Identification And Mechanism Study Of Key GPCRs During Virus Infection

Viral diseases threaten human health seriously.Although a variety of compounds target nucleic acid or protein of the viral life cycle have been developed,it is very easy for virus resist to antiviral drugs because of the high mutagenicity and parasitism of the virus.As the largest membrane receptor family in the organism,there are about 50%clinical drugs targeting G protein coupled receptor(GPCR)directly or indirectly.In recent years,more and more evidences showed that GPCR plays an important role in viral infection,however,with respect to the large number of members of the GPCR family,the study of GPCR in viral infections just begun.Thus,there is an urgent need to establish an effective screening strategy to systematically identify the key GPCRs in viral infection and to lay a good foundation for targeting GPCR antiviral drugs.Therefore,this study attempts to screen the key GPCR promoting viral infection by negative selection using CRISPR-Cas9-sgRNA Library and by positive selection of interferon induced GPCR using RNA-seq.First,CRISPR-Cas9-sgRNA lentivirus library was used for infecting host cells to establish different GPCR knockout cell clones library.Then the LGR4 was found very important in VSV infection by sequencing and analysis of genome in VSV tolerance cells.Then,we verified the important role of Lgr4 in VSV infection both in vivo and in vitro.Lgr4 knockdown or knockout cells significantly inhibited VSV infection and Lgr4 knockout mice were more tolerant than wild-type mice at lethal doses of VSV infection.In addition,overexpression of Lgr4 in RAW264.7 promotes VSV infection.Further,we found that Lgr4 specifically promotes the VSV infection to host in an immune independent manner.Moreover,short-term VSV infection assay showed that the VSV particles on the surface of Lgr4 deficient cells was significantly lower than that in wild-type cells.Therefore,we speculate Lgr4 as a cell surface receptor may be involved in the invasion of VSV virus.Through cell invasion experiments,immunoprecipitation,surface plasmon resonance,we determined that VSV invaded susceptible cells through the extracellular domain of Lgr4.Moreover,blocking Lgr4 with Lgr4 antibody and ligand R-spondin 1 can block the invasion of VSV,and incubating the purified extracellular domain of Lgr4 with VSV also blocked the binding of VSV to cells,thereby reduced the invasion of VSV.Therefore,we identified Lgr4 as a receptor for VSV through Cas9/sgRNA library screening.Identifying Lgr4 not only enrich the understanding of pathogenic mechanism of VS V,but also provide more widely used of the VSV as a gene therapy and oncolytic virus transformation.This part of work also confirmed the power of CRISPR-Cas9-sgRNA library in gene function study,which can be used for the screening of other viral infections.Then,we screened the interferon induced GPCR by gene chip,and found the GPR109A with important antiviral function.First of all,a number of up-regulated and expressed GPCR were found by gene chip technology after IFN treatment in PBMC.Based on this,we confirmed interferon induced expression of GPR109A at RNA and protein level following IFN stimulation and viral stimulation in PBMC-M and mouse derived peritoneal macrophages.Then we found that virus production in Gpr109a knockout macrophages was increased obviously.Meanwhile,overexpression of human GPR109A in 293T could significantly inhibit the production of progeny virus,suggesting that GPR109A restrict viral infection at the cellular level.And then we found that the survival of Gpr109a knockout mice was lower than that of wild-type mice by lethal dose of virus infection at the animal assay.Moreover,the viral infection to peritoneal cells in Gpr109a knockout mice was increased.We subsequently determined the underlying mechanism of Gpr109a and found that the extracellular content of IFN was decreased in Gpr109a knockout mice,tissues and cells.However we observed that Gpr109a deletion inhibits autophagy formation through immunoblotting assay and transmission electron microscopy.Then,reactivation of autophagy in Gpr109a knockout cells rescued IFN production.Furthermore,we found that the ligand Nicotinic acid activated the formation of autophagosome in a Gpr 109a dependent manner.At the same time,the release of IFN enhanced both in vivo and in vitro to fight viral infection after Nicotinic acid activation.It is suggested that GPR109A may be an important target for the regulation of IFN release,and our work lay a foundation for developing new antiviral drugs.To sum up,our two screening strategies are fruitful.High throughput negative screening using CRISPR-Cas9-sgRNA library revealed that LGR4 could serve as a VSV invasive receptor.Gene microarray screening found that GPR109A can regulate IFN release through autophagy.Our work is not only useful for future screening to select key genes by CRISPR-Cas9-sgRNA library but also proved that GPCR played an important role in the process of viral infection,this investigation may open a new field and provides a theoretical basis in the design of novel antiviral drugs targeting GPCR from the view of host immunity.