Host restriction factor SAMHD1 does not restrict seasonal influenza virus replication in human epithelial or macrophage-like cells
Yongyan Xia, Rubaiyea Farrukee, Andrew G. Brooks, Sofía Soler, Rayk Behrendt, Eva Bartok, Sarah L. Londrigan, Patrick C. Reading
TL;DR
This study shows that the protein SAMHD1 does not prevent seasonal influenza virus replication in human epithelial or macrophage-like cells.
Contribution
The study reveals that SAMHD1 does not restrict seasonal influenza virus replication in key cell types, despite restricting DNA viruses.
Findings
SAMHD1 knockout in epithelial and macrophage-like cells did not enhance seasonal influenza virus release.
Inducible SAMHD1 expression failed to restrict seasonal influenza virus growth in these cells.
SAMHD1 did restrict replication of the highly pathogenic avian influenza virus in macrophage-like cells.
Abstract
SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase protein 1 (SAMHD1) is an intracellular protein that regulates a stable deoxynucleoside triphosphates (dNTPs) for normal cellular function. Many studies have documented SAMHD1-mediated restriction of RNA retroviruses and different DNA viruses in non-dividing myeloid cells, where it acts to deplete cellular dNTP pools to inhibit replication of the viral genome. Less is currently known regarding its ability to restrict the replication of other RNA viruses in different cell types. In this study, we investigate the role of SAMHD1 in modulating the replication of RNA viruses associated with respiratory virus infection, with a particular focus on seasonal influenza A virus (IAV). Moreover, our studies have assessed the ability of SAMHD1 to modulate IAV replication in two key cell types associated with the…
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Figure 8- —Global Virus Network Fellowship
- —Germany's Excellence Strategy
- —https://doi.org/10.13039/501100005970Deutsche Stiftung für Herzforschung
- —German Research Foundation
- —https://doi.org/10.13039/501100000925National Health and Medical Research Council
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Taxonomy
Topicsinterferon and immune responses · RNA Research and Splicing · Cytokine Signaling Pathways and Interactions
Background
Host restriction factors are cell-intrinsic host proteins that mediate antiviral activity against one or more viruses and represent an essential component of innate antiviral immunity [1]. SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase protein 1 (SAMHD1) is an important host restriction factor limiting the replication of retroviruses and endogenous retroelements. However, it has also been reported to restrict numerous other DNA viruses as well as some RNA viruses. SAMHD1 is a dNTP triphosphohydrolase (dNTPase) and plays an important physiological role in maintaining stable levels of intracellular deoxynucleoside triphosphates (dNTPs). It has a SAM domain that exhibits DNA and RNA binding abilities and a HD domain that is responsible for its dNTPase activity [2, 3]. SAMHD1 also contains a nuclear localization signal at its N-terminus and predominantly localises to the nucleus, although it has been reported to be expressed in cytoplasmic compartments [4–6]. Furthermore, T592 phosphorylation of SAMHD1 by cyclin A/cdk1 is important for inactivating its dNTPase activity and therefore its ability to regulate the cell cycle [7, 8]. It is well established that terminally differentiated cells show higher overall levels of SAMHD1 protein expression but lower levels of phosphorylated SAMHD1 compared to actively dividing cells [9–11].
SAMHD1 was first reported as a restriction factor against human immunodeficiency virus 1 (HIV-1) in macrophages and dendritic cells (DCs), where its ability to deplete intracellular dNTPs led to inhibition of reverse transcription of the viral genome [12, 13]. Subsequent studies confirmed its ability to inhibit other human retroviruses, including human T cell leukemia virus type 1 (HTLV-1) [14], as well as retroviruses from other species such as feline immunodeficiency virus (FIV), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) [15]. In vivo studies have demonstrated murine SAMHD1 also reduces intracellular dNTP concentrations in various tissues and cell types, including primary macrophages, bone-marrow-derived dendritic cells (BMDCs), splenic B and T cells and embryonic fibroblasts [16, 17]. Furthermore, downregulation of intracellular dNTPs by murine SAMHD1 correlated with restriction of retroviral replication in vitro and in vivo [18]. Of interest, the virion-associated protein (Vpx) expressed by HIV-2 and some SIVs can counteract the antiviral activity of SAMHD1 [12, 13], and Vpx can recruit SAMHD1 to Cullin-RING Ligase 4 (CRL4), a cellular E3 ubiquitin ligase, for proteasomal degradation [19].
SAMHD1 has also been identified as a potent restriction factor against many DNA viruses, including herpes simplex virus type 1 (HSV-1), vaccinia virus (VACV), human cytomegalovirus (HCMV), and hepatitis B virus (HBV) [11, 20–22]. Similar to retroviruses, SAMHD1 depletes cellular dNTP levels in non-dividing myeloid cells during DNA virus infection to block replication of the viral genome [20, 22]. For retroviruses and some DNA viruses, the phosphorylation status of SAMHD1 has been implicated in modulating its antiviral activity. For example, phosphorylation of SAMHD1 at T592 suppressed SAMHD1-mediated restriction of HIV-1 replication in monocytes and macrophages [23]. However, restriction of HSV-1 in macrophages was shown to occur independently of T592 phosphorylation [24]. During HCMV infection, SAMHD1 T592 is rapidly phosphorylated by cellular kinases CdK2, and by the viral kinase pUL97, and phospho-SAMHD1 was shown to relocalize to the cytoplasm, leading the authors to speculate that inactivation and cytoplasmic delocalization by T592 dephosphorylation represents a novel mechanism of HCMV-mediated immune evasion [4]. The ability of viral-encoded kinases to target SAMHD1 for phosphorylation and thereby inhibit its dNTPase activity has been proposed to be a common feature of beta- and gamma-herpesviruses [25].
Recent evidence indicates that in addition to retroviruses, SAMHD1 can also restrict other RNA viruses by mechanisms that are distinct from those described for DNA viruses and retroviruses. For example, An et al.. reported SAMHD1-mediated restriction of Flaviviridae family members, including hepatitis C virus (HCV), Japanese encephalitis virus (JEV), and dengue virus (DENV), via downregulated expression of the sterol regulatory element-binding protein 1 (SREBP1), thereby reducing fatty acid synthesis and lipid droplet formation during infection [26]. SAMHD1 has also been reported to restrict seasonal A(H3N2) [27] IAV, as well as highly pathogenic avian influenza (HPAI) A(H5N1) in A549 cells, although the underlying mechanisms associated with the antiviral activity remain unclear [27, 28]. For HPAI A(H5N1), some evidence has suggested that T592 phosphorylation may contribute to SAMHD1-mediated restriction in A549 cells [28].
In addition to epithelial cells, macrophages are a major cellular target of IAV infection in the respiratory tract and play a key role in shaping disease pathogenesis (reviewed in [29]). Moreover, SAMHD1 is known to act as a restriction factor in macrophages for other viruses, including HSV-1 [20]. Whether SAMHD1 contributes to the control of IAV or other RNA respiratory viruses in macrophages has not yet been examined. In this study, we used PMA-differentiated (d)THP-1 cells as a macrophage model to assess the impact of SAMHD1 overexpression and SAMHD1-knockout (KO) with doxycycline (DOX)-inducible re-expression on the restriction of seasonal IAV strains, and used A549 airway epithelial cells as a comparative model. Moreover, HSV-1 was included as a control to ensure the validity of both the SAMHD1 knockout and inducible overexpression systems in our studies.
Methods
Cells and viruses
Human monocytic THP-1 (ATCC TIB-202) cells and Madin-Darby canine kidney (MDCK) cells (ATCC CCL-34) were maintained and passaged in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum (FCS, Gibco, Thermo Fisher Scientific), 2 mM L-glutamine (Gibco), non-essential amino acids (NEAA, Gibco), 0.55% (v/v) sodium bicarbonate (Gibco), 20 mM HEPES (Gibco), 200 Units (U)/mL penicillin (Gibco), and 200 µg/mL streptomycin (Gibco). Unless indicated, THP-1 were seeded and terminally differentiated (dTHP-1) in 25 ng/ml of PMA in culture media for 24 h prior to use in experiments. Lung carcinoma A549 (CCL-185) cells were maintained in Ham’s F-12K (Kaighn’s) Medium (Gibco) supplemented with 10% (v/v) FCS, and supplements as described above. Human embryonic kidney (HEK) 293T (ATCC CRL-3216), human epithelial type 2 (Hep2), and African Green monkey kidney Vero cells (CSL, Parkville, Australia) were maintained and passaged in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% (v/v) FBS and supplements as described above. All cell lines were cultured at 37°C in a humidified incubator with 5% (v/v) CO_2_.
