Ethics statements and facilities

All experiments involving highly pathogenic avian influenza viruses, such as H5 and H7 viruses, as well as a human-transmissible strain (A/Sichuan/01/2009, H1N1), were conducted in the enhanced animal biosafety level 3 (ABSL3) facility at the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences (HVRI, CAAS). Experiments involving low pathogenic influenza viruses were conducted in the enhanced biosafety level 2 (BSL-2 + ) facility at HVRI, CAAS. The study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of China. The protocol for the animal studies was approved by the Committee on the Ethics of Animal Experiments of HVRI, CAAS (approval number: 240318-01-GJ). The animals used in this study were housed in individually ventilated cage systems.

Cells and viruses

The human embryonic kidney cell line 293T, murine lung epithelial cell line MLE-12, and human lung epithelial cell line A549 were obtained from the American Type Culture Collection (ATCC). 293T and MLE-12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA), whereas A549 cells were cultured in Ham’s F-12K medium (Thermo Fisher Scientific, USA) supplemented with 10% FBS. Madin‒Darby canine kidney (MDCK) cells were cultured in DMEM supplemented with 5% newborn calf serum (NCS; Sigma‒Aldrich, USA) and maintained in minimum essential medium (MEM) supplemented with 0.3% bovine serum albumin (BSA; Sigma‒Aldrich, USA). All culture media were supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Procell, China). All the cell lines were maintained in a humidified incubator at 37 °C with 5% CO₂.

All viruses used in this study are listed in Supplementary Table 1. Avian influenza A viruses were propagated in 10-day-old embryonated chicken eggs, whereas human influenza viruses were grown in MDCK cells cultured in MEM supplemented with 0.3% BSA and 1 µg/mL L-1-tosylamide-2-phenylmethyl chloromethyl ketone (TPCK)-treated trypsin (Sigma‒Aldrich, USA). All viral stocks were stored in our laboratory.

Antibodies, reagents, and chemicals

The NP-specific rabbit monoclonal antibody (mAb) was prepared and stored in our laboratory. Anti-Myc-tag antibody-conjugated agarose beads (CN 20168), goat anti-rabbit IgG (H + L) secondary antibodies, HRP conjugates (31460, 1:10000), goat anti-mouse IgG (H + L) cross-adsorbed secondary antibodies, Alexa Fluor 488 (A-11029, 1:1000), and goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibodies, Alexa Fluor 594 (A-11037, 1:1000) were obtained from Invitrogen. C-Myc-tag Rabbit polyclonal antibody (A00172, 1:1000), anti-HA-tag rabbit polyclonal antibody (51064-2-AP, 1:1000), anti-ATG5 polyclonal antibody (10181-2-AP, 1:500), beta-actin monoclonal antibody (66009-1-Ig, 1:1000), GAPDH rabbit polyclonal antibody (10494-1-AP, 1:5000), and GAPDH mouse monoclonal antibody (60004-1-Ig, 1:5000) were obtained from Proteintech. IRDye 800CW goat anti-rabbit IgG secondary antibody (926-32211, 1:10,000) and IRDye 680RD goat anti-mouse IgG secondary antibody (926-68070, 1:10,000) were obtained from LI-COR Biosciences.

Lipofectamine LTX Reagent (15338100) and Opti-MEM (CN 31985070) were obtained from Invitrogen. Polyethyleneimine (PEI; 24765, stock concentration 1 mg/mL) was purchased from Polysciences. SeaPlaque agarose (50100) was purchased from Lonza. PMSF (ST507), NP-40 lysis buffer (P0013F), and SDS‒PAGE protein loading buffer (P0286) were purchased from Beyotime. DAPI nucleic acid stain (62248, 1:500) and Halt Protease Inhibitor Cocktail (87786) were purchased from Invitrogen (USA). Triton X-100 (9036-19-5) was obtained from Sigma‒Aldrich, and benzonase (C2001) was obtained from HaiGene. A 4%–20% ExpressPlus PAGE gel (M42010C) was purchased from GenScript. The nitrocellulose blotting membrane (10600003) was purchased from Cytiva. Protein G agarose (CN 11243233001) was obtained from Roche. Puromycin (HY-B1743), LY294002 (HY-10108), and MG132 (HY-13259) were purchased from MedChemExpress.

