Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication

Jingting Zhang; Yunhan Wu; Yujie Wang; Penggang Liu; Kaituo Liu; Jing Sun; Pinghu Zhang; Xiaoquan Wang; Xiufan Liu; Xiulong Xu

doi:10.1016/j.isci.2024.110424

. 2024 Jun 29;27(8):110424. doi: 10.1016/j.isci.2024.110424

Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication

Jingting Zhang ¹, Yunhan Wu ¹, Yujie Wang ¹, Penggang Liu ¹, Kaituo Liu ², Jing Sun ¹, Pinghu Zhang ³, Xiaoquan Wang ^1,^4,⁵, Xiufan Liu ^1,^4,⁵, Xiulong Xu ^1,^5,^6,^∗

PMCID: PMC11301072 PMID: 39108727

Summary

Cellular cholesterol plays an important role in influenza A virus (IAV) endocytosis and replication. However, how IAV infection regulates cholesterol biosynthesis remains poorly understood. Here, we report that IAV infection activates SREBP2 and induces the expression of HMGCR, a rate-limiting enzyme in cholesterol synthesis pathway. SREBP2 deficiency suppresses IAV-induced HMGCR expression and virus replication. Mechanistically, IAV infection activates JAK2 and STAT3, inhibition of JAK2 and STAT3 activity by their inhibitors or by gene knockout downregulates IAV-induced SREBP2 and HMGCR expression and IAV replication, reduces the content of cellular cholesterol and virus binding to host cells. Exogenous cholesterol reverses the inhibitory effect of S3I-201 and STAT3 deficiency on virus replication. STAT3 or JAK2 overexpression increases the expression of SREBP2 and its downstream target genes, leading to increased IAV replication. These observations collectively suggest that STAT3 activation facilitates IAV replication by inducing SREBP2 expression and increasing cholesterol biosynthesis.

Subject areas: Genetics, Virology

Graphical abstract

Highlights

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IAV infection activates SREBP2 to upregulate HMGCR expression
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Activation of the JAK2-STAT3 axis increases SREBP2 expression in IAV-infected cells
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Inhibition of JAK2 and STAT3 suppresses IAV replication by blocking HMGCR expression
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STAT3 activation promotes IAV replication in part by enhancing cholesterol biosynthesis

Genetics; Virology

Introduction

Influenza A virus (IAV) is one of the most common pathogens that infect the respiratory tract.¹ Approximately 3–5 million patients infected with IAV worldwide develop severe pulmonary and systemic illness, which causes 250000–500000 deaths annually.¹ Patients with existing comorbidities such as diabetes and cardiovascular disease are at high risk for disease complications.²^,³ Nearly one-third of hospitalized patients infected with pandemic 2009 H1N1 virus are morbidly obese.⁴^,⁵^,⁶ Obesity prolongs hospitalization in the intensive care unit (ICU) and increases the need for invasive mechanical ventilation during influenza virus infection.⁶^,⁷ However, the factors that contribute to the increased disease severity in obese hosts remain incompletely understood. Dyslipidemia, a hallmark feature of obesity, is associated with immunosuppression and increased susceptibility to influenza virus infection.⁸^,⁹ Both circulating lipoproteins and cellular cholesterol metabolism can influence host immune responses and virus replication.⁸^,⁹ Louie et al.¹⁰ recently reported that mice fed with high cholesterol diet develop dyslipidemia characterized by increased levels of total serum cholesterol and display increased morbidity, compared to those fed with normal chow diet. Gao et al.¹¹ recently reported that due to increased cholesterol levels, mice deficient of apolipoprotein E exhibit enhanced IAV replication and severe disease pathology. Cholesterol appears to be a crucial factor in obese individuals that contributes to the severity of IAV infections.

Cholesterol is a main lipid component in the plasma membrane and cytosol and plays important roles in virus binding, endocytosis, trafficking, assembly, and budding.¹²^,¹³^,¹⁴^,¹⁵^,¹⁶ IAV replication requires host cells to provide a variety of nutrients such as amino acids, nucleic acids, carbohydrates, and lipids.¹⁷^,¹⁸ The envelope of IAV is derived from the cell membrane.¹³ Cholesterol accounts for 42% of the total lipid content of the virus.¹³^,¹⁹^,²⁰ Removal of cholesterol from the cell membrane or IAV envelope using methyl-β-cyclodextrin decreases the infectivity of IAV.²¹^,²² The HA and M2 proteins of IAV are modified by cholesterol to facilitate their trafficking and virus budding.¹²^,²³^,²⁴^,²⁵^,²⁶ Statin, a commonly prescribed anti-cholesterol drug, significantly reduces the mortality of both common and severe pneumonia in hospitalized patients.²⁷^,²⁸ Atorvastatin inhibits IAV-induced lipid droplet formation and virus replication.²⁹ Treatment with the cholesterol-lowering drug gemfibrozil extends the survival time of mice infected with H2N2 virus.³⁰ While emerging evidence suggests that virus infection can reprogram lipid biosynthesis to meet their nutritional needs,¹⁷^,¹⁸ how IAV regulates cholesterol metabolism remains poorly understood.

SREBP2 is a transcription factor that plays a key role in regulating cholesterol biosynthesis.²⁰^,³¹ When intracellular cholesterol is depleted, the SREBP2—SCAP (SREBP cleavage-activating protein) complex in the endoplasmic reticulum dissociates from the cholesterol-binding insulin-induced gene (INSIG) and then translocate to the Golgi apparatus.²⁰^,³¹ The precursor of SREBP2 (pSREBP2) is cleaved by the site-1 protease (S1P) and S2P proteases to generate the transcriptionally active nuclear SREBP2 (nSREBP2).²⁰^,³¹ nSREBP2 induces the transcription of cholesterol biosynthesis-related genes such as HMGCR, a rate-limiting enzyme that serves as the molecular target for numerous cholesterol-lowering drugs such as statin and atorvastatin.²⁰^,³¹ Other genes such as HMGCS (3-hydroxy-3-methylglutaryl-CoA synthase 1) and MVK (mevalonate kinase) are also subject to the transcriptional regulation by SREBP2.²⁰^,³¹ In addition, RORγ, an orphan nuclear factor, cooperates with SREBP2 to enhance the transcription of cholesterol biosynthesis-related genes and to promote cholesterol production.³² It has been increasingly recognized that SREBP2 activation is associated with virus replication. For example, Zika virus (ZIKV) infection activates SREBP2 to enhance cholesterol synthesis, inhibition of SREBP2 activity by its inhibitors or gene knockout suppresses ZIKA replication in dendritic cells.³³ The SREBP inhibitor AM580 suppresses fatty acid and cholesterol biosynthesis and the replication of MERS-CoV and IAV.³⁴ SARS-CoV-2 and human coronavirus OC43 (HCoV-OC43) infections upregulate SREBP2 target genes in Huh-7.5.1 cells to promote virus replication.³⁵ Recently, Teo et al.²⁴ reported that SREBP2 activation via the USP25-Erlin1/2 axis facilitates IAV replication. Whether IAV infection regulates SREBP2 expression remains largely unexplored.

STAT3 is a transcription factor that primarily functions to regulate inflammatory responses of host cells.³⁶^,³⁷ It has been well documented that STAT3 activation promotes virus replication.³⁶^,³⁷ However, whether IAV activates STAT3 to enhance IAV replication remains controversial. For example, Ampomah et al.³⁸ reported that the STAT3 inhibitor WP1066 inhibits IAV replication. In contrast, Liu et al.³⁹ recently reported that STAT3 knockdown increases H3N2 replication in A549 cells and in vivo in the lung tissues of IAV-infected mice. How STAT3 regulates virus replication remains ill-defined. Recent evidence suggests that STAT3 is involved in regulating cholesterol biosynthesis. The JAK2 inhibitor tofacitinib reduces the intracellular cholesterol content in macrophages.⁴⁰ Overexpression of STAT3, a transcription factor activated by JAK2, in mouse liver tissues significantly increases plasma cholesterol levels.⁴¹ Chen et al.⁴² recently reported that STAT3 enhances SREBP2 expression and cholesterol biosynthesis in triple-negative breast cancer. Here, we report that IAV infection leads to increased STAT3 phosphorylation and activation, and that STAT3 inhibition represses IAV replication. Mechanistically, STAT3 induces SREBP2 expression and enhances cholesterol synthesis. Our study unveils a previously unrecognized mechanism of STAT3 regulation of IAV replication.