Viruses
Herpes simplex virus type 1 (HSV-1, KOS strain) and a recombinant HSV-1 KOS virus with a green fluorescent protein (HSV-1-GFP) at the TK locus under the control of an HCMV promoter [30] (a kind gift from Prof. Francis Carbone, The University of Melbourne, Australia), we used in this study. Viruses were propagated in Vero cells, and titers of infectious virus were determined on Vero cells by standard plaque assay and expressed as plaque-forming units (PFU)/mL. The influenza A virus (IAV) strains used in this study were A/Udorn/307/72 (A(H3N2); Ud/72), A/Brazil/11/78 (A(H1N1); Braz/78), A/Newcastle/65/2015 (A(H1N1pdm); Newcastle/15), A/New York/55/2004 (A(H3N2); NewYork/04) and A/Anhui/1/2005 (A(H5N1); Anhui/05), as well as influenza B virus (IBV) strain B/Malaysia/2506/2004 (Malaysia/04). Influenza viruses were obtained from the WHO Collaborating Centre for Reference and Research on Influenza (WHO CCRRI), Melbourne, Australia. Titers of infectious influenza virus were determined by standard plaque assay, TCID_50_ assay [31], or by Virospot (VS) immunostain assay on MDCK cells, as described [32] and expressed as plaque-forming units (PFU)/mL, TCID_50_/mL, or VS/mL, respectively. Human parainfluenza virus 3 (hPIV-3, strain C243, ATCC VR-93) was propagated and titrated using Hep2 cells, and human metapneumovirus (HMPV, strain CAN-1993/87, a kind gift from Prof. Kirsten Spann, Queensland University of Technology, Australia) was grown and titrated in LLC-MK2 cells. Titres of infectious hPIV3 and HMPV titres were determined by VS assay using Hep2 cells and titres were expressed as VS/mL [32].
CRISPR/Cas9 knockout of endogenous SAMHD1
Wild type (WT) and SAMHD1-knockout (KO) THP-1 cells kindly provided by Professor Thomas Gramberg (University of Erlangen-Nuremberg, Germany) were cultured in the presence of 100 µg/L G418 and 2.5 µg/mL puromycin [33]. SAMHD1-KO A549 cells were generated via CRISPR/Cas9 endonuclease system using SF Cell Line 4D-Nucleofector™ X Kit (Lonza). A549 cells were seeded, cultured for 24 hr, and then resuspended in a final concentration of 104 pmol Alt-R^®^ S.p. Cas9 Nuclease V3 (IDT) and 180 pmol of two gRNAs targeting SAMHD1 (sgRNA1: 5’-GGGACGCUUGGAGGGCUGCU-3’, sgRNA2: 5’-UCGCAACGGGGACGCUUGGA-3’; Synthego), or scrambled guides obtained from Synthego. Cells were electroporated in Nucleocuvette™ Strip (Lonza) using pre-programmed settings (CM-130), recovered and expanded and then single cell cloning was performed by limiting dilution. SAMHD1-KO clones were validated via genomic sequencing (AGRF, Peter MacCallum Cancer Center, Melbourne, Australia) and western blot (see below).
To generate SAMHD1-KO monocyte-derived macrophages (MDM), peripheral blood mononuclear cells (PBMC) were isolated from buffy coats and enriched for CD14^+^ monocytes using human CD14 Microbeads (Miltenyi Biotec). Cells were then cultured in RPMI 1640 containing 100 U/mL human recombinant M-CSF (ImmunoTools) and supplemented with 10% FCS (Thermo Fisher Scientific), 2 mM Na-pyruvate (Gibco), NEAA (Gibco), and 200 U/mL penicillin-streptomycin (Gibco). After 2 days in culture, the cells were detached using 2 mM EDTA (Gibco) in PBS. After 2 washes with PBS, 1 million cells were resuspended in 10 µL buffer T, containing 1.5 µM Alt-R Cas9 (IDT), 1.8 µM Alt-R CRISPR-Cas9 crRNA: tracrRNA duplex (SAMHD1-KO crRNA sequences: 5’-GTGTATCAATGATTCGGACG-3’ and 5’-TCTTCGATACATCAAACAGC-3’; CTRL control: Alt-R™ Cas9 Negative Control crRNA #1&2; IDT), and 1.8 µM Electroporation Enhancer (IDT). Cells were electroporated using a Neon transfection system (Thermo Fisher) with HBSS as electrode buffer, using the following program: 2200 V, 1 pulse, and 15 msec width. After electroporation, cells were resuspended and cultured in culture media containing 100 U/mL M-CSF, with the media changed every two days. At day 5 after electroporation, some cells were lysed for western blot and genomic DNA extraction for Sanger sequencing, and the rest of the cells were plated into 24-well tissue culture plates in culture media containing M-CSF, cultured overnight and then infected with IAV (see below).
Generation of cell lines with DOX-inducible protein expression
To generate cells with DOX-inducible SAMHD1 expression, a two-step cloning strategy was used. Condon-optimized SAMHD1 (Accession number NP_056289.2) with a C-terminal FLAG-tag was synthesized as a ‘geneblock’ (GeneArt String DNA fragments; Invitrogen). The coding sequence was cloned into the pTRE-Tight plasmid vector, then subsequently cloned into pFUV1-mCherry lentivirus transfer plasmid (kindly provided by Professor Marco Herold, Olivia Newton-John Cancer Research Institute, Melbourne, Australia) [34]. Site-directed mutagenesis was also performed to generate T592A and T592D mutants of SAMHD1 in the pFUV1-mCherry lentivirus transfer plasmid. Control (CTRL) plasmid expressing cytoplasmic hen egg ovalbumin lacking a C-terminal FLAG tag [35] was also prepared. Lentivirus was produced by co-transfecting packaging plasmids pMDL (0.25 µg), pRSV-REV (0.12 µg), pMD2.G (0.15 µg), and pFUV1-mCherry-SAMHD1 or -CTRL (0.49 µg) transfer plasmids into HEK 293 T cells using Lipofectamine 2000 (Invitrogen). Lentivirus supernatant was harvested after 48 h post-transfection and was used to transduce SAMHD1KO A549 and THP-1 cells in media supplemented with 1 µg/mL polybrene, as described above. Lentiviral transduced cells were sorted 72 h later based on mCherry^+^ cells using a BD FACS Aria III cell Sorter (BD Biosciences) and expanded for use.
Flow cytometry for detection of intracellular FLAG expression and HSV-1- or IAV-infected cells
A549 or THP-1 cell lines with DOX-inducible protein expression were seeded and cultured overnight. To induce SAMHD1 expression, cells were treated with 1 µg/mL of doxycycline (DOX, Sigma-Aldrich) for 24 h at 37°C (+ DOX), while uninduced cells were cultured in media alone (-DOX). Cells were detached and stained with fixable viability dye eFlour 450 (eBioscience). Cells were then fixed with 4% (v/v) paraformaldehyde (PFA) in PBS, permeabilized with 0.5% (v/v) Triton X-100, and stained with anti-FLAG-APC mAb (Clone L5, Biolegend). Cells were acquired using a LSR Fortessa Flow Cytometer (BD Bioscience) and analyzed using FlowJo analysis software version 10.9.0.
HSV-1- or IAV-infected cells were detached, washed, and stained with fixable viability dye eFluor 780 (eBioscience). Cells were then fixed with 4% (v/v) PFA in PBS, permeabilized with 0.5% (v/v) Triton X-100, and stained with (i) a mouse mAb specific for HSV-1 ICP4 (Abcam), followed by donkey anti-mouse IgG conjugated to Alexa Fluor 647 (Invitrogen), or (ii) IAV NP-FITC (clone 431; Abcam). Cells infected with recombinant HSV-1-GFP were stained with fixable viability dye eFluor 780 (eBioscience), fixed in 4% (vol/vol) PFA and then washed and analysed by flow cytometry. Samples were acquired and analysed as described above.
Confocal microscopy for detection and localisation of DOX-inducible SAMHD1
FLAG-tagged SAMHD1 protein expression in A549 and dTHP-1 cells was visualised by confocal microscopy. Cells were seeded onto coverslips (Thermo Scientific), cultured overnight in 24-well tissue culture plates, and then cultured for 24 h in the presence (+ DOX) or absence (-DOX) of 1 µg/mL DOX. After 24 h DOX-induction, cells were fixed in PBS containing 4% (v/v) PFA and permeabilized in PBS containing 5% (w/v) BSA, 5% (v/v) FCS, and 0.1% (v/v) Triton X-100. Cells were stained with purified anti-FLAG antibody (Clone L5, Sigma-Aldrich), followed by goat anti-rat immunoglobulin AlexaFluor 647 (Invitrogen). Endoplasmic reticulum (ER) was stained with an anti-calnexin rabbit polyclonal antibody (Abcam), followed by AlexaFluor 488 anti-rabbit IgG (Invitrogen), and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) before mounting with ProLong Gold (Life Technologies). Images were acquired using Zeiss LSM 780 and analyzed using Fiji ImageJ Software (Version 2.1.0/1.54f).
Western blot for detection of endogenous or DOX-inducible SAMHD1
Cells cultured for 24 h in the presence (+ DOX) or absence (-DOX) of 1 µg/mL DOX were lysed using RIPA lysis buffer (1% (v/v) Triton-X-100, 150mM NaCl, 1% (w/v) Na deoxycholate, 0.1% SDS (v/v), and 50 mM Tris (pH 7.5)) supplemented with protease inhibitors (complete Mini Protease Inhibitor Cocktail; Roche, Basel, Switzerland) to generate whole cell lysate. Cell lysates were incubated with 5x SDS-sample buffer (50 mM Tris, 0.05% (w/v) bromophenol blue, 2% (v/v) sodium dodecyl sulfate (SDS), and 20 mM dithiothreitol (DTT)) at 95 °C for 15 min and then separated by gel electrophoresis using a 10% (v/v) SDS-polyacrylamide gel. Samples were subsequently transferred onto a PVDF membrane and incubated in blocking buffer (5% (w/v) BSA, 0.1% Tween-20 (Sigma-Aldrich) in PBS) for 1 h at room temperature (RT). Membranes were probed with anti-SAMHD1 mAb (Clone OTI3F5, Thermo Fisher; Cell Signaling, Cat#49158), anti-phospho-SAMHD1 (Thr592) rabbit pAb (Clone D7O2M, Cell Signaling), anti-calnexin rabbit pAb (Abcam, CAT#22595), or anti-β-actin rabbit mAb (LI-COR) overnight at 4 °C. After washing with PBS containing 0.05% (v/v) Tween-20, the membrane was probed with appropriate secondary antibodies, namely rabbit anti-mouse IgG horseradish peroxidase (HRP, DAKO), donkey anti-rabbit IgG HRP (Abcam), goat anti-rabbit IgG HRP (Bio-Rad), or AlexaFluor 647 anti-rabbit IgG (Invitrogen), for 2 h at RT. After washing, bound antibodies were detected by fluorescence detection or chemiluminescence detection using Amersham Imager 800 and analysed using Fiji ImageJ software.
