Research paperCRISPR-Cas9 mediated knockout of AnxA6 gene enhances influenza A virus replication in low-permissive HEK293FT cell line
Graphical abstract
Introduction
Influenza poses a constant threat to human health worldwide causing seasonal epidemics characterized by increased levels of morbidity and mortality. Annual influenza epidemics infect about 10–20% of the population each season causing illness that can range in severity from mild to life-threatening or even lethal. Various systems are proposed for influenza virus isolation, culture, and characterization. For decades from the 1950 s most influenza viruses were isolated in embryonated chicken eggs (ECE). Developing chicken embryos still are the gold standard technology of influenza vaccine production. However, not all influenza viruses can efficiently replicate in ECE, and adaptation to ECE often leads to the acquisition of mutations altering receptor specificity and antigenic properties of isolated strains (Parker et al., 2016). Among mammalian cell cultures the MDCK cell line derived from the canine kidney is the most widely used standard system for influenza propagation (Eisfeld et al., 2014). It is more convenient and cost-effective for laboratory studies of influenza, but not ideal in vitro model of a human respiratory system. Modern human influenza A/H3N2 viruses poorly replicate in MDCK cells due to the low level of α-2,6-linked sialic acid receptors(Lin et al., 2017), fostering the use of genetically engineered cell lines like MDCK-Siat (Matrosovich et al., 2003). Different human-origin cell lines have been reported to support influenza virus growth - Caco-2 (Zhirnov et al., 2009, Chiapponi et al., 2010), A549 (Li et al., 2009), HEp-2 (El Ahmer et al., 1999), NCI-H292 (Hierholzer et al., 1993), HEK293 (Le Ru et al., 2010). Host-specific patterns of glycosylation required for efficient replication of human viruses and retention of initial antigenic properties can be achieved only in human cell lines (Schwarzer et al., 2009).
HEK293 cell line is initially derived from human embryonic kidney and is widely used in laboratories for the production of recombinant proteins and viral vectors for gene therapy and other applications. Also, HEK293 cells are recognized platform for biopharmaceutical manufacturing. Several products produced in HEK293 cells have been recently approved for human use by regulatory agencies in the US and EU (Dumont et al., 2016). Suspension HEK293 cell line is known to support high influenza virus production (Le Ru et al., 2010). However, adherent HEK293 cells demonstrate low permissiveness to influenza infection in comparison to MDCK cells (Vlecken et al., 2013).
The main challenge for cell culture-based influenza virus isolation (and vaccine production) is low virus yield, and extended passaging is often needed to reach an acceptable level of virus propagation. Virus yield highly depends on host cell permissiveness to influenza infection.
Permissiveness to influenza virus infection is a multigenic trait. Recent developments in omics technologies led to the identification of numerous host factors involved in influenza virus replication acting on different stages of the life cycle (König et al., 2010, Watanabe and Kawaoka, 2015, Zhao et al., 2017). siRNA-mediated gene knockdown is one of the approaches widely used to enhance host-cell permissiveness to influenza virus and increase virus yield. For example, siRNA knockdown of IRF7 gene expression in MDCK cells results in increased production of influenza virus particles (Hamamoto et al., 2013). Attempts to enhance permissiveness of Vero cells to different viruses by simultaneous siRNA knockdown of the set of genes (BTN2A1, CNTD2, EP300, GCGR, PYCR1, and ZNF205) were made (Murray et al., 2017).