Construction of plasmids

The genes encoding the α-domain of the human von Hippel‒Lindau (VHL) tumor suppressor (GenBank accession no. NP_000542.1), mouse VHL (GenBank accession no. NP_033533.1), and anti-NP nanobodies (Nb52, Nb77, Nb135, Nb170, and Nb355) and an anti-SARS-CoV-2 Spike nanobody (GenBank accession no. QNA42481.1) were synthesized and cloned into the lentiviral expression vector pLVX-CMV-Puromycin (Addgene plasmid #164589) and/or the pCMV-Myc vector (Clontech, 635689). The NP gene of the PR8 virus was cloned into the pCAGGS vector with no or an eGFP tag at its C-terminus, resulting in the constructs pCAGGS-NP and pCAGGS-NP-eGFP. The NP genes of 16 different influenza A virus subtypes (H1–H16; see Supplementary Table 1) were subsequently cloned into the pCAGGS vector. The ubiquitin gene was cloned into the pCAGGS vector with an HA tag at its C-terminus.

Degradation analysis

293T cells were seeded into 60-mm dishes or 6-well plates and cultured until they reached approximately 70% confluence. The cells were then cotransfected with pCAGGS-Nb or pCAGGS-VHL-Nb and either pCAGGS-NP-eGFP or pCAGGS-NP. To prepare the transfection complex, we diluted the plasmid DNA in 300 μL of Opti-MEM, followed by the addition of PEI at a ratio of 1 μg of DNA to 4 μL of PEI. The mixture was gently pipetted and incubated at room temperature for 15 minutes. The resulting DNA‒PEI complexes were added dropwise to the cells across the culture surface. Six hours posttransfection, the medium was replaced with fresh Opti-MEM to remove residual transfection reagents. At 48 h posttransfection, fluorescence signals were observed via a fluorescence microscope to assess NP-eGFP degradation, and the NP expression levels were analyzed via Western blotting.

Generation of stable-expressing cells

A second-generation lentiviral packaging system was used to generate stable mammalian cells that expressed the target genes. 293T cells were transfected as described for the transfection assay via a plasmid mixture consisting of the target expression plasmid, psPAX2, and pMD2.G at a ratio of 4:3:2 was diluted in Opti-MEM. Viral supernatants collected from the transfected 293T cells were used to transduce A549 and MLE-12 cells. At 48 h postinfection, the culture medium was replaced with puromycin-containing Opti-MEM selection medium (1 μg/mL for A549 cells and 1.5 μg/mL for MLE-12 cells). After antibiotic selection, the surviving cell population was cultured for further analysis.

Generation of the ATG5-knockout 293T cell line

The CRISPR/Cas9 gene-editing system was used to generate an ATG5-knockout 293T (293T-ATG5^-/-) cell line. Single-guide RNA (sgRNA) oligonucleotides (sgRNA-1: 5′-CACCAACTTGTTTCACGCTATATC-3′ and sgRNA-2: 5′-AAACGATATAGCGTGAAACAAGTT-3′) were cloned into the lentiCRISPRv2 vector (Addgene plasmid #98290). Then, the 293T cells were cotransfected with lentiCRISPRv2-sgRNA, psPAX2, and pMD2.G plasmids at a ratio of 4:3:2 via Lipofectamine LTX. Lentiviral supernatants were collected 48 h posttransfection. The 293T cells were then infected with the collected lentivirus. At 48 h post infection, the transduced cells were selected with puromycin (2 μg/mL) for 3–5 days to eliminate noninfected cells. Following selection, single puromycin-resistant cells were isolated via fluorescence-activated cell sorting (FACS) via a MA900 high-speed cell sorter (Sony, Japan) and seeded into 96-well plates to establish monoclonal populations. The knockout cell clones were validated at the DNA level by PCR and at the protein level by Western blotting.

Viral growth kinetics assay

To analyze viral growth kinetics in vitro, A549 and/or MLE-12 cells were seeded in 24-well plates and infected with viruses at an MOI of 0.01. Cells infected with human seasonal H1N1 and H3N2 viruses were incubated at 33 °C, whereas those infected with PR8, avian H5N1, H7N9, or H9N2 viruses were incubated at 37 °C. After 1 h of viral infection, the supernatant was removed, and the cells were washed three times with MEM. The cells were then maintained at either 33 °C or 37 °C in MEM/BSA supplemented with 0.2 μg/mL TPCK-treated trypsin. Culture supernatants were collected from triplicate wells at 12, 24, 48, and 72 h postinfection. Viral titers were determined via plaque assays in MDCK cells. All the experiments were performed in triplicate.

Viral titration

Plaque assays were performed to determine viral titers in the collected samples. Supernatants from A549 and MLE-12 cells infected with influenza viruses were harvested for titration. Briefly, MDCK cells were seeded in 24-well plates and cultured until they reached 100% confluence. The monolayers were washed three times with MEM and then infected with 10-fold serial dilutions of virus-containing supernatants prepared in MEM/BSA. After 1 h of adsorption at 33 °C or 37 °C, the inoculum was removed, and the cells were washed twice with MEM. The cells were overlaid with 1% Sea Plaque agarose in MEM/BSA containing 1 μg/mL TPCK-treated trypsin. Following incubation for 2–3 days, the cells were fixed with 4% paraformaldehyde (PFA) in PBS, and plaques were counted to determine viral titers.