Virus infection assays
Cells seeded into 24-well tissue culture plates were cultured overnight at 37°C in 5% (v/v) CO_2_ and then used immediately for infection or cultured an additional 24 h in the presence (+ DOX) or absence (-DOX) of 1 µg/mL. For infection assays, cells were washed once with serum-free media and then incubated for 1 h at 37°C with virus diluted in serum-free media to the indicated multiplicity of infection (MOI). After washing, cells were incubated in serum-free media at 37°C in the presence or absence of DOX for various times. For some IAV/IBV infections, 0.5 µg/mL of TPCK trypsin (Worthington Biochemical) was added to cell culture media immediately after infection to promote multicycle virus growth. At 2, 24, or 48 h post-infection (hpi), cell supernatants were collected, clarified by centrifugation, and titres of infectious virus were then determined by plaque, TCID50, or VS assays (described above) with titres expressed as PFU/mL, TCID50/mL, or VS/mL, respectively.
SiRNA knockdown of endogenous SAMHD1
Cells seeded into 24-well tissue culture plates were cultured overnight at 37°C, and then the media was replaced with OPTIM (Gibco) containing 5% FCS. Non-targeting control (NTC, DHA-D-001910-10−05 Accell Non-targeting, Dharmacon™ - Horizon Discovery) or SAMHD1 siRNA (siGENOME SMARTpool, Dharmacon™ - Horizon Discovery) were mixed with serum-free OPTIM and Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher) for 5 min at RT. After incubation, 50 nM of siRNA/LipoRNAiMAX was then added to cells and incubated at 37°C in 5% (v/v) CO2. After 48 h, cells were (i) lysed for extraction of mRNA for qRT-PCR analysis to determine knockdown efficiency, or (ii) infected with IAV or HSV-1 at the MOI indicated and analysed as described above.
Detection of endogenous or DOX-inducible SAMHD1 mRNA expression by qPCR
To detect endogenous SAMHD1 mRNA expression, total RNA was extracted using a RNeasy minikit (Qiagen) and then treated with RQ1 RNase-free DNase (Promega) to remove genomic DNA (gDNA). DNase-treated RNA was then converted to cDNA using SensiFAST cDNA synthesis kit (Bioline). SYBR green-based qRT-PCR (QuantStudio 7) was then performed to analyze the expression of endogenous SAMHD1 (forward 5’-GGATTACTAAAAACCAGGTTTCACAACT-3’ and reverse 5’-TGTCGTTCCATTCCTTTTTTTGA-3’) or DOX-inducible SAMHD1 (forward 5’- CCAAAAGTGCTGCTGGATGT-3’ and reverse 5’- TTCTTGCAGTACACCCGGAT-3’) relative to the housekeeping gene (HKG) GAPDH (forward 5’- TGAAGGTCGGAGTCAACGG-3’ and reverse 5’-GGCAACAATATCCACTTTACCAGAG-3’) using SensiFAST Probe Lo-ROX One-Step kit (Bioline) according to manufacturer’s instructions. The relative gene expression of SAMHD1 was normalized to GAPDH, then expressed as a fold change from mock/No DOX conditions using the 2^−ΔΔCT^ method [36].
Statistical analysis
Graphs and statistical analysis were performed using GraphPad Prism (version 10.1.0. GraphPad Software) and R-studio as indicated in the figure legends. For knockdown studies in primary donors, the standard deviations in the difference in viral titres between conditions (CTRL or KD) was calculated at each MOI. This information was then used alongside R’s pwr package to calculate the statistical power of the test [37]. For representative experiments, statistical analysis was performed using Two-way ANOVA. A mixed effects model (nlme package in R) was used to estimate the fixed effects when assessing differences between +DOX vs. -DOX while controlling for similarities between technical replicates across independent experiments.
Results
Impact of seasonal IAV infection on expression of SAMHD1 mRNA and protein in human epithelial and macrophage-like cells
SAMHD1 is a well-established restriction factor against DNA viruses and RNA retroviruses. However, several viruses have evolved mechanisms to counteract SAMHD1 by downregulating mRNA expression and/or degrading SAMHD1 protein. HCMV infection leads to potent downregulation of SAMHD1 mRNA and protein, as well as decreased SAMHD1 phosphorylation [38]. Recent studies have implicated the polymerase acidic (PA) protein of HPAI A(H5N1) viruses in repressing SAMHD1 promoter activity, thereby reducing SAMHD1 expression [28]. To assess whether seasonal IAV infection similarly affects SAMHD1, we infected PMA-differentiated THP-1 (dTHP-1) macrophages and A549 epithelial cells with the seasonal IAV strains A/Brazil/11/78 (Braz/78, A(H1N1)) or A/Udorn/307/72 (Ud/72, A(H3N2)) and quantified SAMHD1 mRNA (Fig. 1A) and protein (Fig. 1B) expression over time via qPCR and western blot, respectively. Following IAV infection of both cell types, levels of viral nucleoprotein (NP) and PA mRNA increased between 2 and 12 hpi and remained stable up to 48 hpi, confirming infection. In both cell types, SAMHD1 mRNA expression progressively increased during this period (Fig. 1A). Consistent with viral gene expression, both NP and PA protein levels increased between 2 and 12 hpi markedly and remained detectable up to 48 hpi in dTHP-1 and A549 cells, with a plateau or slight decrease at 48 hpi (Fig. 1B(ii)). In A549 cells, SAMHD1 protein expression was largely undetectable in mock conditions but increased progressively after infection up to 48 hpi, consistent with the gene expression data (Fig. 1Bii, lower panel). Levels of SAMHD1 protein increased only slightly in dTHP-1 following IAV infection, noting that there was substantial variability between experiments (Fig. 1Bii, upper panel). Together, these data show increased expression of SAMHD1 mRNA over time following seasonal IAV infection of dTHP-1 and A549 cells and at the protein level, suggesting that these IAV strains do not suppress SAMHD1 expression in these cell lines. Although the data in dTHP-1 cells were subject to large interexperiment variability, it still clearly demonstrated that the protein is present in dTHP-1 during IAV infection.
Fig. 1. Impact of IAV infection on SAMHD1 mRNA and protein expression in dTHP-1 and A549 cells. IAV strains Braz/78 (A(H1N1)) or Ud/72 (A(H3N2)) were used to infect dTHP-1 (MOI 1) or A549 (MOI 0.5) cells for 1 h, before cells were washed and cultured at 37°C. (A) Cells were lysed at 2, 12, 24, and 48 h post-infection (hpi), total RNA was extracted, and then qPCR was performed to determine the expression of viral NP and PA, as well as cellular SAMHD1, relative to the housekeeping gene GAPDH. Levels of mRNA were expressed as a fold change (log_10_) from mock conditions using the 2^−ΔΔCT^ method with a ratio of log_10_0 (i.e., a value of 1, indicating no difference in fold change from mock) indicated by the dashed line on each graph. Individual data points show mean results from each of three independent experiments (each performed in triplicate), and bars indicate the mean ± SEM across all experiments. (B) Expression of viral NP and PA, as well as cellular SAMHD1 protein, was determined by western blot using Abs specific to NP, PA, and SAMHD1, with β-actin included as a loading control. (i) Representative data from one of three independent experiments are shown. (ii) Densitometry was used to determine relative protein expression levels relative to the β-actin control. The ratio of SAMHD1, NP, and PA to β-actin in IAV-infected samples was then standardized to uninfected (mock) samples and expressed on a log_10_ scale. A ratio of 1 (log_10_ 0, i.e., no difference in fold change) is indicated by dashed lines. Individual data points show the mean ± SEM from three independent experiments
CRISPR/Cas9-mediated deletion of SAMHD1 does not modulate seasonal IAV replication in macrophages or in A549 epithelial cells
Given the well-established role of SAMHD1 in mediating antiviral activity against DNA viruses and retroviruses in macrophages [39], we next examined whether SAMHD1 influences the replication of seasonal IAV. To this end, we compared virus infection and growth in wild-type (WT) and SAMHD1-KO THP-1 cells (kindly provided by Prof. Thomas Gramberg, University of Erlangen-Nuremberg, Germany). As expected, SAMHD1 or phospho-SAMHD1 was undetected in SAMHD1-KO THP-1 cells irrespective of PMA-differentiation wvia western blot analysis (Fig. 2Ai). Moreover, we also confirmed higher levels of endogenous SAMHD1 levels in PMA-differentiated (+PMA) dTHP-1 cells compared to non-differentiated WT cells (Fig. 2Ai), as well as reduced levels of phosphorylated SAMHD1 (Supplementary Fig. 1), consistent with a previous finding [9]. Next, we assessed the antiviral activity of SAMHD1 against seasonal IAV and HSV-1 in dTHP-1 WT vs. SAMDH1-KO dTHP-1 cells. HSV-1, known to be restricted by SAMHD1 in macrophages [20, 24], was used as a positive control for SAMHD1 antiviral activity. Following HSV-1 infection, virus titres increased markedly between 2 and 48 hpi, consistent with viral growth. Moreover, while initial titres at 2 hpi were comparable between WT and SAMHD1-KO cells, HSV-1 replicated to significantly higher titres in SAMHD1-KO cells at 48 hpi (Fig. 2Aii, left panel), with a ~ 20-fold increase across multiple experiments in (Fig. 2Aii, right panel), consistent with an antiviral role for SAMHD1. In contrast, after IAV infection, both residual virus titres at 2 hpi and titres associated with virus growth at 24 hpi were not significantly different in WT and SAMHD1-KO cells (Fig. 2Aii, middle panel), which was also consistent across multiple experiments (Fig. 2Aii, right panel). These results demonstrate that endogenous SAMHD1 inhibits HSV-1 replication, but not seasonal IAV replication in dTHP-1 cells. Next, CRISPR-mediated knockdown (KD) of SAMHD1 in primary human MDMs confirmed genome editing in at least 75% of the reads via TIDE analysis of the Sanger sequencing and protein reduction via western blot (Supplementary Fig. 2). Following infection with either a high (Fig. 2Bi) or a low (Fig. 2Bii) MOI of IAV, we observed no significant differences in virus titres recovered from control (CRTL) versus SAMHD1-KD MDMs. Of note, with 5 donors, the study was sufficiently powered (> 99%) to detect a 0.5 log difference at a high MOI, but only had 72% power to detect a 1 log difference at a low MOI.
