COG6 protein is necessary for influenza A virus infection

To systematically identify host factors essential for IAV infection, we conducted a genome-wide CRISPR/Cas9 knockout screen with human GeCKO libraries. Shortly, A549 cells were transduced with human GeCKO lentiviral pooled libraries of 123,411 single-guide RNAs (sgRNAs) targeting 19,050 genes at multiplicity of infection (MOI) of 0.3 , followed by selection in puromycin for 14 days. Next, library cells were subjected to three rounds of stringent selection with PR8 virus at a high MOI of 5. Genomic DNA from surviving populations was sequenced and analyzed for sgRNA enrichment (Fig. 1A; Data S1). Bioinformatic analysis revealed top 10 candidate genes (Fig. 1B), among which polyclonal knockout lines targeting PCDHGA7, HSD17B14, LRRC43, COG6, TTC9C, TTC28, CLEC4C, and ANKHD1 were generated without compromising cellular viability (Fig. S1A). IFIT3 and TYK2, as known interferon-stimulated genes, were excluded from further validation. A subsequent IAV challenge demonstrated that only COG6 knockout significantly reduced viral infection by more than 50% compared to wild-type (WT) cells, prompting focused investigation of this component of the COG complex (Fig. 1C).

Monoclonal COG6-KO cell lines in A549 and H1299 cells (Fig. S1B and C) exhibited normal proliferation (Fig. S1D), yet displayed markedly reduced susceptibility to PR8 and H3N2 strains, with NP expression reduced to ~40% of WT levels by immunofluorescence (Fig. 2A through D). Viral titers in KO cells were consistently reduced by 0.5–1 log across multiple time points (12–60 h.p.i., MOI = 0.01; Fig. 2E). Complementation with Flag-tagged COG6 restored infectivity to near-WT levels (Fig. 2A through D; Fig. S1E). Specificity was confirmed by the unaltered infectivity of Zika virus (ZIKV) and vesicular stomatitis virus expressing GFP (VSV-GFP) in KO cells (Fig. 2F; Fig. S1F). These findings establish COG6 as a pan-cell-line dependency factor for IAV infection.

COG6 is a component of the COG complex, which is essential for maintaining the normal structure and activity of the Golgi apparatus. COG6 congenital disorder of glycosylation is caused by biallelic mutations in COG6, underscoring its critical role in glycosylation processes (21). Given that the early steps of influenza virus replication involve attachment to sialic acid receptors on the cell surface and subsequent internalization, we explored whether COG6 facilitated IAV attachment and internalization.

Briefly, WT and COG6-KO A549 cells were incubated with PR8 virus at an MOI of 10 at 4°C for 1 h (attachment assay) and then at 37°C for 45 min to allow viral entry (internalization assay). RT-qPCR quantification demonstrated that COG6-KO A549 cells exhibited significantly reduced PR8 virus attachment and internalization compared to WT cells (Fig. 3A). Immunofluorescence microscopy further confirmed that depletion of COG6 led to a marked reduction in IAV attachment and internalization (Fig. 3B).

We then detected α2,3- and α2,6-linked sialic acids on the cell surface using Maackia amurensis lectin (MAL) and Sambucus nigra lectin (SNA), respectively. Flow cytometry analysis revealed that depletion of COG6 resulted in a significant reduction in cell surface α2,3-linked sialic acids, while α2,6-linked sialic acids remained unaffected (Fig. 3C). These results indicate that COG6 is required for the proper expression of α2,3-linked sialic acids.

To assess whether COG6 is involved in additional stages of IAV infection, we investigated its role in viral RNA synthesis. WT and COG6-KO H1299 cells were infected with PR8 virus, and levels of NP vRNA, cRNA, and mRNA were quantified at 3 and 6 h.p.i. using RT-qPCR. In COG6-KO cells, all three RNA species were significantly reduced compared to WT cells at both time points (Fig. 4A).

