Identification Of The G-quadruplex Structure Of Nucleic Acid Near The Replication Starting Point Of Pseudorabies Virus And Its Regulatory Effect On Virus Proliferation

Pseudorabies virus(PRV)is a pathogen of pseudorabies,belonging to the herpesviridae family,α-Herpesvirus subfamily.Pigs are the main natural host of the virus,which has caused enormous economic losses to the pig industry worldwide.Since the end of2011,the emergence of pseudorabies virus variants has posed new challenges to the prevention and control of PRV in China.The genome of PRV is double stranded DNA,with a length of approximately 145 kb.An important feature of the virus genome is its high GC content,which can reach 70 to 80%.DNA or RNA sequences rich in guanine bases(G)can form a nucleic acid secondary structure different from the classical double helix structure: a G-quadruplex structure.The sequences that form the G-quadruplex structure are commonly found in the genomes of prokaryotes and eukaryotes,and participate in important biological processes,such as gene recombination,DNA replication,transcription,and RNA translation.In addition to the G-quadruplex structure of eukaryotes,more and more studies have reported that there are sequences forming the G-quadruplex structure in the genome of viruses in recent years.These sequences are mainly distributed in the repetitive sequence regions,gene regulatory regions,or coding regions of the virus genome.The G-quadruplex structure formed affects the replication,transcription,and translation of the virus genome.In this study,through bioinformatics analysis,we found that there are two sequences upstream of the PRV genome replication starting point(Ori L)that tend to form a G-quadruplex structure: Ori L-S and Ori L-A,which are highly conserved among 32 PRV strains.Through circular dichroism spectroscopy,gel electrophoresis and dimethyl sulfate footprint protection experiments,it is proved that Ori L-S and Ori L-A can fold to form a parallel G-quadruplex structure in vitro.Fluorescence resonance energy transfer and Taq polymerase elongation retardation experiments showed that the small molecule ligand Phen DC3 promoted the formation of G-quadruplex structures from Ori L-S and Ori L-A sequences,and improved their thermal stability.In addition,using the PRV genome after incubation and annealing with Phen DC3 as a template for PCR amplification,the results showed that the Ori L-S and Ori L-A regions of the PRV genome can form G-quadruplex structures,and Phen DC3 can stabilize the G-quadruplex structures formed by Ori L-S and Ori L-A.The results of enzyme linked immunosorbent assay(ELISA)and electrophoretic mobility assay(EMT)using BG4 antibodies that target to recognize G-quadruplex further indicate that Ori L-S and Ori L-A can form G-quadruplex structures.Using BG4 antibody for chromatin immunoprecipitation experiments,BG4 antibody was enriched in the Ori L-S and Ori L-A regions of the PRV genome.The results showed that PRV can form a G-quadruplex structure in cells.Since the small molecule ligand Phen DC3 can promote and stabilize the G-quadruplex structure formed by Ori L-S and Ori L-A sequences,and it has been confirmed that Ori L-S and Ori L-A of the PRV genome can form G-quadruplex structures in cells,we further investigate the effect of the small molecule ligand Phen DC3 on the proliferation of PRV in host cells.Immunofluorescence experiments have shown that Phen DC3 can promote the formation of G-quadruplex structures in cells.The results of viral titer testing and Western blot experiments indicate that Phen DC3 inhibits the proliferation of PRV in cells.Flow cytometry analysis shows that Phen DC3 inhibits the proliferation of PRV mainly in the replication stage of cells.The results of dosing experiments at different time points and q PCR detection further confirmed that Phen DC3 inhibited the replication phase of PRV in cells.After PRV infected cells,Phen DC3 was added to extract total RNA from cell samples at different time points.RT-q PCR was used to detect the transcription of IE180,EP0,and UL5 genes of PRV.The results showed that Phen DC3 inhibited the m RNA expression of these genes,indicating that Phen DC3 has potential antiviral effects.Next,we investigate the regulatory mechanism of G-quadruplex structure in viral replication and gene expression from the perspective of binding proteins of G-quadruplex structure.Through Pull down experiments and tandem rustic analysis,we screened nucleolin(NCL),a host protein that binds to the G-quadruplex structure formed by Ori L-S and Ori L-A,from the nuclear proteins of PK-15 cells infected with PRV virus.Western blot and flow cytometry analysis showed that NCL had a negative regulatory effect on PRV proliferation through overexpression of NCL gene and si RNA knockdown.Next,we investigate the regulatory mechanism of G-quadruplex structure in viral replication and gene expression from the perspective of binding proteins of G-quadruplex structure.Through Pull down experiments and tandem mass analysis,we screened nucleolin(NCL),a host protein that binds to the G-quadruplex structure formed by Ori L-S and Ori L-A,from the nuclear proteins of PK-15 cells infected with PRV virus.Through the experiments overexpression and si RNA knockdown the NCL gene,Western blot and flow cytometry analysis showed that NCL had a negative regulatory effect on PRV proliferation.In summary,this study found and identified that the Ori L-A and Ori L-S sequences upstream of the replication starting point(Ori L)of the PRV genome can form a G-quadruplex structure,and the small molecule ligand Phen DC3 can promote and stabilize the G-quadruplex structure formed by the Ori L-S and Ori L-A sequences.Meanwhile,Phen DC3 inhibits the replication phase of the PRV genome,thereby inhibiting the proliferation of the virus within the cell.Finally,we explored the molecular mechanism of G-quadruplex regulating PRV proliferation from the perspective of G-quadruplex structure binding proteins.This study not only provides a new understanding of the structure and gene expression regulation of the PRV genome,but also provides potential molecular targets for the development of new PRV vaccines and drugs.