Compared to control-treated A549 cells, Silva et al.. reported that siRNA-mediated knockdown of SAMHD1 resulted in a very modest (a ~2-fold) enhancement in replication of an A(H3N2) IAV strain [40]. To recapitulate these results, we used qPCR to confirm efficient siRNA-mediated knockdown of SAMHD1 in A549 cells both prior to (0 hpi) and 24 h after infection (24 hpi) with a seasonal A(H3N2) strain (Fig. 2Ci). In contrast to the findings of Silva et al.., our results indicated a modest but significant reduction in virus titres recovered from A549 cells infected with an A(H3N2) IAV following knockdown of SAMHD1 (Fig. 2Cii), while knockdown of SAMHD1 resulted in enhanced titres of HSV-1 at 48 hpi (Supplementary Fig. 3). Given potential off-target effects associated with siRNA, we used CRISPR/Cas9 to generate SAMHD1-KO A549 cells and confirmed gene editing at the genomic level using Sanger sequencing (data not shown) and western blotting (Fig. 2D(i)). Since A549 cells only express low basal levels of SAMHD1 protein, WT and SAMHD1-KO cells were treated with IFN-α to induce SAMHD1 expression before western blot analysis. We observed upregulation of endogenous SAMHD1 protein in WT cells (consistent with its established role as an interferon-stimulated gene (ISG) [41]) but not in SAMHD1-KO cells (Fig. 2D(i)). WT and SAMHD1-KO A549 cells were then infected with A(H1N1) IAV or HSV-1, and virus titres were determined at 24 or 48 hpi, respectively. In contrast to our previous results in dTHP-1 (Fig. 2A(ii)), no significant differences were observed in titres of HSV-1 recovered from WT versus SAMHD1-KO A549 cells at 48 hpi (Fig. 2D(ii) left panel). There was no marked fold increase in virus titres recovered from SAMHD1-KO cells across multiple experiments (Fig. 2D(ii) right panel). Similarly, SAMHD1-KO did not result in significant differences in IAV titres recovered from A549 cells following infection with an A(H1N1) IAV strain. Together, these data indicate that in A549 cells, endogenous SAMHD1 does not act as a potent restriction factor against either HSV-1 or seasonal IAV.
Fig. 2. Endogenous SAMHD1 restricts HSV-1 in dTHP-1, but not A549 cells, but does not restrict seasonal IAV in either cell type. (A(i)) Wild type (WT) or SAMHD1-KO dTHP-1 cells cultured in the presence (+PMA) or absence (-PMA) of 25 ng/mL of PMA for 24 h to generate differentiated (d)THP-1 were analysed by western blot using an anti-SAMHD1 mAb or an anti-calnexin pAb as a loading control, followed by staining with donkey anti-mouse HRP (DAKO) and goat anti-rabbit HRP (Abcam) secondary Abs, respectively. (A(ii)) WT and SAMHD1KO dTHP-1 cells were infected with (ii) HSV-1 KOS (MOI 5), cultured for 48 h, and virus titres in clarified supernatants determined by plaque assay on Vero cells, or IAV Braz/78 (MOI = 1), cultured for 24 h in the presence of exogenous trypsin and virus titres determined by Virospot (VS) assay on MDCK cells. Representative data from 1 of 3 independent experiments (each performed in triplicate) are shown and the limit of detection is indicated as a dotted line. Fold changes in virus titre were determined by calculating fold change between 2 vs. 24/48hpi, and then comparing differences between KO and WT cells across multiple experiments. A ratio of 1 (i.e., no difference in fold change) is indicated by the dashed line. (B) CD14^+^ monocytes were purified from primary human PBMC and cultured with M-CSF at 37°C. On day 3, cells were electroporated with non-targeting control (NTC) or SAMHD1-specific guide RNAs and then returned to culture in the presence of M-CSF. On day 8, aliquots of cells were collected for genotyping via Sanger sequencing (Supplementary Fig. 2) or for western blot analyses, while remaining cells were re-seeded, cultured overnight, and then infected with IAV Braz/78 (MOI 3 or 0.01) for 1 h at 37°C. After washing, cells were cultured in the presence of exogenous trypsin, and virus titres in clarified supernatants collected at 24 or 48 hpi were determined. Data from 5 independent donors is shown, with each point representing the mean of triplicate samples from each donor. (C) A549 cells were treated with 50 nM NTC or SAMHD1 siRNA for 48 h, then infected with Ud/72 (A(H3N2), MOI 0.01) for 1 h, washed and incubated at 37°C. (i) Cells were lysed before (0 hpi) and 24 h after infection (24 hpi), total RNA was extracted, and then qPCR was performed to determine the expression of endogenous SAMHD1. Statistical analysis for qPCR data was performed using a Two-way ANOVA. (ii) Virus titres in clarified cell supernatants collected at 24 hpi were determined via plaque assay on MDCK cells. Representative data from one of two independent experiments are shown. (D(i)) A549 WT or SAMHD1-KO cells treated with 1000 U/mL of IFN for 24 h were analysed by western blot for the detection of endogenous SAMHD1 with calnexin as a loading control. (ii) WT and SAMHD1KO dTHP-1 cells were infected with (ii) HSV-1 KOS (MOI 0.01), cultured for 48 h and virus titres in clarified supernatants determined by plaque assay on Vero cells, or IAV Braz/78 (MOI = 0.01), cultured for 24 h in the presence of exogenous trypsin and virus titres determined by plaque assay on MDCK cells. Representative virus titre data from 1 of 3 independent experiments is shown and the limit of detection is indicated as a dotted line. Results are also expressed as the fold change between WT and KO cells across multiple experiments with a ratio of 1 (i.e., no difference in fold change), as indicated by the dashed line. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. For donor-derived data, power calculations were done using R as described in the Methods
Generation of human epithelial and macrophage-like cells with doxycycline-inducible expression of SAMHD1
Next, we used SAMHD1-KO THP-1 and SAMHD1-KO A549 cells to generate cell lines with doxycycline (DOX)-inducible overexpression of FLAG-tagged SAMHD1 (KO + SAMHD1) or cytoplasmic hen egg ovalbumin lacking a FLAG tag (KO + CTRL) as an irrelevant protein control. Cells were cultured in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX, for 8–24 h and then fixed, permeabilised, and stained with an anti-FLAG mAb prior to analysis by flow cytometry. In contrast to KO + CTRL cells, culture of KO + SAMHD1 cells for 8 h in the presence of DOX resulted in upregulation of FLAG-tagged SAMHD1 in dTHP-1 (Fig. 3A(i)) and A549 cells (Fig. 3A(ii)), and levels were further enhanced at 24 h. Therefore, cells were cultured for 24 h in the presence or absence of DOX (+/-DOX) in all subsequent experiments. Next, a western blot was used to confirm expression of a DOX-inducible SAMHD1 protein of ~ 72 kDa in lysates from dTHP-1 (Fig. 3B(i)) or A549 cells (Fig. 3B(ii)). To understand the relationship between expression levels of endogenous (-IFNα vs. + IFNα) and DOX-inducible SAMHD1 (-DOX vs. +DOX), we used qPCR to assess relative mRNA expression levels, noting that different qPCR primer sets were used to detect endogenous or inducible SAMHD1. When calculating the relative expression, defined as the difference in Ct values of endogenous SAMHD1 relative to GAPDH (2^−ΔCt^) [36], we observed that dTHP-1 cells expressed higher basal levels (-IFNα) of SAMHD1 mRNA than A549 cells (Fig. 3C(i), left panel). Despite these differences, mRNA levels increased significantly in both cell types following IFNα treatment. In KO + CTRL and KO + SAMHD1 cells, +DOX conditions significantly increased SAMHD1 levels in KO + SAMHD1 cells but, as expected, not in KO + CTRL cells, demonstrating that the DOX-inducible promoter was functional (Fig. 3C(i), right panel). When using the 2^−ΔΔCt^ method to calculate the fold change of test relative to control conditions (i.e. -IFNα to + IFNα or -DOX to +DOX for endogenous or DOX-inducible SAMHD1, respectively), and then normalising to GAPDH [36]. We observed a markedly higher induction of endogenous SAMHD1 in A549 compared to dTHP-1 cells in response to IFNα (Fig. 3C(ii), left panel), consistent with low-level expression in A549 cells in mock conditions. Moreover, the fold increase of DOX-induced SAMHD1 was markedly higher (~ 1000-fold relative to -DOX) than the induction of endogenous SAMHD1 in response to IFNα treatment (2–6 fold relative to -IFNα) in both dTHP-1 and A549 cells.