Results

IAV infection induces SREBP2 activation

IAV replication requires host cells to supply a large quantity of cholesterol.¹⁷^,¹⁸ SREBP2 is a key transcription factor that regulates cholesterol biosynthesis.²⁰^,³¹ To determine if IAV infection activated SREBP2, we evaluated the relative levels of the inactive pSREBP2 and the active nSREBP2 in NL20 cells, a noncancerous human bronchial epithelial cell line, and 293T cells, a human kidney epithelial-like cell line. Two H5N1 (SY and CK10) and H1N1 (PR8) strains dose- and time-dependently lowered the levels of pSREBP2 but increased the levels of nSREBP2 and its downstream target gene HMGCR in NL20 (Figure 1A) and 293T cells (Figure 1B). Of note, HMGCR induced by PR8 virus infection was presented as a processed 55-kDa protein, which is also reported by others.⁴³ The viral NS1 protein was detected as the indicator of virus replication (Figure 1). H5N1 virus infection also activated SREBP1 in NL20 and 293T cells (Figures S1A and S1B), a transcription factor that primarily functions to regulate the expression of the genes involved fatty acid biosynthesis.

SREBP2 activation is required for IAV replication

We next investigated if the SREBP2 inhibitor affected IAV replication. Fatostatin, an SREBP-specific inhibitor, dose-dependently lowered the levels of the viral PB2, NP, and NS1 proteins (Figure 2A) and virus titers (Figure 2B) in NL20 cells infected with the H5N1 virus (SY) and the H1N1 virus (PR8) viruses. Fatostatin also dose-dependently decreased SREBP2 activation and HMGCR expression in NL20 cells infected with either SY or PR8 virus (Figure 2A). The EC₅₀ values of fatostatin for SY and PR8 viruses in NL20 cells are 0.3 and 1.2 μM, respectively. The CC₅₀ (cytotoxic to 50% of a population of cells) value of fatostatin for NL20 cells is 13.1 μM (Figure 2B). Inhibition of virus replication was not due to the cytotoxic effects of fatostatin since the selective index (S.I.) values for H5N1 (SY) and H1N1 (PR8) viruses in NL20 cells are both >10. Consistently, SREBP2 deficiency remarkably lowered the levels of three viral proteins and HMGCR (Figures 2C and 2D) as well as virus titers (Figures 2E and 2F) in the conditioned media of NL20 cells infected with SY and PR8 viruses. To rule out the possibility that decreased HMGCR expression in SREBP2 knockout cells was due to decreased virus replication, SREBP2-deficient cells were infected with much higher MOIs of H5N1 (SY) virus than wild-type cells to ensure equal virus replication. Under this setting, almost equal levels of viral proteins were detected in wild-type and SREBP2-deficient cells (Figure 2G). The protein levels of HMGCR and other cholesterol biosynthesis-related enzymes were much lower in IAV-infected SREBP2-deficient NL20 cells than that in the control cells (Figure 2G). The protein and mRNA levels of cholesterol biosynthesis-related enzymes in uninfected SREBP2-deficient NL20 cells were also much lower than that in wild-type cells (Figures 2G and 2H). Of note, SREBP2 knockout did not completely block the induction of HMGCR expression in IAV-infected NL20 cells (Figures 2C, 2D, and 2G), suggesting that IAV infection can induce HMGCR expression by other transcription factors such as SREBP1a and RORγ. SREBP2 overexpression increased the levels of three viral proteins (PB2, NP, NS1) and HMGCR (Figure 2I) as well as the virus titers in the conditional media of IAV-infected NL20 cells (Figure 2J). These observations collectively suggest that SREBP2 promotes IAV replication by enhancing the expression of cholesterol biosynthesis-related genes.

SREBP2 promotes IAV replication

(A) NL20 cells pretreated with the indicated concentrations of Fatostatin (Fato) for 8 h were infected with 0.01 MOI H5N1 (SY) or H1N1 (PR8) virus and then incubated for 24 h in the presence of the same concentrations of Fatostatin. Untreated control cells were treated with 0.1% dimethyl sulfoxide (DMSO). Cell lysates were prepared and analyzed for the levels of indicated proteins by western blot. GAPDH was detected as a loading control.

(B) NL20 cells seeded in a 96-well plate (3.5×10⁴ cells/well) were incubated in the absence or presence of the indicated concentrations of Fatostatin in triplicate for 48 h. Cell viability was measured by using the CellTiter-Glo kit. The CC₅₀ values were calculated based on the mean ± standard deviation (SD) of three experiments. To determine the EC₅₀ values, NL20 cells seeded in a 24-well plate were pretreated with the indicated concentrations of Fatostatin for 8 h. After infection with 0.01 MOI H5N1 or H1N1 virus, the cells were incubated in the absence or presence of the same concentrations of Fatostatin for 24 h. The conditioned media were collected and analyzed for virus titers by measuring TCID₅₀ values. The results represent the mean ± SD of three experiments. The selective index (S.I.) values were calculated by dividing the CC₅₀ values with the EC₅₀ values.

(C–F) Control and SREBP2-deficient NL20 cells were infected with the indicated MOI of H5N1 (SY) or H1N1 (PR8) virus and then incubated for 16 h. Cell lysates were prepared and analyzed for the expression of indicated proteins (C and D). Conditioned medial were collected for measuring TCID₅₀ values (E and F). WT, wild type; ΔSREBP2, SREBP2 deficiency. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01.

(G) Control and SREBP2-deficient NL20 cells infected with indicated MOI of H5N1 (SY) were incubated for 16 h. Cell lysates were analyzed for the expression of cholesterol biosynthesis-related and viral proteins.

(H) Total RNAs from control and two SREBP2-deficient clones were extracted and analyzed for the levels of cholesterol biosynthesis-related mRNA levels by RT-qPCR. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01.

(I and J) NL20 cells transiently transfected with the pCAGGS empty vector or the vector encoding the nSREBP2 gene. After incubation for 48 h, the cells were infected with the indicated MOI of H5N1 (SY) and then incubated for 16 h. Cell lysates were prepared and analyzed for the expression of indicated proteins (I). Conditioned medial were collected for measuring TCID₅₀ values (J). The results represent one of three independent experiments with similar results. Data are the mean ± SD of three experiments. ∗p < 0.05.

IAV infection activates STAT3 to enhance virus replication

STAT3 has been implicated in enhancing the replication of a variety of viruses.³⁶^,³⁷ Emerging evidence indicates that STAT3 can upregulate SREBP2 expression and promote cholesterol biosynthesis.⁴² We hypothesized that STAT3 activation may promote virus replication by inducing SREBP2 expression. We first show that the levels of JAK2 and STAT3 phosphorylation were indeed elevated in NL20 (Figures 3A and 3B) and 293T (Figures 3C and 3D) cells infected with two H5N1 strains in a dose- and time-dependent manner.

IAV activates the JAK2/STAT3 signaling pathway

NL20 (A and B) and 293T (C and D) cells infected with 0.1 MOI of H5N1 (SY and CK10) viruses were incubated for the indicated lengths of time or infected with the indicated MOI of IAV for 16 h (NL20 cells) or 24 h (293T cells). Cell lysates were prepared and analyzed for JAK2 and STAT3 phosphorylation. After stripping, the membranes were re-probed with antibodies against their total proteins. The NS1 protein was detected as an infection control. β-actin was detected as a loading control. The relative phosphorylation levels were analyzed by quantifying the density of the phosphorylated protein bands normalized to their corresponding total proteins. The results were presented as bar graphs. Data represents the mean ± SD of three independent experiments. ∗p < 0.05, *∗∗p* < 0.01, ns, not significant, compared to the uninfected controls.

We then tested if STAT3 inhibition could inhibit IAV replication. S3I-201, an STAT3-specific inhibitor, dose- and time-dependently decreased the levels of viral PB2, NP, and NS1 proteins (Figures 4A and 4C) and virus titers (Figures 4B and 4C) in NL20 and 293T cells infected with two H5N1 and one H1N1 viruses. The EC₅₀ values of S3I-201 for SY, CK10, and PR8 viruses in NL20 cells are 17.6, 12.9, and 31.6 μM, respectively. S3I-201 had little cytotoxicity on NL20 cell proliferation, with a CC₅₀ value of 203.7 μM. The S.I. values of S3I-201 for SY, CK10, and PR8 virus in NL20 cells are 11.6, 15.8, and 6.4 respectively. The EC₅₀ values of S3I-201 for SY, CK10, and PR8 in 293T cells are 22.7, 19.8, and 10.0 μM, respectively. The CC₅₀ value of S3I-201 on 293T cells is 271.6 μM. The S.I. values of S3I-201 to inhibit SY, CK10, and PR8 in 293T are 12.0, 13.7, and 27.2, respectively.

