Genome-wide CRISPR/Cas9 High-throughput Screening For Host Factors Regulating Influenza Virus Replication

Influenza A virus is one of major causes of upper respiratory tract infections and can cause serious acute respiratory diseases.There are tens of thousands of deaths caused by influenza A virus infection every year in worldwide,which have an inestimable impact on public health.Avian influenza is a zoonotic disease caused by influenza A virus that can be transmitted from poultry to human.Currently,preventive and control measures against influenza A viruses are mainly vaccines and drugs,but influenza viruses are capable of acquiring drug resistance through constant mutation and evolution,and in order to counter this property of the virus,we need to explore new host proteins to target avian influenza viruses.Since the influenza virus life cycle involves various steps,numerous different host factors contribute to these steps and affect the susceptibility of cells to influenza virus.Therefore,the screening and identification of host factors that important for virus replication can insights that will support the development of antiviral therapies.Selecting the appropriate screening strategy is an important step to explore antiviral host factors.CRISPR/Cas9 is modified from the antiviral immune mechanism existing in many bacterial and archaeal species.CRISPR/Cas9 has the characteristics of lower cost,less off-target,easier to preparation and storage,and more efficient knockout at DNA level.The A549 cell line was screened and optimized using CRISPR/Cas9technology,which has high Cas9 cleavage activity and can be widely used for in vitro gene knockout,transgene knock-in,mutagenesis,transgene integration or other applications related to CRISRP/Cas9 genome editing.Then the lentivirus knock-out library of human genome was successfully packaged by lentivirus system,which is highly efficiency in infection and can be used for large-scale screening work.This lentiviral library was successfully used to construct a library containing human genome-wide single gene knockout in A549 cells,which laid the foundation for the screening of host factors associated with different subtypes of influenza viruses.In this study,we performed genome-wide CRISPR knockout（Ge CKO）screening with H5N1 highly pathogenic avian influenza virus and identified a key host factor IGDCC4（Immunoglobulin superfamily DCC subclass member 4）for influenza virus to enter host cells.IGDCC4 has not been reported to be associated with any virus infection previously.Our study found that IGDCC4-KO A549 cells can significantly reduce virus replication.To further confirm these results,we synthesized and tested an IGDCC4 si RNA,and found that the replication of H5N1 virus in IGDCC4si RNA-treated cells was lower than that of the control A549 cells at12,24,36 and 48 h post-infection.To determine which stage of the influenza virus life cycle is mediated by IGDCC4,IGDCC4-KO and control A549 cells were infected with H5N1 virus and the levels of v RNA and m RNA of the viral NP gene in the cells were measured at 2,4,6,and8 h p.i.by using quantitative reverse transcription PCR（q RT-PCR）.At these four timepoints,the levels of both types of viral RNA in IGDCC4-KO cells were significantly lower than those in the control A549 cells.At2 h p.i.,as the early stage of the viral life cycle,when the v RNA in the cells was mainly derived from infecting virus rather than progeny virus,a lower level of v RNA in IGDCC4-KO cells at this timepoint indicates that the host factor IGDCC4 was most likely involved in the early stage of the viral life cycle.In order to determine the specific stage of IAV replication in which IGDCC4 is involved,we first explore the process of virus infection in cells using laser confocal.We determined that the expression of sialic acid receptors had no effect,nor did it affect the adsorption of virus on the cell surface.However,viruses attached to the surface of IGDCC4-KO cells were removed by PBS-HCL（p H=1.5）elution during internalization,resulting in a significant reduction in the NP level of the viruses internalized in the infected cells.Overexpression of IGDCC4could restore the endocytosis of influenza virus in IGDCC4-KO cells.In addition,we found that the extracellular domain of IGDCC4 interacts with HA protein of IAV by CO-IP.The interaction of IGDCC4 and HA was further confirmed by using a colocalization assay.IGDCC4 does not affect the internalization of vesicular stomatitis virus（VSV）and transferrin suggesting that IGDCC4-mediated endocytosis may be cargo-specific.The above results indicate that IGDCC4 is an important factor for IAV to invade host cells and mainly affects the internalization stage of the virus.In addition,we established a model of IGDCC4^-/-mice.After infection with the H5N1 virus,it was found that compared with wild-type group mice,the IGDCC4^-/-mice did not decrease more weight and partially survived,while the wild-type mice all died.We also measured virus titers in nasal turbinates and lungs,and found that the viral loads of nasal turbinates and lungs were significantly lower in IGDCC4^-/-mice than that in wild-type mice.Our results show that IGDCC4 facilitates H5N1 virus infection in vivo and that IGDCC4^-/-mice exhibit increased resistance to H5N1 virus infection.In addition to identifying a new host factor IGDCC4,our research also screened host factors that have been reported to be involved in influenza virus replication such as SLC35A1 and ST3GAL4.In summary,this study identifies IGDCC4 as a novel host factor that promotes influenza virus infection by facilitating internalization and provides insights that will support the development of antiviral therapies.This study demonstrates that CRISPR/Cas9 screening is a reliable tool for discovering the host factors necessary for various replication stages of intracellular pathogens,and provides a theoretical basis for identifying host-targeted drug development.