Fig. 3. Characterisation of SAMHD1-KO A549 and THP-1 cells with doxycycline-inducible expression of SAMHD1. SAMHD1-KO THP-1 or A549 cells, as well as SAMHD1-KO with DOX-inducible expression of either FLAG-tagged SAMHD1 (KO + SAMHD1) or an irrelevant intracellular control protein lacking a FLAG tag (KO + CTRL), were cultured in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX. (A) Cells cultured for 8–24 h were fixed, permeabilised and stained with an anti-FLAG mAb prior to analysis by flow cytometry. Representative histograms are shown. (B) Cells cultured for 24 h were lysed, subjected to SDS-PAGE and then analysed by western blot using an anti-SAMHD1 mAb or an anti-calnexin pAb as a loading control, followed by staining with donkey anti-mouse HRP (DAKO) and goat anti-rabbit HRP (Abcam) secondary Abs, respectively. (C) To examine expression of endogenous or DOX-inducible SAMHD1 expression (i) parental dTHP-1 and A549 cells were cultured in the presence (+ IFNα) or absence (-IFNα) of 1000 U/mL recombinant IFNα, or (ii) dTHP-1/A549 KO + CTRL or dTHP-1/A549 KO + SAMHD1 cells were cultured in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX. After 8 h in culture, cells were lysed, and total mRNA was extracted for qPCR using specific primers for the detection of endogenous or DOX-inducible (codon-optimised) SAMHD1. (i) Relative expression of endogenous SAMHD1 relative to housekeeping gene (HKG) GAPDH (left panel) or DOX-inducible SAMHD1 relative to GAPDH (right panel) and (ii) fold change in endogenous SAMHD1 between -IFNα and +IFNα cells (left panel) or between -DOX and + DOX cells (right panel) with a ratio of 1 (i.e., no difference in fold change) indicated by the dashed line. Representative data from 1 of 2 independent experiments are shown. For (i), statistical analysis was performed using an unpaired Student’s t-test. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (D) dTHP-1/A549 KO + CTRL or dTHP-1/A549 KO + SAMHD1 cells were cultured for 24 h in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX. Cells were then fixed, permeabilized, and stained with primary antibodies to FLAG (to detect DOX-induced SAMHD1) or calnexin (ER marker), followed by appropriate secondary antibodies, as well as with DAPI to detect cell nuclei. Cells were analysed using Zeiss Confocal LSM 780 microscopy at 63X magnification
To examine the cellular localisation of DOX-induced SAMHD1, cells cultured for 24 h +/- DOX were analysed by confocal microscopy. Following culture in +DOX conditions, FLAG-tagged SAMHD1 was detected predominantly in the nucleus of dTHP-1 (Fig. 3D(i)) and A549 (Fig. 3D(ii)) cells, consistent with the cellular localisation reported for endogenous SAMHD1 [42]. Of note, no FLAG signal was observed in any cell type cultured under -DOX conditions (Supplementary Fig. 4). Together, these data confirm the generation of macrophage- and epithelial cells with DOX-inducible expression of FLAG-tagged SAMHD1 protein.
DOX-inducible SAMHD1 does not inhibit HSV-1 entry but does inhibit HSV-1 replication in human macrophage-like or epithelial cells
We next assessed the impact of DOX-inducible SAMHD1 overexpression on HSV-1 replication in dTHP-1 and A549 cells. First, dTHP-1 cells cultured +/- DOX, for 24 h, were infected with HSV-1 (KOS) then, at 5 hpi, cells were fixed, permeabilised and stained for expression of intracellular HSV-1 infected cell protein 4 (ICP4), an immediate early gene expressed prior to viral genomic replication [43]. The gating strategy and representative dot plots are shown in Supplementary Fig. 5A(i/ii). For dTHP-1 KO + CTRL and KO + SAMHD1 cells, we did not observe any reduction in the percentage (Fig. 4A(i), left panel) or the geometric mean fluorescence intensity (gMFI) of ICP4^+^ cells (Fig. 4A(i), right panel) after culture in +DOX vs. -DOX conditions. Rather, culture +DOX resulted in a modest, but significant, increase in the percentage of ICP4^+^ cells at 5 hpi, suggesting that DOX enhanced HSV-1 infection in a manner independent of transgene expression, as has been described previously [44]. Despite this, when virus titres were determined at 48 hpi, we observed that infection in the presence of DOX-inducible SAMHD1 resulted in potent inhibition of HSV-1 replication in dTHP-1 cells (Fig. 4B(i), left panel) which was consistent across multiple experiments (Fig. 4B(i), right panel) and in line with previous published data on HSV-1 in macrophages [24].
Fig. 4DOX-induced SAMHD1 restricts replication of HSV-1, but not of IAV in dTHP-1 and A549 cells. dTHP-1/A549 KO + CTRL or dTHP-1/A549 KO + SAMHD1 cells were cultured for 24 h in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX. (A) To investigate the impact of DOX-induced SAMHD1 on the early stages of HSV-1 infection (i) dTHP-1 cells were infected with HSV-1 KOS (MOI = 5), fixed at 5 hpi, permeabilized and then stained with an anti-ICP4 mAb, followed by anti-mouse Alexa Fluor 647 secondary antibody and analysed via flow cytometry. The mean percentage of GFP^+^ cells (left panel) and the geometric mean fluorescence (gMFI) of GFP^+^ cells from triplicate samples are shown (right panels). Data from one of two independent experiments are shown. (ii) A549 cells were infected with HSV-1-GFP (MOI = 1), fixed at 8 hpi and GFP expression was determined by flow cytometry. The percentage of GFP^+^ cells (right panels) and the gMFI of GFP^+^ cells (left panel) of triplicate samples from 1 of 2 independent experiments are shown. (B) To investigate the impact of DOX-induced SAMHD1 on the late stages of HSV-1 infection (i) dTHP-1 and (ii) A549 cells were infected with HSV-1 KOS at MOI 5 and 0.01, respectively. At 48 hpi, supernatants were collected and clarified and titres of infectious HSV-1 were determined by plaque assay on Vero cells. The limit of detection is shown as a dotted line. Representative virus titres from one experiment are shown (left panels), as well as pooled data from multiple experiments showing the fold change in virus titres between cells cultured in + DOX versus -DOX conditions (right panels, n = 3 for dTHP-1, n = 5 for A549). (C) To investigate the impact of DOX-induced SAMHD1 on the early stages of IAV infection (i) dTHP-1 cells or (ii) A549 cells were infected with IAV (Braz/78, A(H1N1), MOI 5, fixed at 8 hpi, then permeabilized and stained with a FITC-conjugated mAb to the viral NP and analyzed via flow cytometry. The mean percentage of NP^+^ cells (left panel) and the gMFI of NP^+^ cells from triplicate samples (right panels) from one of two independent experiments for each cell line are shown. (D) To assess the impact of DOX-induced SAMHD1 on the late stages of IAV infection (i) dTHP-1 and (ii) A549 cells were infected with IAV (Braz/78, A(H1N1), MOI 1) and cultured in the presence of exogenous trypsin. At 24 hpi, supernatants were collected and titres of infectious in clarified supernatants were determined by Virospot (VS). Representative virus titres from one experiment are shown (left panels) as well as pooled data from multiple experiments showing the fold change in virus titres between cells cultured in +DOX versus -DOX conditions (right panels, n = 3 for dTHP and A549 cells). Statistical analysis for representative virus titre data was performed using Two-way ANOVA and an unpaired Student’s t-test for fold change data between -DOX versus +DOX conditions. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
For A549 cells, we assessed the impact of DOX-inducible SAMHD1 on early events in HSV-1 infection using a recombinant HSV-1 virus with a disrupted thymidine kinase (TK) gene that expresses green fluorescent protein (GFP) under the control of the HCMV promoter [30]. Since GFP expression is independent of HSV-1 genomic replication, it serves as a reliable indicator of successful viral genome delivery into the nucleus, noting that this approach was not suitable for dTHP-1 cells due to autofluorescence interfering with the GFP signal (data not shown). Following culture for 24 h in +/- DOX, A549 cells were infected with HSV-1-GFP and then analysed by flow cytometry at 8 hpi. Gating strategy and representative dot plots are shown in (Supplementary Fig. 5A(iii)). For A549 KO + CTRL and KO + SAMHD1 cells, we did not observe any reduction in the percentage of GFP^+^ cells (Fig. 4A(ii), left panel) or in the gMFI of ICP4^+^ cells (Fig. 4A(ii), right panel) after culture in +DOX vs. -DOX conditions. Despite this, when virus titres were determined at 48 hpi, we observed that infection in the presence of DOX-inducible SAMHD1 resulted in significant inhibition of HSV-1 titres at 48 hpi (Fig. 4B(ii), left panel) and this was observed across multiple experiments (Fig. 4B(ii), right panel). Together, these data indicate that DOX-inducible SAMHD1 expression in dTHP-1 and A549 SAMHD1-KO cells does not inhibit early events in HSV-1 replication but does restrict titres of infectious HSV-1 released from virus-infected cells.