STAT3 promotes IAV replication through SREBP2

NL20 (A) and 293T (C) cells were pretreated with the indicated concentrations of S3I-201 (S3I) for 8 h. Untreated control cells were treated with 0.5% DMSO. After infection with 0.01 MOI H5N1 or H1N1 viruses, the cells were incubated for 24 h in the absence or presence of the same concentration of S3I-201. Viral proteins were detected with their specific antibody by western blot. NL20 (B) and 293T (D) cells seeded in a 96-well plate (3.5×10⁴ cells/well) were incubated in the absence or presence of the indicated concentrations of S3I-201 in triplicate for 48 h. Cell viability was measured by using the CellTiter-Glo kit. The CC₅₀ values were calculated based on the mean ± SD of three experiments. To determine the EC₅₀ values, NL20 cells seeded in a 24-well plate were pretreated with the indicated concentrations of S3I-201 for 8 h. After infection with 0.01 MOI H5N1 or H1N1, the cells were incubated in the absence or presence of the same concentrations of S3I-201 for 24 h. The conditioned media were collected and analyzed for virus titers by measuring TCID₅₀ values. The results represent the mean ± SD of three experiments. The S.I. values were calculated by dividing the CC₅₀ values with the EC₅₀ values.

(E) Control and STAT3-deficient 293T cells infected with the indicated MOI of H5N1 (SY) virus were incubated for 16 h. Cell lysates were prepared and analyzed for cholesterol biosynthesis-related and viral protein levels. WT, wild type; ΔSTAT3, STAT3 deficiency.

(F) 293T cells were transfected with the empty vector or the vector encoding STAT3. After incubation for 48 h, the cells were infected with the indicated MOI of H5N1 (SY) virus and then incubated for 16 h. Cell lysates were analyzed for the indicated proteins by Western blot.

(G and H) Conditioned media from control or STAT3-deficient or overexpression 293T cells were collected and analyzed for virus titers by measuring the TCID₅₀ values. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01.

To confirm the specificity of S3I-201 to inhibit virus replication by targeting STAT3, we tested if STAT3 deficiency also suppressed IAV replication. Indeed, STAT3 deficiency significantly decreased the levels of nSREBP2, HMGCR, and the viral PB2 and NS1 proteins (Figure 4E) and the virus titers (Figure 4G) in the conditioned media of IAV-infected 293T cells. Similar observations were made in STAT3-deficient NL20 cells (Figure S2). In contrast, STAT3 overexpression in 293T cells increased the levels of nSREBP2, HMGCR, viral proteins (Figure 4F), and virus titers (Figure 4H) in the conditioned media of IAV-infected cells.

STAT3 upregulates SREBP2 expression

To rule out the possibility that regulation of SREBP2 and HMGCR expression by STAT3 was secondary to virus replication, we tested if STAT3 suppression could still inhibit SREBP2 and HMGCR expression when virus replication was not affected. S3I-201 was added into cell culture 4 h post infection (hpi). In this experimental setting, S3I-201 did not decrease the levels of the viral NS1 proteins and virus titers but dose-dependently decreased the levels of nSREBP2 and HMGCR (Figures 5A and 5B). Of note, all pSREBP2 protein molecules were cleaved to produce nSREBP2 (Figure 5A), suggesting that S3I-201 did not affect SREBP2 activation. Decreased nSREBP2 expression by S3I-201 suggests that STAT3 inhibition leads to decreased SREBP2 expression.

STAT3 activation increases SREBP2 expression and cholesterol levels

(A) NL20 cells were first infected with 0.1 MOI H5N1 (SY) virus. After incubation for 4 h, cells were treated with the indicated concentration of S3I-201 and then incubated for 20 h. Untreated control cells were treated with 0.5% DMSO.

(B) Conditioned media were collected and analyzed for virus titers. Data are the mean ± SD of three experiments. ns, not significant.

(C) Control and STAT3-deficient NL20 cells infected with the indicated MOI of H5N1 (SY) virus were incubated for 16 h. Cell lysates were prepared and analyzed for cholesterol biosynthesis-related and viral proteins.

(D) Conditioned media were collected and analyzed for virus titers. Data are the mean ± SD of three experiments. ns, not significant.

(E) Total cellular RNAs from control and STAT3-deficient NL20 cells were extracted and analyzed for SREBP2 and HMGCR mRNA levels by RT-qPCR. The results represent the mean ± SD of three experiments. ∗∗p < 0.01.

(F) Control and STAT3-deficient NL20 cells seeded on coverslips were stained with Filipin III and visualized under a confocal microscope. Scale bar, 10 μm.

(G) The monolayers of control and STAT3-deficient NL20 cells in a 96-well plate (3.5×10⁴ cells/well) were incubated in serum-free medium for 8 h. Filipin and Sytox Green fluorescent intensity was quantified in a plate reader. The arbitrary units of Filipin fluorescence intensity were normalized with that of Sytox Green intensity. The results represent the mean ± SD of three experiments. ∗p < 0.05.

(H) NL20 cells transiently transfected with the pcDNA empty vector or the vector encoding the STAT3 gene. After incubation for 48 h, the cells were infected with the indicated MOI of H5N1 (SY) and then incubated for 16 h. Cell lysates were prepared and analyzed for the expression of indicated proteins.

(I) Total cellular RNAs from NL20 cells transfected with the empty vector or the vector encoding STAT3 were extracted and analyzed for SREBP2 and HMGCR mRNA levels by RT-qPCR. The results represent the mean ± SD of three experiments. ∗∗p < 0.01.

(J) NL20 cells seeded on coverslips were transiently transfected with the empty vector or the STAT3 expression vector. After incubation for 40 h, the cells were replenished with serum-free medium and then incubated for 8 h. The cells were stained with Filipin III and visualized under a confocal microscope. Scale bar, 10 μm.

(K) NL20 cells seeded in a 96-well plate (3.5×10⁴ cells/well) were transiently transfected with the empty vector or the STAT3 expression vector. After incubation for 40 h, the cells were replenished with serum-free medium and then incubated for 8 h. Filipin and Sytox Green fluorescent intensity was quantified in a plate reader. The arbitrary units of Filipin intensity were normalized with that of Sytox Green. The results represent the mean ± SD of three experiments. ∗p < 0.05.

To further confirm the ability of STAT3 to regulate SREBP2 expression, STAT3-deficient cells were infected with 2- to 10-fold higher MOI of IAV than wild-type NL20 cells (Figure 5C). The levels of viral NP and NS1 proteins and virus titers in wild-type and STAT3-deficient NL20 cells were almost equal, indicating equal virus replication (Figures 5C and 5D). However, the levels of nSREBP2 and HMGCR were significantly lower in STAT3-deficient cells than in the wild-type control NL20 cells (Figure 5C). STAT3 deficiency also significantly decreased the basal levels of pSREBP2 and its target genes HMGCS, SQLE, MVD, and MVK (Figure 5C). STAT3 deficiency also decreased SREBP2 and HMGCR mRNA levels in uninfected NL20 cells (Figure 5E). Of note, IAV infection induced HMGCR expression but did not increase or even slightly decreased the levels of several other enzymes such as FDFT1 in NL20 cells infected with 1 MOI of IAV. The mechanisms underlying this remain to be investigated. Filipin staining revealed that STAT3 deficiency significantly deficiency decreased the content of cholesterol in the plasma membrane and cytosols, compared to that in their wild-type control (Figure 5F) and the levels of cellular cholesterol (Figure 5G). In contrast, STAT3 overexpression also significantly increased the levels of the viral NP and NS1 proteins and the levels of SREBP2 and its several target genes including HMGCS, SQLE, FDFT1, and MVD (Figure 5H). STAT3 overexpression increased SREBP2 and HMGCR mRNA levels in uninfected NL20 cells, compared to that in the vector-transfected control cells (Figure 5I). Filipin staining revealed that STAT3 overexpression significantly increased the content of cholesterol in the plasma membrane and cytosols (Figure 5J) and the levels of cellular cholesterol (Figure 5K), compared to that in their vector-transfected control cells.

