Systemic Removal of Host Restriction Factors Enables Rapid, Scalable Virus Production for Cell Culture-Based Vaccines High-Yield Virus Production System generated by gene knockout of multiple anti-viral host factors

Systemic Removal of Host Restriction Factors Enables Rapid, Scalable Virus Production for Cell Culture-Based Vaccines High-Yield Virus Production System generated by gene knockout of multiple anti-viral host factors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Systemic Removal of Host Restriction Factors Enables Rapid, Scalable Virus Production for Cell Culture-Based Vaccines High-Yield Virus Production System generated by gene knockout of multiple anti-viral host factors Se-ho Park, Zhengmei XU, Jaemyeong Jeon, Youngki Shin, Jinsoo Oh This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9184882/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The efficiency of cell culture-based vaccine production is fundamentally constrained by the antiviral defenses of host cells, creating a major bottleneck for rapid and large-scale vaccine manufacturing. Key antiviral proteins- such as RNase L, PKR, and JAK1 act as intrinsic brakes on viral replication, limiting efficient propagation of many clinically relevant viruses. To overcome this challenge, we generated HEK293T, Vero, and MDCK cell lines with targeted knockouts of multiple antiviral genes. Notably, these engineered cells maintained normal growth and viability while supporting markedly increased viral yields. Multi-gene deletions enhance the replication of both enveloped viruses, including influenza A virus, pseudotyped lentivirus, and porcine epidemic diarrhea virus (PEDV), and non-enveloped viruses such as coxsackievirus. The magnitude of enhancement scaled proportionally with the number of genes disrupted. By systematically removing host restriction factors, this platform provides a versatile and powerful strategy for accelerating viral propagation, offering a strong foundation for more efficient development and large-scale production of cell culture-based anti-viral vaccines. Biological sciences/Biotechnology Biological sciences/Immunology Biological sciences/Microbiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Vaccines are one of the most effective tools for preventing infectious diseases 1 . However, the efficiency of vaccine manufacturing remains a critical bottleneck in global health preparedness 2 , 3 . Cell culture-based systems, which form the foundation of most modern vaccine production, face substantial challenges in achieving high viral yields 4 . Commonly used cell lines, such as Vero and MDCK, often have limited viral productivity, primarily due to the host cells' potent intrinsic antiviral defense mechanisms 5 . These defense mechanisms, including interferon (IFN) signaling and restriction factor expression, actively suppress viral replication 6 . Advances in gene editing, particularly CRISPR-Cas9, provide a powerful means to overcome this barrier by engineering host cells to be more permissive to viral replication 7 . Promising results have been obtained from the first proof-of-concept study disrupting BST2, which tethers the host cells and viral membrane thus limiting virus release from the host cells 8 . However, because many viruses possess viral elements that counteract specific host restriction factors, it is difficult to restrict a wide range of viruses with a single restriction factor. Therefore, host cells possess a variety of restriction factors throughout the viral life cycle, effectively suppressing viral replication. While proof-of-concept studies, including BST2 knockout to enhance the release of enveloped viruses, have shown promise, the redundancy of the host antiviral network inherently limits such single-target approaches 8 . In fact, BST2 can only act on a subset of enveloped viruses. Non-enveloped viruses are not restricted by BST2 9 , and certain enveloped viruses, such as human immunodeficiency virus 1 (HIV-1), degrade host BST2 with viral Vpu protein 10 . To systematically dismantle this redundant defense network, we targeted key nodes of the innate immune system: PKR 11 , RNase L 12 , and JAK1 genes in addition to BST2. Protein kinase R (PKR) is a host enzyme activated by double-stranded RNA (dsRNA) during viral infection. Activated PKR phosphorylates the translation factor eIF2α, which blocks viral replication by halting protein synthesis. It can also induce host cell apoptosis and activate NF-κB signaling to enhance antiviral gene expression 13 , 14 . RNase L is a host enzyme activated by 2′,5′-oligoadenylate (2-5A), which is produced by oligoadenylate synthetase (OAS) in response to viral double-stranded RNA (dsRNA). RNase L degrades viral and host RNA, halting viral replication and producing small RNA fragments that amplify interferon signaling 12 . Janus Kinase 1 (JAK1) is a key host kinase in the interferon signaling pathway initiated by viral infection. When interferon binds to its receptor, JAK1 along with TYK2 phosphorylates STAT1/STAT2, which forms the ISGF3 complex, establishing an antiviral state within the cell by inducing the expression of interferon-stimulated genes (ISGs) 15 , 16 . BST2 (also known as tetherin or CD317) is an interferon-induced antiviral protein that restricts the release of many enveloped viruses from infected cells. It acts by physically “tethering” budding virions to the cell membrane, preventing their release and subsequent infection of new cells 17 . We generated knockout cell lines for these genes in three cell models with industrial applications: HEK293T, a human kidney cell line used to produce pseudotyped lentivirus for gene transfer; Vero, a monkey kidney cell line used to produce various antiviral vaccines; and MDCK, a canine kidney cell line which is the major cell line used to produce anti-influenza vaccines. In all these cell lines, multiple antiviral gene knockouts significantly increased virus production for a wide range of viruses, including both enveloped and non-enveloped viruses. This cross-species systematic multi-gene knockout virus production platform (collectively, the platform) will provide robust productivity to produce antiviral vaccines and therapeutic vectors 18 , 19 . 2. Results 2.1. Generation and Validation of Antiviral Gene Knockout Cell Lines We employed the CRISPR-Cas9 system to generate targeted knockouts of key antiviral genes in HEK293T, Vero, and MDCK cells. Specific sgRNAs were cloned into the pX458 vector, and monoclonal cell lines were subsequently isolated through sorting GFP-positive cells and limited dilution (Fig. 1 A). The following knockout combinations were established: BST2 (B), BST2 and PKR (BP), BST2 and RNase L (BR), BST2, PKR, and RNase L (BPR), and BST2, PKR, RNase L, and JAK1 (BPRJ), with nomenclature reflecting the targeted genes in each cell line. Targeted mutations were verified by T7 endonuclease 1(T7E1) digestion (Fig. 1 B), and Sanger sequencing of representative clones confirmed homozygous frameshift mutations (Table S1 ). To validate the functional disruption of target genes, we examined protein expression in 293T cells after knocking out RNase L. Western blot analysis confirmed the disappearance of RNase L protein upon interferon γ (IFN-γ) stimulation in RNase L-deficient cells (Figure S1 A). To verify the function of JAK1, we assessed the phosphorylation pattern of Signal transducer and Activator of Transcription 1 (STAT1), a key downstream signaling molecule of JAK1. Western blot analysis revealed significantly reduced STAT1 phosphorylation (p-STAT1) in JAK1-deficient cells following IFN stimulation, confirming inhibition of the JAK-STAT signaling pathway (Fig. 1 C). Despite the targeted mutations, cell proliferation assays revealed that these gene knockouts did not impair normal cell growth or viability (Fig. 1 D). 2.2. Enhanced Production of Enveloped Viruses in Multi-Gene Knockout Cells We first assessed the platform's ability to enhance the production of enveloped viruses, using the influenza virus as a primary model. Upon infection with influenza A/H1N1 (A/Brisbane/59/2007) or H3N2 (A/Brisbane/10/2007), hemagglutinin (HA) protein expression increased in gene knock-out number manner across 293T, VERO and MDCK engineered vaccine production cell lines. The triple or quadruple gene knockout cell lines showed increased viral output by 3 to 8-fold relative to wild-type (WT) cells, with the magnitude of enhancement being cell-specific (Fig. 2 A and 2 B). This enhancement was not influenza A virus-specific, as the production of influenza B/Yamagata (B/Wisconsin/2010) in MDCK BPRJ cells also resulted in significantly higher cytopathic effect (CPE) percentage than in those of WT cells (Figure S2 B). To evaluate the platform's utility in veterinary vaccinology, we infected Vero background cells with the classical porcine epidemic diarrhea virus (PEDV) strain CV777. Western blot analysis of the viral nucleocapsid (N) protein demonstrated a substantial increase in viral production in the knockout cells (Fig. 2 C). These results collectively suggest that a targeted multi-gene knockout strategy can be applied to enhance the production of diverse enveloped viruses. Viral infection of VERO-derived antiviral knockout cell lines demonstrated that stepwise removal of interferon-induced restriction factors markedly enhances viral production while uncoupling STAT1 activation from functional antiviral control. Following infection, BPR cells (BST2/PKR/RNase L triple knockout) exhibited substantially increased STAT1 phosphorylation compared with wild-type cells, reflecting heightened innate immune sensing driven by elevated viral replication. Despite this robust STAT1 activation, viral production in BPR cells was significantly increased compared to wild-type cells, indicating that the deletion of key antiviral effector proteins compromises the effective restriction of viral replication and release. In contrast, BPRJ cells (BST2/PKR/RNase L/JAK1 quadruple knockout) displayed minimal STAT1 phosphorylation, consistent with disruption of JAK–STAT signaling and supported the highest levels of viral production among all cell lines tested (Fig. 2 D). These findings demonstrate that elimination of antiviral effector pathways is sufficient to increase viral yield substantially, and that additional abrogation of upstream interferon signaling further maximizes viral replication and release. 2.3. Enhanced Lentiviral Production in Multi-Gene Knockout HEK293T Cells HEK293T cells are the industry standard for producing lentiviral vectors (LVs) used in cell therapies. We produced VSV-G pseudotyped LVs carrying a copGFP reporter gene in the engineered HEK293T background cell lines. Produced viruses were transduced into HEK293T WT to evaluate the virus titer. Flow cytometry analysis of transduced HEK293T WT cells revealed that viruses produced from the BR knockout cells exhibited significantly higher functional titers (TU/mL) than those from WT cells (Fig. 3 A). Additionally, we evaluated the preparation of more complex LVs carrying the anti-CD19 chimeric antigen receptor (CAR). The HEK293T BR cell line consistently produced higher levels of LVs than HEK293T WT cells (Fig. 3 B). Functionally active CD19-CAR LV underscores the platform's potential for advanced manufacturing of complex therapeutic vectors. 