Distribution Analysis And Function Exploration Of G₂-Quadruplex Of Pseudorabies Virus

Pseudorabies virus?PRV?which belongs to the herpesviruses is an important pathogen that harms the swine industry and has caused huge economic losses in China.In recent years,the farm inoculated with the Bartha-K61 vaccine had developed a PRV outbreak in northern and eastern parts of China,and confirmed that the Bartha-K61vaccine did not provide complete protection.The G-quadruplex is a special nucleic acid secondary structure formed by a guanine rich sequences,and plays an important role in the replication,transcription and translation of genes.G-quadruplex structures present in many viral genomes and can affect pathogenesis by affecting the expression of key proteins.It was found that the G-quadruplex ligand could exert antiviral effects against the key G-quadruplex structure.The PRV genome has a very high GC content,and a large number of G-quadruplex sequences are expected to affect the interaction of PRV with the host.Studying the role of PQS in the life cycle of PRV is expected to lay the foundation for vaccine research in PRV.The G₃-quadruplex typically present in the G₃₊N_1-7G₃₊N_1-7G₃₊N_1-7G₃₊mode.Wherein G is guanine and N is any nucleotide comprising G.The G₂-quadruplex is usually present in the G₂₊N_1-7G₂₊N_1-7G₂₊N_1-7G₂₊mode.Compared with the widely-studied G₃-quadruplexes,the dynamic G₂-quadruplexes are more sensitive to the cell microenvironment,but they attract less attention.Systematic analysis of G₂-quadruple is expected to reveal more functions of G-rich sequences in herpesviruses.The main contents of this research are as follows:1.Bioinformatics analysis of putative G-quadruplex sequences?G₂-PQSs?in PRV.1)Analysis of G₂-PQSs distribution in the PRV genome:The distribution of G₂-PQS in the PRV genome was analyzed by the Quadparser program,and G₂-PQS was found to be widely distributed throughout the genome.In the UTR region,the 3'UTR region has a higher G₂-PQS density than the 5'UTR region.;2)Evaluation of the conservation of G₂-PQSs in PRV:After comparing the same genes of different viruses in Varicellovirus,the conservation scores of 12 G₂-PQS in PRV CDS were higher than 0.7.Among the different PRV strains,38 G₂-PQS in the repeat region between IE180 and US1 showed greater than 90%conservation.;3)Predict G₂-PQSs with function:Genes involved in the regulation of gene expression,genomic DNA replication and viral maturation are rich and conserved G₂-PQS in their miRNA sequences.The intron of LLT has a microRNA cluster containing 15 G₃-PQS and 35 G₂-PQS involved in transcriptional regulation of microRNA during PRV latency.2.Experimental verification of structure and function of G₂-quadruplex.1)Structural analysis in vitro:Circular dichroline determined that G₂-PQS can form G-quadruplexs and study on the effect of K⁺,Na⁺and G-quadruplex ligands on G₂-quadruplex structure;2)functional analysis in vitro:Based on the results of structural studies in vitro,including dual luciferase assay to verify the regulation of G₂-quadruplex expression in 3'UTR?G₂-PQS in UL9-3'UTR can inhibit gene expression;G₂-PQS in UL5-3'UTR promotes gene expression?and the effect of G₂-quadruplex on miRNA and mRNA interaction?G₂-PQS in the non-seed sequence pairing region currently did not show an effect on the interaction between UL5-3'UTR and PRV-miR-LLT3?;3)virus level:The inhibitory effect of G-quadruplex ligand NMM on PRV virus was preliminarily verified.The sequences predicted in this paper lay the foundation for the future functional study of PQS in the virus;Study on the effect of ions and G-quadruplex ligands on G₂-quadruplex structure is convenient for the study on the function of environmental regulation G₂-quadruplex in further;functional studies of G₂-PQS in 3'UTR provide guidance for the developing PRV vaccine;studies at the viral level indicate that G-quadruplex ligands have great antiviral potential.