Influenza A virus (IAV) infection provokes an antiviral response involving the expression of type I and III interferons (IFN) and Influenza A viruses (IAVs) are prototypic members of the Orthomyxoviridae family, featuring a segmented RNA genome composed of eight single-stranded RNAs that have negative polarity (1). IAVs circulate in the human population, causing periodic epidemic outbreaks and occasional pandemic waves of respiratory disease (2). Moreover, there is a large natural IAV host reservoir in wild aquatic birds, such as ducks and geese, in which the viruses cause mainly mild or no apparent symptoms. IAV strains are usually well adapted to their particular host species, which is reflected not only in the existence of stable virus lineages but also in polymorphic amino acid positions in viral proteins distinctively found in human or avian strains (3).IAVs target the epithelial cell layers lining the human respiratory tract, in which they are subject to immune control in infected cells, mediated by the antiviral type I interferon (IFN) response (4). Many of the key events and factors driving the IFN response have been identified and involve initial recognition of the viral genomic 5=-triphosphorylated RNA by the intracellular RNA helicase RIG-I, which governs a signaling module culminating in the activation of transcription factors, such as IRF-3 and NF-B, thereby inducing the transcription of type I IFN genes (5, 6). Type I IFNs comprise 14 subtypes of IFN-␣ and one IFN-␤ that are secreted from virus-infected cells and exert antiviral effects against many virus families, including IAV (4, 7). Type I IFNs secreted by

In recent years genome-wide RNAi screens have revealed hundreds of cellular factors required for influenza virus infections in human cells. The long-term goal is to establish some of them as drug targets for the development of the next generation of antivirals against influenza. We found that several members of the polo-like kinases (PLK), a family of serine/threonine kinases with well-known roles in cell cycle regulation, were identified as hits in four different RNAi screens and we therefore studied their potential as drug target for influenza. We show that knockdown of PLK1, PLK3, and PLK4, as well as inhibition of PLK kinase activity by four different compounds, leads to reduced influenza virus replication, and we map the requirement of PLK activity to early stages of the viral replication cycle. We also tested the impact of the PLK inhibitor BI2536 on influenza virus replication in a human lung tissue culture model and observed strong inhibition of virus replication with no measurable toxicity. This study establishes the PLKs as potential drug targets for influenza and contributes to a more detailed understanding of the intricate interactions between influenza viruses and their host cells.

Influenza A virus (IAV) is a human respiratory pathogen that causes yearly global epidemics, as well as sporadic pandemics due to human adaptation of pathogenic strains. Efficient replication of IAV in different species is, in part, dictated by its ability to exploit the genetic environment of the host cell. To investigate IAV tropism in human cells, we evaluated the replication of IAV strains in a diverse subset of epithelial cell lines. HeLa cells were refractory to the growth of human H1N1 and H3N2 viruses and low-pathogenic avian influenza (LPAI) viruses. Interestingly, a human isolate of the highly pathogenic avian influenza (HPAI) H5N1 virus successfully propagated in HeLa cells to levels comparable to those in a human lung cell line. Heterokaryon cells generated by fusion of HeLa and permissive cells supported H1N1 virus growth, suggesting the absence of a host factor(s) required for the replication of H1N1, but not H5N1, viruses in HeLa cells. The absence of this factor(s) was mapped to reduced nuclear import, replication, and translation, as well as deficient viral budding. Using reassortant H1N1:H5N1 viruses, we found that the combined introduction of nucleoprotein (NP) and hemagglutinin (HA) from an H5N1 virus was necessary and sufficient to enable H1N1 virus growth. Overall, this study suggests that the absence of one or more cellular factors in HeLa cells results in abortive replication of H1N1, H3N2, and LPAI viruses, which can be circumvented upon the introduction of H5N1 virus NP and HA. Further understanding of the molecular basis of this restriction will provide important insights into the virus-host interactions that underlie IAV pathogenesis and tropism. IMPORTANCE Many zoonotic avian influenza A viruses have successfully crossed the species barrier and caused mild to life-threatening disease in humans. While human-to-human transmission is limited, there is a risk that these zoonotic viruses may acquire adaptive mutations enabling them to propagate efficiently and cause devastating human pandemics. Therefore, it is important to identify viral determinants that provide these viruses with a replicative advantage in human cells. Here, we tested the growth of influenza A virus in a subset of human cell lines and found that abortive replication of H1N1 viruses in HeLa cells can be circumvented upon the introduction of H5N1 virus HA and NP. Overall, this work leverages the genetic diversity of multiple human cell lines to highlight viral determinants that could contribute to H5N1 virus pathogenesis and tropism.