STAT3 promotes IAV replication by enhancing cholesterol biosynthesis

STAT3 is a pleiotropic transcription factor that may regulate virus replication by multiple mechanisms.³⁶ To test if STAT3 activation promoted IAV replication by enhancing cholesterol biosynthesis, we tested if exogenous cholesterol could ameliorate the antiviral activity of S3I-201. S3I-201 lowered the levels of the viral PB2 and NP proteins, which was largely blocked by pretreatment with exogenous cholesterol (5 & 10 μg/mL) (Figures 6A and 6B). Exogenous cholesterol also restored virus titers in S3I-201-treated NL20 cells, compared to that in the untreated control (Figure 6C). Consistently, cholesterol also increased the levels of viral proteins (Figures 6D and 6E) and virus titers (Figure 6F) in STAT3-deficient NL20 cells. Cholesterol in the lipid raft of the cell membrane has been implicated in IAV endocytosis.¹²^,¹⁵^,¹⁶^,²² Verma et al.⁴⁴ reported that IAV bound to the cell membrane is co-localized with the lipid raft, and that raft disruption using methyl-β-cyclodextrin decreases IAV binding to MDCK cells, suggesting that cholesterol is also required for IAV binding. Here, we tested whether inhibition of STAT3 activity by S3I-201 impaired virus binding. NL20 cells pretreated with S3I-201 for 8 h were inoculated with 2.5 MOI of the SY virus and incubated at 4°C for 1 h for virus binding. S3I-201 pretreatment dose-dependently decreased virus binding to NL20 cell surface (Figure 6G). Consistently, STAT3 deficiency in NL20 cells also decreased virus binding to host cells (Figure 6H). To further confirm the effect of cellular cholesterol in virus attachment, NL20 cells were treated with S3I-201 at different time points before and after virus infection. The inhibitory effect of S3I-201 on virus replication was strengthened with longer pretreatment time (Figures 6I–6K). S3I-201 added during or 2 h post virus infection had minimal or modest effect on the levels of viral proteins and virus titers in the conditioned media of NL20 cells (Figures 6I–6K).

STAT3 enhances IAV replication by increasing cholesterol biosynthesis

(A–C) NL20 cells were pretreated without or with cholesterol (5 or 10 μg/mL) minus or plus S3I-201 (25 μM) for 8 h and then infected with 0.01 MOI of H5N1 (SY) virus and incubated for 24 h. Cell lysates were prepared and analyzed for the PB2 and NP proteins (A). The relative viral protein levels were analyzed by quantifying the density of the protein bands using NIH ImageJ software and normalized to the density of the GAPDH band as a loading control. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01, ns, not significant, compared to the untreated control; ^##p < 0.01, compared to H5N1 virus infection (B).

(C) Conditioned media were collected and analyzed for virus titers. Data are the mean ± SD of three experiments. ∗∗p < 0.01, compared to the untreated control; ^##p < 0.01, compared to H5N1 virus infection.

(D–F) Control and STAT3-deficient NL20 cells were infected with the indicated MOIs of H5N1 (SY) virus and then incubated in the absence or presence of cholesterol (5 μg/mL) for 16 h. Cell lysates were prepared and analyzed for viral proteins (D). The relative viral protein levels were analyzed by quantifying the density of the protein bands using NIH ImageJ software and normalized to the density of the GAPDH band as a loading control. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01 (E). Conditioned media were collected and analyzed for virus titers. Data are the mean ± SD of three experiments. ∗p < 0.05 (F).

(G) NL20 cells were pretreated in serum-free media containing S3I-201 for 8 h. Cells were then infected with 2.5 MOI H5N1 (SY) virus and incubated at 4°C for 1 h. After removing unattached viruses, total cellular RNAs were extracted and analyzed for viral RNAs by RT-qPCR. Data are the mean ± SD of three experiments. ∗∗p < 0.01.

(H) Wild-type and STAT3-deficient NL20 cells were starved in serum-free media for 8 h. Cells were then infected with 2.5 MOI of H5N1 (SY) virus and incubated at 4°C for 1 h. After removing unattached viruses, total cellular RNAs were extracted and analyzed for viral RNAs by RT-qPCR. Data are the mean ± SD of three experiments. ∗∗p < 0.01.

(I–K) NL20 cells were treated with SI3-201 (25 μM) for the indicated time points before or after H5N1 (SY) virus infection (0.01 MOI). Cell lysates were prepared and analyzed for viral proteins (I). The relative protein levels were analyzed by quantifying the density of the protein bands using NIH ImageJ software and normalized to the density of the β-actin band as a loading control (E and J). The results were presented as bar graphs. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01, ns, not significant (J). Conditioned media were collected 24 h post infection and analyzed for virus titers. Data are the mean ± SD of three experiments. ∗p < 0.05, ∗∗p < 0.01, ns, not significant (K).

JAK2 activates STAT3 to promote cholesterol biosynthesis

JAK2 phosphorylates and activates STAT3.³⁶ Having shown that STAT3 was involved in enhancing IAV replication by promoting cholesterol biosynthesis, we finally tested whether JAK2 inhibition could decrease the expression of cholesterol biosynthesis-related genes and virus replication. Consistent with previous observations that ruxolitinib (Rux), a JAK-specific inhibitor, lowers virus titers in chicken embryonic fibroblast cells,⁴⁵ Rux dose-dependently lowered the levels of the viral proteins PB2, NP, and NS1 in NL20 (Figure 7A) and 293T cells (Figure 7B) infected with SY, CK10, and PR8 viruses. To determine the ability of Rux to regulate cholesterol biosynthesis, Rux was added into NL20 cells 10 hpi so that virus replication was no longer affected. Indeed, equal levels of the NS1 protein were observed in untreated and Rux-treated cells (Figure 7C). However, Rux dose-dependently lowered the levels of nSREBP2 and several target genes in IAV-infected NL20 cells (Figure 7C). Rux also dose-dependently inhibited the expression of pSREBP2 and other cholesterol biosynthesis-related genes in uninfected NL20 cells (Figure 7C). To confirm the role of JAK (Janus kinase) in regulating IAV replication, we tested if JAK2 deficiency or overexpression also affected IAV replication. JAK2 deficiency partially blocked IAV-induced STAT3 phosphorylation and decreased the levels of pSREBP2 in uninfected NL20 cells and the levels of nSREBP2 in IAV-infected cells (Figure 7D). JAK2 deficiency also decreased IAV-induced HMGCR expression and lowered the levels of viral PB2 and NS1 proteins (Figure 7D) and virus titers in the conditioned media (Figure 7E). Of note, JAK2 knockout did not completely block the induction of HMGCR expression in IAV-infected NL20 cells, suggesting that IAV infection can induce HMGCR expression by other transcription factors such as RORγ. In contrast, JAK2 overexpression increased IAV-induced STAT3 phosphorylation and pSREBP2 levels in uninfected NL20 cells and nSREBP2 levels in IAV-infected cells (Figure 7F). JAK2 overexpression increased IAV-induced HMGCR expression and increased the levels of the viral PB2 and NS1 proteins (Figure 7F) and virus titers in the conditioned media (Figure 7G). These observations collectively suggest that JAK2 plays an important role in activating STAT3 and enhancing the expression of cholesterol biosynthesis-related genes.

JAK2 promotes IAV replication by upregulating cholesterol biosynthesis

(A and B) After incubation with the indicated concentrations of Rux for 8 h, NL20 and 293T cells were infected with 0.01 MOI H5N1 or H1N1 virus and then incubated for 24 h in the absence or presence of the same concentrations of Rux. Viral proteins were detected by western blot with the indicated antibodies. β-actin was detected as a loading control. Untreated control cells were treated with 0.2% DMSO.

(C) NL20 cells were first infected with 0.2 MOI H5N1 (SY) virus After incubation for 10 h, the cells were incubated in the absence or presence of the indicated concentrations of with Rux for 14 h. Cell lysates were analyzed for the indicated proteins by Western blotting. The experiments in Figures 7A–7C were carried out three times with similar results.

(D) Control and JAK2-deficient NL20 cells were infected with the indicated MOIs of H5N1 (SY) virus and then incubated for 16 h. Cell lysates were prepared and analyzed for cholesterol biosynthesis-related proteins and viral proteins. WT, wild type; ΔJAK2, JAK2 deficiency.

(E) Conditioned media were collected and analyzed for virus titers.

(F) NL20 cells were transfected with the empty vector or the vector encoding JAK2. After incubation for 48 h, the cells were infected with the indicated MOIs of H5N1 (SY) virus and then incubated for 16 h. Cell lysates were prepared and analyzed for cholesterol biosynthesis-related and viral proteins.

(G) Conditioned media were collected and analyzed for virus titers.

(E and G) Data represent the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01.