To elucidate whether COG6 regulates viral RNA levels, we first examined its effect on IAV polymerase activity. WT or COG6-KO H1299 cells were co-transfected with vRNP components (PB2, PB1, PA, and NP) and a reporter plasmid encoding Firefly luciferase flanked by the 5′ and 3′ untranslated regions of the IAV NS segment. A Renilla luciferase-expressing plasmid was included as an internal control to normalize transfection efficiency. As shown in Fig. 4B, COG6 depletion significantly impaired viral polymerase activity, indicating that COG6 functions at a post-entry stage of infection.

Furthermore, COG6 knockout markedly decreased the expression levels of all vRNP components in the reconstituted IAV minigenome replicon system (Fig. 4C). Since the expression of each viral gene in this system is driven by an exogenous plasmid promoter and does not rely on viral RNA polymerase activity, the observed reduction in viral protein levels in COG6-KO cells was not attributable to impaired viral transcription or replication.

To further examine the effect of COG6 on individual vRNP subunits, PB2, PB1, PA, and NP were expressed separately. As shown in Fig. 4D through G, COG6 deletion significantly reduced the protein levels of all four components. In contrast, the expression levels of ZIKV NS1 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD) were unaffected in COG6-KO cells relative to WT controls (Fig. 4H and I). These results suggest that in the absence of COG6, IAV proteins are targeted for degradation, indicating that COG6 contributes to the stabilization of viral proteins during infection.

The ubiquitin-proteasome system and the autophagy-lysosome pathway represent the two major protein degradation mechanisms in eukaryotic cells (22). To elucidate how COG6 stabilizes IAV proteins, WT and COG6-KO H1299 cells were individually transfected with each vRNP component (PB2, PB1, PA, or NP), followed by treatment with the proteasome inhibitor MG132, lysosomal inhibitors bafilomycin A1 (BafA1), or chloroquine (CQ). As shown in Fig. 5A through D, treatment with BafA1 or CQ effectively prevented the degradation of vRNP proteins, consistent with the observation that complementation of COG6 in COG6-KO cells restored vRNP protein expression.

We next examined whether COG6 modulates lysosome biogenesis or function. In COG6-KO H1299 cells, we observed a marked increase in LAMP1-positive puncta, indicative of an elevated number of lysosomes, regardless of IAV infection (Fig. 5E). Collectively, these findings suggest that COG6 deficiency disrupts lysosomal homeostasis and enhances lysosomal activity by promoting lysosome biogenesis, which functionally facilitates the degradation of IAV proteins.

To investigate the biological mechanism by which COG6 protects IAV proteins from lysosomal degradation, we tested its potential interactions with IAV proteins, including PB2, PB1, PA, and NP. Co-immunoprecipitation (Co-IP) assays were conducted in 293T cells co-transfected with COG6 and each of these viral proteins. Immunoblot analysis revealed a strong interaction between COG6 and NP, and no detectable interactions with PB2, PB1, or PA (Fig. 6A and D through F). Consistently, COG6 was also effectively pulled down by NP (Fig. 6B). Furthermore, the interaction between COG6 and NP was RNA-independent, as treatment of cell lysates with RNase A did not disrupt their association (Fig. 6C). Collectively, these findings demonstrate that COG6 robustly interacts with NP, while its interactions with other viral proteins are weak or undetectable, indicating that COG6 may not broadly bind to IAV proteins to prevent their lysosomal degradation.

The COG complex is comprised of COG1, COG2, COG3, COG4, COG5, COG6, COG7, and COG8 subunits (23–25). Our study demonstrated that COG6 is a critical host factor that facilitates IAV infection by presenting sialic acid on the cell surface and protecting viral proteins from lysosomal degradation. To determine whether other subunits of the COG complex share this function, we generated seven knockout H1299 cell lines for COG1-KO, COG2-KO, COG3-KO, COG4-KO, COG5-KO, COG7-KO, and COG8-KO (Fig. S2A through G). These cell lines showed no significant impact on cell viability (Fig. S2H) and were subsequently infected with the PR8 virus. The results showed that knockout of any individual COG subunit significantly impaired PR8 virus infection (Fig. 7A).