2.4. Broadened Applicability to Non-Enveloped Virus Production Among viruses infecting animal hosts, many possess envelopes, but non-enveloped viruses also constitute a significant proportion, and their modes of infection differ 20 – 22 . Having established the efficacy of our platform for enveloped viruses, we next examined whether the multi-gene knockout strategy could enhance the production of non-enveloped viruses, which BST2 does not restrict 8 . Coxsackievirus B was produced in VERO WT and VERO BPRJ cells, and the harvested virus was subsequently used to infect VERO WT cells. Infection with Coxsackievirus B produced in VERO BPRJ cells resulted in a significant increase in viral titers compared to virus derived from WT cells, as determined by the CPE percentage (Fig. 4 A). These findings demonstrate that disrupting downstream effector pathways (PKR, RNase L) along with core signaling (JAK1) can overcome antiviral defenses of host cells against a broader range of viruses, including non-enveloped types. Furthermore, Coxsackievirus B3 (CVB3) replicons generated in 293 T-based cells induced progressively more severe cytopathic effects (CPE) in infected VERO WT cells as the number of gene knockouts increased, indicating a positive correlation between replicon replication efficiency and the extent of host gene depletion (Fig. 4 A). 2.5. Rescue of Poorly Replicating Viruses in Knockout Cells Next, we tested our platform using the varicella-zoster virus (VZV). Due to its cell-associated nature and weak replication capacity, VZV is difficult to amplify in traditional cell cultures. We first infected VERO WT and VERO BPR cells with VZV-Oka virus, then passaged the infected cells to detect glycoprotein E expression at each time point. Although Vero cells were known to poorly support VZV replication, expression of VZV-Oka glycoprotein E progressively increased in passaged Vero BPR cells at each passage. In contrast, VZV-Oka glycoprotein E expression in VERO WT cells was extremely low (Fig. 4 B, Figure S1 C). These results demonstrate the significant potential of host cell engineering to produce difficult-to-culture human pathogens. 2.6. Integration with Suspension Culture for Scalable Manufacturing To address the scalability limitations of adherent culture systems, we adapted the multi-gene knockout cells to suspension growth. Both MDCK WT and BRJ cells adapted in serum-free conditions until cells proliferate stably in suspension (Fig. 5 A). Upon H1N1 infection, HA expression in suspension-adapted MDCK BRJ cells remained significantly higher than in suspension-adapted WT cells (Fig. 5 B). Process optimization in shake flask cultures achieved HA yields of ≥ 60 µg/mL (Figure S1 D), confirming that the productivity advantage is retained in scalable formats. Similarly, suspension-adapted Vero BPR cells infected with PEDV exhibited higher viral yields, as indicated by N protein expression, compared to that of adherent cultures. This enhancement was more pronounced under serum-free conditions (Fig. 5 C, D). The successful adaptation of multiple engineered cell lines to high-yield suspension culture underscores the industrial robustness and broad applicability of this synergistic platform. 3. Discussion The development of robust and high-yield viral production platforms is a cornerstone of effective vaccine manufacturing and virological research 23 , 24 . In this study, we established a versatile and highly efficient platform by systematically engineering industrially relevant cell lines—HEK293T, Vero, and MDCK 25 , 26 —through targeted knockout of key intrinsic antiviral defense genes, including BST2, JAK1, RNase L, and PKR. This multi-target knockout approach resulted in a substantial enhancement of replication for a wide spectrum of viruses. A central finding of our work is the consistent, knockout-gene-number-dependent enhancement of viral replication across all tested cell lines and virus families. The replication of multiple enveloped viruses, including influenza A (H1N1 and H3N2), influenza B (Yamagata), PEDV (CV777), and pseudotyped lentivirus, was significantly enhanced in the multi-gene knockout cells. This demonstrates that the redundancy inherent in the host antiviral network can be effectively overcome by a coordinated, multi-target knockout strategy, thereby surpassing the limited efficacy of single-gene edits like BST2 8 . These findings position BST2 as a context-dependent modulator of viral replication rather than a universal restriction factor and underscore that dismantling the PKR-RNase L-JAK1 axis is pivotal for creating a broadly permissive cellular environment for viral replication 27 . Critically, the enhancement provided by our platform was not confined to enveloped viruses. The significantly increased production of Coxsackievirus B, a non-enveloped picornavirus, in Vero BPRJ cells indicates that the restrictions mediated by PKR, RNase L, and JAK-STAT signaling constitute a broad-spectrum antiviral barrier, independent of viral structure or budding mechanism. This finding significantly expands the potential utility of our platform for producing a broader range of viral biologics. A particularly compelling result was the successful enhancement of Varicella-zoster virus (VZV) replication, a human pathogen notoriously difficult to propagate in conventional cell cultures due to its highly cell-associated nature 28 . The progressive increase in VZV glycoprotein E expression over passages in our engineered Vero BPR—in stark contrast to the limited replication in WT controls—highlights the unique capacity of this host cell engineering strategy to enable the propagation of fastidious pathogens. This outcome holds significant promise for improving the manufacturing efficiency of live-attenuated VZV vaccines 29 . Furthermore, the marked increase in the functional titer of complex therapeutic lentiviral vectors, including a CD19-CAR construct, produced in engineered HEK293T BR and BRJ cells, underscores the direct clinical relevance of our platform. The ability to achieve higher vector titers without compromising integrity addresses a major challenge in the cost-effective and scalable manufacturing of cell therapies, such as CAR-T, thereby enhancing their clinical and commercial translation 30 . Finally, a crucial step for industrial application is the transition from a proof-of-concept in adherent culture to a scalable, industry-ready format. We demonstrated that the genetic advantages of our multi-gene knockout cells were fully retained following adaptation to suspension culture. The high yields of influenza HA protein (reaching 60µg/mL in shake flasks) and PEDV in suspension-adapted cells confirm that our strategy successfully integrates intrinsic cellular enhancements with the bioprocess. This synergy between cellular engineering and bioprocess optimization may present a comprehensive solution to long-standing production bottlenecks 31 . In summary, this study establishes a broadly applicable and robust framework for engineering high-efficiency viral production by engineering cell lines. By systematically targeting conserved and redundant nodes of the innate immune system, we have created a platform that significantly boosts the yield of diverse viruses, ranging from vaccine targets and therapeutic vectors to hard-to-culture pathogens. The successful adaptation of these engineered cells to suspension culture paves the way for industrial-scale manufacturing. 4. Materials and methods Cell lines and gene knockout MDCK.1(ATCC-CRL-2935), Vero(ATCC-CCL-81), HEK293T(ATCC-CRL-3216) cells, and their respective multi gene deficient cell lines-HEK293T (B, BR, BRJ), VERO(B, BP, BPR, BPRJ), MDCK(B, BR, BRJ) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS(Hyclone), 2mML-glutamine(Gibco), 1% penicillin-streptomycin(Gibco), 10µg/ml Gentamycin(Gibco), and 50mMb-mercaptoethanol(Gibco) at 37℃ with 5% CO2. Generation of BST2-deficient cell lines HEK293T-B, VERO-B, and MDCK-B has been described previously 32 . Gene knockouts were generated using the pSpCas9(BB)-2A-GFP (PX458) backbone. For each target gene, sgRNA expression plasmids (pX458-sgRNA(Target gene]-Cas9(BB)-T2A-GFP) were constructed and transfected into cells with Turbofect (Thermo Fisher Scientific). GFP-positive cells were enriched by fluorescence-activated cell sorting (BD FACSAria Ⅱ) and seeded into 96-well plates for single-cell cloning. Genomic DNA from individual clones was analyzed by T7 endonuclease I (T7E1) assay to detect indels. Clones showing cleavage patterns were further validated by Sanger sequencing (Bioneer), and homozygous knockout lines were confirmed. Cells used for growth analysis were passaged approximately 20 times before the assay. The cells were seeded into 96-well plates, and cell viability was measured daily using the CCK-8 assay. The optical density (OD) was recorded to determine the growth rate of each cell line. Virus production and viral titer determination Seasonal influenza H1N1 (A/Brisbane/59/2007) and H3N2 (A/Brisbane/10/2007) virus was obtained from the Korea Centers for Disease Control and Prevention (KCDC), and Yamagata(B/Wiscosin/1/2010) was obtained from the National Biobank of Korea (NBK). Coxsackievirus B4 was obtained from the Korea Disease Control and Prevention Agency (KDCA). For infection by the influenza virus, we followed the previous protocol 32 . Porcine epidemic diarrhea virus (strain CV777), provided by the Korea Veterinary Culture Collection (KVCC), was used to infect cells. In brief, cells were seeded 20-24h before infection. When cells reached 90% confluence, they were washed with PBS. Next, cells were inoculated with 0.001-0.1 MOI of virus containing 0.3% BSA and 1–2µg/mL of TPCK-trypsin (Sigma) in MEM medium for 1h. Finally, the virus inoculum was removed and replaced with a fresh medium containing 0.3% BSA and TPCK-trypsin for 48 hours at 37°C under 5% CO₂. Following incubation, the culture supernatant, containing progeny viruses, was collected, centrifuged to remove cellular debris, and stored at -80°C until analysis. VERO cells were seeded in 24-well plates (or specified culture vessel) and allowed to adhere for 20–24 hours. At the time of infection, when cells had reached approximately 90% confluency, the culture medium was aspirated, and the cell monolayer was gently rinsed with PBS to remove residual serum. Infection was carried out by incubating the cells with Coxsackievirus B4 at the indicated dose (1.25–2.5 µL of viral stock) in serum-free DMEM. The virus-cell mixture was