Discussion

Cholesterol, a main lipid component in the cell membrane and intracellular compartments, plays a key role in influenza virus infection and replication.⁴⁶^,⁴⁷ Our present study focuses on the mechanisms of SREBP2 regulation by STAT3 and their role in virus replication. We provide several lines of evidence that STAT3 activation increases SREBP2 expression and cholesterol biosynthesis to promote virus replication: (1) STAT3 deficiency and the STAT3 inhibitor S3I-201 downregulated SREBP2 and HMGCR expression even in the experimental setting when virus replication was not affected; (2) STAT3 deficiency lowered the basal levels of SREBP2 and other cholesterol synthesis-related genes and lowered cellular cholesterol levels in uninfected cells; (3) exogenous cholesterol reversed the antiviral effect of the STAT3 inhibitor and STAT3 deficiency; (4) JAK2 inhibition suppressed STAT3 activation and virus replication, and inhibited the expression of SREBP2 and HMGCR even when virus replication was not affected; (5) SREBP2 inhibition decreased cholesterol biosynthesis and IAV replication. Our study sheds light on the mechanisms of IAV-induced cholesterol biosynthesis and improves our understanding of how STAT3 activation supports virus replication.

Virus infection activates STAT3 through multiple mechanisms.³⁶ STAT3 becomes phosphorylated at Y705 by a tyrosine kinase such as JAK2 and Syk and at S727 by the mTOR and MAPK (Mitogen-activated protein kinase) signaling pathways.³⁹^,⁴⁸ Hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV induce IL-6 expression and activate STAT3 in hepatocytes through ROS (reactive oxygen species) and in renal tubular epithelial cells through JAK2.⁴⁹ African swine fever virus (ASFV) activates JAK2 and STAT3 through the interaction of its transmembrane protein CD2v with the colony-stimulating factor 2 receptor subunit alpha (CSF2RA).⁵⁰ Whether and how IAV activates STAT3 remains controversial. Hui et al.⁵¹ reported that IAV inhibits STAT3 phosphorylation at Y705 in human respiratory epithelial cells. In contrast, Wang et al. showed that H5N1 virus increased STAT3 phosphorylation in 293T cells.⁵² Liu recently reported that IAV infection activates Syk to phosphorylate STAT3 at Y705 in A549 cells via the RIG-I-MAVS pathway.³⁹ Syk knockout partially blocks IAV-induced STAT3 phosphorylation, suggesting that IAV infection could phosphorylate STAT3 by other tyrosine kinases.³⁹ In support of this notion, our present study shows that IAV infection activated JAK2 in NL20 and 293T cells. Rux and JAK2 deficiency blocked STAT3 phosphorylation. These observations suggest that IAV infection induces STAT3 phosphorylation largely by JAK2 in NL20 and 293T cells.

It has been well documented that STAT3 activation promotes virus replication.³⁶^,³⁷ For example, STAT3 inhibitors such as STA-21, S3I-201, and Cpd188 suppress the replication of HCV, Varicella-Zoster virus (VZV), and human cytomegalovirus (HCMV).⁵³^,⁵⁴^,⁵⁵ STAT3 inhibits OASL and PKR expression and blocks IFN-α-induced STAT1 activation.⁵⁶ STAT3 inhibition suppresses enterovirus 71 (EV71) replication.⁵⁷ Consistent with these observations, Ampomah et al.³⁸ reported that the STAT3 inhibitor WP1066 inhibits IAV replication in A549 cells. Our study reveals that S3I-201 and STAT3 deficiency inhibited IAV replication in NL20 and 293T cells, whereas STAT3 overexpression promotes IAV replication. In contrast, Liu et al.³⁹ recently reported that STAT3 knockdown increases H3N2 replication in A549 cells and in vivo in the lung tissues of IAV-infected mice. It is not clear why STAT3 knockdown, which is supposed to enhance antiviral immunity, would reduce virus replication, in particular, in vitro in A549 cells.³⁹

How STAT3 regulates virus replication remains incompletely understood.³⁶^,³⁷ STAT3 downregulates antiviral immunity by suppressing STAT1 nuclear translocation and ISG expression.³⁶^,³⁷ Epstein-Barr virus (EBV) and HCMV activate STAT3 to inhibit cell death, thereby enhancing virus replication or maintaining persistent infection.³⁶^,³⁷ Ampomah et al.³⁸ reported that STAT3 activation increases IAV replication by inducing the expression of formyl peptide receptor 2 (FRP2), a receptor that recognizes numerous proinflammatory and anti-inflammatory stimuli. Our present study reveals that STAT3 deficiency and S3I-201 decreased the expression of SREBP2 and its target genes such as HMGCR and other cholesterol biosynthesis-related genes (Figure 5). Exogenous cholesterol partially antagonized the inhibitory effect of S3I-201 or STAT3 deficiency (Figures 6A–6D) on virus replication. These observations suggest that STAT3 promotes IAV replication in part by enhancing SREBP2 expression and cholesterol biosynthesis. In support of this notion, several other studies have also implicated STAT3 in upregulating SREBP2 expression and cholesterol biosynthesis. For example, Chen et al.⁴² reported that STAT3 activation by endosulfine alpha (ENSA) cooperates with protein phosphatase 2A (PP2A) to increase SREPB2 expression, cholesterol biosynthesis, and cell proliferation in triple-negative breast cancer. STAT3 overexpression in mouse liver tissue leads to a significant increase in plasma triglyceride and cholesterol levels.⁴¹ Diosgenin (DG) inhibits the STAT3 transcriptional activity, decreases NPC1L1 expression and intestinal cholesterol uptake, resulting in decreased incidence of cholesterol gallstones in mice.⁵⁸ IL-5 activates the JAK2/STAT3 pathway in macrophage, leading to increased ABCA1 expression and intracellular cholesterol efflux as well as atherosclerosis.⁵⁹ Tofacitinib, a JAK inhibitor, inhibits cholesterol biosynthesis and uptake but increases cholesterol efflux.⁴⁰ These observations collectively suggest STAT3 enhances intracellular cholesterol levels by multiple mechanisms, and that STAT3 enhances virus replication by increasing cholesterol biosynthesis.

SREBP2 plays a crucial role in virus replication. ZIKV infection increases SREBP2 activation and the transcription of genes related to cholesterol synthesis.³³ SREBP2 inhibitors and gene knockout both suppress ZIKV replication in dendritic cells.³³ The SREBP inhibitor AM580 inhibits fatty acid and cholesterol biosynthesis, effectively suppressing the replication of MERS-CoV and IAV.³⁴ SREBP2 target genes are upregulated in Huh-7.5.1 cells infected with SARS-CoV-2 and human coronavirus OC43 (HCoV-OC43); Deletion of the SREBP2 gene significantly reduces virus replication.³⁵ S1P inactivation, which blocks SREBP2 activation and cholesterol synthesis, inhibits the replication of Andes-Hantavirus recombinant virus, which could be reversed by exogenous cholesterol.⁶⁰ Teo et al.²⁴ recently reported that USP25 stabilizes the ER proteins Erlin1/2 to block SREBP2 activation and restrict IAV replication. Consistent with these observations, our present study shows that IAV infection enhanced the expression of nSREBP2 and HMGCR in two epithelial cell lines. SREBP2 deficiency led to decreased expression of cholesterol biosynthesis-related genes and virus replication. These observations suggest that IAV infection promotes cholesterol biosynthesis not only by STAT3-mediated SREBP2 upregulation but also by SREBP2 activation, probably due to decreased availability of cytoplasmic cholesterol.

Several prior studies have identified JAK1 and JAK2 as crucial cellular factors that support IAV replication.⁶¹^,⁶² Inhibition of JAK1 and JAK2 by siRNA or by Rux and other JAK inhibitors such as A77 1726 suppresses IAV replication.⁴⁵^,⁶¹^,⁶² How JAK regulates IAV replication remains incompletely understood. Two prior studies showed that JAK phosphorylates the M1 protein of IAV at Y132 and facilitate its nuclear import and virus assembly.⁶³^,⁶⁴ Our present study suggests that JAK2 may promote IAV replication in part by activating STAT3 and enhancing cholesterol biosynthesis, leading to increased virus replication.

In summary, our study provides evidence that IAV infection induces HMGCR and promotes cholesterol biosynthesis by activating STAT3 and SREBP2. Inhibition of STAT3 and JAK2 by their inhibitors or gene deficiency inhibits IAV replication by blocking SREBP2 expression and cholesterol synthesis. Our study unveils a previously unrecognized role of STAT3 in enhancing virus replication and provides mechanistic insights into how IAV infection regulates cholesterol biosynthesis.