To further explore this effect, COG1-KO, COG2-KO, COG3-KO, COG4-KO, COG5-KO, COG6-KO, COG7-KO, and COG8-KO H1299 cells were co-transfected with vRNP components (PB2, PB1, PA, and NP). Notably, the absence of nearly any subunit of the COG complex led to destabilization of vRNP proteins, resulting in their degradation via the lysosomal autophagy pathway (Fig. 7B). However, knockout of COG3 predominantly affected PA stability (Fig. 7B).

We next evaluated the role of COG subunits in viral binding. WT, COG1-KO, COG2-KO, COG3-KO, COG4-KO, COG5-KO, COG6-KO, COG7-KO, and COG8-KO H1299 cells were incubated with PR8 virus at an MOI of 10 at 4°C for 1 h for viral attachment. RT-qPCR analysis revealed that the deletion of any subunit of the COG complex significantly reduced viral attachment (Fig. 7C). Consistently, we used MAL and SNA lectins to assess the expression of α2,3- and α2,6-linked sialic acids on the cell surface. Flow cytometry analysis revealed that, similar to COG6-KO cells, knockout of COG1, COG2, COG4, COG5, COG7, or COG8 reduced the expression of α2,3-linked sialic acids on the cell surface. In contrast, COG3 knockout had minimal effect on either of the sialic acids, suggesting that COG3 may not regulate sialylation but instead affects other glycosylation modifications of membrane proteins (Fig. S3A and B).

Collectively, these findings underscore the critical role of the COG complex in maintaining host cell glycosylation and preventing lysosomal degradation of IAV proteins.

Human embryonic kidney (HEK) 293T cells, human lung carcinoma A549 cells, human lung carcinoma H1299 cells, and MDCK cells were purchased from the American Type Culture Collection. 293T, A549, and MDCK cells were cultured in Dulbecco's modified Eagle medium (10-013-CV, Corning) with 10% fetal bovine serum (FBS) (C04001-500, Vivacell) and 1% penicillin-streptomycin (PS, 100 IU/mL of penicillin and 100 µg/mL of streptomycin) (C100C5, NCM Biotech). H1299 cells were cultured in Roswell Park Memorial Institute 1640 (10-040-CV, Corning) with 10% FBS and 1% PS. All cells were maintained at 37℃ in a 5% CO₂ humidified incubator.

Antibodies used in the study included mouse anti-Flag M2 (F1804, Sigma), mouse anti-GAPDH (AC002, ABclonal), β-actin Rabbit mAb (AC026, ABclonal), rabbit anti-influenza A virus NP antibody (GTX125989, GeneTex), rabbit anti-influenza A virus PA antibody (GTX125932, GeneTex), rabbit anti-influenza A virus PB1 antibody (GTX125923, GeneTex), rabbit anti-influenza A virus PB2 antibody (GTX125926, GeneTex), Zika virus envelope protein antibody (GTX133314, GeneTex), S-Tag rabbit pAb (A24465, ABclonal), goat anti-rabbit IgG-HRP (B2615, Santa Cruz Biotechnology), goat anti-mouse IgG-HRP (31,430, Invitrogen), Alexa Fluor 568-conjugated donkey anti-mouse IgG (H+L) (ab175472, Abcam), and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (H+L) (ab150077, Abcam).

To construct the mammalian transient expression vector pCAGGS encoding COG6 fused with 3×Flag tag at C-terminus, 3×Flag was added to the 3′ end of COG6 by polymerase chain reaction (PCR). The gene-targeting sgRNA fromwas synthesized and cloned into lentiCRISPRv2 vector (Addgene, 52961). The open reading frames of NP, PB1, PB2, and PA derived from influenza virus A/Puerto Rico/8/1934 (PR8) were cloned into pCAGGS vectors. A reporter plasmid carrying Firefly luciferase in the negative sense flanked by 5′ and 3′ terminal ends of IAV NS and a control reporter plasmid carrying Renilla luciferase driven by a thymidine kinase promoter for constitutive expression were generated. All plasmid constructs were confirmed by Sanger sequencing. Table S1

