to promote their own viral replication;however,the potential manipulation of the host cell cycle by EV-D68,which is associated with higher lethality in recent large-scale outbreaks,has not been previously characterized.Object:In this study,we assessed the effect of the host cell cycle on EV-D68 viral production,as well as the ability of EV-D68 to manipulate host cell cycle progression.And try to increase the understanding of the pathogenic mechanisms of enteroviruses and provide a potential target for the treatment and prevention of enterovirus-related diseases.Methods:To examined the effects of the cell cycle status on EV-D68 viral replication,as well as the impact of EV-D68 virus on the host cell cycle,RD cells were treated with serum starvation for G0/G1 synchronization,The cells were synchronized in S phase by culture with 0.85 m M thymidine,and to assess the effects of G2/M phase synchronization,cells were treated with 25 ng/ml nocodazole.Cell-cycle profiles were determined by flow cytometry and the percentage of cells in each phase of the cell cycle was analyzed by the Mod Fit LT program.Levels of intracellular EV-D68 RNA and the m RNA levels of cell cycle-related proteins were detected in RD cells by quantitative real-time PCR.To determine the virulence of the virus,progeny viruses in the supernatants were titrated using RD cells.The regulation and protein concentration of cell cycle-related proteins were analyzed by western blot analysis and Elisa kit,respectively.Results:The results suggest that synchronization in G0/G1 phase,but not S phase,promotes viral production,while synchronization in G2/M inhibits viral production.Both an early EV-D68 isolate and currently circulating strains of EV-D68 can manipulate the host cell cycle to arrest cells in the G0/G1 phase,thus providing favorable conditions for virus production.Cell cycle regulation by EV-D68 was associated with corresponding effects on the expression of cyclins and CDKs,which were observed at the level of the protein and/or m RNA.Furthermore,the viral non-structural protein 3D of EV-D68 prevents progression from G0/G1 to S.Interestingly,another member of the Picornaviridae family,EV-A71,differs from EV-D68 in that G0/G1 synchronization inhibits,rather than promotes,EV-A71 viral replication.However,these viruses are similar in that G2/M synchronization inhibits the production and activity of both viruses,which is suggestive of a common therapeutic target for both types of enterovirus.These results further clarify the pathogenic mechanisms of enteroviruses and provide a potential strategy for the treatment and prevention of EV-D68-related disease.Conclusion:Different with EV-A71,these results indicate that the EV-D68 displayed significant ability to increase the percentage of cells in G0/G1 phase,and that G0/G1 phase is most favorable for EV-D68 replication.But both EV-D68 and EV-A71 can be inhibited by the medicines that induce G2/M arrest.Therefore,medicines that induce G2/M arrest might be considered as a common approach for inhibiting different types of anti-enterovirus infection,which provides a new direction for anti-enterovirus drug development.