Human alveolar epithelial cells, A549 and Madin-Darby canine kidney (MDCK) cells were grown in minimum essential medium (MEM) supplemented with 10% foetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin (Life Technologies) at 37°C under 5% CO₂. Human bronchial epithelial cells, HBEC3-KT (ATCC) were grown in HBEC3-KT growth kit (ATCC). Plasmid expressing human NAA60 (kindly gifted by Prof Thomas Arnesen and Dr Henriette Aksnes, University of Bergen, Norway) was amplified in Escherichia coli DH5α cells and extracted using a plasmid purification kit (Qiagen).

Influenza virus A/Puerto Rico/8/1934/(H1N1) and A/California/07/2009/(H1N1) were propagated in 10-day old embryonated chicken eggs and titrated on MDCK cells. For infection, cell monolayers grown in individual wells of a cell culture plate were washed twice with serum-free MEM and incubated with virus inoculum (prepared in serum-free MEM) for 1 hour at 35°C. Following incubation, the inoculum was removed, cells were washed once with serum-free MEM and replaced with fresh serum-free MEM. This point was considered as ‘0 hour’ infection timepoint, and the cells were incubated back at 35°C for desired hours before further processing.

Pre-designed human NAA60 gene-targeting small-interfering ribonucleic acid (siRNA) (5’-CCA AGA GUG GCA UCG AGU A-3’) and a non-targeting MISSION^® control siRNA were purchased from Sigma-Aldrich. The siRNA was delivered to cells by reverse transfection using Lipofectamine RNAiMax (Invitrogen). For this, 10 nM siRNA and 2 µL RNAiMax were diluted separately in Opti-MEM (Gibco) and incubated for 5 minutes at room temperature. The solutions were then mixed and incubated for further 30 minutes at room temperature. Finally, the siRNA-RNAiMax complex was mixed with cell suspension and cells were seeded in a cell culture plate. The cells were incubated at 37°C for 72 hours before further processing.

The plasmid expressing human NAA60 was delivered to cells by reverse transfection using Lipofectamine 2000 reagent (Invitrogen). For this, 1 µg plasmid DNA and 3 µL Lipofectamine 2000 were diluted separately in Opti-MEM and incubated for 5 minutes at room temperature. The solutions were then mixed and incubated for further 30 minutes at room temperature. Finally, the DNA-Lipofectamine 2000 complex was mixed with cell suspension and cells were seeded in a cell culture plate. The cells were incubated at 37°C for 24 hours before further processing.

Total RNA was extracted from cells by using Nucleospin RNA isolation kit (Macherey-Nagel) and used as template to synthesize the cDNA by using PrimeScript RT reagent kit (Takara) by following the manufacturers’ instructions. The reverse transcription quantitative real-time PCR (RT-qPCR) was performed by using pre-designed KiCqStart primers or custom-designed primers (Sigma-Aldrich) and SYBR select master mix (Applied Biosystems) on ViiA7 Real-time PCR system (Applied biosystems). The custom-designed primers for β-actin were: Forward 5′-GACGACATGGAGAAAATCTG-3’, Reverse 5′-ATGATCTGGGTCATCTTCTC-3′. The actin mRNA level was used as a reference and the fold or percent change in the mRNA level of target genes was measured using standard 2^−ΔΔCT method.

