A genome-wide RNAi screen identified 295 human host factors required for early-stage influenza virus replication. These factors include those involved in kinase-regulated signaling, ubiquitination, and phosphatase activity, and they form a significant host-pathogen interaction network. Of these, 219 were confirmed to be essential for efficient influenza virus growth. The study also identified 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Additionally, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication, including nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B). The study also showed that swine-origin H1N1 influenza virus is dependent on these host factors, and small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication. Influenza viruses are a major cause of morbidity and mortality, and influenza A viruses have the potential to cause pandemic outbreaks. Current antiviral drugs targeting neuraminidase (NA) and the M2 ion channel protein have widespread resistance, highlighting the need for new therapeutic strategies. Targeting host factors required for viral replication is an alternative strategy that may reduce the emergence of viral resistance. The study used a genome-wide RNAi screen with human lung epithelial (A549) cells to identify host factors required for influenza virus replication. The study confirmed 295 genes for which at least two siRNAs reduced viral infection by 35% or greater without significant cellular toxicity. Analysis of over-represented biological annotations identified over 170 statistically enriched categories, which fell into 11 broadly related functional groups. Signaling molecules, including those involved in the PI3K/AKT pathway, molecules that regulate cytoskeletal dynamics, and proteins involved in ubiquitination, phosphatase, and protease activities were overrepresented among the 295 factors. The study also identified the COPI coat complex as a key factor in influenza virus entry. Depletion of COPI subunits blocked WSN-PP infection, confirming their role in low-pH-dependent virus entry. The study further demonstrated that CSE1L specifically inhibited influenza virus gene expression in a minigenome replicon assay, suggesting that CSE1L activity is required for the nuclear import of vRNPs as well as newly synthesized viral proteins. The study also showed that a specific inhibitor of CAMK2B, KN-93, inhibits influenza virus growth, suggesting that pharmacological targeting of this kinase may be an effective strategy for the development of host factor-directed antivirals. The studyA genome-wide RNAi screen identified 295 human host factors required for early-stage influenza virus replication. These factors include those involved in kinase-regulated signaling, ubiquitination, and phosphatase activity, and they form a significant host-pathogen interaction network. Of these, 219 were confirmed to be essential for efficient influenza virus growth. The study also identified 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-beta. Additionally, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication, including nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) II beta (CAMK2B). The study also showed that swine-origin H1N1 influenza virus is dependent on these host factors, and small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication. Influenza viruses are a major cause of morbidity and mortality, and influenza A viruses have the potential to cause pandemic outbreaks. Current antiviral drugs targeting neuraminidase (NA) and the M2 ion channel protein have widespread resistance, highlighting the need for new therapeutic strategies. Targeting host factors required for viral replication is an alternative strategy that may reduce the emergence of viral resistance. The study used a genome-wide RNAi screen with human lung epithelial (A549) cells to identify host factors required for influenza virus replication. The study confirmed 295 genes for which at least two siRNAs reduced viral infection by 35% or greater without significant cellular toxicity. Analysis of over-represented biological annotations identified over 170 statistically enriched categories, which fell into 11 broadly related functional groups. Signaling molecules, including those involved in the PI3K/AKT pathway, molecules that regulate cytoskeletal dynamics, and proteins involved in ubiquitination, phosphatase, and protease activities were overrepresented among the 295 factors. The study also identified the COPI coat complex as a key factor in influenza virus entry. Depletion of COPI subunits blocked WSN-PP infection, confirming their role in low-pH-dependent virus entry. The study further demonstrated that CSE1L specifically inhibited influenza virus gene expression in a minigenome replicon assay, suggesting that CSE1L activity is required for the nuclear import of vRNPs as well as newly synthesized viral proteins. The study also showed that a specific inhibitor of CAMK2B, KN-93, inhibits influenza virus growth, suggesting that pharmacological targeting of this kinase may be an effective strategy for the development of host factor-directed antivirals. The study