incubated for 72 hours at 37°C under 5% CO₂. Following incubation, the culture supernatant, containing progeny viruses, was collected, centrifuged to remove cellular debris, and stored at -80°C until analysis. Cytopathic effects (CPE) induced by virus infection were monitored by light microscopy. Infected and mock-infected VERO WT cells were observed daily for CPE using an inverted phase-contrast microscope. At 48–72 h post-infection, representative images were captured at identical magnifications for comparison. For each well, the extent of CPE was estimated by determining the percentage of the cell monolayer exhibiting characteristic cytopathic changes, including cell rounding, detachment, and loss of monolayer integrity. CPE (%) was calculated as the proportion of affected cells relative to the total cell population in each well. The mean CPE percentage ± SEM was calculated from four wells per condition. Lentiviral particles were produced using a three-plasmid packaging system. The plasmids included pCDH-VSV G as the envelope plasmid, psPAX2 as the packaging plasmid, and either pCDH-copGFP or pCDH-CD19-CAR-mCherry as the transfer plasmid. Viral supernatants were harvested 48 hours post-transfection and stored at − 80°C. Viral titers (TU/mL) were determined by infecting HEK293T WT cells with serial dilutions of the harvested supernatants. Following infection, the proportion of reporter gene–positive cells (copGFP + or Protein L + ) was quantified by flow cytometry, and the viral titers were calculated as follows: TU/mL = (Number of seeded cells × Percentage of positive cells × Dilution factor) / Volume of virus used (ml). VERO cells were allowed to adhere in 6-well plates for 20 hours before being infected with VZV (4×10 3 PFU/well in 2% DMEM). Infected cells were subsequently passaged once a week, and the cells were monitored for the VZV glycoprotein E surface expression by flow cytometry at each passage. Replicon transfection. Coxsackievirus CVB3 replicons was amplified in Escherichia coli and purified using a plasmid purification kit (Bioneer) according to the manufacturer’s instructions. Purified DNA was quantified by spectrophotometry and stored at − 20°C until use. Cells were seeded 20–24 h before transfection and allowed to reach approximately 70–80% confluence at the time of transfection. Plasmid DNA was transfected into cells using a lipid-based transfection reagent (Invitrogen Lipofectamine™ 2000) following the manufacturer’s protocol. Briefly, plasmid DNA and transfection reagent were diluted separately in Opti-MEM (Gibco), mixed gently, and incubated at room temperature for 10–20 min to allow complex formation. The DNA–lipid complexes were then added dropwise to the cells in serum-free medium. After 48h of incubation at 37°C under 5% CO₂, the culture supernatant, containing progeny virus replicon, was collected, centrifuged to remove cellular debris, and stored at -80°C until analysis. Establishment of Suspension Cell Lines and Virus Production. MDCK and VERO cells were adapted to serum-free suspension culture in 125-mL Erlenmeyer shake flasks (Corning-431143) containing 30 mL of medium, and the cultures were agitated at 110 rpm on Lab companion SK-300 orbital shaker (Jeiotech, Daejeon, Korea). MDCK cells were maintained in serum-free CD MDCK 244 medium (KCell Biosciences), while VERO cells were cultured in serum-free DMEM/F12 medium (Gibco-12500062). Adaptation was initiated at 5 × 10 6 cells and achieved through serial passaging, with the removal of adherent cells, until stable, uniformly growing suspension clones were obtained. For Vero cells adaptation, all cells harvested in the early stage of adaptation were re-inoculated into fresh medium due to poor cell growth, until the total harvested cell number exceeded 2 × 10 6 cells. 2 × 10 6 cells were then subcultured into each new culture thereafter. Following infection with H1N1 influenza virus (A/Brisbane/59/2007) or porcine epidemic diarrhea virus (PEDV, CV777), suspension cells were cultured in serum-free medium under constant agitation at 110 rpm. Supernatants were collected at intervals between 48 and 92-hours post-infection, clarified by centrifugation, and analyzed by Western blotting. Western blotting. Cells were lysed with RIPA Cell Lysis buffer (GenDEPOT) supplemented with Xpert Protease Inhibitor Cocktail Solution (GenDEPOT). Then 10–30µg of extracts or virus sup were electrophoresed onto a 12% gradient gel (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with primary antibodies (dilutions 1:1000) in Tris-buffered saline with 5% BSA and 0.1% Tween 20(TBST). Antibodies were listed in the Supplementary Table I. Blot images were obtained using LAS4000 mini (GE Healthcare). Band intensities were analyzed with the ImageJ program. Levels of target proteins were normalized against the level of GAPDH as a loading control. Statistical analysis GraphPad Prism Version 6.0 (GraphPad Software) was used to perform statistical analysis. Differences in data were compared using an unpaired two-tailed t-test or a two-way ANOVA with Sidak’s multiple comparison test. Data are expressed as means ± standard errors of the mean (SEM). Differences were considered significant if p < 0.05. Declarations Author contributions SP conceived the project, coordinated collaborations, and supervised the overall study design and data interpretation. ZX and JJ designed and performed experiments related to antiviral gene knockouts, conducted viral infection assays, analyzed cytopathic effects, and wrote the manuscript. JO completed part of the experiments and data analysis. YS participated in the experimental process. All authors have read and approved the final manuscript. Acknowledgements This study received no funding. Competing interests All authors declare no financial or non-financial competing interests. Data availability The datasets generated and analysed during the current study are available from the corresponding author upon reasonable request. Additional raw data supporting the findings of this study are included in the Supplementary Information file. Code availability No custom computer code was used in this study. Standard software packages (GraphPad Prism, ImageJ) were employed for statistical analysis and image quantification. Any minor scripts used for sgRNA design or viral titer calculations are available from the corresponding author upon reasonable request. References Gallagher, T. & Lipsitch, M. Postexposure Effects of Vaccines on Infectious Diseases. Epidemiol Rev 41, 13–27, doi: 10.1093/epirev/mxz014 (2019). Williams, B. A., Jones, C. H., Welch, V. & True, J. M. Outlook of pandemic preparedness in a post-COVID-19 world. NPJ Vaccines 8, 178, doi: 10.1038/s41541-023-00773-0 (2023). Peletta, A., Lemoine, C., Courant, T., Collin, N. & Borchard, G. Meeting vaccine formulation challenges in an emergency setting: Towards the development of accessible vaccines. Pharmacol Res 189, 106699, doi: 10.1016/j.phrs.2023.106699 (2023). Zinnecker, T., Reichl, U. & Genzel, Y. Innovations in cell culture-based influenza vaccine manufacturing - from static cultures to high cell density cultivations. Hum Vaccin Immunother 20, 2373521, doi: 10.1080/21645515.2024.2373521 (2024). Duggal, N. K. & Emerman, M. Evolutionary conflicts between viruses and restriction factors shape immunity. Nat Rev Immunol 12, 687–695, doi: 10.1038/nri3295 (2012). Randall, R. E. & Goodbourn, S. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89, 1–47, doi: 10.1099/vir.0.83391-0 (2008). Hu, Y. et al. Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection. Front Immunol 15, 1408510, doi: 10.3389/fimmu.2024.1408510 (2024). Yi, E., Oh, J., Kang, H. R., Song, M. J. & Park, S. H. BST2 inhibits infection of influenza A virus by promoting apoptosis of infected cells. Biochem Biophys Res Commun 509, 414–420, doi: 10.1016/j.bbrc.2018.12.110 (2019). Le Tortorec, A., Willey, S. & Neil, S. J. Antiviral inhibition of enveloped virus release by tetherin/BST-2: action and counteraction. Viruses 3, 520–540, doi: 10.3390/v3050520 (2011). Van Damme, N. et al. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. Cell Host Microbe 3, 245–252, doi: 10.1016/j.chom.2008.03.001 (2008). García, M. A. et al. Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. Microbiol Mol Biol Rev 70, 1032–1060, doi: 10.1128/mmbr.00027-06 (2006). Silverman, R. H. Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during the interferon antiviral response. J Virol 81, 12720–12729, doi: 10.1128/jvi.01471-07 (2007). Sadler, A. J., Latchoumanin, O., Hawkes, D., Mak, J. & Williams, B. R. An antiviral response directed by PKR phosphorylation of the RNA helicase A. PLoS Pathog 5, e1000311, doi: 10.1371/journal.ppat.1000311 (2009). Martínez, N. W. et al. PKR-driven ISR signaling controls synaptic translation and structural plasticity in an age-dependent manner. Neurobiol Dis 216, 107113, doi: 10.1016/j.nbd.2025.107113 (2025). Au-Yeung, N., Mandhana, R. & Horvath, C. M. Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway. Jakstat 2, e23931, doi: 10.4161/jkst.23931 (2013). Hu, X., Li, J., Fu, M., Zhao, X. & Wang, W. The JAK/STAT signaling pathway: from bench to clinic. Signal Transduct Target Ther 6, 402, doi: 10.1038/s41392-021-00791-1 (2021). Evans, D. T., Serra-Moreno, R., Singh, R. K. & Guatelli, J. C. BST-2/tetherin: a new component of the innate immune response to enveloped viruses. Trends Microbiol 18, 388–396, doi: 10.1016/j.tim.2010.06.010 (2010). Jones, C. E., Tan, W. S., Grey, F. & Hughes, D. J. Discovering antiviral restriction factors and pathways using genetic screens. J Gen Virol 102, doi: 10.1099/jgv.0.001603 (2021). Liu, R. & Moss, B. Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants. J Virol 90, 7864–7879, doi: 10.1128/jvi.00869-16 (2016). Valero-Rello, A. & Sanjuán, R. Enveloped viruses show increased propensity to cross-species transmission and zoonosis. Proc Natl Acad Sci U S A 119, e2215600119, doi: 10.1073/pnas.2215600119 (2022). Dimitrov, D. S. Virus entry: molecular mechanisms and biomedical applications. Nat Rev Microbiol 2, 109–122, doi: 10.1038/nrmicro817 (2004). Tenge, V. R., Gounder, A. P., Wiens, M. E., Lu, W. & Smith, J. G. Delineation of interfaces on human alpha-defensins critical for human adenovirus and human papillomavirus inhibition. PLoS Pathog 10, e1004360, doi: 10.1371/journal.ppat.1004360 (2014). Joe, C. C. D. et al. Accelerated and intensified manufacturing of an adenovirus-vectored vaccine to enable rapid outbreak response. Biotechnol Bioeng 121, 176–191, doi: 10.1002/bit.28553 (2024). Kis, Z. et al. Pandemic-response adenoviral vector and RNA vaccine manufacturing. NPJ Vaccines 7, 29, doi: 10.1038/s41541-022-00447-3 (2022). Yang, J. et al. Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors. AMB Express 9, 70, doi: 10.1186/s13568-019-0794-5 (2019). Genzel, Y. Designing cell lines for viral vaccine production: Where do we stand? Biotechnol J 10, 728–740, doi: 10.1002/biot.201400388 (2015). Kane, M. et al. Identification of Interferon-Stimulated Genes with Antiretroviral Activity. Cell Host Microbe 20, 392–405, doi: 10.1016/j.chom.2016.08.005 (2016). Laemmle, L., Goldstein, R. S. & Kinchington, P. R. Modeling Varicella Zoster Virus Persistence and Reactivation - Closer to Resolving a Perplexing Persistent State. Front Microbiol 10, 1634, doi: 10.3389/fmicb.2019.01634 (2019). Liu, H. et al. Research Progress on Varicella-Zoster Virus Vaccines. Vaccines (Basel) 13, doi: 10.3390/vaccines13070730 (2025). Michels, A., Ho, N. & Buchholz, C. J. Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms. Mol Ther 30, 2401–2415, doi: 10.1016/j.ymthe.2022.05.018 (2022). Hegde, N. R. Cell culture-based influenza vaccines: A necessary and indispensable investment for the future. Hum Vaccin Immunother 11, 1223–1234, doi: 10.1080/21645515.2015.1016666 (2015). Yi, E., Oh, J., Giao, N. Q., Oh, S. & Park, S. H. Enhanced production of enveloped viruses in BST-2-deficient cell lines. Biotechnol Bioeng 114, 2289–2297, doi: 10.1002/bit.26338 (2017). Additional Declarations No competing interests reported. Supplementary Files Supplementary.pdf Supplementary information Supplementary Information is available as a single merged PDF file accompanying this manuscript. It includes additional figures and tables. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9184882","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":612477079,"identity":"ad963b8c-a5e3-4a28-b94c-aecff2cbd77e","order_by":0,"name":"Se-ho Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYFACHjBpwM8DF2EjUotkD8laDM4Qq0W+vffg44Kae8bGZw4f+/ChhkGev4Et7QM+LQZnziUbzzhWbGZ2ti155oxjDIYzDrAdnoFXi0SOmTQPW4KN2XkeY2YeNgbGDQzszfgdNv8NUMu/BBvjfqCWP/8Y7AlqYbjBYybN25ZgZsDbY8zM2MaQuIGB7TBeHQZncoyNZ/YlGEucOZbM2NsnkTzjMFsyfoe1nzF8XPAtwbC/J/kww49vNrb97W3G+B0GBMxIbAlULjFaRsEoGAWjYBRgAgAhtj+bDCliwAAAAABJRU5ErkJggg==","orcid":"","institution":"Korea University","correspondingAuthor":true,"prefix":"","firstName":"Se-ho","middleName":"","lastName":"Park","suffix":""},{"id":612477080,"identity":"aa994cba-3f93-441c-bf57-750418477964","order_by":1,"name":"Zhengmei XU","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Zhengmei","middleName":"","lastName":"XU","suffix":""},{"id":612477081,"identity":"3b4b7bde-b80f-4c05-98a2-4ab16200ab23","order_by":2,"name":"Jaemyeong Jeon","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Jaemyeong","middleName":"","lastName":"Jeon","suffix":""},{"id":612477082,"identity":"40f7990d-71d3-4c26-b1b9-2a9186019f78","order_by":3,"name":"Youngki Shin","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Youngki","middleName":"","lastName":"Shin","suffix":""},{"id":612477083,"identity":"98d739c2-f754-46ed-a080-c651d691f472","order_by":4,"name":"Jinsoo Oh","email":"","orcid":"","institution":"Korea University","correspondingAuthor":false,"prefix":"","firstName":"Jinsoo","middleName":"","lastName":"Oh","suffix":""}],"badges":[],"createdAt":"2026-03-21 09:53:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9184882/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9184882/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105729306,"identity":"e840dbbe-7170-4906-a044-eb18e67735e4","added_by":"auto","created_at":"2026-03-30 11:14:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":195087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGeneration and functional characterization of antiviral gene knockout cell models.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Workflow for generating single-clone knockout cell lines. Guide RNA for the target gene was cloned into pSpCas9(BB)-2A-GFP(PX458) vector. After transfection with the gene-targeting vector, GFP-positive cells were isolated via FACS and single-cell cultured.\u003c/p\u003e\n\u003cp\u003e(B) Effective mutagenesis was confirmed by T7E1 analysis, and the presence of a homozygous frameshift mutation was verified by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e(C) JAK1 knockout abolished IFNγ-induced STAT1 phosphorylation.\u003c/p\u003e\n\u003cp\u003e(D) Normal cell growth of mutant cell lines. CCK8 assay optical density (O.D.) values indicate normal proliferation capacity of knockout cells compared to wild-type cells. Data represent mean ± standard deviation (n = 3); ns, statistically insignificant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/12ce8e5728603b67398003da.png"},{"id":105712884,"identity":"95b4b3be-cf4c-4a11-9168-8f1478e04bf4","added_by":"auto","created_at":"2026-03-30 08:18:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":173615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced Envelop virus production in multi-gene knockout cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A), (B) Influenza HA expression in HEK293T, Vero, and MDCK cells increased stepwise depending on the number of knocked-out genes. Results were obtained 48 hours after infection with (A) influenza H1N1 (A/Brisbane/59/2007) and (B) H3N2 (A/Brisbane/10/2007).\u003c/p\u003e\n\u003cp\u003e(C) In VERO knockout cell lines infected with PED virus (CV777), PED N expression increased stepwise depending on the number of knocked-out genes after 48 hours post-infection.\u003c/p\u003e\n\u003cp\u003e(D) Expression of phosphorylated STAT1 (p-STAT1) 24 hours after infection with influenza H1N1 virus (left panels) and PED virus (right panels).\u003c/p\u003e\n\u003cp\u003eBottom panels: Western blot quantification results are expressed as fold change relative to wild-type cells. HA; hemagglutinin, PEDV N; PED virus nucleocapsid.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/930632c99340777727c14556.png"},{"id":105712890,"identity":"b1bab0bc-7417-4f59-9a27-30daf78fba1f","added_by":"auto","created_at":"2026-03-30 08:18:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":112884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced lentivirus production in multi-gene knockout 293T cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Pseudo-typed lentiviruses carrying cop-GFP were produced in 293T WT and gene-knockout cell lines (293T B \u0026amp; 293T BR). Each lentivirus was transduced into 7.5 ×10\u003csup\u003e4\u003c/sup\u003e of WT 293T cells at the same volume (10 μl), and the transduction units were measured. Left panels: Representative images of FACS analysis. Right panel: Transduction units calculated based on the results from left panel.\u003c/p\u003e\n\u003cp\u003e(B) Productivity of lentiviruses containing the CD19-CAR gene in 293T WT and gene-knockout cell lines (293T B \u0026amp; 293T BR). Each lentivirus was transduced into 7.5 ×10\u003csup\u003e4\u003c/sup\u003e of WT 293T cells at the same volume (5 μl), and the transduction units were measured. CD19-CAR was stained with protein-L-PE. Right panel: Transduction units calculated based on the results from left panel.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/12d761f09adc690122434d4a.png"},{"id":105712886,"identity":"9d7b2faf-02a1-49d3-be13-9a13928f86ba","added_by":"auto","created_at":"2026-03-30 08:18:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136052,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced production of non-enveloped virus and varicella zoster virus (VZV) in multi-gene knocked-out cell lines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Production of Coxsackievirus B4 (CVB4) in gene-deficient cell lines. Left panel: Coxsackievirus B4 was produced in Vero background cell lines and their cytopathic effect (CPE) were measured in Vero WT cells. Right panel: The Coxsackievirus B3 (CVB3) replicon was transfected into 293T background cell lines, and the CPE of the produced viruses was measured in Vero WT cells. TCID50; Tissue Culture Infectious Dose 50%.\u003c/p\u003e\n\u003cp\u003e(B) VERO WT and BPR cells were infected with VZV-Oka virus and passaged weekly. Cell surface expression of glycoprotein E was measured in both cell types during passage using flow cytometry.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/a36578cb59563e7113a73cbb.png"},{"id":105729541,"identity":"a42d4db4-bcaa-45b6-a811-0a12496b2a5c","added_by":"auto","created_at":"2026-03-30 11:17:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":111273,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuspension culture of multi-gene knockout cells enhances viral production\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Growth kinetics of suspension-adapted WT and MDCK BRJ cells during suspension adaptation in serum-free conditions.\u003c/p\u003e\n\u003cp\u003e(B) HA expression levels in suspension culture were measured following H1N1 infection. 8 μl of culture supernatants were analyzed by Western blotting.\u003c/p\u003e\n\u003cp\u003e(C) Growth kinetics of Vero WT and Vero BPR cells during suspension adaptation in serum-free conditions.\u003c/p\u003e\n\u003cp\u003e(D) Western blot analysis of PEDV N protein in Vero BPR cells cultured in DMEM media under adherent or suspension conditions, with or without fetal bovine serum (FBS). 10 μl of culture supernatants were analyzed by Western blotting.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/841819ef53955d03e5218a26.png"},{"id":106336952,"identity":"d9faff68-d691-4276-aefe-f7476556401b","added_by":"auto","created_at":"2026-04-07 14:57:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1495761,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/f092a219-9d8a-4901-8012-90c1c6422509.pdf"},{"id":105729305,"identity":"a5a02fd4-4900-49ae-a414-219a4bc24017","added_by":"auto","created_at":"2026-03-30 11:14:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":475174,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Information is available as a single merged PDF file accompanying this manuscript. It includes additional figures and tables.