{'fraction_non_alphanumeric': 0.03531660295828486, 'fraction_numerical': 0.03037227854412498, 'mean_word_length': 5.222595656670114, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 2, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Nonclassical autophagy activation pathways are essential for production of infectious Influenza A virus in vitro

Abstract Autophagy is a critical mechanism deployed by eukaryotic cells in response to stress, including viral infection, to boost the innate antimicrobial responses. However, an increasing number of pathogens hijack the autophagic machinery to facilitate their own replication. Influenza A virus (IAV), responsible for several global pandemics, has an intricate dependence on autophagy for successful replication in mammalian cells. To elucidate key chokepoints in the host stress responses facilitating IAV replication, we constructed a meta‐transcriptome of IAV and host gene expression dynamics during early (1–3 hpi), mid (4–6 hpi), and late (8–12 hpi) stages of the viral replication cycle at two multiplicities of infection (MOI): 1 and 5. We supplemented the global transcriptome study with phosphoproteomic analysis of stress‐activated protein kinase (SAPK/JNK) signaling in lung carcinoma (predominantly used as an in vitro model of IAV replication) and normal human bronchial epithelial cells. We report significant differences in the activation profiles of autophagy regulating genes upon IAV infection at the two MOI as well as divergent dependence on ULK1 signaling within the normal and cancer cells. Regardless of the cell model, JNK‐Thr187 signaling was crucial for the production of infectious viral particles.

