Structure And Function Analysis Of G-quadruplex Forming Sequences In The 3’UTR Of IE180 Gene Of Pseudorabies Virus

The spread of pseudorabies causes great economic losses to the swine industry in China.The current vaccines failed to completely protect the swines from the variant strains of pseudorabies virus(PRV).The mechanism studies on PRV infection,spread,latency and reactivated infection could benifit for the development of the new vaccines and anti-viral drugs.As current mechanism studies are focusing on important proteins,to reveal the mechanisms basing on the function of nucleic acid will provide new insight into the anti-PRV research.Guanine rich sequences can form a special nucleic acid secondary structure called G-quadruplex,which plays important roles in the replication of virus genomes,and the transcription or translation of virus genes.The G-quadruplex in the genome of PRV has the potential role in affecting PRV genome replication,which will provide new strategies in PRV control.The IE180 gene encoding the only immediate early gene in PRV,is the transcriptional activator of other viral genes.We discovered clusters of putative G-quadruplex forming sequences in the 3’UTR of IE180 gene.Regulating the transcription or translation of IE180 through G-quadruplex forming sequences will illustrate the function of G-quadruplex in PRV,which also can be a target for anti-PRV drug development.The main contents of this research are as follows:1.The structure and function analysis of G-quadruplex forming sequence in the 3’UTR of IE180 gene.1)The Bioinformatics analysis showed two G-rich regions,157-170 and 256-366,in the IE180 gene 3’UTR;2)Though the G-rich sequence 157-170 was predicted to be putative G-quadruplex sequence,the circular dichroism spectroscopy showed that sequence 157-170 formed duplex structure.Dual-luciferase assay showed that the RNA duplex structure negatively regulated reporter gene translation;3)G-quadruplex structure of sequence PQS3 was confirmed with circular dichroism spectroscopy,but it did not regulate reporter gene translation level in cells;4)G-quadruplex forming sequence PQS18 can form parallel G-quadruplex according to the circular dichroism spectroscopy,and dual-luciferase assay indicated that it suppressed reporter gene translation in cells.2.G-quadruplex ligand TmPyP4 shows anti-PRV activity.1)G-quadruplex ligand TmPyP4 can stabilize the PQS18 G-quadruplex;2)TmPyP4 can enhance the repressive effect of the PQS18 G-quadruplex to the reporter gene translation;3)Plaque assay showed that TmPyP4 displayed anti-PRV activity in PK-15 cells through inhibiting the early replication of PRV.Conclusions: This study discovered a conserved G-quadruplex forming sequence PQS18 in the 3’UTR of PRV IE180 gene.PQS18 can fold into G-quadruplex and suppress the reporter gene translation.The G-quadruplex ligand TmPyP4 can enhance the repressive effect of PQS18 in gene translation through stabilizing the PQS18 G-quadruplex,which result in inhibiting the early replication of PRV.