Annexin protein family is a group of Ca2 + -dependent membrane binding proteins conserved across a wide range of species from protists to vertebrates. Twelve proteins have been identified in humans (AnxA1-AnxA13, AnxA12 is unassigned). Annexins are linked to Ca2 + -regulated membrane organization (Shaw et al., 2008). Several annexins were reported to be incorporated into influenza virus particles probably as a result of budding from annexin-rich microdomains of cell plasma membrane (Cornely et al., 2011). Annexin A6 (AnxA6) is known as an organizer of membrane microdomains (lipid rafts) regulating cholesterol homeostasis, receptor localization and signalling (Takeda et al., 2003). Lipid rafts play a significant role in the influenza virus life cycle - serving as a site for the gathering of viral proteins. Lack of interaction with raft microdomains impairs influenza virus infectivity (Zhang et al., 2000) and budding (Musiol et al., 2013). It was shown that an AnxA6 overexpression leads to reduced reproduction of influenza A virus in cell culture accompanied by a significant decrease in cholesterol levels both in the cell plasma membrane and the membrane of progeny virions (Ma et al., 2012). AnxA6 also interacts with M2 proton channel (CRISPR design tool, 2017). It is important to note that the lipid composition of virion membrane influences the infectious activity of influenza virus, and virions depleted in cholesterol are less infectious. The suppression of AnxA6 in A549 cells by siRNA leads to a significant increase in the production of influenza virus particles (Ma et al., 2012, CRISPR design tool, 2017).
CRISPR-Cas9 genome editing is a promising technology for generation of highly permissive cell lines by knockout of host genes aborting or restricting influenza infection. Here we report the use of CRISPR-Cas9 genome editing system to enhance influenza virus replication in HEK293FT cells by stable knockout of ANXA6 gene.
Section snippets
RNA-seq analysis
cDNA libraries were prepared from two biological replicates of each time point (0 h (mock-infected) and 48 h (influenza-A/Puerto Rico/8/1934-infected) HEK293FT). The construction of cDNA libraries was conducted according to a standard protocol using a NEBNext Ultra II Directional RNA library preparation kit (New England Biolabs, UK) and NEBNext mRNA Magnetic Isolation Module (New England Biolabs, UK). Massive parallel sequencing was performed on a NextSeq Illumina 500 platform. The raw data
Generation of the HEK293FT AnxA6-/- cell lines
Previously, using RNA-seq analysis differentially expressed genes (DEGs) were identified in HEK293FT cells infected with influenza A (ArrayExpress E-MTAB-9511). Gene Ontology enrichment with ClueGO indicated that these DEGs were mainly involved in regulation of steroid metabolic process and regulation of ion channel activity (Fig. S1). The obtained results allowed us to select ANXA6 gene as a target for the CRISPR-Cas9-mediated knockout, furthermore the suppression AnxA6 was earlier shown to
Discussion
The CRISPR-Cas9-mediated genomic knockout is a breakthrough technology that has been successfully used as a potent research tool for probing viral-host interactions (Heaton et al., 2017). In contrast to siRNA-knockdown that is transient CRISPR-Cas9 genome editing can be used for generation of stable modified cell lines. Up to date several host genes acting on different stages of virus life cycle were targeted using CRISPR-Cas9 knockout to increase influenza virus replication in human cell
Conclusions
To increase the permissiveness of the cell line to influenza infection, it is crucial to identify bottleneck limiting virus replication in this particular cell type. We used CRISPR-Cas9-mediated knockout of ANXA6 gene involved in regulation of influenza virus budding to enhance influenza infection of HEK293FT cells. Taken together, our results demonstrate that AnxA6 depletion increased influenza virus yield at 60 hpi. However, bottleneck defining low permissiveness of HEK293FT cells seems to be
Funding
This work was supported by the Russian Science Foundation [grant number 18-75-10069]. Work was partially supported by State Budget Program (0245-2019-0001). RNAseq analysis was supported by the Russian Foundation for Basic Research [project number 19-34-90168].
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Authors would like to thank Dr. Irina V. Amosova (Research Institute of Influenza) for anti-influenza A NP monoclonal antibodies, Kirill A. Vasiliev (Research Institute of Influenza) for assistance in flow cytometry experiments and Dr. Marina A. Stukova (Research Institute of Influenza) for support in conducting of this work. IVR-165 influenza virus was obtained from WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention,
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Cited by (2)
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These authors contributed equally to this work.