\u003c/p\u003e","description":"","filename":"Supplementary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/736cd476c88aa7178afbe6e7.pdf"},{"id":105712891,"identity":"d9f89113-c7ad-4a06-8145-612ac5fa180d","added_by":"auto","created_at":"2026-03-30 08:18:34","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":473673,"visible":true,"origin":"","legend":"","description":"","filename":"RAWDATAgelandblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9184882/v1/da7a3dd8b738d8400026fefd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Systemic Removal of Host Restriction Factors Enables Rapid, Scalable Virus Production for Cell Culture-Based Vaccines High-Yield Virus Production System generated by gene knockout of multiple anti-viral host factors","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eVaccines are one of the most effective tools for preventing infectious diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. However, the efficiency of vaccine manufacturing remains a critical bottleneck in global health preparedness\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Cell culture-based systems, which form the foundation of most modern vaccine production, face substantial challenges in achieving high viral yields\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Commonly used cell lines, such as Vero and MDCK, often have limited viral productivity, primarily due to the host cells' potent intrinsic antiviral defense mechanisms\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. These defense mechanisms, including interferon (IFN) signaling and restriction factor expression, actively suppress viral replication\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Advances in gene editing, particularly CRISPR-Cas9, provide a powerful means to overcome this barrier by engineering host cells to be more permissive to viral replication\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Promising results have been obtained from the first proof-of-concept study disrupting BST2, which tethers the host cells and viral membrane thus limiting virus release from the host cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, because many viruses possess viral elements that counteract specific host restriction factors, it is difficult to restrict a wide range of viruses with a single restriction factor. Therefore, host cells possess a variety of restriction factors throughout the viral life cycle, effectively suppressing viral replication.\u003c/p\u003e \u003cp\u003eWhile proof-of-concept studies, including BST2 knockout to enhance the release of enveloped viruses, have shown promise, the redundancy of the host antiviral network inherently limits such single-target approaches\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In fact, BST2 can only act on a subset of enveloped viruses. Non-enveloped viruses are not restricted by BST2\u003csup\u003e9\u003c/sup\u003e, and certain enveloped viruses, such as human immunodeficiency virus 1 (HIV-1), degrade host BST2 with viral Vpu protein\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To systematically dismantle this redundant defense network, we targeted key nodes of the innate immune system: PKR\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, RNase L\u003csup\u003e12\u003c/sup\u003e, and JAK1 genes in addition to BST2. Protein kinase R (PKR) is a host enzyme activated by double-stranded RNA (dsRNA) during viral infection. Activated PKR phosphorylates the translation factor eIF2α, which blocks viral replication by halting protein synthesis. It can also induce host cell apoptosis and activate NF-κB signaling to enhance antiviral gene expression\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. RNase L is a host enzyme activated by 2\u0026prime;,5\u0026prime;-oligoadenylate (2-5A), which is produced by oligoadenylate synthetase (OAS) in response to viral double-stranded RNA (dsRNA). RNase L degrades viral and host RNA, halting viral replication and producing small RNA fragments that amplify interferon signaling\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Janus Kinase 1 (JAK1) is a key host kinase in the interferon signaling pathway initiated by viral infection. When interferon binds to its receptor, JAK1 along with TYK2 phosphorylates STAT1/STAT2, which forms the ISGF3 complex, establishing an antiviral state within the cell by inducing the expression of interferon-stimulated genes (ISGs)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. BST2 (also known as tetherin or CD317) is an interferon-induced antiviral protein that restricts the release of many enveloped viruses from infected cells. It acts by physically \u0026ldquo;tethering\u0026rdquo; budding virions to the cell membrane, preventing their release and subsequent infection of new cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe generated knockout cell lines for these genes in three cell models with industrial applications: HEK293T, a human kidney cell line used to produce pseudotyped lentivirus for gene transfer; Vero, a monkey kidney cell line used to produce various antiviral vaccines; and MDCK, a canine kidney cell line which is the major cell line used to produce anti-influenza vaccines. In all these cell lines, multiple antiviral gene knockouts significantly increased virus production for a wide range of viruses, including both enveloped and non-enveloped viruses. This cross-species systematic multi-gene knockout virus production platform (collectively, the platform) will provide robust productivity to produce antiviral vaccines and therapeutic vectors\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Generation and Validation of Antiviral Gene Knockout Cell Lines\u003c/h2\u003e \u003cp\u003eWe employed the CRISPR-Cas9 system to generate targeted knockouts of key antiviral genes in HEK293T, Vero, and MDCK cells. Specific sgRNAs were cloned into the pX458 vector, and monoclonal cell lines were subsequently isolated through sorting GFP-positive cells and limited dilution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The following knockout combinations were established: BST2 (B), BST2 and PKR (BP), BST2 and RNase L (BR), BST2, PKR, and RNase L (BPR), and BST2, PKR, RNase L, and JAK1 (BPRJ), with nomenclature reflecting the targeted genes in each cell line. Targeted mutations were verified by T7 endonuclease 1(T7E1) digestion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and Sanger sequencing of representative clones confirmed homozygous frameshift mutations (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To validate the functional disruption of target genes, we examined protein expression in 293T cells after knocking out RNase L. Western blot analysis confirmed the disappearance of RNase L protein upon interferon γ (IFN-γ) stimulation in RNase L-deficient cells (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). To verify the function of JAK1, we assessed the phosphorylation pattern of Signal transducer and Activator of Transcription 1 (STAT1), a key downstream signaling molecule of JAK1. Western blot analysis revealed significantly reduced STAT1 phosphorylation (p-STAT1) in JAK1-deficient cells following IFN stimulation, confirming inhibition of the JAK-STAT signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Despite the targeted mutations, cell proliferation assays revealed that these gene knockouts did not impair normal cell growth or viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Enhanced Production of Enveloped Viruses in Multi-Gene Knockout Cells\u003c/h2\u003e \u003cp\u003eWe first assessed the platform's ability to enhance the production of enveloped viruses, using the influenza virus as a primary model. Upon infection with influenza A/H1N1 (A/Brisbane/59/2007) or H3N2 (A/Brisbane/10/2007), hemagglutinin (HA) protein expression increased in gene knock-out number manner across 293T, VERO and MDCK engineered vaccine production cell lines. The triple or quadruple gene knockout cell lines showed increased viral output by 3 to 8-fold relative to wild-type (WT) cells, with the magnitude of enhancement being cell-specific (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This enhancement was not influenza A virus-specific, as the production of influenza B/Yamagata (B/Wisconsin/2010) in MDCK BPRJ cells also resulted in significantly higher cytopathic effect (CPE) percentage than in those of WT cells (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the platform's utility in veterinary vaccinology, we infected Vero background cells with the classical porcine epidemic diarrhea virus (PEDV) strain CV777. Western blot analysis of the viral nucleocapsid (N) protein demonstrated a substantial increase in viral production in the knockout cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results collectively suggest that a targeted multi-gene knockout strategy can be applied to enhance the production of diverse enveloped viruses.\u003c/p\u003e \u003cp\u003eViral infection of VERO-derived antiviral knockout cell lines demonstrated that stepwise removal of interferon-induced restriction factors markedly enhances viral production while uncoupling STAT1 activation from functional antiviral control. Following infection, BPR cells (BST2/PKR/RNase L triple knockout) exhibited substantially increased STAT1 phosphorylation compared with wild-type cells, reflecting heightened innate immune sensing driven by elevated viral replication. Despite this robust STAT1 activation, viral production in BPR cells was significantly increased compared to wild-type cells, indicating that the deletion of key antiviral effector proteins compromises the effective restriction of viral replication and release. In contrast, BPRJ cells (BST2/PKR/RNase L/JAK1 quadruple knockout) displayed minimal STAT1 phosphorylation, consistent with disruption of JAK\u0026ndash;STAT signaling and supported the highest levels of viral production among all cell lines tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). These findings demonstrate that elimination of antiviral effector pathways is sufficient to increase viral yield substantially, and that additional abrogation of upstream interferon signaling further maximizes viral replication and release.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Enhanced Lentiviral Production in Multi-Gene Knockout HEK293T Cells\u003c/h2\u003e \u003cp\u003eHEK293T cells are the industry standard for producing lentiviral vectors (LVs) used in cell therapies. We produced VSV-G pseudotyped LVs carrying a copGFP reporter gene in the engineered HEK293T background cell lines. Produced viruses were transduced into HEK293T WT to evaluate the virus titer. Flow cytometry analysis of transduced HEK293T WT cells revealed that viruses produced from the BR knockout cells exhibited significantly higher functional titers (TU/mL) than those from WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, we evaluated the preparation of more complex LVs carrying the anti-CD19 chimeric antigen receptor (CAR). The HEK293T BR cell line consistently produced higher levels of LVs than HEK293T WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Functionally active CD19-CAR LV underscores the platform's potential for advanced manufacturing of complex therapeutic vectors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Broadened Applicability to Non-Enveloped Virus Production\u003c/h2\u003e \u003cp\u003eAmong viruses infecting animal hosts, many possess envelopes, but non-enveloped viruses also constitute a significant proportion, and their modes of infection differ\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Having established the efficacy of our platform for enveloped viruses, we next examined whether the multi-gene knockout strategy could enhance the production of non-enveloped viruses, which BST2 does not restrict\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Coxsackievirus B was produced in VERO WT and VERO BPRJ cells, and the harvested virus was subsequently used to infect VERO WT cells. Infection with Coxsackievirus B produced in VERO BPRJ cells resulted in a significant increase in viral titers compared to virus derived from WT cells, as determined by the CPE percentage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). These findings demonstrate that disrupting downstream effector pathways (PKR, RNase L) along with core signaling (JAK1) can overcome antiviral defenses of host cells against a broader range of viruses, including non-enveloped types.