The Molecular Mechanism Of NDRG1 In The Nuclear Import Of Pseudorabies Virus UL31 And UL34 Protein

Pseudorabies(PR)is a major infectious disease threatening the swine industry worldwide.The causative agent of this disease is pseudorabies virus(PRV),an enveloped,double-stranded DNA virus.It has a broad host range and can infect humans and a variety of other vertebrates,causing acute death and severe clinical symptoms in animals.Pseudorabies virus can also remain latent in the infected host for life,causing serious damage to human health.In recent years,a pseudorabies virus has mutated in many areas,leading to poor vaccination results and causing huge economic losses to the pig industry.In order to understand the molecular mechanism of PRV pathogenesis and its interaction with the host,this study focused on the host gene N-myc downstream regulatory gene 1(NDRG1),and analyzed the molecular mechanism of NDRG1 promoting PRV UL31/UL34 nuclear translocation.This study provides a scientific and theoretical basis for the prevention and control of PRV.The PRV UL31 protein plays a vital role in the translocation of the viral nucleocapsid from the nucleus to the cytoplasm.This is achieved through its interaction with the UL34 protein,forming a heterodimer known as the nuclear export complex(NEC).The UL31/UL34 protein complex facilitates capsid docking,budding,and unencapsulation.The absence of either UL31 or UL34,or both,significantly hinders viral replication and capsid transfer from the nucleus to the cytoplasm.Hence,the UL31/UL34 heterodimer is crucial for nuclear export and virus proliferation.However,the nuclear import process of UL31/UL34 remains unclear.In this study,the impact of PRV-QXX infection on PK-15,MARC-145,HEK293,and RAW264.7 cells was investigated.The results showed that PRV infection led to an increase in the expression of NDRG1 m RNA and protein.To elucidate the underlying molecular mechanism of this up-regulation,the NDRG1 promoter was analyzed and it was discovered that P53 binds to the promoter and regulates NDRG1 transcription.To confirm the role of P53 in PRV-induced up-regulation of NDRG1 expression,the NDRG1 promoter was mutated.The findings confirmed that PRV induces the up-regulation of NDRG1 expression through P53.Furthermore,it was demonstrated that PRV infection causes DNA damage in cells,as evidenced by immunofluorescence and other experimental methods.Finally,the study revealed that PRV induces DNA damage,leading to P53 activation and subsequent up-regulation of NDRG1 expression.In order to further explore the molecular mechanism of NDRG1 promoting PRV proliferation.The study revealed that infection with Pseudorabies virus(PRV)causes the subcellular localization of N-myc downstream-regulated gene 1(NDRG1)to shift from the cytoplasm to the nucleus,while PRV UL31/UL34 is localized to the nuclear membrane.However,in the absence of NDRG1,UL31/UL34 remains in the cytoplasm.To understand the relationship between NDRG1 and PRV UL31/UL34,the interaction between the two was examined using indirect immunofluorescence(IFA)and co-immunoprecipitation(Co-IP).The results indicated that NDRG1 interacts with PRV UL31/UL34,with UL31/UL34 binding to the N-terminus of NDRG1.Furthermore,GST-Pull down experiments demonstrated that NDRG1 directly interacts with PRV UL31/UL34,suggesting that NDRG1 may facilitate the nuclear import and translocation of UL31/UL34.Although UL31 can translocate to the nucleus in the absence of a nuclear localization signal(NLS),NDRG1 lacks NLS,indicating that other proteins may assist in the nuclear translocation process of UL31/UL34.The study identified heat shock cognate protein 70(HSC70)as a key protein that facilitates the nuclear translocation process of NDRG1 and PRV UL31/UL34.Immunofluorescence experiments were conducted to investigate the subcellular localization of HSC70 after PRV infection.The results showed that UL31/UL34 interacts with the N-terminal domain of NDRG1,while the C-terminal domain binds to HSC70.The nuclear translocation of UL31,UL34,and NDRG1 was abolished when HSC70ΔNLS was supplemented in HSC70 knockdown cells or when the expression of importinα was interfered with.These findings suggest that NDRG1 assists in the nuclear import of PRV UL31/UL34 through HSC70,promoting virus proliferation.In summary,this study systematically elucidated the molecular mechanism by which NDRG1 promotes nuclear translocation of UL31/UL34 protein to promote viral proliferation after PRV infection.P53 can bind to the NDRG1 promoter,regulate the transcription of NDRG1,and up-regulate the expression of NDRG1.As a bridge between HSC70 and PRV UL31/UL34 protein,NDRG1 promotes the translocation of PRV UL31/UL34 into the nucleus and ensures the smooth proliferation of PRV.