Western blotting

Plasmid-transfected cells were washed twice with cold PBS and lysed in NP-40 lysis buffer supplemented with Halt protease inhibitor cocktail and benzonase for 1 h at 4 °C. The cell lysates were subsequently centrifuged at 13,000 × g for 10 minutes at 4 °C. The resulting supernatants were mixed with SDS‒PAGE loading buffer and boiled for 10 minutes. Protein samples were separated by electrophoresis on a 4%–20% ExpressPlus PAGE gel and transferred onto a nitrocellulose membrane (0.45 μm). The membrane was blocked with 5% skim milk in PBS for 2 h at room temperature and then incubated with primary antibodies overnight at 4 °C. After five washes with PBST (PBS containing 0.1% Tween-20), the membrane was incubated for 1 h at room temperature with IRDye 800CW goat anti-rabbit IgG (1:10,000) or IRDye 680RD goat anti-mouse IgG (1:10,000) secondary antibodies. After three additional washes with PBST, the immunolabeled proteins were visualized via an Odyssey infrared imaging system (LI-COR Biosciences, USA). Band intensities were quantified via ImageJ software.

Coimmunoprecipitation assay

For coimmunoprecipitation assays, 293T cells transfected with the indicated plasmids were lysed as described in the Western blotting procedure. The supernatants from the cell lysates were first incubated with 30 μL of Protein G agarose beads for 4 h at 4 °C on a flip shaker to preclear nonspecific binding proteins. After this incubation, the beads were removed via centrifugation, and the resulting supernatants were collected. The precleared supernatants were then incubated with 30 μL of anti-His-tag or anti-Myc-tag antibody-conjugated agarose beads overnight (i.e., > 12 h) at 4 °C on a shaker. Following this incubation, the beads were washed five times with cold NP-40 lysis buffer to remove nonspecific proteins. The washed beads were resuspended in PBS, mixed with SDS‒PAGE loading buffer, and boiled for 10 minutes. The samples were then analyzed by Western blotting.

Drug treatment assay

For the MG132 and LY294002 treatment assays, 293T cells were seeded in 6-well plates and transfected with the indicated expression plasmids. After a 24 h incubation at 37 °C, the cells were treated with either DMSO (control), MG132 (10 μM), or LY294002 (20 μM) for 8 h. Following treatment, the cells were lysed, and the NP protein expression levels were analyzed via Western blotting. Band intensities were quantified via ImageJ software.

Intratracheal administration of AAV-LungM3 to mice

AAV-LungM3 constructs, including the AAV-LungM3-eGFP vector and AAV-LungM3-eGFP-mVHL-Nb170 (1 × 10¹³ vg/mL each), were obtained from OBiO Technology (Shanghai Co., Ltd.). Groups of 14 five-week-old female BALB/c mice (Vital River Laboratories, Beijing, China) were anesthetized with 3–5% isoflurane delivered via a gas anesthesia system (Yuyan Instruments Co., Ltd., China) and maintained under anesthesia with continuous isoflurane inhalation. For AAV-LungM3-mediated gene delivery, each mouse was positioned on an angled surgical platform and received two intratracheal administrations of AAV-LungM3 at 5 × 10¹⁰ vg per dose in 50 μL of PBS, with administrations spaced 3 days apart. The AAV-LungM3 suspension was delivered intratracheally via a microsprayer aerosolizer device (Yuyan Instruments Co., Ltd., China). Three mice per group were euthanized at 3 weeks post-administration. The efficacy of intratracheal delivery was validated by immunohistochemistry (Supplementary Fig. 3).

Mouse experiment

Three weeks after AAV-LungM3 administration, groups of mice (14 per group for AAV-LungM3-VHL-Nb170 and AAV-LungM3-eGFP vectors) were lightly anesthetized with 3–5% isoflurane and intranasally challenged with 5 MLD₅₀ of H1N1 (PR8) or H5N1 (SD012) virus in a total volume of 50 μL. At 3 days postchallenge, four mice from each group were euthanized, and their organs were collected for virus titration in 10-day-old embryonated chicken eggs. The remaining mice were monitored daily for changes in body weight and survival for up to 14 days. The criterion for determining mortality in mice was defined as a 25% reduction in body weight.

Statistical analysis

Statistical analyses were performed via GraphPad Prism version 9.0. Viral replication kinetics were assessed via a two-tailed unpaired Student’s t test, and degradation efficacy was analyzed via one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A p value