The current concepts in cellular biology attribute plurality of functions to autophagy, including sustenance of healthy cellular physiology and energy homeostasis; regulation of inflammation; and support of innate and adaptive immunity through pathogen clearance and antigen presentation (Abdulrahman et al., 2011;Deretic et al., 2013;Harris et al., 2011;Kim et al., 2019b;Mintern et al., 2015;Paul & Münz, 2016;Saitoh et al., 2008;Siqueira et al., 2018;Valečka et al., 2018). While the cell signaling pathways regulating autophagy initiation are stress factor specific, the core mechanism of autophagosome biogenesis invariably leads to the formation of double-membraned vesicles that randomly or selectively engulf cytoplasmic cargo. For the degradation of the captured cargo, autophagosomes fuse with lysosomes forming autolysosomes where low pH and abundance of proteolytic enzymes aid the recycling of damaged organelles into anabolic building blocks and energy source molecules. Additionally, microbes engulfed by the eukaryotic cells via endocytosis are targeted to the autolysosomes for degradation (Levine & Kroemer, 2019;Mizushima et al., 2010).
The role of autophagy as an intrinsic mechanism of eukaryotic cell resistance to pathogens has sparked interest in host-directed therapeutics development (Kim et al., 2019b;Liang et al., 1998).
Due to the great diversity of DNA and RNA virus replication mechanisms, development of broad-spectrum antiviral therapeutics remains an impossible task. To address this problem, anti-viral treatment protocols based on augmentation of host cell antimicrobial mechanisms, such as autophagy, can be implemented (Campbell & Spector, 2011;Kobayashi et al., 2020;Shoji-Kawata et al., 2013). Paradoxically, a growing list of viruses and intracellular bacteria take advantage of host autophagic machinery to promote their own replication (Deretic et al., 2013;Levine et al., 2011;Li et al., 2011;Starr et al., 2012). To further complicate the issue, in discrete cases, autophagy activation has both pro-and anti-viral function (Campbell & Spector, 2011;Kyei et al., 2009). Therefore, identification of pharmacological targets for broad-spectrum antiviral therapeutics demands in depth understanding of the autophagic machinery components that enhance host defenses and those co-opted for virus replication.
Influenza A virus (IAV) is an enveloped respiratory pathogen causing seasonal infections with morbidity and mortality among human and animal populations. Historically, influenza viruses have been responsible for several devastating global pandemics (Smith et al., 2009;Sriwilaijaroen & Suzuki, 2020). Periodic cycling of influenza subtypes and strains requires the design of seasonal vaccines that often miss unexpected rearrangements of viral antigens (Viboud et al., 2020).
IAVs evolve resistance to antiviral drugs and genomic re-assortment events further accelerate the global dissemination of resistant viruses (Nelson et al., 2009;Simonsen et al., 2007).
When traditional anti-viral treatments fail, therapeutics against host regulatory proteins may come to the rescue. It has been previously shown that chemical inhibition of autophagy improved the survival rate of the H5N1-infected host by reducing inflammation and severe lung damage (Sun et al., 2012). Notwithstanding the evidence, the relationship between IAV and autophagy is quite complicated. The autophagy machinery was found to facilitate influenza virus replication, while at the same time most IAV strains hijack host translational machinery by activating the mechanistic target of rapamycin (mTOR) complex 1 to ensure viral protein synthesis (Kuss-Duerkop et al., 2017;Zhirnov & Klenk, 2013;Zhou et al., 2009).
The IAV causing seasonal epidemics in humans (strains H1N1 and H3N2) utilize their nonstructural protein 1 (NS1) as an anti-apoptotic agent via direct interaction with the p85β subunit of PI3K and activation of AKT1 signaling pathway (Ehrhardt et al., 2007;Shin et al., 2007;Zhirnov & Klenk, 2007). Thus, the viral NS1 stimulates mTORC1 kinase function to facilitate protein synthesis and cap-dependent translation initiation through cascade of phosphorylation events, including p70 S6 kinase and 4E-BP1 (Kuss-Duerkop et al., 2017;Ma et al., 2011). Activated mTORC1 is supposed to inhibit autophagy through ULK1 signaling; however, it supports the synthesis of viral proteins acting as autophagy stimulation factors, including hemagglutinin (HA) and ion channel matrix 2 (M2) (Zhirnov & Klenk, 2013). Specifically, the influenza virus M2 protein interaction with the microtubule-associated protein light chain 3 (LC3), a core structural protein of the autophagosomes, was found to inhibit autolysosome formation while redirecting the LC3-M2 complex to the plasma membranes (Beale et al., 2014;Zhirnov & Klenk, 2013). This process is associated with delays in apoptosis initiation and facilitation of virion assembly and budding (Beale et al., 2014;Gannagé et al., 2009). Current models describe autophagy as a host response mechanism activated during the late stage of IAV replication (Gannagé et al., 2009), while mTORC1 and mTORC2 activation by IAV infection was reported to occur midway through the IAV replication cycle (Kuss-Duerkop et al., 2017). These models, based on Western blot (WB) phosphoproteomic data analysis, are influenced by two main experimental shortcomings: sensitivity limitations of WB and use of cancer cells as host models for in vitro IAV infection studies. Here, we sought to elucidate the autophagy-regulating component that is essential for IAV replication in lung epithelial cells. We applied a highly sensitive analytical approach and validated our findings in a noncancer cell model. We performed a RNA-sequencing-based meta-transcriptomic study of IAV and host gene activation dynamics throughout the virus replication cycle at two multiplicities of infection (MOI), 1 and 5. We found that viral load has a strong influence on the mode and dynamics of host cell protective responses and autophagy activation. With a protocol that does not induce autophagy due to serum starvation of the host cells during the virus absorption stage, we detected simultaneous activation of mTOR complex 1 genes and key autophagy regulating genes early upon virus infection (1-3 hr post infection [hpi] at MOI 1). Subsequent phosphoproteomic analysis verified that the classical autophagy activation pathway through ULK1 signaling did not play a significant role in the autophagy activation upon IAV infection, while the activation of c-jun N-terminal kinase (JNK) was essential for viral protein accumulation. JNK, a stress-activated protein kinase (SAPK) activated by various viruses (Clarke et al., 2001;Eliopoulos et al., 1999;McLean & Bachenheimer, 1999), is stimulated by direct interaction with the IAV NS1 protein (Nacken et al., 2014). We show that chemical inhibition of JNK abrogated viral protein accumulation in lung carcinoma A549 cells and significantly inhibited infectious viral titer in noncancer cells. Host-directed antiviral therapy targeting key regulators of nonclassical autophagy pathways, such as JNK/SAP, could show less toxicity in vivo compared with a pharmacological inhibition of the classical autophagy pathways that are essential for proper host immune defenses.