DOX-inducible SAMHD1 does not inhibit seasonal IAV entry or replication in human macrophage-like or epithelial cells
Given that DOX-inducible SAMHD1 restricts HSV-1 growth in dTHP-1 and A549 cells, we next assessed its ability to restrict seasonal IAV replication. To examine the early stages of IAV replication, dTHP-1 cells were cultured +/- DOX and then infected with Braz/78 (A(H1N1)). At 8 hpi, cells were fixed, permeabilised and stained for intracellular expression of newly-synthesized viral nucleoprotein (NP), with the gating strategy and representative dot plots shown in (Supplementary Fig. 5B(i/ii)). When analysed by flow cytometry, no significant differences were observed in the percentage (Fig. 4C(i), left panel) or gMFI (Fig. 4C(i), right panel) of NP^+^ cells cultured in +DOX vs. -DOX conditions. Furthermore, we observed no differences in virus titres recovered from dTHP-1 cells cultured in +DOX vs. -DOX conditions following infection with Braz/78 A(H1N1) (Fig. 4D(i), left panel) across multiple experiments (Fig. 4D(i), right panel). Similar results were obtained using a second IAV strain Ud/72 A(H2N3) (Supplementary Fig. 6A/B(i)). Thus, while DOX-inducible SAMHD1 expression in dTHP-1 SAMHD1-KO cells did inhibit the growth of HSV-1, it did not impact the entry or growth of seasonal IAV.
Next, A549 cells cultured +/- DOX were infected with Braz/78 (A(H1N1)), and the percentage of NP^+^ cells was determined at 8 hpi. Gating strategy and representative dot plots are shown in (Supplementary Fig. 5B(iii)). When analysed by flow cytometry, DOX-inducible SAMHD1 resulted in a modest, but significant, enhancement in the percentage (Fig. 4C(ii) left panel) and the gMFI (Fig. 4C(ii) right panel) of NP^+^ A549 cells, similar to results observed using HSV-1, and was also associated with a significant increase in the gMFI of NP^+^ cells. Moreover, when examining IAV titres we observed a general enhancement of viral titres in supernatants recovered from cells cultured under +DOX conditions, which was more pronounced in A549 cells expressing DOX-inducible SAMHD1 (Fig. 4D(ii) left panel), and this was observed across multiple experiments (Fig. 5B(ii) right panel). Similar results were obtained using a second IAV strain Ud/72 A(H2N3) (Supplementary Fig. 6A/B(ii)). Thus, while DOX-inducible SAMHD1 expression in A549 SAMHD1-KO cells inhibited the growth of HSV-1, it did not inhibit the growth of seasonal IAV strains.
Fig. 5. Inducible overexpression of SAMHD1 does not modulate the replication of human metapneumovirus (HMPV) or parainfluenza virus type 3 (hPIV-3) in dTHP-1 or A549 cells. (A) Parental dTHP-1 and A549 cells were infected with HMPV and hPIV-3 at two different MOI (as indicated) for 1 h at 37°C, washed, and then returned to culture. At 2 and 48 hpi, supernatants were collected, clarified, and virus titres determined by Virospot assay on Hep2 cells. The dashed line represents the limit of detection and samples below this were assigned a value of 99 VS/mL for statistical analyses. (B/C) dTHP-1/A549 SAMHD1-KO with DOX-inducible overexpression of CTRL or FLAG-tagged SAMHD1 were cultured for 24 h in the presence (+ DOX) or absence (-DOX) of 1 µg/mL DOX. Cells were then infected with (B) HMPV (MOI 1 for dTHP-1, MOI 0.1 for A549), or (C) hPIV-3 (MOI 0.1 for dTHP-1, MOI 0.01 for A549) and, after washing and incubation at 37°C, virus titres in clarified supernatant were determined at 48 hpi via Virospot assay on Hep2 cells. Representative data from one of three independent experiments are shown. The limit of detection is shown as a dotted line. Statistical analysis of representative virus titre data was performed using a Two-way ANOVA. Data from all three experiments are also shown as fold change in virus titre between -DOX versus +DOX with a ratio of 1 (i.e. no change) indicated as a dashed line. For statistical analyses an unpaired Student’s *t-*test was performed. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Overexpression of SAMHD1 does not inhibit the replication of human metapneumovirus and parainfluenza virus type 3
We extended our study to examine two other human RNA respiratory viruses, namely human metapneumovirus (HMPV) and human parainfluenza virus type 3 (hPIV-3), for their sensitivity to restriction by DOX-inducible SAMHD1. We first confirmed that HMPV and hPIV-3 replicate productively in parental dTHP-1 and A549 cells, as evidenced by increased virus titres between 2 and 48 hpi (Fig. 5A). Following culture +/- DOX, cells with DOX-inducible SAMHD1 expression were infected with HMPV or hPIV-3, and titres of infectious virus were determined at 48 hpi. Similar to results with IAV, infection in the presence of DOX-induced WT SAMHD1 did not reduce titres of HMPV (Fig. 5B) or hPIV-3 (Fig. 5C) in dTHP-1 or A549 cells. Together, these results suggest that while inducible expression of SAMHD1 in dTHP-1 or A549 cells can inhibit growth of HSV-1, it does not inhibit seasonal influenza viruses, HMPV or hPIV-3 in either cell type.
Generation of human epithelial and macrophage-like cells with doxycycline-inducible expression of SAMHD1 phosphorylation mutants
In addition to modulating restriction of DNA viruses and RNA retroviruses, the phosphorylation status of SAMHD1 at residue 592 can also modulate the potency of SAMHD1-mediated restriction of other RNA viruses, including enteroviruses (by inhibiting viral protein interactions, and therefore virus assembly [45]), and hepatitis C virus (by inhibiting genomic replication, likely by limiting availability of cholesterol and lipids produced by de novo synthesis [26]). Given that we did not observe SAMHD1-mediated restriction of seasonal IAV, we hypothesized that modulation of SAMHD1 phosphorylation status might enhance its potency against these viruses, similar to that recently reported for SAMHD1-mediated restriction of A(H5N1) viruses in A549 cells [28]. To determine if altering the T592 phosphorylation status of SAMHD1 might enable antiviral activity against seasonal IAV, we generated two FLAG-tagged SAMHD1 mutants, a non-phosphorylation mutant (T592A) and a phosphomimetic mutant (T592D), inTHP-1 and A549 cells with DOX-inducible expression of each SAMHD1 mutant. Protein expression was confirmed by flow cytometry (Fig. 6A) and western blot (Fig. 6B(i)). We also used a phospho-specific SAMHD1 antibody to confirm phosphorylation at T592 in dTHP-1 (Fig. 6B(i), lower panel) or A549 cells (Fig. 6B(i)i, lower panel) expressing wild-type (WT) SAMHD1, but not for T592A and T592D mutant proteins. Of note, the phospho-specific antibody does not bind to the phosphomimetic mutant as it specifically recognises the phosphate group and epitope structure of the protein [46].
Fig. 6. Generation and characterization of THP-1 and A549 cells with DOX-inducible SAMHD1 phosphorylation mutants. THP-1/A549 SAMHD1-KO cells with DOX-inducible overexpression of FLAG-tagged SAMHD1 T592 or SAMHD1 T592A/D mutants were cultured in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX for 24 h. (A) Cells were then fixed, permeabilised, and stained with an anti-FLAG mAb prior to analysis by flow cytometry. Representative histograms are shown. (B) Cells were then lysed by RIPA buffer, and proteins resolved by SDS PAGE and analysed by western blot using antibodies specific for SAMHD1 or phospho-SAMHD1(T592), or with antibodies to calnexin as a loading control. Representative blots for (i) THP-1 and (ii) A549 cells are shown
SAMHD1 does not mediate antiviral activity against seasonal influenza viruses, regardless of phosphorylation status at residue 592
Previous studies have demonstrated that SAMHD1 T592 phosphorylation mutants retained their ability to restrict HSV-1 in macrophage-like cells [24]. Therefore, dTHP-1 cells cultured for 24 h in +/- DOX were infected with HSV-1, and virus titres were determined at 48 hpi. In addition to WT SAMHD1, DOX-inducible SAMHD1 T592A retained the ability to restrict HSV-1 replication in dTHP-1 (Fig. 7A(i), left panel). Although the T592D mutant showed evidence of modest restriction of HSV-1 in some experiments, this was not significant across multiple experiments (Fig. 7A(i), right panel). In A549 cells, DOX-inducible WT and T592A SAMHD1 both restricted HSV-1 replication, whereas the T592D mutant did not (Fig. 7A(ii). When cells were infected with seasonal IAV Braz/78 A(H1N1) and virus titres determined at 24 (Supplementary Fig. 7A/Bi) or 48 hpi, no evidence of virus restriction was observed in dTHP-1 cells (Fig. 7B(i)) or in A549 cells (Fig. 7B(ii)) expressing SAMHD1 with T592 phosphorylation mutations. Similar results were obtained following infection of dTHP-1 and A549 cells with IAV Udorn/72 (A(H3N2)) (Supplementary Fig. 7A/Bii). Given that our experiments to date utilised historical A(H1N1) and A(H3N2) strains from the 1970 s, we next tested more contemporary IAV strains, as well as an influenza B virus (IBV) strain for sensitivity to SAMHD1-mediated restriction. In these studies, dTHP-1 and A549 cells cultured +/- DOX were infected with A/Newcastle/2015 (A(H1N1pdm)), A/NewYork/2004 (A(H3N2)) or B/Malaysia/2004, and virus titres were determined at 48 hpi. However, DOX-induced overexpression of SAMHD1 or SAMHD1 phosphorylation mutants in (i) dTHP-1 or (ii) A549 cells did not result in significant reductions in titres for any of the virus strains tested (Fig. 7C). Together, these findings indicate that the phosphorylation status of SAMHD1 T592 does not enhance its ability to restrict seasonal IAV replication.
