Function And Mechanism Of Cell Autophagy And Host Genes Screened By Genome-wide CRISPR/Cas9 In Porcine Virus Replication

Pathogen diseases in swine not only cause production performance and huge losses to pig industry,but also pose a predominant threat to environment,meat product and human health.The major reasons why these diseases con not be control are that the mechanisms of the interaction between virus and host,virus pathogenic and swine antivirus are not clear,therefore,host genes and cellular response mechanism that play important roles for viral replication have great potential to be developed in disease resistance breeding and antiviral vaccines,as well as to control the spread of the virus.CRISPR/Cas9 genome editing system is a microbial adaptive immune system that use a nuclease and a guided small RNA to cleave foreign genetic elements through base pairing with target DNA sequence.This system is highly specific,markedly easier to design,efficient and well-suited for multiplexed gene editing for a variety of species and high-throughput.Cell autophagy is a well-controlled biological process and plays essential roles in development,intracellular homeostasis and diseases.In addition,as an important protective mechanism in host,autophagy is very important for defending against the virus infection.Hence,this research identified host genes related with porcine virus infection by porcine genome-wide CRISPR/Cas9 library and investigated the function and mechanism of cell autophagy in regulating PRRSV replication.The main research results included as follow:1.Verifying that CRISPR/Cas9 system can efficiently induce porcine gene knockout in porcine cell linesTo determine that the CRISPR/Cas9 genome editing technology can knockout successfully the gene in pig cells,we first adapted this system in two porcine cell lines(PK15 and C?2(+)cells)by synthesizing the human-codon optimized Cas9 and demonstrated whether it could sit-specifically cut target DPM1 gene in two porcine cell lines by transiently co-transfecting of Cas9 and sgRNA expression vector.Flow cytometry analyses of DPM1 gene using FLAER showed that 10%cells transfected with Cas9 and gRNA were mutant,suggesting that CRISPR/Cas9 system is capable of inducing gene silencing.We then examined whether constitutive expression of Cas9 and gRNA is sufficient for DSB induction.We generated two porcine cell lines that constitutively express Cas9 and then transfected these porcine cell lines with DPM1 gRNA.All Cas9-expressing porcine cell lines showed higher knockout frequencies(20%-30%)than the control porcine cell transfected with the same vectors.These results indicate that the expression levels of Cas9 in these stable clones are sufficient to induce DSBs.We next introduced the gRNA expression cassette into two Cas9-expressing porcine cell lines by piggyBac transposition.All stable transfectants were analyzed by flow cytometry and the result showed that most colonies almost completely lacked DPM1 gene expression(80%).Taken together,our results pointed out that stable expression of Cas9 and gRNA in porcine cell lines were adequate to induce CRISPR/Cas-mediated target DSBs and the CRISPR/Cas9 can be used for the research of gene knockout and function screen.2.Constructing and validating the pig genome-wide CRISPR/Cas9 library for carrying out the large-scale genetic screening by using high-throughput sequencingIn order to take full advantage of the value of CRISPR/Cas9 system in genome editing,we designed and built the porcine genomw-wide gRNA library.We then cloned these gRNAs into the lentiviral vector to form a genome-wide lentiviral gRNAs library.High-throughput sequencing showed that lentivirus plasmid library contained 65,316 gRNAs and all of the genes in the library contained at least two gRNAs,indicating that the gRNAs library is diversity and better quality.We further generated two different Cas9-expressing porcine cells,resulting in mutant porcine cells libraries.We deep sequenced all the gRNAs in the porcine cell libraries and found that 13,000 genes with at least one gRNAs per gene were almost present in the libraries,determining that genome-wide gRNAs library for pig has higher quality and can be carried out for large-scale loss-of-functin screening.3.Screening and identifying host genes involved in swine influenza virus by using the constructed genome-wide mutant cell librariesIn this study,we utilized swine influenza virus as the research object and conducted recessive screens to identity host genes that modulate susceptibility to swine influenza virus via using these constructed mutant porcine cell libraries.For identification of primary hits depended on the selected parameters,we identified 140 genes in virus-resistant cells that were targeted by at least two gRNAs in two independent infection of two different cell libraries.In