| Serum depletion activates autophagy prior to Influenza A virus replication in vitro
Cleavage of IAV hemagglutinin (HA) is required for fusion of the viral capsid with host-cell endosomal membranes. Proteases secreted by the respiratory epithelium are responsible for HA cleavage in vivo IAV infection (Klenk & Garten, 1994;Steinhauer, 1999;Zhirnov et al., 2002). The majority of in vitro cell models (MDCK, HEK293, or A549) utilized to study the role of autophagy in IAV replication do not produce proteases that cleave HA necessitating the addition of trypsin to promote virus infectivity. Typically, during the 1-hr virus absorption stage, the host cells are incubated in serum-free media containing the infectious virus and TPCK-trypsin (Beale et al., 2014;Gannagé et al., 2009;Kuss-Duerkop et al., 2017;Wang et al., 2019;Zhirnov & Klenk, 2013). Applying time-lapse microscopy, we monitored the cellular localization of transcription factor EB (TFEB) in live cells, as a biomarker of autophagy activation (Napolitano & Ballabio, 2016), and registered TFEB translocation into the nucleus of mock-treated A549 cells within 40 min of incubation in serum and virus-free media ( Figure S1a). Consistent with TFEB nuclear localization, we observed a significant drop in the levels of phosphorylated ribosomal protein S6 kinase β-1 (p70S6K1) indicating inhibition of mTORC1 activity ( Figure S1b).
Thus, serum depravation protocols for in vitro infection of cancer cell lines with IAV generate data that are biased to a condition where mTORC1 function is downregulated and autophagy is induced prior to the initiation of viral replication. To investigate the role of IAV infection on autophagy activation, we adapted a protocol that did not include a serum starvation step. With this protocol, we were able to study the normal course of autophagy activation upon IAV infection ( Figure S1c) and found that H3N2 strain, A/Port Chalmers/1/73, showed higher tropism to A549 cells compared with the H1N1 strain, A/PR/8/1934 ( Figure S1d). Therefore, in this study we used the H3N2 strain to investigate the role of autophagy in IAV replication in lung epithelial cells.

| Autophagy activation correlates with Influenza virus A load
Employing strand-specific RNA-sequencing (RNA-seq) of infected A549 cells, we simultaneously measured the kinetics of A/PCh/1/73 replication and the expression of host genes affected by IAV replication at 1, 3, 4, 6, 8, and 12 hpi. In order to measure virus replication, we generated RNA-based growth curves by normalizing the total virus reads to the total host read coverage (Figure 1a). The viral RNA accumulation profile was consistent with the virus titration growth curves ( Figure S1d). To reproducibly detect reads from genes with low transcription rates, we performed infections with moderate (1) and high (5) multiplicities of infection (MOI) representing the ratios of host cells to infectious virus particles. At MOI5, we observed a rapid increase in the viral transcript levels that accounted for 10% of the total RNA reads within 2 hr of infection and plateaued between 4 and 8 hpi, accounting for 75% of the total RNA reads ( Figure 1a). In contrast, we observed exponential viral growth within 4 hr of infection at MOI1. By 12 hpi, viral RNA levels started to decline in both infection conditions.
The objective of our study was to evaluate the effect of IAV replication on the expression profiles of genes involved in the regulation of autophagy, mTOR signaling pathways, and innate immu- Additionally, MAPK3 activates mTORC1 by phosphorylation of RAPTOR and TSC complex (Carriere et al., 2011;Zoncu et al., 2011).  Growth factors activate mTORC1 through the phosphoinositide-3 kinase (PI3K)-AKT or SOS-MAPK3/ERK signaling axis (Mendoza et al., 2011;Xu et al., 2020). Additionally, the LRP-GSK axis neutralizes the inhibitory action of the TSC1/2 complex (Inoki et al., 2006).
Previous studies have shown that H1N1 IAV ramps up the produc-

| IAV replication activates autophagy in lung epithelium cells independently of the ULK1 signaling pathway
Our global transcriptome analysis revealed previously uncharacterized correlation between IAV load and activation of ULK gene transcription. While ULK1 gene was consistently transcribed in A549 cells throughout the IAV virus replication cycle at MOI1, its expression dynamics remained mostly unchanged at MOI5 (Figure 2, Figure S2).
Therefore, we further investigated the role ULK1 plays in autophagy activation and IAV replication at a post-transcriptional level.
Applying Western blot (WB) analysis, we could barely detect viral We further validated our WB results by applying immunocytofluorescence with anti-LC3B antibodies. We observed autophagosome vesicle formations in IAV-infected A549 cells when ULK1 signaling was inhibited with a small molecule (Figure 3c) thus confirming the WB-based proteome data. Collectively our data indicated that autophagy was activated through an alternative to ULK1 signaling pathway during IAV infection.

