Regulation Of G-quadruplex On The Expression Of E165R Gene Of African Swine Fever Virus

African swine fever(ASF)is a highly contagious and deadly disease that affects pigs.It is caused by the African swine fever virus(ASFV),which is a double-stranded DNA arbovirus that encodes over 60 structural proteins and more than 100non-structural proteins.One of the non-structural proteins,E165 R,is responsible for providing energy in viral replication and plays a crucial role in ASFV replication.However,despite the characteristics of rapid transmission,high mortality,and difficulty in prevention and control,there is still a lack of effective vaccines and drugs for the disease,resulting in significant economic losses to China’s pig industry.G-quadruplex(G4)structures are special nucleic acid secondary structures formed by guanine-rich DNA or RNA,are ubiquitous in eukaryotes,prokaryotes,and viral genomes,and play an important regulatory role in DNA replication,gene transcription,and protein expression.Additionally,studies have reported that G4 structure regulates viral replication and becomes a potential antiviral target.In this study,bioinformatics analysis showed that there was a guanine-rich sequence in the protein coding region(CDS)of the E165 R gene,which was predicted to form a G4 structure.Experimental results confirmed that the sequence can indeed form a G4 structure,suggesting that targeting this structure may be a potential antiviral strategy.To experimentally verify the formation of G4 structure in the E165 R gene,this paper first synthesized the G-rich sequence of the gene and its mutant sequences E165R-G4 and E165R-G4 M.The formation of G4 structure was identified by acrylamide gel electrophoresis and circular dichroism(CD).The results of the experiments showed that G4 and G4M have the same mobility in the modified gum containing urea,while G4 had a faster mobility in the non-modified gum because of its more compact structure.The CD experimental results revealed that G4 with K+ had a negative peak at 240 nm and a positive peak at 260 nm,which is a typical phenomenon of parallel G4 structure.G4 with K+ was more stable due to the presence of positive ions.Moreover,to further verify the structure formation,a G4-specific small molecule stabilizer,NMM,was used for fluorescence analysis.The results showed that the fluorescence of NMM was stronger in the presence of K+,and the fluorescence signals of G4 and G4 M in the presence of Li+were relatively weaker.These results indicate that the G4 structure of the E165 R gene can be formed in vitro.In this paper,we investigated whether G4 stabilizers PDS and TMPyP4 can bind and stabilize the G4 structure.For this purpose,the DNA was combined with different concentrations of stabilizers for fluorescence resonance energy transfer(FRET)experiments.The results of the experiments showed that with the increase of stabilizers,the energy required to open the G4 structure also increased,indicating that PDS and TMPyP4 can improve the thermal stability of the G4 structure.Moreover,the results of Taq enzyme extension block experiments revealed that PDS and TMPyP4 can stabilize the G4 structure and hinder the extension of Taq enzyme on the DNA template.In this paper,the biological function of G4 structure formed by E165 R gene was studied.With the help of G4 structure antibody BG4,immunofluorescence experiments confirmed that E165R-G4 could form G4 structure in cells.The G4-WT-GFP and G4-MUT-GFP plasmids were constructed by inserting the G4 and G4 M sequences into the start codon of the GFP protein,respectively.The results of flow cytometry and cell fluorescence detection showed that PDS and TMPyP4 inhibited the expression of GFP protein.Then,in order to study the effect of G4 on the expression of E165 R gene,the FLAG tag was inserted after the E165 R gene to construct the E165R-FLAG plasmid.The results of WB and RT-q PCR showed that E165 R protein was inhibited by G4 structure but the m RNA level did not change,indicating that G4 stabilizer did not affect protein transcription.Then,the RNA sequence of E165R-G4 was synthesized.CD and NMM fluorescence turn-on experiments confirmed that the sequence could form a stable parallel RNA G4 structure.The above results showed that ASFV E165 R gene can form RNA G4 structure,and G4 stabilizer PDS and TMPyP4 can regulate the expression of E165 R gene through this structure.In this paper,we found that the G-rich sequence of E165 R gene can form G4 structure in vitro and in cells,and G4 stabilizer PDS and TMPyP4 can bind and stabilize the formed G4 structure.Further studies have found that G4 stabilizer regulates E165 R protein expression by affecting the post-transcriptional stage.This finding elucidates the regulatory effect of G4 structure on the expression of E165 R gene,and provides a new idea for the prevention and control of ASFV.