addition,to improve data quality and accurately identify host factors required for influenza virus infection,we performed a second round of screening using a sub-library constructed with the genes identified in the first round that contains 217 gene.The mutant cell lines containing sub-library were established and subsequently infection with two virus strains,respectively.Analysis of the HST sequence comparing with different virus strains and cell lines resulted in 61 genes containing at least one gRNA were simultaneously found in four infection experiments.We than selected 32 important host genes to construct gRNA expression vectors for each gene and tested the gRNA individually to see whether they could give rise to resistant cells through plaque assay and confocal microscopy.The results showed that candidate genes in screen were able to affect swine influenza virus replication under different degrees,further evidencing that CRISPR/Cas9 system is more valuable in swine genetic screening.4.Cells autophagy and apoptosis were involved in PRRSV infection,and Autophagy was triggered earlier than cell apoptosisTo study the mechanism of cellular biological process such as autophagy in response to virus infection,we used porcine reproductive and respiratory syndrome virus(PRRSV)as research object and MARC 145 as research model to analyze how virus utilizes autophagy for its replication.A significant increase in punctate LC3 was observed in PRRSV-infected cells and an increase in the levels of LC3-II in PRRSV infected cells was also measured significantly until 36h post infection(hpi)via western blot assay.We also detected p62 protein levels in PRRSV infected cells which showed a continuous decline till 36 hpi,demonstrating that PRRSV can enhance autophagic flux as onset of the early events in virus infection.MARC 145 cells infected by PRRSV exhibited characteristics of apoptosis such as chromatin condensation and cell nucleus cleavage though hoechst staining analysis.Taken together,both cell autophagy and apoptosis can participate in PRRSV replication.In addition,to investigate whether PRRSV can delineate the temporal relationship between autophagy and apoptosis,Transmission electron microscope(TEM)was used.Autophagic-like vesicles formation were observed after 12 hpi and reached its peak at 36 hpi,while there was no significant change in membrane structure,mitochondria or nucleus at 12hpi.But a series of apoptotic phenomenon,such as cytoplasmic shrinkage and nuclear fragmentation were observed in PRRSV-infected MARC-145 cells after 24hpi.Moreover,we found that the expression of PRRSV ORF7 was increased during PRRSV infection.The above results in combination concluded that cell autophagy was induced earlier that PRRSV-mediated apoptosis5.Autophagy inhibited apoptotic death of MARC-145 cells and increased PRRSV replication in PRRSV infectionBoth autophagy and apoptosis were induced by PRRSV infection to regulate viral replication,it was very critical to reveal the interaction between cell autophagy and apoptosis at the early stages of virus infection.We used an autophagy inhibitor(3-MA)and RNAi to inhibit autophagy process.The result suggested that the inhibition of autophagy by 3-MA could increase the number of apoptotic cells during PRRSV infection.Moreover,3-MA also inhibited the expression of PRRSV Nsp2 via western blotting.In addition,when cells were transfected with Beclinl siRNA that led to a significant decrease of Beclin1 expression,knockdown of Beclin1 resulted in the increase of the cleavage of caspase3 protein.Our results suggested that autophagy inhibition strengthened PRRSV-induced cell apoptosis and reduced PRRSV replication.6.Bad regulated PRRSV-induced autophagy through interacting with BeclinlTo study whether Bad and Beclin1 are related with PRRSV infection,we observed that the expression of Bad and Beclinl mRNA increased with the progression of PRRSV infection.Western blot analysis of Bad protein did not show significant change during PRRSV infection in MARC-145 cells.However,PRRSV infection robustly increased p-Bad(S112)and Beclinl protein expression with maximal production at 36hpi,suggesting that Bad phosphorylation and Beclinl may play a critical role in PRRSV-inducted autophagy.In addition,we found that Bad knockdown in PRRSV-infected MARC-145 cells significantly resulted in the increased expression of LC3.Moreover,silencing of Bad decreased the cleavage of caspase3 and increased the punctate accumulation of LC3.Moreover,intracellular distribution of Bad protein in control and infected MARC-145 cells ware varied by fluorescence microscopy.Co-immunoprecipitation analysis or immunofluorescence assay for the Bad and Beclinl interaction was performed that showed PRRSV infection promoted the association between Bad and beclinl and increased its replication before 36 h infection.