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, Coxsackievirus B3 (CVB3) replicons generated in 293 T-based cells induced progressively more severe cytopathic effects (CPE) in infected VERO WT cells as the number of gene knockouts increased, indicating a positive correlation between replicon replication efficiency and the extent of host gene depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Rescue of Poorly Replicating Viruses in Knockout Cells\u003c/h2\u003e \u003cp\u003eNext, we tested our platform using the varicella-zoster virus (VZV). Due to its cell-associated nature and weak replication capacity, VZV is difficult to amplify in traditional cell cultures. We first infected VERO WT and VERO BPR cells with VZV-Oka virus, then passaged the infected cells to detect glycoprotein E expression at each time point. Although Vero cells were known to poorly support VZV replication, expression of VZV-Oka glycoprotein E progressively increased in passaged Vero BPR cells at each passage. In contrast, VZV-Oka glycoprotein E expression in VERO WT cells was extremely low (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). These results demonstrate the significant potential of host cell engineering to produce difficult-to-culture human pathogens.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Integration with Suspension Culture for Scalable Manufacturing\u003c/h2\u003e \u003cp\u003eTo address the scalability limitations of adherent culture systems, we adapted the multi-gene knockout cells to suspension growth. Both MDCK WT and BRJ cells adapted in serum-free conditions until cells proliferate stably in suspension (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Upon H1N1 infection, HA expression in suspension-adapted MDCK BRJ cells remained significantly higher than in suspension-adapted WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Process optimization in shake flask cultures achieved HA yields of \u0026ge;\u0026thinsp;60 \u0026micro;g/mL (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD), confirming that the productivity advantage is retained in scalable formats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, suspension-adapted Vero BPR cells infected with PEDV exhibited higher viral yields, as indicated by N protein expression, compared to that of adherent cultures. This enhancement was more pronounced under serum-free conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). The successful adaptation of multiple engineered cell lines to high-yield suspension culture underscores the industrial robustness and broad applicability of this synergistic platform.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThe development of robust and high-yield viral production platforms is a cornerstone of effective vaccine manufacturing and virological research\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In this study, we established a versatile and highly efficient platform by systematically engineering industrially relevant cell lines\u0026mdash;HEK293T, Vero, and MDCK\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u0026mdash;through targeted knockout of key intrinsic antiviral defense genes, including BST2, JAK1, RNase L, and PKR. This multi-target knockout approach resulted in a substantial enhancement of replication for a wide spectrum of viruses.\u003c/p\u003e \u003cp\u003eA central finding of our work is the consistent, knockout-gene-number-dependent enhancement of viral replication across all tested cell lines and virus families. The replication of multiple enveloped viruses, including influenza A (H1N1 and H3N2), influenza B (Yamagata), PEDV (CV777), and pseudotyped lentivirus, was significantly enhanced in the multi-gene knockout cells. This demonstrates that the redundancy inherent in the host antiviral network can be effectively overcome by a coordinated, multi-target knockout strategy, thereby surpassing the limited efficacy of single-gene edits like BST2\u003csup\u003e8\u003c/sup\u003e. These findings position BST2 as a context-dependent modulator of viral replication rather than a universal restriction factor and underscore that dismantling the PKR-RNase L-JAK1 axis is pivotal for creating a broadly permissive cellular environment for viral replication\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCritically, the enhancement provided by our platform was not confined to enveloped viruses. The significantly increased production of Coxsackievirus B, a non-enveloped picornavirus, in Vero BPRJ cells indicates that the restrictions mediated by PKR, RNase L, and JAK-STAT signaling constitute a broad-spectrum antiviral barrier, independent of viral structure or budding mechanism. This finding significantly expands the potential utility of our platform for producing a broader range of viral biologics.\u003c/p\u003e \u003cp\u003eA particularly compelling result was the successful enhancement of Varicella-zoster virus (VZV) replication, a human pathogen notoriously difficult to propagate in conventional cell cultures due to its highly cell-associated nature\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The progressive increase in VZV glycoprotein E expression over passages in our engineered Vero BPR\u0026mdash;in stark contrast to the limited replication in WT controls\u0026mdash;highlights the unique capacity of this host cell engineering strategy to enable the propagation of fastidious pathogens. This outcome holds significant promise for improving the manufacturing efficiency of live-attenuated VZV vaccines\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurthermore, the marked increase in the functional titer of complex therapeutic lentiviral vectors, including a CD19-CAR construct, produced in engineered HEK293T BR and BRJ cells, underscores the direct clinical relevance of our platform. The ability to achieve higher vector titers without compromising integrity addresses a major challenge in the cost-effective and scalable manufacturing of cell therapies, such as CAR-T, thereby enhancing their clinical and commercial translation\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFinally, a crucial step for industrial application is the transition from a proof-of-concept in adherent culture to a scalable, industry-ready format. We demonstrated that the genetic advantages of our multi-gene knockout cells were fully retained following adaptation to suspension culture. The high yields of influenza HA protein (reaching 60\u0026micro;g/mL in shake flasks) and PEDV in suspension-adapted cells confirm that our strategy successfully integrates intrinsic cellular enhancements with the bioprocess. This synergy between cellular engineering and bioprocess optimization may present a comprehensive solution to long-standing production bottlenecks\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn summary, this study establishes a broadly applicable and robust framework for engineering high-efficiency viral production by engineering cell lines. By systematically targeting conserved and redundant nodes of the innate immune system, we have created a platform that significantly boosts the yield of diverse viruses, ranging from vaccine targets and therapeutic vectors to hard-to-culture pathogens. The successful adaptation of these engineered cells to suspension culture paves the way for industrial-scale manufacturing.\u003c/p\u003e"},{"header":"4. Materials and methods","content":"\u003cp\u003e \u003cb\u003eCell lines and gene knockout\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMDCK.1(ATCC-CRL-2935), Vero(ATCC-CCL-81), HEK293T(ATCC-CRL-3216) cells, and their respective multi gene deficient cell lines-HEK293T (B, BR, BRJ), VERO(B, BP, BPR, BPRJ), MDCK(B, BR, BRJ) were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS(Hyclone), 2mML-glutamine(Gibco), 1% penicillin-streptomycin(Gibco), 10\u0026micro;g/ml Gentamycin(Gibco), and 50mMb-mercaptoethanol(Gibco) at 37℃ with 5% CO2. Generation of BST2-deficient cell lines HEK293T-B, VERO-B, and MDCK-B has been described previously\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGene knockouts were generated using the pSpCas9(BB)-2A-GFP (PX458) backbone. For each target gene, sgRNA expression plasmids (pX458-sgRNA(Target gene]-Cas9(BB)-T2A-GFP) were constructed and transfected into cells with Turbofect (Thermo Fisher Scientific). GFP-positive cells were enriched by fluorescence-activated cell sorting (BD FACSAria Ⅱ) and seeded into 96-well plates for single-cell cloning. Genomic DNA from individual clones was analyzed by T7 endonuclease I (T7E1) assay to detect indels. Clones showing cleavage patterns were further validated by Sanger sequencing (Bioneer), and homozygous knockout lines were confirmed.\u003c/p\u003e \u003cp\u003eCells used for growth analysis were passaged approximately 20 times before the assay. The cells were seeded into 96-well plates, and cell viability was measured daily using the CCK-8 assay. The optical density (OD) was recorded to determine the growth rate of each cell line.\u003c/p\u003e \u003cp\u003e \u003cb\u003eVirus production and viral titer determination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSeasonal influenza H1N1 (A/Brisbane/59/2007) and H3N2 (A/Brisbane/10/2007) virus was obtained from the Korea Centers for Disease Control and Prevention (KCDC), and Yamagata(B/Wiscosin/1/2010) was obtained from the National Biobank of Korea (NBK). Coxsackievirus B4 was obtained from the Korea Disease Control and Prevention Agency (KDCA). For infection by the influenza virus, we followed the previous protocol\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Porcine epidemic diarrhea virus (strain CV777), provided by the Korea Veterinary Culture Collection (KVCC), was used to infect cells. In brief, cells were seeded 20-24h before infection. When cells reached 90% confluence, they were washed with PBS. Next, cells were inoculated with 0.001-0.1 MOI of virus containing 0.3% BSA and 1\u0026ndash;2\u0026micro;g/mL of TPCK-trypsin (Sigma) in MEM medium for 1h. Finally, the virus inoculum was removed and replaced with a fresh medium containing 0.3% BSA and TPCK-trypsin for 48 hours at 37\u0026deg;C under 5% CO₂. Following incubation, the culture supernatant, containing progeny viruses, was collected, centrifuged to remove cellular debris, and stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eVERO cells were seeded in 24-well plates (or specified culture vessel) and allowed to adhere for 20\u0026ndash;24 hours. At the time of infection, when cells had reached approximately 90% confluency, the culture medium was aspirated, and the cell monolayer was gently rinsed with PBS to remove residual serum. Infection was carried out by incubating the cells with Coxsackievirus B4 at the indicated dose (1.25\u0026ndash;2.5 \u0026micro;L of viral stock) in serum-free DMEM. The virus-cell mixture was incubated for 72 hours at 37\u0026deg;C under 5% CO₂. Following incubation, the culture supernatant, containing progeny viruses, was collected, centrifuged to remove cellular debris, and stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003eCytopathic effects (CPE) induced by virus infection were monitored by light microscopy. Infected and mock-infected VERO WT cells were observed daily for CPE using an inverted phase-contrast microscope. At 48\u0026ndash;72 h post-infection, representative images were captured at identical magnifications for comparison. For each well, the extent of CPE was estimated by determining the percentage of the cell monolayer exhibiting characteristic cytopathic changes, including cell rounding, detachment, and loss of monolayer integrity. CPE (%) was calculated as the proportion of affected cells relative to the total cell population in each well. The mean CPE percentage\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM was calculated from four wells per condition.