Fig. 7DOX-inducible SAMHD1 phosphorylation mutants do not restrict replication of contemporary seasonal influenza viruses in dTHP-1 or A549 cells. dTHP-1/A549 SAMHDI KO cells with DOX-inducible expression of CTRL, SAMHD1 or SAMHD1 mutants were infected with (A) HSV-1 KOS at (i) MOI 5 or (ii) 0.01, (B) IAV Braz/78 (A(H1N1)) at MOI 0.01, or (C) IAV NewCastle/2015 (A(H1N1pdm)), NewYork/2004 (A(H3N2)) or influenza B virus B/Malaysia/2004 at MOI 0.01 in the presence of exogenous trypsin to promote multicycle virus replication. Supernatants collected at 48 hpi were clarified and virus titres determined by plaque assay on Vero cells (HSV-1) or Virospot on MDCK cells (IAV). Representative data from one of three independent experiments are shown, and the limit of detection is shown as a dotted line. For all panels, statistical analysis of virus titre data was performed using a Two-way ANOVA. Data from three experiments are also shown as fold changes in virus titre between -DOX and +DOX, with a ratio of 1 (i.e., no change) indicated by a dashed line. An unpaired Student’s t-test for fold changes from +DOX to -DOX was performed. (C) Graphs show mean titres ± SEM (from triplicate samples) from three independent experiments. Statistical analysis for representative virus titre data was performed using a Two-way ANOVA. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Overexpression of SAMHD1 can restrict IAV A(H5N1) virus replication in human macrophage-like cells
In a recent study, expression of the viral NP in A(H5N1)-infected A549 cells was reduced in lysates from cells stably overexpressing the T592A, but not the T592D mutant of SAMHD1 [28]. However, this study did not directly assess the impact of the SAMHD1 phosphorylation mutants on virus growth. Thus, we assessed the ability of DOX-inducible WT and phosphorylation mutants of SAMHD1 to modulate the growth of an HPAI A(H5N1) virus in dTHP-1 and A549 cells. Following culture +/- DOX, cells were infected with A/Anhui/1/2005 (Anhui/05, A(H5N1)) and virus titres were determined at 24 hpi. For dTHP-1 cells, titres of A(H5N1) were significantly reduced in the presence of DOX-induced WT and T592A SAMHD1, but not in the presence of the T592D mutant (Fig. 8), consistent with previous data reporting that WT/T592A SAMHD1 inhibited viral NP expression more strongly than the SAMHD1 T592D mutant in A(H5N1)-infected A549 cells [28]. However, in our hands, we obtained no evidence of SAMHD1-mediated restriction of A(H5N1) replication in A549 cells, and virus titres in samples cultured in +DOX conditions were actually enhanced relative to -DOX conditions for each cell line tested. Consistent with our previous results (Fig. 4D(ii)), these data indicate that DOX treatment exerts a proviral effect on IAV replication in A549 cells. Together, these results did not confirm the previously reported effect of SAMHD1 expression on IAV A(H5N1) in A549 cells [28], although we cannot rule out a potentially confounding effect resulting from DOX application to the cells. However, it does provide evidence for a similar effect for DOX-induced SAMHD1 in macrophage-like cells, which is indeed dependent of T592 phosphorylation status.
Fig. 8DOX-inducible SAMHD1 and a SAMHD1 mutant lacking phosphorylation at residue 592 restrict A(H5N1) IAV replication in dTHP-1 cells. A549 and dTHP-1 SAMHD1-KO with DOX-inducible overexpression of CTRL, or FLAG-tagged SAMHD1, SAMHD1 T592A or SAMHD1 T592D were cultured for 24 h in the presence (+DOX) or absence (-DOX) of 1 µg/mL DOX. Cells were then infected with Anhui/05 (A(H5N1), MOI 0.01) for 1 h at 37°C, washed and cultured at 37°C in the presence of exogenous trypsin. At 24 hpi, supernatants were collected, clarified and titres of infectious virus determined by TCID_50_ assay on MDCK cells. Pooled data from two independent experiments were shown with distinct symbols, showing triplicate samples from each experiment. The limit of detection is shown as a dotted line. Statistical analysis was performed using a mixed-effects model with data from two experiments, as described in Materials and Methods. The p-values are indicated as follows: ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001
Discussion
Many studies have documented SAMHD1-mediated restriction of RNA retroviruses and different DNA viruses in non-dividing myeloid cells, including macrophages and dendritic cells (reviewed in [39]). Macrophages, along with airway epithelial cells, are the first cells to encounter IAV infection in the respiratory tract and play a key role in modulating IAV pathogenesis (reviewed in [29]). Therefore, in our current study, we investigated the ability of SAMHD1 to modulate influenza and other RNA respiratory viruses in macrophages, the cell type most commonly associated with SAMHD1-mediated restriction of DNA viruses (reviewed in [39]). Using dTHP-1 cells, we demonstrate that neither endogenous nor overexpressed SAMHD1 restricts seasonal IAV, IBV, HMPV or hPIV-3 in macrophages under conditions where restriction of HSV-1 replication was observed. Preliminary studies in primary human MDM also demonstrated that knocking out endogenous SAMHD1 did not modulate seasonal IAV growth, although further studies with additional virus strains are required to conclude this definitively. A more detailed examination of the expression and role of endogenous SAMHD1 in response to different seasonal IAV in physiologically-relevant primary macrophages, including alveolar macrophages, would also be of interest.
Previous studies have investigated the role of SAMHD1 in the restriction of seasonal and A(H5N1) IAV in A549 epithelial cells [27, 28]. Using siRNA-mediated knockdown of endogenous SAMHD1 in A549 cells, Silva et al.. reported a modest (~ 2-fold) enhancement in virus titres at 24 hpi and that knockdown also abolished CCL5 agonist-mediated inhibition of viral replication [27]. Subsequently, Zhao et al.. reported that knockdown of endogenous SAMHD1 or stable overexpression of SAMHD1 in A549 cells resulted in increased or reduced growth of a A(H5N1) strain, respectively [28]. Overexpression studies in A549 cells also indicated that the phosphorylation status of the T592 of SAMHD1 modulated expression of the viral NP during A(H5N1) infection, although its impact of restricting virus growth was not examined. The viral PA protein of an A(H5N1) strain has also been implicated in inhibiting SAMHD1 transcription by binding to IRF3 to downregulate the TBK1-IRF3 signalling pathway during infection [28]. Our current study has focused on a comparison between two distinct cell types relevant to the infection and replication of respiratory viruses, namely dTHP-1 cells (representing terminally differentiated macrophages) and A549 airway epithelial cells. These cells differ markedly in regard to lineage, proliferative capacity, and responses they elicit following infection with IAV and other respiratory viruses.
It is well established that terminally differentiated myeloid cells express high levels of SAMHD1, resulting in low levels of cellular dNTPs due to the triphosphohydrolase activity of the SAMHD1 protein [47]. This, in turn, is a key factor contributing to SAMHD1-mediated restriction of DNA viruses and retroviruses that require dNTPs for transcription of the viral genome [20, 47]. While HSV-1 served as an effective control to confirm SAMHD1-mediated restriction in macrophages, studies examining intracellular dNTP pools would provide additional confidence regarding the expression of functional SAMHD1 in our studies. Moreover, less is known regarding its ability to inhibit HSV-1 in different cell types and under a range of proliferative conditions. In addition to macrophages, it is well established that HSV-1 infects subsets of parenchymal cells, including epithelial cells, stromal cells, keratinocytes, and neurons (reviewed in [48]). Moreover, HSV-1 infection of the respiratory tract, including airway epithelial cells, has been associated with HSV-1-induced pneumonia in immunocompromised patients [49, 50]. Given the low basal levels of SAMHD1 in A549 cells and the importance of the dNTP triphosphohydrolase activity of SAMHD1 in mediating anti-HSV-1 activity in macrophages [24], it was not surprising that knockout of endogenous SAMHD1 in A549 cells did not result in enhanced HSV-1 replication (Fig. 2D(ii)). However, HSV-1 growth was significantly suppressed in A549 cells in the presence of DOX-induced SAMHD1, confirming the functionality of overexpressed SAMHD1. While we cannot directly compare SAMHD1 levels due to the different qPCR assays used, it is noteworthy that DOX-induced SAMHD1 expression (~ 800-fold higher between -DOX vs. + DOX) was much higher than IFNα-induced expression of endogenous SAMHD1 (~ 6-fold higher between -IFNα vs. +IFNα) in A549 cells (Fig. 3C). Furthermore, IFNα treatment upregulates not only endogenous SAMHD1 but also hundreds of additional ISGs whereas DOX treatment specifically upregulates FLAG-tagged SAMHD1, allowing for focused studies assessing the antiviral effects of this protein. Despite its antiviral activity against HSV-1, DOX-inducible SAMHD1 did not inhibit replication of any of the seasonal IAVs tested (Fig. 4D), IBV (Fig. 6A), HMPV (Fig. 6B), or hPIV-3 (Fig. 6C) in either dTHP-1 or A549 cells.