Cells were lysed in a lysis buffer (150 mM NaCl, 50mM Tris-HCl [pH 7.4], 0.5% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 1% Triton X-100 and protease inhibitor cocktail [Roche]), and total protein was estimated using BCA protein assay kit (Pierce) as per the manufacturers’ instructions. Equal protein amounts were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) along with SeeBlue Plus2 pre-stained protein standards (Invitrogen). The protein was transferred to Protran nitrocellulose membrane (Amersham), which was then blocked with 5% milk or 3% bovine serum albumin (BSA) diluted in tris-buffered saline (TBS). The membrane was probed with primary antibodies: rabbit anti-NAA60 (1:1000; HPA040916, Sigma-Aldrich), rabbit anti-GFP (1:1000; #632592, Takara), mouse anti-NP (1:5000; EMD Millipore or 1:10000; NR-19868, obtained through BEI resources, NIAID, NIH), goat anti-NP (1:10000; kindly provided by Richard Webby, St Jude Children’s Research Hospital, USA), mouse anti-pSTAT1 (pY701, 1:1000; clone 14/P-STAT1; BD Biosciences), mouse anti-STAT1 (1:1000; clone 42/STAT1; BD Biosciences), mouse anti-viperin (1:1000; clone D5T2X; Cell Signaling), rabbit anti-ISG15 (1:1000; Cell Signaling), rabbit anti-IFITM3 (1:1000; Abcam), rabbit anti-acetyl histone H4-Lys8 (1:1000; Cell Signaling), mouse anti-acetyl-lysine (1:1000; AAC03, Cytoskeleton Inc) and rabbit anti-PDI (1:10000; Sigma-Aldrich) antibodies followed by the horseradish peroxidase (HRP)-conjugated anti-mouse, anti-goat, or anti-rabbit (1:5000; Life Technologies or Sigma-Aldrich) IgG antibodies. Some blots were re-probed by stripping with 0.5 M sodium hydroxide for 5-7 minutes at room temperature. The protein bands were visualized with ECL or ECL Prime western blotting detection reagent (Amersham), and the images were acquired on Odyssey Fc imaging system (Li-COR) equipped with Image Studio Lite version 5.2. The images were exported as TIFF and compiled in figures using Adobe Photoshop 2022. If needed, the changes in brightness and/or contrast were applied to whole image. For quantification, the intensity of protein bands was measured in Image Studio Lite software and normalized with the loading control, protein disulfide isomerase (PDI).

Cells were incubated with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reagent (Sigma-Aldrich) for 1 hour at 37°C. Then, dimethyl sulfoxide (DMSO) (Calbiochem) was added to the cells and cells were further incubated for 15 minutes at room temperature on a rocking platform. Then, the absorbance was measured at 570 nm in 680-microplate reader (Bio-Rad).

The infected cells were harvested in culture medium and centrifuged at 12,000 x g for 1 minute. The supernatant was collected and supplemented with 0.3% BSA (Sigma-Aldrich) and titrated on confluent MDCK cell monolayers by microplaque assay. Briefly, the MDCK monolayers were infected with 10-fold serial dilutions of the culture medium for 1 hour at 35°C. The inoculum was replaced with overlay medium containing 2X serum-free MEM, 0.8% Avicel (RC-185; FMC Biopolymer) and 1 μg/ml TPCK-treated trypsin (Sigma-Aldrich), and cells were incubated for 22-24 hours at 35°C. The overlay medium was removed, and cells were fixed with 4% formalin (Sigma-Aldrich) and permeabilized with 0.5% Triton X-100 in 20 mM glycine. Then, cells were stained with mouse anti-NP (1:1000; EMD Millipore or BEI Resources) followed by the HRP-conjugated anti-mouse IgG antibody (1:1000). The plaques were visualized by TrueBlue substrate (KPL Biosciences).

The graphs were plotted and prepared in Prism 9.0 (GraphPad). Each graph was exported as TIFF and compiled in figures using Adobe Photoshop 2022. The statistical analyses were also performed in the Prism. The P values were calculated using student t test for two datasets and ANOVA for more than two datasets. When using ANOVA, the Geisser-Greenhouse correction was applied. A P value of ≤0.05 was considered significant.