| IAV infection upregulates AKT and Stress Response Kinase JNK activity in A549 cells midway through the virus replication cycle
Previous in vitro studies of IAV infection have demonstrated that viral proteins stimulate mTORC1 activity to ensure virus protein expression and promote successful replication (Zhirnov & Klenk, 2007. Additionally, mTORC2 function supports host survival in the later stages of IAV replication when cells undergo significant stress (Kuss-Duerkop et al., 2017).
To validate our findings from the global transcriptome analysis of mTOR-regulatory complex differential gene expression, we measured mTORC1 and mTORC2 activity in IAV infected A549 cells. We The phosphoproteomic data presented here demonstrated that pro-survival mechanisms dependent on AKT-Ser473 phosphorylation were activated at mid throughout the late stage of the virus replication cycle, while mTORC1 activity was not affected in cells infected with IAV at MOI1. We detected mTORC1 activation in the later stages of IAV replication specifically at very high MOI, when mTORC1 function was required to satiate the high demand for protein synthesis (Figure 4b: 9 hpi vs. 6 hpi).
Together with the activation of AKT anti-apoptotic signaling, we investigated the JNK stress-response regulator upon virus infection. JNK activation is a common response to many stress stimuli, including endoplasmic reticulum (ER) excessive stress, causing apoptotic cell death through regulation of the BCL2 family proteins (Raciti et al., 2012;Wei et al., 2008;Yamamoto et al., 1999). Pharmacological inhibition of JNK signaling dramatically reduced viral protein synthesis in IAV-infected lung carcinoma A549 cells.

| Pharmacological inhibition of JNK-Thr187 signaling decreased IAV titer in normal human bronchial epithelial cells (NHBE) in vitro
Since the cancer cells, including the A549 line, used as models to study IAV replication may regulate stress response and survival   The JNK-Thr187 signaling appeared as a stress regulatory mechanism activated by the rampant synthesis of viral proteins. Therefore, inhibition of JNK-Thr187 signaling decreased the host cell capacity as a virus "factory" and resulted in a significant reduction of infectious IAV titer in vitro.

| IAV matrix protein M2 interaction with the host LC3B protein activates autophagosome formation
Our Western blot results have consistently indicated that IAV infection activates autophagy in strict correlation to accumulation of viral proteins (Figures 3a, 4d, and 5a). Since detection of the lipidated LC3B-II form was contingent to high viral protein load, we designed experiments to test whether direct interaction between the viral M2 protein and the autophagosome core protein occurs in situ and activates autophagy independently of the classical (ULK1) or alternative (JNK) signaling cascades. Previous studies have inferred interaction between the two proteins from in vitro binding studies and infections with M2-deficient H1N1 virus (Beale et al., 2014). Here, we utilized split green fluorescent protein (GFP) methodology (Cabantous et al., 2005) to directly test M2-LC3 interaction in live cells. We tagged each LC3B and M2 proteins with GFP fragments 10 (G10) and 11 (G11).
Plasmids expressing proteins tagged with the GFP fragments were co-transfected in HEK293T cells. No fluorescence was detected in transfected cells prior to addition of the GFP1-9 scaffold domain (data not shown). Since the GFP1-9 utilized in this study was not optimized to fold properly in mammalian cells, we supplied purified and folded GFP1-9 peptide in trans similar to the antibody staining techniques.
Thus, we indirectly registered protein-protein interactions between G10 and G11 fragments that occurred in live cells after we visualized this interaction in fixed cells supplemented with the GFP1-9 scaffold.
Guided by previous knowledge that IAV M2 protein forms homodimer and tetramer complexes (Holsinger & Lamb, 1991), we co- We observed global host transcriptome changes (LFC ≥2) that varied between the early stages of IAV replication and later timepoints depending on the ratio between infectious virus and host cells, indicating that virus load played a significant role in host gene expression dynamics (Figures 1 and 2). Despite host cells' efforts to shut down protein synthesis, IAV replication activated mTORC1 signaling, positively regulating protein synthesis in nutrient-rich conditions.
Both mTORC1 and mTORC2 carry out distinct functions: activation of mTOR1 facilitates protein synthesis and inhibits autophagy via repression of ULK1 signaling, while mTORC2 regulates cytoskeleton dynamics and cell survival (Oh & Jacinto, 2011;Sabatini, 2017).
Despite the activation of mTORC1, IAV replication also simultaneously activated autophagy independent of ULK1 signaling. The ULK1 gene executes a feedback loop between mTORC1 signaling and the autophagy pathway, inhibiting mTORC1 via interaction with RPTOR during stress (Dunlop et al., 2011;Jung et al., 2011;Luo et al., 2018). RPTOR gene activation was limited to the early stages of IAV infection and may not have had the capacity to inhibit ULK1 function, particularly given the continuous ULK1 transcript accumulation throughout the virus replication cycle (Figure 2 and Figure S2).
Regardless, the IAV replication and production of infectious virus particles remained unchallenged when compared with the experimental group treated with JNK-specific inhibitor SP600125 ( Figure 5) indicating that even at reduced activity JNK signaling could support IAV infection. Only upon complete inhibition of the JNK function, the IAV titer was reduced. Collectively, our data indicate that lung carcinoma A549 show reduced sensitivity to inhibition of JNK signaling during IAV replication compared with NHBEC, supporting the idea that cell models utilized to study host-pathogen interaction would produce subjective results. Our study solidified the link between IAV infection and JNK-Thr187 signaling as a stress regulatory mechanism accompanying the rampant synthesis of viral proteins and activating alternative autophagy pathways (Clarke et al., 2001;Eliopoulos et al., 1999;McLean & Bachenheimer, 1999;Nacken et al., 2014;Zhang et al., 2019). Therefore, therapies that decrease the host cell capacity as a viral particle "factory" through inhibition of JNK-Thr187 signaling can result in a significant reduction of infectious IAV titer.
In conclusion, our findings indicate that the IAV load is responsible for differential gene expression dynamics within the cell host.
We demonstrate that IAV replication simultaneously activated tran- All cells were cultured at 37°C and 5% CO 2 .
To inhibit ULK1 signaling, A549 cells were cultured in 10%-DMEM media supplemented with 10 μM SBI0206965 (Selleckchem, Pittsburgh, PA) for 6 hr prior to IAV infection, and drug concentration was maintained in the media post-virus infection. Since BEAS-2B and NHBE have lower expression levels of ULK1, SBI0206965 was added at 1 μM to the BEGM growth media. We observed high toxicity at 10 μM SBI0206965 for both noncarcinoma cell types, which could be caused by an off-target drug effect at this concentration.
To inhibit JNK-Thr187 phosphorylation, we added 10 μM SP600125 (Selleckchem, Pittsburgh, PA) to A549 growth media 6 hr prior to IAV infection, while 5 and 1 μM were added to the culture media of NHBE and BEAS-2B, respectively, due to sensitivity issues resulting from JNK inhibition.