A human GeCKO v.2.0 library containing 123,411 unique sgRNAs targeting 19,050 genes was obtained from Addgene (#1000000048). The sgRNA plasmid library was amplified, purified, and packaged into lentiviral particles in HEK293T cells via co-transfection with psPAX2 and pVSV-G at a 4:3:2 ratio using TransIT-LT1 Transfection Reagent (#2306, Mirus). At 36–48 h post-transfection, the viral supernatant was collected, clarified by centrifugation at 3,000 rpm–5,000 rpm for 30 min, concentrated, aliquoted, and stored at −80°C.

For CRISPR sgRNA screening, A549-GeCKO cell library was generated as previously described (34). Briefly, A549 cells were transduced with the sgRNA lentiviral library at a low MOI of 0.1–0.3 to ensure that most cells received a single sgRNA. Transduced cells were centrifuged at 2,000 rpm for 1 h. After 24 h, cells were selected with puromycin for approximately 2 weeks and subsequently pooled. To maintain a 300-fold coverage of the sgRNA library, sufficient cells were seeded and infected with PR8 virus at an MOI of 5, followed by incubation until nearly all cells were eliminated. The surviving cells were harvested and replated. After a 3 to 5 day recovery period, a second round of screening was performed in the same procedure. Surviving cells were expanded, and genomic DNA was extracted using QIAamp DNA Mini Kit (51306, Qiagen). Genomic DNA from uninfected, mutagenized cells was also extracted to serve as an unselected control.

sgRNA sequences were amplified from genomic DNA by PCR as previously described (35). PCR products containing sgRNA sequences were purified and subjected to next-generation sequencing using Illumina HiSeq 3000 platform. Sequencing data were processed by normalizing sgRNA read counts to counts per million total reads; counts fewer than five were adjusted to five to reduce noise from low-abundance reads. sgRNA-level P-values were calculated using Fisher's method. Log₂ fold change (log₂FC) values for each sgRNA were determined by comparing their distributions to those of control sgRNAs from the uninfected population. Finally, enrichment scores were calculated by integrating both statistical significance and expression change magnitude using the formula:

Enrichment score = −log₁₀(P-value) × sign(log₂FC)

And enrichment score plots were generated based on upregulated genes.

For generation of KO cell lines, sgRNA was annealed into double-strand pair, phosphorylated by T4 polynucleotide kinase (EK0031, Thermo Scientific) and inserted into BsmBI-digested lentiCRISPRv2 by T4 DNA Ligase (EL0011, Thermo Scientific). LentiCRISPRv2 vectors carrying gene-targeting sgRNA or non-targeting control, psPAX2, and pMD2.G were co-transfected into 293T cells, and lentiviruses were collected 48 h post-transfection by filtering the supernatant of 293T with 0.22 µm filters and used to infect WT A549 or H1299 cells in the presence of polybrene (107689, Sigma). Transduced cells were treated with puromycin for 14 days and isolated by limiting dilution to generate monoclonal knockout cell lines. Knockout cells were validated by DNA sequencing or western blotting.

For the generation of complemented and overexpression cell lines, cDNA was synthesized from total RNA isolated from WT A549 cells using GoScript Reverse Transcriptase (Promega, A5003). The coding sequence of COG6, fused with either a Flag or Myc tag, was cloned into pCAGGS vector for transient expression or into pHAGE-CMV-IRES-Puro/Hygro for stable expression. The tagged COG6 constructs in pHAGE were packaged into lentiviral particles and used to infect WT A549 or H1299 cells. Stable cell lines were established through puromycin/Hygro selection. The expression of COG6 was confirmed by western blotting.