We employed RNA interference to investigate the involvement of human NAA60 in IAV infection. For this, we obtained a predesigned siRNA and RT-qPCR primer pair and an antibody for NAA60. There are nine NAA60 transcript variants (generated via alternative splicing or promoters) and five polypeptide isoforms in National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/) and UniProt (https://www.uniprot.org/) database, respectively. The siRNA binds to all NAA60 transcript variants and the primer pair binds to five of them (Supplementary Figure 1). The antibody, which has been raised against an N-terminal peptide (Sigma-Aldrich), binds to three isoforms (Supplementary Figure 1). The A549 cells and HBECs were transfected with optimized concentration (10 nM) of NAA60 or non-targeting control (CTRL) siRNA, and the depletion of NAA60 was confirmed after 72 hours (h) by RT-qPCR and western blotting. Under these conditions, the NAA60 mRNA was depleted by a significant 89.5% (P=<0.0001) and 75.1% (P=<0.0001) in A549 cells and HBECs, respectively (Figure 1A). Furthermore, the NAA60 polypeptide level was also depleted (Figure 1B), by a significant 48.8% (n=3, P=0.001); for this, NAA60 polypeptide level in control cells was considered 100% to determine its level in depleted cells. In addition, such depletion of NAA60 reduced the lysine acetylation of histone H4 considerably (Supplementary Figure 2A) and other cellular proteins (mostly high molecular weight) slightly (Supplementary Figure 2B), without affecting the cell viability (Figure 1C). Next, NAA60-depleted and control A549 cells or HBECs were infected with influenza virus A/Puerto Rico/8/1934/(H1N1) (hereafter referred to as PR8) or influenza virus A/California/07/2009/(H1N1) (hereafter referred to as CA09) strains at a multiplicity of infection (MOI) of 1.0. After 24 h, the infectious progeny released in the culture medium was quantified by microplaque assay. We found that, compared to the control, there was a significant 52.5% (P=0.04) and 44.8% (P=0.014) reduction in the release of PR8 and CA09 infectious progeny, respectively, from NAA60-depleted A549 cells (Figure 1D). Similarly, there was a significant 46.7% (P=0.019) reduction in the release of infectious PR8 progeny from NAA60-depleted HBECs compared to the control (Figure 1D). Next, we assessed IAV growth kinetics in the absence of NAA60 over 24 h. For this, control and NAA60-depleted A549 cells were infected with PR8 at MOI of 0.1. The culture medium was collected at 6 h, 12 h and 24 h of infection and titrated by microplaque assay to measure total viral progeny release. Consistent with above data, the IAV growth kinetics was slower in NAA60-depleted cells than in control cells (Figure 1E). The NAA60-depleted cells released 61% and a significant 64% (P=0.038, determined by two-way ANOVA Bonferroni’s test) less infectious viral progeny after 12 h and 24 h of infection, respectively, compared to the control cells (Figure 1E).

To support above data, we analyzed the IAV progeny release from the cells overexpressing NAA60. For this, we used HEK-293T cells for their higher transfection efficiency (Supplementary Figure 3). HEK-293T cells were transfected with a plasmid (pNAA60) expressing NAA60 as green fluorescent protein (GFP) fusion protein and an empty plasmid (pEGFP) expressing only GFP for 24 h. Then, cells were infected with PR8 at an MOI of 1.0. After 24 h, the culture medium was titrated by microplaque assay and the cells were processed for western blotting to confirm the NAA60 overexpression (Figure 2A). Consistent with RNA interference data, the overexpression of NAA60 enhanced the release of infectious IAV progeny release by a significant 2.5-fold (P=0.003) when compared to the control (Figure 2B). Taken together, these data indicated a pro-IAV role for human NAA60.

To gain insight into the pro-viral mechanism of NAA60, we investigated its role in IAV-induced host innate antiviral response, because, previously, we identified that the HDACs exerts their anti-IAV function via this pathway (9–11, 13). Furthermore, the IAV-induced expression of IFNα was downregulated in HDAC11-depleted cells (13) and HDAC6-knockout mice (14). To determine if the opposite was true in NAA60-depleted cells, A549 cells were transfected with the control or NAA60 siRNA and infected with PR8 at an MOI 1.0. After 3 h and 6 h of infection, cells were processed for RT-qPCR to detect the mRNA levels of IFNα, IFNβ, IFNγ and NAA60. Indeed, compared to control, the expression of IFNα mRNA was upregulated in NAA60-depleted cells by a significant 3.2-fold (P=0.0036) and 2.2-fold (P=0.0413) after 3 h and 6 h of infection, respectively (Figure 3A). However, under the same conditions, no significant change was observed in the expression of IFNβ or IFNγ in NAA60-depleted cells (Figures 3B, C). This data indicated that NAA60 suppresses the expression of IFNα in IAV-infected cells. The 3 h and 6 h post-infection timepoints were chosen because the transcription of IFNs and IFN-stimulated genes (ISGs) occurs and peaks within few hours of infection and then starts subsiding due to negative feedback mechanism (21, 22). The same is evident here as the expression of IFN mRNAs is either not changing or subsiding from 3 h to 6 h post-infection in control or NAA60-depleted cells. The transcription of different IFNs is temporal and differential in response to virus infection, with IFNα transcription and IFNα-induced transcription of ISGs occurring early during infection compared to IFNβ or IFNγ (21–23). It seems that NAA60 is involved early in the host anti-IAV signaling events mediated by IFNα, not IFNβ or IFNγ.