\u003c/p\u003e \u003cp\u003eLentiviral particles were produced using a three-plasmid packaging system. The plasmids included pCDH-VSV G as the envelope plasmid, psPAX2 as the packaging plasmid, and either pCDH-copGFP or pCDH-CD19-CAR-mCherry as the transfer plasmid. Viral supernatants were harvested 48 hours post-transfection and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003cp\u003eViral titers (TU/mL) were determined by infecting HEK293T WT cells with serial dilutions of the harvested supernatants. Following infection, the proportion of reporter gene\u0026ndash;positive cells (copGFP\u003csup\u003e+\u003c/sup\u003e or Protein L\u003csup\u003e+\u003c/sup\u003e) was quantified by flow cytometry, and the viral titers were calculated as follows: TU/mL = (Number of seeded cells \u0026times; Percentage of positive cells \u0026times; Dilution factor) / Volume of virus used (ml).\u003c/p\u003e \u003cp\u003eVERO cells were allowed to adhere in 6-well plates for 20 hours before being infected with VZV (4\u0026times;10\u003csup\u003e3\u003c/sup\u003ePFU/well in 2% DMEM). Infected cells were subsequently passaged once a week, and the cells were monitored for the VZV glycoprotein E surface expression by flow cytometry at each passage.\u003c/p\u003e \u003cp\u003e \u003cb\u003eReplicon transfection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCoxsackievirus CVB3 replicons was amplified in Escherichia coli and purified using a plasmid purification kit (Bioneer) according to the manufacturer\u0026rsquo;s instructions. Purified DNA was quantified by spectrophotometry and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eCells were seeded 20\u0026ndash;24 h before transfection and allowed to reach approximately 70\u0026ndash;80% confluence at the time of transfection. Plasmid DNA was transfected into cells using a lipid-based transfection reagent (Invitrogen Lipofectamine\u0026trade; 2000) following the manufacturer\u0026rsquo;s protocol. Briefly, plasmid DNA and transfection reagent were diluted separately in Opti-MEM (Gibco), mixed gently, and incubated at room temperature for 10\u0026ndash;20 min to allow complex formation. The DNA\u0026ndash;lipid complexes were then added dropwise to the cells in serum-free medium.\u003c/p\u003e \u003cp\u003eAfter 48h of incubation at 37\u0026deg;C under 5% CO₂, the culture supernatant, containing progeny virus replicon, was collected, centrifuged to remove cellular debris, and stored at -80\u0026deg;C until analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEstablishment of Suspension Cell Lines and Virus Production.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMDCK and VERO cells were adapted to serum-free suspension culture in 125-mL Erlenmeyer shake flasks (Corning-431143) containing 30 mL of medium, and the cultures were agitated at 110 rpm on Lab companion SK-300 orbital shaker (Jeiotech, Daejeon, Korea). MDCK cells were maintained in serum-free CD MDCK 244 medium (KCell Biosciences), while VERO cells were cultured in serum-free DMEM/F12 medium (Gibco-12500062). Adaptation was initiated at 5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells and achieved through serial passaging, with the removal of adherent cells, until stable, uniformly growing suspension clones were obtained. For Vero cells adaptation, all cells harvested in the early stage of adaptation were re-inoculated into fresh medium due to poor cell growth, until the total harvested cell number exceeded 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells. 2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells were then subcultured into each new culture thereafter.\u003c/p\u003e \u003cp\u003eFollowing infection with H1N1 influenza virus (A/Brisbane/59/2007) or porcine epidemic diarrhea virus (PEDV, CV777), suspension cells were cultured in serum-free medium under constant agitation at 110 rpm. Supernatants were collected at intervals between 48 and 92-hours post-infection, clarified by centrifugation, and analyzed by Western blotting.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blotting.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCells were lysed with RIPA Cell Lysis buffer (GenDEPOT) supplemented with Xpert Protease Inhibitor Cocktail Solution (GenDEPOT). Then 10\u0026ndash;30\u0026micro;g of extracts or virus sup were electrophoresed onto a 12% gradient gel (Bio-Rad), transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with primary antibodies (dilutions 1:1000) in Tris-buffered saline with 5% BSA and 0.1% Tween 20(TBST). Antibodies were listed in the Supplementary Table I. Blot images were obtained using LAS4000 mini (GE Healthcare). Band intensities were analyzed with the ImageJ program. Levels of target proteins were normalized against the level of GAPDH as a loading control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGraphPad Prism Version 6.0 (GraphPad Software) was used to perform statistical analysis. Differences in data were compared using an unpaired two-tailed t-test or a two-way ANOVA with Sidak\u0026rsquo;s multiple comparison test. Data are expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard errors of the mean (SEM). Differences were considered significant if p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSP conceived the project, coordinated collaborations, and supervised the overall study design and data interpretation. ZX and JJ designed and performed experiments related to antiviral gene knockouts, conducted viral infection assays, analyzed cytopathic effects, and wrote the manuscript. JO completed part of the experiments and data analysis. YS participated in the experimental process. All authors have read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received no funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no financial or non-financial competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are available from the corresponding author upon reasonable request. Additional raw data supporting the findings of this study are included in the Supplementary Information file.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo custom computer code was used in this study. \u0026nbsp;Standard software packages (GraphPad Prism, ImageJ) were employed for statistical analysis and image quantification. \u0026nbsp;Any minor scripts used for sgRNA design or viral titer calculations are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGallagher, T. \u0026amp; Lipsitch, M. Postexposure Effects of Vaccines on Infectious Diseases. \u003cem\u003eEpidemiol Rev\u003c/em\u003e 41, 13\u0026ndash;27, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/epirev/mxz014\u003c/span\u003e\u003cspan address=\"10.1093/epirev/mxz014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, B. A., Jones, C. H., Welch, V. \u0026amp; True, J. M. Outlook of pandemic preparedness in a post-COVID-19 world. \u003cem\u003eNPJ Vaccines\u003c/em\u003e 8, 178, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41541-023-00773-0\u003c/span\u003e\u003cspan address=\"10.1038/s41541-023-00773-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePeletta, A., Lemoine, C., Courant, T., Collin, N. \u0026amp; Borchard, G. Meeting vaccine formulation challenges in an emergency setting: Towards the development of accessible vaccines. \u003cem\u003ePharmacol Res\u003c/em\u003e 189, 106699, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.phrs.2023.106699\u003c/span\u003e\u003cspan address=\"10.1016/j.phrs.2023.106699\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZinnecker, T., Reichl, U. \u0026amp; Genzel, Y. Innovations in cell culture-based influenza vaccine manufacturing - from static cultures to high cell density cultivations. \u003cem\u003eHum Vaccin Immunother\u003c/em\u003e 20, 2373521, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21645515.2024.2373521\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2024.2373521\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuggal, N. K. \u0026amp; Emerman, M. Evolutionary conflicts between viruses and restriction factors shape immunity. \u003cem\u003eNat Rev Immunol\u003c/em\u003e 12, 687\u0026ndash;695, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nri3295\u003c/span\u003e\u003cspan address=\"10.1038/nri3295\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRandall, R. E. \u0026amp; Goodbourn, S. Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures. \u003cem\u003eJ Gen Virol\u003c/em\u003e 89, 1\u0026ndash;47, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/vir.0.83391-0\u003c/span\u003e\u003cspan address=\"10.1099/vir.0.83391-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, Y. \u003cem\u003eet al.\u003c/em\u003e Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection. \u003cem\u003eFront Immunol\u003c/em\u003e 15, 1408510, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2024.1408510\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2024.1408510\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi, E., Oh, J., Kang, H. R., Song, M. J. \u0026amp; Park, S. H. BST2 inhibits infection of influenza A virus by promoting apoptosis of infected cells. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e 509, 414\u0026ndash;420, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.bbrc.2018.12.110\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2018.12.110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLe Tortorec, A., Willey, S. \u0026amp; Neil, S. J. Antiviral inhibition of enveloped virus release by tetherin/BST-2: action and counteraction. \u003cem\u003eViruses\u003c/em\u003e 3, 520\u0026ndash;540, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/v3050520\u003c/span\u003e\u003cspan address=\"10.3390/v3050520\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Damme, N. \u003cem\u003eet al.