Limitations of the study

While our study has demonstrated that STAT3 promotes IAV replication by enhancing cholesterol biosynthesis, several issues remain unsolved: (1) it is not clear how IAV infection activates JAK2. We speculate that JAK2 could be activated initially through a receptor tyrosine kinase and later through IL-6 and IFN receptors³⁹^,⁶⁵; (2) whether Syk also contributes to STAT3 activation and SREBP2 expression remains to be investigated; (3) STAT3 is also activated in vivo in the lungs of IAV-infected mice.³⁹ Since STAT3 can regulate inflammatory and immune response, STAT3 is likely not to be a molecular target for antivirals. Indeed, Yao et al.⁶⁶ recently reported that STAT3 inhibition exacerbates IAV infection; and (4) our study has not been confirmed in primary human bronchial or alveolar epithelial cells nor in vivo in a STAT3-deficient mouse model. Great cautions should be taken in interpreting these conclusions.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse monoclonal anti-NS1	Santa Cruz	Cat# sc-130568; RRID:AB_2011757
Mouse monoclonal anti-HMGCR	Santa Cruz	Cat# sc-271595; RRID:AB_10650274
Mouse monoclonal anti-HMGCS	Santa Cruz	Cat# sc-373681; RRID:AB_10947237
Mouse monoclonal anti-MVK	Santa Cruz	Cat# sc-390669
Mouse monoclonal anti-MVD	Santa Cruz	Cat# sc-376975
Mouse monoclonal anti-FDFT1	Santa Cruz	Cat# sc-271602; RRID:AB_10676824
Mouse monoclonal anti-SQLE	Santa Cruz	Cat# sc-271651; RRID:AB_10708249
Mouse monoclonal anti-SREBP1	Santa Cruz	Cat# sc-13551; RRID:AB_628282
Mouse monoclonal anti-GAPDH	Santa Cruz	Cat# sc-47724; RRID:AB_627678
Mouse monoclonal anti-β-actin	Santa Cruz	Cat# sc-8432; RRID:AB_626630
Rabbit monoclonal anti-JAK2	Cell Signaling Technology	Cat# 3230; RRID:AB_2128522
Mouse monoclonal anti-STAT3	Cell Signaling Technology	Cat# 9139; RRID:AB_331757
Rabbit monoclonal anti-phospho-JAK2(Tyr1007/1008)	Cell Signaling Technology	Cat# 3771; RRID:AB_330403
Rabbit monoclonal anti-phospho-STAT3(Tyr705)	Cell Signaling Technology	Cat# 9145; RRID:AB_2491009
Mouse monoclonal anti-SREBP2 (Detection of pSREBP2)	R & D Systems	Cat# MAB7119
Rabbit monoclonal anti-SREBP2 (Detection of nSREBP2)	Abcam	Cat# ab30682; RRID:AB_779079
Rabbit polyclonal anti-PB2	GeneTex	Cat# GTX125926; RRID:AB_11162999
Rabbit monoclonal anti-NP	GeneTex	Cat# GTX636247; RRID:AB_2909956

Bacterical and virus strains

H5N1 influenza virus strain A/mallard/Huadong/S/2005 (SY)	Ref.⁶⁷^,⁶⁸^,⁶⁹	N/A
H5N1 influenza virus strain A/chicken/Jiangsu/K0402/2010 (CK10)	Ref.⁶⁷^,⁶⁸^,⁶⁹	N/A
H1N1 influenza virus strain A/PR/8/1934 (PR8)	Dr. Liqian Zhu, Yangzhou University	N/A

Chemicals, peptides, and recombinant proteins

Fatostatin	MedChemExpress	Cat# 125B11
S3I-201	Selleck Chemicals	Cat#S1155
Ruxolitinib	Selleck Chemicals	Cat# S1378
Water-soluble cholesterol	Sigma-Aldrich	Cat# C4951
Filipin III	Cayman Chemical	Cat# 480-49-9
Sytox Green nucleic acid stain	Invitrogen	Cat# S7020
Fetal bovine serum	EallBio	Cat# 03.U16001DC
DMEM	GIBCO	Cat# 12100-046
F12	GIBCO	Cat# 21700-075
RNA isolater Total RNA Extraction Reagent	Vazyme	Cat# R401-01
HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper)	Vazyme	Cat# R212-02
ChamQ Blue Universal SYBR qPCR Master Mix	Vazyme	Cat# Q312-02

Chemicals, peptides, and recombinant proteins

2×EasyTaq PCR SuperMix (+dye )	TransGen Biotech	Cat# AS111-11
OPTI-MEM® medium	GIBCO	Cat# 31985-070
Dimethyl sulfoxide (DMSO)	Sigma	Cat# D2650
TurboFect Transfection Reagent	Thermo Scientific	Cat#00596562

Experimental models: Cell lines

NL20	ATCC	CRL-2503; RRID:CVCL_3756
293T	ATCC	CRL-11268

Critical commercial assays

Seamless Cloning and Assembly Kit	Transgen	Cat# CU101-01
CellTiter-Glo® Luminescent Cell Viability Assay	Promega	Cat# 7572

Recombinant DNA

pCAGGS-nSREBP2	This paper	N/A
pRc/CMV-STAT3-FLAG	Addgene	Cat# 8707; RRID:DGRC_8707
pBABE-JAK2	Dr. Eric Chang ( Baylor College of Medicine, Houston)	N/A

Oligonucleotides

PCR primers for SREBP2 (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers for HMGCR (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers for HMGCS (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers SQLE (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers for MVK (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers for IAV M1 (see Table S1)	Anhui General Bio, Inc.	N/A
PCR primers for ACTB (see Table S1)	Anhui General Bio, Inc.	N/A
gRNA for SREBP2 (see Table S1)	Anhui General Bio, Inc.	N/A
gRNA for STAT3 (see Table S1)	Anhui General Bio, Inc.	N/A
gRNA for JAK2 (see Table S1)	Anhui General Bio, Inc.	N/A

Software and algorithms

Benchling CRISPR Guide Design Software	Benchling	https://www.benchling.com/crispr
ImageJ	NIH	https://imagej.nih.gov/ij/
GraphPad prism	GraphPad software 8.0	https://www.graphpad.com/scientific-software/prism
Biorender	Biorender	https://www.biorender.com/

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Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Xiulong Xu (xxl@yzu.edu.cn).

Materials availability

Plasmids and the viruses used in this study can be obtained from the lead contact after the permission of original distributors.

Data and code availability

•
All data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
•
The authors declare that the data supporting the findings of this study are available within the article and from the corresponding author upon request.

Experimental model and study participant details

Cell culture and virus

NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line) and 293T (a human embryonic kidney cell line) cells were purchased from the American Tissue Culture Collection (Manassas, VA, USA). NL20 cells were grown in Ham's F12 medium with 1.5 g/L sodium bicarbonate, 2.7 g/L glucose, 2.0 mM L-glutamine, 0.1 mM nonessential amino acids, 0.005 mg/ml insulin, 10 ng/ml epidermal growth factor, 0.001 mg/ml transferrin, 500 ng/ml hydrocortisone and 4% fetal bovine serum. 293T cells were grown in DMEM containing 10 % fetal bovine serum. Both cell lines were periodically tested for mycoplasma negative. Experiments with the H5 subtype highly pathogenic avian influenza virus A/mallard/Huadong/S/2005 (H5N1) (SY) and A/chicken/Jiangsu/K0402/2010 (H5N1) (CK10) were conducted in a BSL-3 level facility. Experiments with the H1N1 virus A/PR/8/1934 virus (PR8) were carried out in our BSL-2 laboratory. The IAV stocks were prepared by inoculating 10-day-old specific-pathogen-free embryonic chicken eggs. Virus titers were determined by infecting MDCK cells with 10-fold serially diluted samples (10¹ to 10⁹). The Reed and Muench method was used to determine the 50% tissue culture infection dose (TCID₅₀/100 μl).

Method details

EC₅₀ and CC₅₀

NL20 and 293T cells seeded in a 24-well plate were pretreated with various concentrations of Fatostatin or S3I-201 for 8 h. After infection with 0.01 MOI of SY, CK10, or PR8 virus, the cells were incubated for 24 h in the absence or presence of the same concentration of Fatostatin or S3I-201. The conditioned media were collected analyzed for the TCID₅₀ values. The results from three experiments were pooled and used to calculate the EC₅₀ values. The Reed and Muench method⁴⁵ was used to determine the 50% tissue culture infection dose (TCID₅₀ per 100 μl). To determine the cytotoxicity of Fatostatin and S3I-201, NL20 cells seeded in 96-well plates (3.5×10⁴ cells/well) were incubated with various concentrations of Fatostatin or S3I-201 and then incubated for 48 h. Cell viability was measured by using a CellTiter-Glo kit (Promega, Madison, WI, USA). Data are the mean ± SD of three independent experiments.