One of the consequences of IFNα expression in IAV-infected cells is the phosphorylation-mediated activation of transcription factor, signal transducer and activator of transcription 1 (STAT1) (24). Next, we investigated if the NAA60-mediated suppression of IFNα expression affects STAT1 signaling. For this, A549 cells, transfected with the control or NAA60 siRNA, were infected with PR8 at an MOI of 1.0. Then, cells were harvested after 0 h, 6 h, 12 h and 24 h of infection and the level of the phosphorylated (p) and total (t) STAT1 was detected by western blotting. As expected, in control cells, the pSTAT1 was detected after 6 h of infection and its level gradually increased as the infection progressed (Figure 4A) (24). In NAA60-depleted cells, the level of pSTAT1 increased even further after 6 h, 12 h and 24 h of infection compared to controls cells (Figure 4A). However, the level of tSTAT1 remained almost steady between control and NAA60-depleted cells after 6 h, 12 h and 24 h of infection (Figure 4A). To quantify this change, the intensity of pSTAT1, tSTAT1 and corresponding PDI (loading control) bands from three separate blots (raw images are shown as Supplementary Figure 4) was quantified at 6 h, 12 h, and 24 h timepoints. First, the amount of pSTAT1 and tSTAT1 was normalized by PDI amount at corresponding timepoints. Second, the amount of PDI-normalized pSTAT1 was normalized by the PDI-normalized tSTAT1 amount at each corresponding timepoints. Finally, such double-normalized amount of pSTAT1 in control cells at each timepoint (e.g., 12 h) was considered 1-fold to determine its amount in NAA60-depleted cells at the corresponding timepoint (i.e., 12 h). Based on this analysis, we found that the level of pSTAT1 in NAA60-depleted cells after 12 h and 24 h of infection was a significant 1.48-fold (P=0.044) and 2.0-fold (P=0.048) higher, respectively, than the control cells (Figure 4B).

In the next step of IFN signaling, pSTAT1 is translocated to the nucleus and induces the expression of ISGs. Therefore, we next compared the magnitude of the expression of ISGs involved in IAV infection (25), such as cholesterol 25-hydroxylase (CH25H), IFN‐induced transmembrane (IFITM) protein 1, 2 and 3, ISG15, mitochondrial antiviral signaling protein (MAVS), myxovirus resistance protein 1 (MX1), tripartite motif protein 22 (TRIM22), and viperin in control and NAA60-depleted cells in response to the IAV infection. A549 cells were transfected with the siRNAs and infected with PR8 for 3 h, and the mRNA levels of above ISGs were analyzed by RT-qPCR. Consistent with pSTAT1 level, compared to control cells, there was a significant increase in the mRNA levels of CH25H (3.2-fold, P=0.0006), IFITM1 (3.0-fold, P=0.048), IFITM2 (2.2-fold, P=0.006), IFITM3 (2.0-fold, P=0.003), ISG15 (4.0-fold, P=0.003), MX1 (2.8-fold, P=0.022), and viperin (3.2-fold, P=0.012) in NAA60-depleted cells in response to IAV infection (Figures 5A–E, G, I). Whereas no change was evident in the MAVS and TRIM22 mRNA levels between control and NAA60-depleted infected cells (Figures 5F, H). Also, no change in the basal mRNA levels of above ISGs (except IFITM2) between control and NAA60-depleted cells was observed without the infection (Supplementary Figures 5A–I). We also measured the mRNA levels of above ISGs at 6 h post-infection and found a significant increase in the mRNA levels of CH25H (2.3-fold, P=0.028) and viperin (2.5-fold, P= 0.019) in NAA60-depleted cells (Supplementarys Figure 6A, I). However, no significant change was observed in the mRNA levels of other ISGs between control and NAA60-depleted cells (Supplementary Figures 6B–H). This data seems consistent with the change in IAV-induced IFNα mRNA level between control and NAA60-depleted cells (Figure 3A), where increase in the IFNα mRNA level subsided at 6 h post-infection.