\u003c/em\u003e The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from the cell surface by the viral Vpu protein. \u003cem\u003eCell Host Microbe\u003c/em\u003e 3, 245\u0026ndash;252, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2008.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2008.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarc\u0026iacute;a, M. A. \u003cem\u003eet al.\u003c/em\u003e Impact of protein kinase PKR in cell biology: from antiviral to antiproliferative action. \u003cem\u003eMicrobiol Mol Biol Rev\u003c/em\u003e 70, 1032\u0026ndash;1060, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/mmbr.00027-06\u003c/span\u003e\u003cspan address=\"10.1128/mmbr.00027-06\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSilverman, R. H. Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during the interferon antiviral response. \u003cem\u003eJ Virol\u003c/em\u003e 81, 12720\u0026ndash;12729, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jvi.01471-07\u003c/span\u003e\u003cspan address=\"10.1128/jvi.01471-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSadler, A. J., Latchoumanin, O., Hawkes, D., Mak, J. \u0026amp; Williams, B. R. An antiviral response directed by PKR phosphorylation of the RNA helicase A. \u003cem\u003ePLoS Pathog\u003c/em\u003e 5, e1000311, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1000311\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1000311\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez, N. W. \u003cem\u003eet al.\u003c/em\u003e PKR-driven ISR signaling controls synaptic translation and structural plasticity in an age-dependent manner. \u003cem\u003eNeurobiol Dis\u003c/em\u003e 216, 107113, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.nbd.2025.107113\u003c/span\u003e\u003cspan address=\"10.1016/j.nbd.2025.107113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAu-Yeung, N., Mandhana, R. \u0026amp; Horvath, C. M. Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway. \u003cem\u003eJakstat\u003c/em\u003e 2, e23931, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4161/jkst.23931\u003c/span\u003e\u003cspan address=\"10.4161/jkst.23931\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, X., Li, J., Fu, M., Zhao, X. \u0026amp; Wang, W. The JAK/STAT signaling pathway: from bench to clinic. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 6, 402, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41392-021-00791-1\u003c/span\u003e\u003cspan address=\"10.1038/s41392-021-00791-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEvans, D. T., Serra-Moreno, R., Singh, R. K. \u0026amp; Guatelli, J. C. BST-2/tetherin: a new component of the innate immune response to enveloped viruses. \u003cem\u003eTrends Microbiol\u003c/em\u003e 18, 388\u0026ndash;396, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tim.2010.06.010\u003c/span\u003e\u003cspan address=\"10.1016/j.tim.2010.06.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones, C. E., Tan, W. S., Grey, F. \u0026amp; Hughes, D. J. Discovering antiviral restriction factors and pathways using genetic screens. \u003cem\u003eJ Gen Virol\u003c/em\u003e 102, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/jgv.0.001603\u003c/span\u003e\u003cspan address=\"10.1099/jgv.0.001603\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, R. \u0026amp; Moss, B. Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants. \u003cem\u003eJ Virol\u003c/em\u003e 90, 7864\u0026ndash;7879, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/jvi.00869-16\u003c/span\u003e\u003cspan address=\"10.1128/jvi.00869-16\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValero-Rello, A. \u0026amp; Sanju\u0026aacute;n, R. Enveloped viruses show increased propensity to cross-species transmission and zoonosis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e 119, e2215600119, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.2215600119\u003c/span\u003e\u003cspan address=\"10.1073/pnas.2215600119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDimitrov, D. S. Virus entry: molecular mechanisms and biomedical applications. \u003cem\u003eNat Rev Microbiol\u003c/em\u003e 2, 109\u0026ndash;122, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nrmicro817\u003c/span\u003e\u003cspan address=\"10.1038/nrmicro817\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTenge, V. R., Gounder, A. P., Wiens, M. E., Lu, W. \u0026amp; Smith, J. G. Delineation of interfaces on human alpha-defensins critical for human adenovirus and human papillomavirus inhibition. \u003cem\u003ePLoS Pathog\u003c/em\u003e 10, e1004360, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.ppat.1004360\u003c/span\u003e\u003cspan address=\"10.1371/journal.ppat.1004360\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJoe, C. C. D. \u003cem\u003eet al.\u003c/em\u003e Accelerated and intensified manufacturing of an adenovirus-vectored vaccine to enable rapid outbreak response. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e 121, 176\u0026ndash;191, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/bit.28553\u003c/span\u003e\u003cspan address=\"10.1002/bit.28553\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKis, Z. \u003cem\u003eet al.\u003c/em\u003e Pandemic-response adenoviral vector and RNA vaccine manufacturing. \u003cem\u003eNPJ Vaccines\u003c/em\u003e 7, 29, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41541-022-00447-3\u003c/span\u003e\u003cspan address=\"10.1038/s41541-022-00447-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J. \u003cem\u003eet al.\u003c/em\u003e Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors. \u003cem\u003eAMB Express\u003c/em\u003e 9, 70, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13568-019-0794-5\u003c/span\u003e\u003cspan address=\"10.1186/s13568-019-0794-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGenzel, Y. Designing cell lines for viral vaccine production: Where do we stand? \u003cem\u003eBiotechnol J\u003c/em\u003e 10, 728\u0026ndash;740, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/biot.201400388\u003c/span\u003e\u003cspan address=\"10.1002/biot.201400388\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKane, M. \u003cem\u003eet al.\u003c/em\u003e Identification of Interferon-Stimulated Genes with Antiretroviral Activity. \u003cem\u003eCell Host Microbe\u003c/em\u003e 20, 392\u0026ndash;405, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.chom.2016.08.005\u003c/span\u003e\u003cspan address=\"10.1016/j.chom.2016.08.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaemmle, L., Goldstein, R. S. \u0026amp; Kinchington, P. R. Modeling Varicella Zoster Virus Persistence and Reactivation - Closer to Resolving a Perplexing Persistent State. \u003cem\u003eFront Microbiol\u003c/em\u003e 10, 1634, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmicb.2019.01634\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.01634\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, H. \u003cem\u003eet al.\u003c/em\u003e Research Progress on Varicella-Zoster Virus Vaccines. \u003cem\u003eVaccines (Basel)\u003c/em\u003e 13, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/vaccines13070730\u003c/span\u003e\u003cspan address=\"10.3390/vaccines13070730\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichels, A., Ho, N. \u0026amp; Buchholz, C. J. Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms. \u003cem\u003eMol Ther\u003c/em\u003e 30, 2401\u0026ndash;2415, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.ymthe.2022.05.018\u003c/span\u003e\u003cspan address=\"10.1016/j.ymthe.2022.05.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHegde, N. R. Cell culture-based influenza vaccines: A necessary and indispensable investment for the future. \u003cem\u003eHum Vaccin Immunother\u003c/em\u003e 11, 1223\u0026ndash;1234, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/21645515.2015.1016666\u003c/span\u003e\u003cspan address=\"10.1080/21645515.2015.1016666\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi, E., Oh, J., Giao, N. Q., Oh, S. \u0026amp; Park, S. H. Enhanced production of enveloped viruses in BST-2-deficient cell lines. \u003cem\u003eBiotechnol Bioeng\u003c/em\u003e 114, 2289\u0026ndash;2297, doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/bit.26338\u003c/span\u003e\u003cspan address=\"10.1002/bit.26338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-9184882/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9184882/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe efficiency of cell culture-based vaccine production is fundamentally constrained by the antiviral defenses of host cells, creating a major bottleneck for rapid and large-scale vaccine manufacturing. Key antiviral proteins- such as RNase L, PKR, and JAK1 act as intrinsic brakes on viral replication, limiting efficient propagation of many clinically relevant viruses. To overcome this challenge, we generated HEK293T, Vero, and MDCK cell lines with targeted knockouts of multiple antiviral genes. Notably, these engineered cells maintained normal growth and viability while supporting markedly increased viral yields. Multi-gene deletions enhance the replication of both enveloped viruses, including influenza A virus, pseudotyped lentivirus, and porcine epidemic diarrhea virus (PEDV), and non-enveloped viruses such as coxsackievirus. The magnitude of enhancement scaled proportionally with the number of genes disrupted. By systematically removing host restriction factors, this platform provides a versatile and powerful strategy for accelerating viral propagation, offering a strong foundation for more efficient development and large-scale production of cell culture-based anti-viral vaccines.\u003c/p\u003e","manuscriptTitle":"Systemic Removal of Host Restriction Factors Enables Rapid, Scalable Virus Production for Cell Culture-Based Vaccines High-Yield Virus Production System generated by gene knockout of multiple anti-viral host factors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-30 08:18:29","doi":"10.21203/rs.3.rs-9184882/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6d03399-bf0d-47d1-be8b-f90a853e5af5","owner":[],"postedDate":"March 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65169126,"name":"Biological sciences/Biotechnology"},{"id":65169127,"name":"Biological sciences/Immunology"},{"id":65169128,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-07T14:56:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-30 08:18:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9184882","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9184882","identity":"rs-9184882","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}