Influenza A/Puerto Rico/8/1934 (H1N1) and influenza A/Aichi/2/1968 (H3N2) virus were grown in specific pathogen free chicken embryos for propagation. Virus titers were determined in MDCK cells using 50% tissue culture infective dose assay. For infection assays, plated cells were infected with PR8 or H3N2 virus in maintenance medium containing 1% FBS and 1 µg/mL tosyl phenylalanyl chloromethyl ketone-treated trypsin. Plated cells were infected with ZIKV or VSV-GFP in maintenance medium containing 2% FBS.

Cells were seeded in Millicell EZ SLIDES (PEZGS0816, Millipore) 12 h prior to infection. Plated cells were infected with IAV, ZIKV, or VSV-GFP in maintenance medium for 1 h at 37°C. Supernatants were replaced into serum-free medium for subsequent incubation. The slides were then blocked with phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 2% donkey serum for 30 min at room temperature (RT) and processed to incubation of primary antibodies diluted in blocking buffer for 1 h at RT. After three-time washes with phosphate-buffered saline with tween-20 (PBST), the slides were incubated with secondary antibody diluted in blocking buffer for 30 min at RT. Finally, cells were stained with ProLong Gold Antifade with DAPI (P36931, Life Technologies) for another 1 min for cell nucleus positioning. Immunofluorescence images were visualized by TissueFAXS 200 flow-type tissue cytometer (TissueGnostics GmbH, Vienna, Austria).

WT and COG6-KO A549/H1299 cells were infected with PR8 or H3N2 virus at an MOI of 0.01, and supernatants were collected at the indicated time points for viral titration in MDCK cells. Similarly, WT and COG6-KO A549 cells were infected with ZIKV (MOI = 0.5) or VSV-GFP (MOI = 0.01), and supernatants were collected at the indicated time points for titration in A549 cells. Viral titers were determined using the Reed-Muench method.

Cell viability was determined by Cell Counting Kit-8 (CCK-8, A311-01, Vazyme). Briefly, cells were pre-seeded in opaque-walled 96-well plates. A total of 10 µL of CCK-8 solution was added into each well and incubated at 37°C for 4 h, followed by measurement of absorbance at 450 nm. Data were from three or six biological replicates.

Cells were lysed using 4×Protein SDS-PAGE Loading Buffer (9173, Takara) and denatured at 95°C for 5 min. Protein samples were resolved by SDS-PAGE and transferred to polyvinylidene fluoride membranes (10600021, Cytiva). Membranes were blocked with 5% non-fat milk in PBST for 1 h at RT and incubated with 5% non-fat milk-diluted primary antibodies overnight at 4°C. Membranes bound with primary antibodies were washed thrice with PBST and incubated with 5% non-fat milk-diluted secondary antibodies for 1 h at RT. Membranes were washed thrice with PBST and visualized by a enhanced chemiluminescent substrate using Bio-Rad ChemiDoc MP Imaging System.

Biotinylated MAL II (B-1265-1) and SNA (B-1305-2) were purchased from VectorLabs. WT and COG6-KO A549 cells were washed with PBS twice and incubated with biotinylated lectins for 1 h on ice, followed by incubation with 1 µg/mL PE streptavidin (405203, BioLegend). The levels of lectin binding were determined by flow cytometry.

To assess viral NP levels, total RNA from virus-infected cells was extracted using Direct-zol RNA Miniprep (R2052, Zymo Research). Relative amounts of NP vRNA, mRNA, and cRNA were quantified by RT-qPCR as previously described (18). RNA levels were normalized to GAPDH mRNA copies.

WT and COG6-KO H1299 cells pre-seeded in 12-well plates were washed five times by cold PBS and infected with PR8 virus (MOI = 10) for 1 h on ice. For the attachment assay, virus-bound cells were washed five times with ice-cold PBS (pH = 7.2) and lysed with RNAzol. For the internalization assay, virus-bound cells continued to an additional 45 min incubation at 37°C and, subsequently, cells were washed three times with ice-cold acidic PBS (pH = 1.5) and lysed with RNAzol. Viral RNA was quantified by RT-qPCR.

