CEND1 Inhibits The Replication Of ZIKV By Binding To NS4B

AIM: Zika virus is an arthropod-borne(arbovirus)and a single stranded positive-sense RNA virus in the genus Flavivirus and the family Flaviviridae.In 2015,the Zika virus broke out in Brazil and spread rapidly to more than 50 countries and regions in the Americas and It is seriously harmful to Infected pregnant women who gave birth to babies with abnormal brain development.The non-structural zika virus protein NS4 B can help the Replication of Zika virus in the host by interfering with the production of IFN-? which is the initial and basic defence mechanism of the host against the virus.Cell cycle exit and neuronal differentiation 1(CEND1)is a neuronal lineage-specific modulator that plays a role in synchronization of cell cycle exit and differentiation of neuronal progenitors in the developing nervous system.It is an integral membrane protein composed of two 22–23 k Da polypeptide chains linked together by disulphide bridges.This paper aims to explore the effect of CEND1 on the function of Zika virus and the specific mechanism by which CEND1 exerts its antiviral function by interacting with NS4 B.Currently,No Zika virus vaccine exists.This study provides experimental basis for further research on the molecular mechanism of antiviral function of CEND1 protein,and lays a foundation for finding new drug targets against Zika virus.Methods:(1)The fragments encoding full-length(FL)or peptides of CEND1 were amplified from c DNA fused in tandem with an HA tag by molecular cloning.The plasmids were transferred to DH5? and extracted by using extraction kit.The plasmid HA-CEND1 was transfected into U251,A549 and Hela cells gradiently and zika virus infection was added.q RT-PCR,Western Blot and Confocal experiment were used to study the effect of CEND1 on the function of Zika virus.(2)The U251-CEND1 cell line with stable expression of CEND1 was constructed and added with Zika virus infection.q RT-PCR and Western Blot were used to further verify the effect of CEND1 on the function of Zika virus.(3)N2A-sh CEND1 cell line that interfered with CEND1 expression was constructed and added with zika virus.q RT-PCR experiment was conducted to investigate the effect of interfering CEND1 expression on the function of Zika virus.(4)The plasmids encoding full-length or peptides of CEND1 were co-transferred into HEK293 T cells with Flag-NS4 B plasmids,respectively,and the specific region where CEND1 interacts with NS4 B was determined by co-immunoprecipitation(CO-IP).(5)q RT-PCR was used to investigate the effect of interference with CEND1 on IFN-? production in cells infected with N2A-sh CND1 by Zika virus.Results:(1)q RT-PCR,Western Blot and Confocal experiments showed that CEND1 could inhibit the m RNA and protein levels of Zika virus in cell lines expressing CEND1 by transient transfer.(2)q RT-PCR and Western Blot results showed that CEND1 could inhibit the m RNA and protein levels of Zika virus in cell lines with stable expression CEND1.(3)q RT-PCR results showed that CEND1 could promote the m RNA level of Zika virus in cell lines that interfered with CEND1 expression.(4)CO-IP experiments showed that the 130-150 aa fragment of CEND1 could interact with NS4 B protein of zika virus.(5)q RT-PCR results showed that interference with CEND1 could inhibit the intracellular rise of IFN-?.Conclusion: Cell cycle exit and neuronal differentiation protein 1(CEND1)can inhibit the m RNA and protein levels of Zika virus in cells.CEND1 can bind to the zika virus non-structural protein NS4 B and the binding region is 130-150 aa.Interference with CEND1 can inhibit the intracellular rise of IFN-?.These results provide experimental basis for further research on the molecular mechanism of antiviral function of CEND1 protein.