Plasmids

The nSREBP2 (1443-bp) gene was amplified with a human cDNA template that was reverse-transcribed from cellular total mRNAs extracted from NL20 cells. The sequences of the forward and reverse primers used to amplify nSREBP2 are 5-GCTCATCGATGCATGGTACCATGGACGACAGCGGCGAGC-3 and GAGGGAAAAAGAT-CTGCTAGTCACCGTGAGCGGTCTACCATGC, respectively. The PCR fragment was then cloned into the Kpn I- and Nhe I-cleaved pCAGGS vector by using the Seamless Cloning and Assembly Kit (Transgen, Beijing, China). The expression vector designated as pCAGGS-nSREBP2 was sequenced to confirm the insertion of the nSREBP2 gene. pRc/CMV-STAT3-FLAG was purchased from Addgene (Beijing Zhongyuan, Beijing China). pBABE-JAK2 plasmid pBABE-JAK2, a retroviral vector encoding JAK2 tagged with a fragment of yellow fluorescence protein (YFP), was kindly provided by Dr. Eric Chang, Baylor College of Medicine, Houston.⁷⁰

CRISPR/Cas9 sgRNA design

sgRNAs were designed by using the Benchling CRISPR Guide Design Software (San Francisco, CA, USA). Paired oligos corresponding to the sgRNAs were synthesized and cloned into the LentiCRISPR V2 vector. The sgRNA sequences are listed in Table S1. NL20 and 293T cells plated in 24-well plates were transfected with recombinant plasmids that target SREBP2, STAT3, or JAK2. After incubation for 48 h, the monolayers were trypsinized and re-seeded in 6-well plates in the medium containing puromycin (1-2 μg/ml). After incubation for 14 days, individual colonies were picked, expanded, and analyzed for the expression of SREBP2, STAT3, and JAK2 by Western blot. The colonies screened from the cells transfected with a pLenti-V2 empty vector were used as controls. All experiments were carried out with at least two colonies.

SREBP2, STAT3, and JAK2 transfection

293T and NL20 cells were transiently transfected with the empty vector or pCAGGS-nSREBP2, pRc/CMV-STAT3-FLAG, and pBABE-JAK2 or their corresponding empty vectors as a control. After incubation for 48 h, the cells were left uninfected or infected with the indicated MOI of the H5N1 virus and then incubated for 16 h. Cell lysates were prepared and analyzed for the proteins of interest. Virus titers in the conditioned media were collected and analyzed for the TCID₅₀ values. The results represent the mean ± SD of three independent experiments.

Western blot

NL20 and 293T cells were harvested and lysed in NP-40 lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 5 mm EGTA, 1 mM NaF, 2 mM sodium vanadate, the cocktail of protease inhibitors (1X) (Pierce Chemical Co., Rockford, IL), and 2 mM sodium pervandate). Cell lysates were prepared and analyzed for the expression of viral proteins (PB2, NP, and NS1) and cellular proteins by Western blot with their specific antibodies. Blots were detected by horseradish peroxidase-conjugated goat anti-rabbit IgG or goat-anti-mouse IgG followed by chemiluminoscence with a SuperSignal Western Pico substrate (Pierce Chemical Co., Rockford, IL). To detect the phosphorylation of JAK2 and STAT3, blots were first probed with an antibody against phosphorylated protein. The membrane was then stripped and re-probed with an antibody against their total proteins. Full-length and truncated SREBP2 were detected by probing sliced blots from the same membrane. All Western blot experiments were repeated at least twice with similar results, each with the detection of β-actin or GAPDH as a loading control. The relative phosphorylation levels were analyzed by quantifying the density of the phosphorylated protein bands normalized to their corresponding total proteins. The relative levels of viral proteins were analyzed by quantifying the density of protein bands normalized to the bands of β-actin or GAPDH. The results were presented as bar graphs.

RT-qPCR analyses

Total cellular RNA from wild-type, SREBP2- (Figure 2F) or STAT3-deficient or overexpressing (Figures 5E and 5H) NL20 cells was extracted using TRIzol (Vazyme, Nanjing, China). Reverse transcription was performed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s protocol. The cDNA was subjected to quantitative real-time PCR using a ChamQ Universal SYBR qPCR Master Mix kit (Vazyme, Nanjing, China). The sequences of the primers used for RT-qPCR are shown in Table S1. The viral mRNA levels were quantified by using a pair of primers that amplify the M gene. The PCR reaction was set with an initial denaturation of 30 s at 95°C and subsequent 40 cycles of denaturation for 5 s at 95°C, annealing for 30 s at 60°C, and extension for 15 s at 72°C. All expression levels were normalized to the β-actin mRNA levels in the same sample. Fold change was calculated by the ΔΔCT method. Percent expression was calculated as the ratio of the normalized value of each sample to that of the corresponding untreated control sample. All Real-Time RT-PCR analyses were performed in triplicate. Results from three independent experiments were pooled and statistically analyzed.

Virus binding assay

NL20 cells were chilled at 4°C for 10 min and then inoculated with 2.5 MOI of SY virus. After incubation at 4°C for 1 h, unbound virions were removed by rinsing three times with cold PBS 3 times. Total RNAs were extracted by directly lysing the cells in TRIzol (Vazyme, Nanjing, China). Viral mRNA levels of the M gene were quantified by RT-PCR. Results from three independent experiments were pooled and statistically analyzed.

Filipin staining

NL20 cells grown in 96-well plates in triplicate or coverslips were rinsed twice with PBS and then fixed with 4% paraformaldehyde at room temperature for 30 min. After rinsing twice with PBS, the cells were incubated in PBS containing 1.5 mg glycine/ml at room temperature for 10 min to quench paraformaldehyde. The cells were then stained in Filipin III working solution (50 μg/ml in PBS containing 10% FBS) at room temperature for 2 h. After removal of the Filipin solution and rinse twice with Hank’s balanced salt solution (HBSS), the cells were stained in HBSS containing Sytox Green (167 nM) for 15 min. After removing the Sytox Green solution and wash twice with HBSS, the cells were visualized under a Leica confocal microscope (DMI6000 B) or quantified for Filipin (Excitation, 360 nm; Emission, 480 nm) and Sytox Green (Excitation, 504 nm; Emission, 523 nm) fluorescence in a Tecan plate reader (Infinite 200 PRO). The arbitrary units of Filipin intensity were normalized with that of Sytox Green. The results represent the mean ± SD of three experiments.

Quantification and statistical analysis

Differences in virus titers, mRNA levels, the density of protein bands, and Filipin fluorescent intensity were statistically analyzed by using an unpaired Student t test. A p value of <0.05 was considered statistically significant. All statistics were analyzed with GraphPad Prism 8 (GraphPad, San Diego, CA).

Acknowledgments

This work was supported in part by the National Key Research and Development Project of China (2021YFD1800202) (X.W.), the Priority Academic Program Development of Jiangsu Higher Education Institutions (X.X.), and the State Key Laboratory for Animal Disease Control and Prevention, Langzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (SKLVEB-KFKT-02) (P.L.). We thank Dr. Liqian Zhu (College of Veterinary Medicine, Yangzhou University) for kindly providing the H1N1 (PR8) virus, Dr. Eric Chang (Baylor College of Medicine, Houston) for kindly providing pBABE-JAK2 plasmid, and BioRender (biorender.com) for providing items for drawing graphic abstract. We greatly appreciate all supports including research reagents and technical advice kindly provided Drs. Daxin Peng, Hui Yang, and Sujuan Chen.

Author contributions

X.X. and J. Z. conceptualization, J.Z., Y.W., and Y.W. investigation, P.L, K.L., and J.S. supervision and coordination, P.Z., D.C., X.W., X.L., and P.L. critical resources and fund acquisition, J.Z, manuscript drafting. X.X. manuscript writing and editing.

Declaration of interests

All authors declare no competing interests.

Published: June 29, 2024

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2024.110424.