| Viruses and virus infection procedures
The following IAV strains were used in this study: The virus titer was determined in MDCK cells by plaque assays, as described previously (Szretter et al., 2006), and immunoplaque analysis was performed with anti-NP antibody (ThermoFisher, PA5-32242) and alkaline phosphatase-conjugated anti-rabbit antibody.
To study the dynamics of viral RNA replication, A549 cells were plated 18 hr prior to infection in high glucose DMEM supplemented with 5% or 10% fetal clone III serum at 1 × 10 6 per well in six-well format dishes. In order to standardize the multiplicity of virus in-

Uninfected cells and cells infected with
To determine the percentage of infected cells, images with fluorescently labeled anti-IVA/M2 antibody were obtained with 20× objective choosing fields with >300 cells. For each culture condition, five images were taken from nonoverlapping fields and were analyzed with ImageJ. A cell counter plugin was created with the following parameters: (a) manual threshold, (b) fill holes, (c) enhance contrast and saturated pixels at 0.3%, (d) watershed separation, and (e) particle analysis at size 100-infinity and circularity at 0.2-0.1. The fraction of cells labeled with anti-viral antibody was calculated as percent of total cell population labeled with DAPI.

| Plasmids and reagents for split-GFP in situ protein-protein interactions
To study direct interaction of IAV M2 and host LC3B proteins, we used a split green fluorescent protein (GFP) assay, as previously described (Cabantous et al., 2005). gBLOCK gene fragments, encoding for GFP11-and GFP10-tagged M2 and LC3B, were chemically synthesized by Twist Biosciences Inc. The synthetic DNA contained a set of primer sequences for amplification and Hind III and EcoR V restriction enzyme sites. The genes also contained methionine required for the start and a stop codon. The 5′ end of all gene constructs con- (15 min at room temperature) and permeabilized with 0.3% Triton X100 (10 min at room temperature). After blocking with 2% BSA in phosphate buffer saline (PBS), the cells were incubated overnight at 4°C with purified protein fraction of GFP1-9 (10 μg/ml). Only when in very close proximity, the GFP11 and GFP10 peptides can be complimented with the larger fraction GFP1-9 to produce folded and fluorescent GFP (Cabantous et al., 2013). Imaging was performed on Zeiss Axio Observer.Z1 and processed with ZenPro software.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All transcriptomics data have been submitted to GEO under accession number GSE16 2295 (https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE16 2295) and NCBI tracking system #21497852.