The mechanisms associated with SAMHD1-mediated restriction of HIV-1 and other retroviruses have been particularly well studied, highlighting its dNTPase activity as a critical determinant for restriction of HIV-1 [8, 12, 51]. However, other retroviruses, including HIV-2 and simian immunodeficiency viruses (SIVs), counteract SAMHD1-mediated virus restriction via expression of the viral accessory protein Vpx, which induces SAMHD1 protein degradation via the ubiquitin-proteosome system (UPS) [12]. Beyond retroviruses, SAMHD1 has also been reported to restrict other RNA viruses, including flaviviruses via mechanisms that are distinct from those associated with restriction of DNA viruses and retroviruses [26]. Specifically, knockout and overexpression approaches demonstrated SAMHD1-mediated restriction of HCV, Japanese encephalitis virus (JEV), and dengue virus 2 (DENV2) in hepatoma cells by downregulating expression of host genes essential for lipid bio-metabolic pathways and lipid droplets formation, both of which are required for flavivirus replication [26]. While dNTPase activity is relevant for SAMHD1-mediated restriction of many DNA viruses and retroviruses [47], this activity is likely to be less relevant to genomic replication of RNA viruses, which require cellular ribonucleoside triphosphates (rNTPs) rather than dNTPs. In addition to influenza viruses, we also tested the impact of DOX-induced SAMHD1 on HMPV and hPIV-3 replication, as these represent additional RNA viruses from the Pneumoviridae and Paramyxoviridae families, respectively [52], which are also associated with respiratory virus infections. Of interest, while replication of the viral genome occurs within the nucleus during influenza virus infection [53], HMPV and hPIV-3 replicate in cytoplasmic inclusion bodies [54, 55]. Despite reports that SAMHD1, although it localises predominantly within the nucleus [56] (Fig. 3D), can be detected in the cytoplasm [42], expression of DOX-inducible SAMHD1 in either dTHP-1 or A549 cells did not significantly impact the growth of either HMPV or hPIV-3.
Beyond retroviruses, addition RNA viruses such as enteroviruses (e.g. EV-71 [45]) and flaviruses (e.g. HCV [26]) have been reported to be sensitive to SAMHD1-mediated restriction. However, these viruses have a positive-sense non-segmented RNA genome (the IAV genome is negative-sense segmented RNA) and replicate exclusively within the cytoplasm (genomic replication of IAV occurs within the nucleus). Moreover, SAMHD1-mediated restriction of these viruses occurs via very different mechanisms, involving inhibition of VP1/VP2 viral protein interactions for EV-71, compared to limiting the availability of cholesterol and lipids produced by de novo synthesis for HCV. While lipid metabolism also plays a critical regulatory role during IAV infection (reviewed in [57]), it is likely that intrinsic differences in the steps of the virus replication cycle, the particular metabolic processes affected by SAMHD1 and/or the cell types studied are relevant to the lack of IAV restriction observed in our study. When considering flaviviruses, one study reported restriction of HCV, JEV, and DENV2 in hepatoma cells whereas an independent study identified SAMHD1 as a proviral factor for Zika virus (ZIKV), another flavivirus, when studied in a different cell type [58]. These findings highlight the need for caution when drawing broad conclusions regarding sensitivity to SAMHD1 restriction, as differences may be observed when viruses from the same family are studied under different experimental conditions. In terms of seasonal IAV, it is possible that one or more viral proteins might somehow antagonise the functional activity of SAMHD1, hence attenuating its ability to mediate IAV restriction. Further studies using pull-down assays coupled with mass spectrometry approaches could be used to identify potential interactions between SAMHD1 and specific IAV proteins.
Seasonal and HPAI IAVs interact differently with components of host innate immunity and therefore elicit distinct host responses. For example, IRF3 has been reported to activate IFNβ signalling downstream of a RIG-I-MAVS-TBK1-dependent pathway to increase IFN production during seasonal IAV infection (reviewed in [59]). IRF3 then binds to different ISREs promoters to induce ISG transcription, including SAMHD1, as IRF3 is known to bind to its IRSE promoter during infection [60]. In contrast, the viral PA protein of HPAI A(H5N1) and A(H7N9) viruses acts to suppress IRF3 phosphorylation, dimerization, and nuclear translocation, thereby inhibiting IFNβ signalling [61]. By inhibiting IRF3-dependent ISG activation [28], HPAI may also reduce SAMHD1 expression during infection, given that upregulation of SAMHD1 has been reported to be IRF3-dependent [60]. Restriction of HPAI A(H5N1) replication has previously been associated with the phosphorylation status at site T592 of SAMHD1, as A(H5N1) NP protein levels were reduced in A549 cells constitutively overexpressing SAMHD1 T592 or T592A; however, this inhibition was lost in cells expressing T592D [28]. Herein, we used a DOX-inducible system to assess the impact of SAMHD1 T592 phosphorylation on A(H5N1) replication in different cell types. In dTHP-1, expression of DOX-inducible WT (T592) and dephosphorylated (T592A) SAMHD1 resulted in a modest, but significant, inhibition of A(H5N1) growth which was not observed using the phosphomimetic SAMHD1 mutant (T592D) (Fig. 8), in seeming contradiction to this previous publication. However, it should be noted that a DOX-induced enhancement of A(H5N1) growth was observed in A549 cells expressing CTRL or SAMHD1 proteins (Fig. 8), and this effect may well have masked any inhibitory effects of SAMHD1 in our experiments. In future studies, systematic testing of specific clads of avian HPAI, as well as HPAI H7Nx and other avian viruses, would be of interest to determine if sensitivity to SAMHD1-mediated restriction might contribute to a species barrier limiting the introduction of avian IAV into humans, as described for human MxA (reviewed in [62]). Of note, in a previous study we reported a modest but reproducible proviral effect of DOX against some viruses in particular cell lines (e.g. IAV and HSV-1 in A549 cells and HeLa/Hep2 cells, respectively), whereas culture in DOX did not affect growth of other viruses (e.g. RSV in various cell lines) [44]. Other studies have reported that DOX itself inhibits replication of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) [63] and dengue virus [64], noting that this was not an issue in our study as we did not observe any antiviral effect of DOX itself in our CTRL cell lines. However, these findings do highlight the need for complementary approaches to when investigating whether a putative restriction factor does or does not mediate antiviral activity.
Our in vitro studies in human dTHP-1 and A549 cells demonstrate that neither endogenous nor DOX-inducible SAMHD1 mediates potent antiviral activity against seasonal IAV. Despite this, in future studies it would be of interest to examine the impact of SAMHD1 in modulating IAV infection in vivo using SAMHD1-KO mice. Murine (m)SAMHD1 has been reported to reduce cellular dNTP concentrations and to restrict retroviral replication in various mouse cells, including lymphocytes, macrophages, and DCs [18]. Moreover, MCMV infection of SAMHD1-KO mice resulted in enhanced virus replication in multiple organs, consistent with the fact that MCMV encodes the M97 kinase, which counteracts the antiviral function of SAMHD1 [65]. While SAMHD1 may not impact seasonal IAV replication effectively in vitro, the mouse model of influenza infection is well established and would be useful for studies to determine if SAMHD1 modulates other aspects of virus-induced inflammation and immunity in vivo.
Conclusion
Studies presented herein demonstrate that neither endogenous nor overexpressed SAMHD1 effectively restricted replication of A(H1N1), A(H1N1pdm), A(H3N2) seasonal IAVs or influenza B viruses (IBVs) in macrophage-like cells under conditions where SAMHD1-mediated restriction of HSV-1 was observed. Furthermore, endogenous SAMHD1 did not restrict replication of A(H1N1) seasonal IAV in primary human MDMs. Similarly, neither endogenous nor overexpressed SAMHD1 inhibited replication of seasonal IAV in A549 epithelial cells. Overexpression of SAMHD1 in macrophage-like or epithelial cells also did not inhibit replication of other human RNA viruses associated with respiratory disease, namely human metapneumovirus (HMPV) and human parainfluenza virus type 3 (hPIV3). However, our findings do suggest that SAMHD1 may contribute to the inhibition of A(H5N1) IAV replication in macrophage-like cells and that phosphorylation of SAMHD1 at T592 modulates this antiviral activity. Together, our data indicate that SAMHD1 does not function as a potent antiviral restriction factor against seasonal IAV, IBV, HMPV, or hPIV3 in macrophages, but may exhibit a subtype-specific effect against HPAI A(H5N1) viruses.
Supplementary Information
Supplementary Material 1.
Supplementary Material 2.
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