Structural And Functional Research Of G-Quadruplex In The Genome Of PRV&TBSV Viruses And Regulation Of Porphyrin Derivatives

G-quadruplex is a special nucleic acid structure folded by guanine-enriched sequence.The structure which is folded or unfolded affects the process of gene replication,transcription,translation and gene recombination.Livestock,poultry and crop virus diseases caused by animal and plant viruses seriously endanger agricultural farming,livestock farming and food production.This has caused significant economic losses.Therefore,we need new prevention and control strategies.Exploring the effects of special nucleic acid structures on the replication and pathogenic capacity of animal and plant viruses,and developing molecular design of antiviral drugs targeting G-quadruplexes are expected to provide technical support for the green prevention of animal and plant virus diseases.Porphyrin derivatives are natural products widely present in nature,and their modifications are expected to become good ecological pesticides.In this study,DNA viruses——Pseudorabies virus?PRV?and RNA viruses——Tomato bushy stunt virus?TBSV?are two important animal and plant viruses.We systematically analyzed the putative G-quadruplex sequences?PQS?in the two viral genomes.We screened out porphyrin derivatives that inhibit the proliferation of viruses,providing a theoretical basis for G-quadruplex functions and applications in agricultural viruses.The specific results are as follows:1. Research on the structure and function of G-quadruplex in the 3'UTR of PRV IE180gene and the regulation of porphyrin derivativesPseudorabies virus?PRV?is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily,with neurotropism and latent infectivity.Pseudorabies caused by PRV are acute and infectious diseases coexisting with many animals.PRV can cause symptoms such as fever,vomiting and miscarriage in the host,which brings huge economic losses to agricultural livestock and poultry farming.At present,there is still a lack of vaccines and effective anti-PRV virus drugs.The most typical feature of PRV genome composition is high GC content,which results in the existence of a large number of PQS in the genome.The IE180 gene of PRV is the only immediate early gene of the virus.Inhibiting the expression of IE180 can prevent the replication and proliferation of viruses.However,due to the complex mutations in the IE180 protein sequence in different virus isolates,it is difficult to screen antiviral drugs targeting this protein.In this study,we identified a conserved G₂-quadruplex forming sequence named PQS18-1?5'-GGCUCGGCGGCGGA-3'?in the 3'UTR of the unique immediate early gene IE180of PRV.The PQS18-1 was folded into parallel G-quadruplex structure.Dual-luciferase assay suggested that the formation of the G-quadruplex in 3?UTR enhancing reporter gene expression.TMPy P4 destroyed PQS18-1 G₂-quadruplex and suppressed reporter gene expression,accordingly reducing PRV replication.Further,we used potassium ion long-wavelength anomalous scattering technology to analyze the DNA/RNA PQS18-1G-quadruplex structure and the crystal structure of the complex with the inhibitor TMPy P4.In this study,using biophysical and chemical methods,we speculated that TMPy P4 might unwind the DNA/RNA PQS18-1 G-quadruplex structure through the"contact-binding-dynamic oscillation-unwinding"mechanism.2. Research on the structure and function of G-quadruplex in TBSV virus genome and preliminary study on the regulation of porphyrin derivativesTomato bushy stunt virus is a positive single-stranded RNA virus,belonging to Tombusviridae,Tombusvirus.After infecting plants,TBSV will cause the phenomenon that the leaves appear faded or necrotic spots.In turn,it causes symptoms such as plant growth retardation,dwarfing and fruit deformation.TBSV causes serious losses of the output and quality of vegetables,flowers and other crops.However,there is currently no effective anti-TBSV virus drug.There are many reports about the molecular mechanism of TBSV replication,transcription and translation,which is a model system to study the interaction between plant viruses and hosts.Similar to other positive single-stranded RNA viruses,TBSV viral genome replication involves a multi-step reaction,and the normal expression of proteins p33 and p92 is the most critical step.This study identified two sequences TBSV-PQS2?5'-GGAUGGAGUGGAGG-3'?and TBSV-PQS4?5'-GGCGGUU GGAAGAUGG-3'?that can form stable G-quadruplex structures in the coding regions of genes.The p33 and p92 replication proteins,the viral?+?RNA and host proteins are recruited to assemble the tombusvirus viral replicase complex to regulate viral proliferation.In this work,it was found that the porphyrin derivative NMM stabilizes these two G-quadruplex structures,and the binding ability of NMM to TBSV-PQS2 is stronger than TBSV-PQS4.We deduced from the results of the Job Plot experiment that the combination ratio of TBSV-PQS2 and TBSV-PQS4 to NMM is 1:1.Through the TBSV-GFP infectious clone experiment,we found that NMM can significantly reduce the proliferation of TBSV in the host.NMM showed a good effect on inhibiting virus replication and potential to be developed into new pesticide formulation.This study introduces G-quadruplexes in the field of chemical biology into the research of two agricultural viruses?PRV and TBSV?.We found that PRV IE180 3'UTR G₂-quadruplex shows a new function to enhance protein expression,which enriches the understanding of the function of G-quadruplex.This work identified that the porphyrin derivative TMPy P4 may inhibit PRV proliferation by unfolding the G-quadruplex structure,while the porphyrin derivative NMM inhibits the proliferation of TBSV by stabilizing the G-quadruplex structure.The two analogs inhibit the proliferation of different viruses by regulating the structure of G-quadruplexes with different mechanisms.This provides an example for dialectically designing antiviral drugs targeting G-quadruplexes.