Further, we also compared the expression kinetics of viperin, IFITM3 and ISG15 at polypeptide level in control and NAA60-depleted cells in response to IAV infection. These three ISGs have been determined to target three different stages of the IAV life cycle (26–28). For this, A549 cells transfected with the siRNAs and infected with PR8 as above were harvested at 0 h, 6 h, 12 h and 24 h post-infection, and viperin, IFITM3 and ISG15 polypeptides were detected by western blotting. Like their mRNA levels, the polypeptide levels of all three ISGs were further upregulated, albeit at different kinetics, in NAA60-depleted cells (Figure 6A). We quantified their upregulation using three separate blots (raw images are shown as Supplementary Figure 7) as pSTAT1 above and found that the IAV-induced expression of viperin polypeptide was further enhanced in NAA60-depleted cells by a significant 4.2-fold (P=0.014), 4.6-fold (P=0.007) and 2.5-fold (P=0.045) at 6 h, 12 h and 24 h post-infection, respectively, compared to control cells (Figure 6B). Likewise, the IAV-induced expression of IFITM3 polypeptide was further enhanced in NAA60-depleted cells by a significant 1.6-fold (P=0.022) and 1.7-fold (P=0.041) at 12 h and 24 h post-infection, respectively, compared to control cells (Figure 6C). Finally, after 24 h of infection, the IAV-induced expression of ISG15 polypeptide in NAA60-depleted cells was further increased by a significant 4.3-fold (P=0.016) compared to control cells (Figure 6D).

Next, we sought to confirm whether ectopic NAA60 expression equally affects the IFNα signaling and produces the results opposite to NAA60 depletion. To do this, HEK293T cells transfected with pEGFP or pNAA60 plasmids as in Figure 2, were infected with PR8 at an MOI of 1.0 for 3 h and 6 h. Then, expression of IFNα, IFNβ, and NAA60 mRNA was measured by RT-qPCR (Figures 7A–C). Consistent with results in NAA60-depleted cells, the expression of IFNα mRNA in NAA60-overexpressing cells was reduced by a significant 51% (P=0.036) and 39.2% (P=0.013) at 3 h and 6 h post-infection, respectively, when compared to control cells (Figure 7A). Further, no significant change was observed in IFNβ expression (Figure 7B). In HEK293T cells, we failed to detect the expression of viperin, IFITM3, or ISG15 polypeptides in response to IAV infection. Hence, we revert to A549 cells, and transfected them with pEGFP or pNAA60 plasmid and infected them with PR8 at an MOI of 1.0 for 24 h and analyzed the expression of viperin polypeptide by western blotting. Opposite to its expression in NAA60-depleted cells, the expression of viperin polypeptide in NAA60-overexpressing cells was decreased (Figure 7D), by a significant 56.5% (P=0.001) when compared to GFP-expressing cells (Figure 7E).

The data presented above strongly indicated that NAA60 is involved in IAV-induced IFNα signaling. We sought to further confirm these observations in IFNα-treated cells (in the absence of IAV infection). For this, we transfected A549 cells with control and NAA60 siRNA for 72 h as above, and then treated them with purified IFNα (Human Recombinant Alpha 2a, NR–3083; BEI Resources, NIAID, NIH) at 10 IU/mL for 0 h, 3 h, 6 h, and 12 h. Subsequently, the level of ISG15, IFITM3, and viperin polypeptides was analyzed by western blotting. Like IAV infection, the IFNα treatment also further enhanced the expression kinetics of viperin (Figure 8A) and ISG15 and IFITM3 (Figure 8B) in NAA60-depleted cells. When quantified, compared to control cells, the level of viperin polypeptide was increased by a significant 3.8-fold (P=0.0006), 14.7-fold (P=0.011), and 6.6-fold (P=0.039) after 3 h, 6 h, and 12 h of IFNα treatment, respectively, in NAA60-depleted cells (Figure 8C). Similarly, compared to control cells, the level of ISG15 (Figure 8D) and IFITM3 (Figure 8E) polypeptides in NAA60-depleted cells was increased by a significant 2.0-fold (P=0.001) and 1.72-fold (P=0.035) after 6 h and 12 h of IFNα treatment, respectively.