In order to measure the activity of reconstituted vRNP, WT and COG6-KO H1299 cells were seeded into a 24-well plate at 2 × 10⁵ cells per well 1 day prior to transfection. After 12 h, cells were co-transfected with pCAGGS expression plasmid encoding PB2 (0.1 µg), PB1 (0.1 µg), PA (0.1 µg), NP (0.1 µg), 0.1 µg of a reporter plasmid carrying Firefly luciferase in negative sense flanked by 5′ and 3′ terminal ends of IAV NS gene, and 0.02 µg of control reporter plasmid with Renilla luciferase as a transfection and toxicity control using TurboFect Transfection Reagent (R0533, Thermo Scientific). Cells were co-transfected with the same plasmids excluding pCAGGS-NP as a blank control. At 24 h post-transfection, cells were lysed, and the luminescence was measured with Dual-Luciferase Reporter Assay System (E1910, Promega) according to the manufacturer's instructions on GloMax 96 microplate luminometer (E6501, Promega). The levels of vRNP luminescence were normalized to those of Renilla luminescence.

Co-IP assays were performed as previously described (36). For COG6 immunoprecipitating the PB2, PB1, PA, or NP protein of PR8 virus, pCAGGS-COG6-Flag was co-transfected to 293T cells pre-seeded in 10 cm dishes with either pCAGGS-PB2, PB1, PA, or NP, respectively, using TransIT-LT1 Transfection Reagent. Cells were washed thrice with prechilled PBS and lysed with Pierce IP Lysis Buffer (87787, Thermo Scientific) supplied with 1% (vol/vol) protease inhibitor cocktail (P8340, Sigma) for 30 min on ice. The lysates were incubated with protein A/G agarose (20422, Sigma-Aldrich) for 1 h at 4°C and were then subjected to centrifugation at 12,000 × g for 10 min at 4°C. The supernatants were transferred to a new tube and incubated with anti-Flag M2 affinity gel (A2220, Sigma) overnight at 4°C. Subsequently, the agarose was washed thrice by prechilled wash buffer and incubated with 3×Flag peptide (F4799, Sigma) overnight at 4°C. The eluted supernatants were transferred into new 1.5 mL tubes, lysed with 4×Protein SDS-PAGE Loading Buffer, and inactivated at 95°C for 5 min. The samples were processed to SDS-PAGE and western blotting. For RNase A treatment followed by Co-IP, supernatants were collected and divided into two parts; one part was supplied with RNase A (EN0531, Thermo Scientific) at a final concentration of 100 µg/mL, while the other part was kept untreated. After rotation of the samples at 4°C for 1 h and centrifugation at 14,000 g for 10 min, supernatants were harvested, and other experimental procedures were performed as previously described. For viral NP proteins immunoprecipitating COG6, pCAGGS-NP-FS were co-transfected to 293T cells pre-seeded in 10 cm dishes with pCAGGS-COG6-Myc, followed by Co-IP assay as described above, and cell lysates were immunoprecipitated using anti-S tag affinity gel (69704, Millipore).

WT and COG6-KO H1299 cells were pre-seeded in 12-well plates and transfected with pCAGGS-NP, PB1, PB2, or PA for 24 h. Cells were treated with MG132 (25 µM, HY-13259, MedChemExpress), BafA1 (100 nM, HY-100558, MedChemExpress), or CQ (50 µM, HY-17589A, MedChemExpress). Cells were lysed with 4×Protein SDS PAGE Loading Buffer, denatured at 95°C for 5 min, and analyzed by western blotting.

All statistical analyses were performed with Prism 9.0 software (GraphPad). Quantitative data are shown as mean ± SD from at least three independent biological replicates. Statistical significance was analyzed using two-tailed unpaired t-test, one-way or two-way analysis of variance, followed by Sidak's or Dunnett's multiple comparison tests. For quantification of infected cells, at least 1,000 fluorescent cells were imaged and counted with a flow-type tissue cytometer. In accordance with current standard practice, P-values ≤0.05 were considered statistically significant and denoted with an asterisk. Non-significant values were denoted as ns.