Supplemental information

Document S1. Figures S1 and S2 and Table S1

mmc1.pdf^{(301.2KB, pdf)}

References

1.Paules C., Subbarao K. Influenza. Lancet. 2017;390:697–708. doi: 10.1016/S0140-6736(17)30129-0. [DOI] [PubMed] [Google Scholar]
2.Nguyen J.L., Yang W., Ito K., Matte T.D., Shaman J., Kinney P.L. Seasonal Influenza Infections and Cardiovascular Disease Mortality. JAMA Cardiol. 2016;1:274–281. doi: 10.1001/jamacardio.2016.0433. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Allard R., Leclerc P., Tremblay C., Tannenbaum T.N. Diabetes and the severity of pandemic influenza A (H1N1) infection. Diabetes Care. 2010;33:1491–1493. doi: 10.2337/dc09-2215. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Smith M., Honce R., Schultz-Cherry S. Metabolic Syndrome and Viral Pathogenesis: Lessons from Influenza and Coronaviruses. J. Virol. 2020;94 doi: 10.1128/JVI.00665-20. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Louie J.K., Acosta M., Samuel M.C., Schechter R., Vugia D.J., Harriman K., Matyas B.T., California Pandemic H1N1 Working Group A novel risk factor for a novel virus: obesity and 2009 pandemic influenza A (H1N1) Clin. Infect. Dis. 2011;52:301–312. doi: 10.1093/cid/ciq152. [DOI] [PubMed] [Google Scholar]
6.Hancková M., Betakova T. Pandemics of the 21st Century: The Risk Factor for Obese People. Viruses. 2021;14:25. doi: 10.3390/v14010025. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Schultz B.M., Paduro C.A., Salazar G.A., Salazar-Echegarai F.J., Sebastian V.P., Riedel C.A., Kalergis A.M., Alvarez-Lobos M., Bueno S.M. A Potential Role of Salmonella Infection in the Onset of Inflammatory Bowel Diseases. Front. Immunol. 2017;8:191. doi: 10.3389/fimmu.2017.00191. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Griffiths W.J., Wang Y. Cholesterol metabolism: from lipidomics to immunology. J. Lipid Res. 2022;63 doi: 10.1016/j.jlr.2021.100165. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Reboldi A., Dang E. Cholesterol metabolism in innate and adaptive response. F1000Res. 2018;7 doi: 10.12688/f1000research.15500.1. F1000 Faculty Rev-1647. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Louie A.Y., Tingling J., Dray E., Hussain J., McKim D.B., Swanson K.S., Steelman A.J. Dietary Cholesterol Causes Inflammatory Imbalance and Exacerbates Morbidity in Mice Infected with Influenza A Virus. J. Immunol. 2022;208:2523–2539. doi: 10.4049/jimmunol.2100927. [DOI] [PubMed] [Google Scholar]
11.Gao P., Ji M., Liu X., Chen X., Liu H., Li S., Jia B., Li C., Ren L., Zhao X., et al. Apolipoprotein E mediates cell resistance to influenza virus infection. Sci. Adv. 2022;8 doi: 10.1126/sciadv.abm6668. [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Hu B., Höfer C.T., Thiele C., Veit M. Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity. J. Virol. 2019;93 doi: 10.1128/JVI.00555-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Li Y.J., Chen C.Y., Yang J.H., Chiu Y.F. Modulating cholesterol-rich lipid rafts to disrupt influenza A virus infection. Front. Immunol. 2022;13 doi: 10.3389/fimmu.2022.982264. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Ripa I., Andreu S., Lopez-Guerrero J.A., Bello-Morales R. Membrane Rafts: Portals for Viral Entry. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.631274. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Tang B., Sun E.Z., Zhang Z.L., Liu S.L., Liu J., Kusumi A., Hu Z.H., Zeng T., Kang Y.F., Tang H.W., Pang D.W. Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry. J. Virol. 2022;96 doi: 10.1128/jvi.01813-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Li G., Su B., Fu P., Bai Y., Ding G., Li D., Wang J., Yang G., Chu B. NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry. Sci. China Life Sci. 2022;65:341–361. doi: 10.1007/s11427-021-1929-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Motsa B.B., Stahelin R.V. Lipid-protein interactions in virus assembly and budding from the host cell plasma membrane. Biochem. Soc. Trans. 2021;49:1633–1641. doi: 10.1042/BST20200854. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Kleinehr J., Wilden J.J., Boergeling Y., Ludwig S., Hrincius E.R. Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets. Viruses. 2021;13 doi: 10.3390/v13102068. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Bukrinsky M.I., Mukhamedova N., Sviridov D. Lipid rafts and pathogens: the art of deception and exploitation. J. Lipid Res. 2020;61:601–610. doi: 10.1194/jlr.TR119000391. [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Duan Y., Gong K., Xu S., Zhang F., Meng X., Han J. Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics. Signal Transduct. Targeted Ther. 2022;7:265. doi: 10.1038/s41392-022-01125-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Sun X., Whittaker G.R. Role for influenza virus envelope cholesterol in virus entry and infection. J. Virol. 2003;77:12543–12551. doi: 10.1128/JVI.77.23.12543-12551.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Barman S., Nayak D.P. Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells. J. Virol. 2007;81:12169–12178. doi: 10.1128/JVI.00835-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Elkins M.R., Williams J.K., Gelenter M.D., Dai P., Kwon B., Sergeyev I.V., Pentelute B.L., Hong M. Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR. Proc. Natl. Acad. Sci. USA. 2017;114:12946–12951. doi: 10.1073/pnas.1715127114. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Teo Q.W., Wong H.H., Heunis T., Stancheva V., Hachim A., Lv H., Siu L., Ho J., Lan Y., Mok C.K.P., et al. Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection. Dev. Cell. 2023;58:2495–2509.e6. doi: 10.1016/j.devcel.2023.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.de Vries M., Herrmann A., Veit M. A cholesterol consensus motif is required for efficient intracellular transport and raft association of a group 2 HA from influenza virus. Biochem. J. 2015;465:305–314. doi: 10.1042/BJ20141114. [DOI] [PubMed] [Google Scholar]
26.Thaa B., Levental I., Herrmann A., Veit M. Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation. Biochem. J. 2011;437:389–397. doi: 10.1042/BJ20110706. [DOI] [PubMed] [Google Scholar]
27.Vandermeer M.L., Thomas A.R., Kamimoto L., Reingold A., Gershman K., Meek J., Farley M.M., Ryan P., Lynfield R., Baumbach J., et al. Association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study. J. Infect. Dis. 2012;205:13–19. doi: 10.1093/infdis/jir695. [DOI] [PubMed] [Google Scholar]
28.Fedson D.S. Pandemic influenza: a potential role for statins in treatment and prophylaxis. Clin. Infect. Dis. 2006;43:199–205. doi: 10.1086/505116. [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Episcopio D., Aminov S., Benjamin S., Germain G., Datan E., Landazuri J., Lockshin R.A., Zakeri Z. Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus. FASEB J. 2019;33:9516–9525. doi: 10.1096/fj.201900428RR. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Budd A., Alleva L., Alsharifi M., Koskinen A., Smythe V., Mullbacher A., Wood J., Clark I. Increased survival after gemfibrozil treatment of severe mouse influenza. Antimicrob. Agents Chemother. 2007;51:2965–2968. doi: 10.1128/AAC.00219-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Luo J., Yang H., Song B.L. Mechanisms and regulation of cholesterol homeostasis. Nat. Rev. Mol. Cell Biol. 2020;21:225–245. doi: 10.1038/s41580-019-0190-7. [DOI] [PubMed] [Google Scholar]
32.Cai D., Wang J., Gao B., Li J., Wu F., Zou J.X., Xu J., Jiang Y., Zou H., Huang Z., et al. RORγ is a targetable master regulator of cholesterol biosynthesis in a cancer subtype. Nat. Commun. 2019;10:4621. doi: 10.1038/s41467-019-12529-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Branche E., Wang Y.T., Viramontes K.M., Valls Cuevas J.M., Xie J., Ana-Sosa-Batiz F., Shafee N., Duttke S.H., McMillan R.E., Clark A.E., et al. SREBP2-dependent lipid gene transcription enhances the infection of human dendritic cells by Zika virus. Nat. Commun. 2022;13:5341. doi: 10.1038/s41467-022-33041-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Yuan S., Chu H., Chan J.F., Ye Z.W., Wen L., Yan B., Lai P.M., Tee K.M., Huang J., Chen D., et al. SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target. Nat. Commun. 2019;10:120. doi: 10.1038/s41467-018-08015-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Schneider W.M., Luna J.M., Hoffmann H.H., Sanchez-Rivera F.J., Leal A.A., Ashbrook A.W., Le Pen J., Ricardo-Lax I., Michailidis E., Peace A., et al. Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks. Cell. 2021;184:120–132.e14. doi: 10.1016/j.cell.2020.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Roca Suarez A.A., Van Renne N., Baumert T.F., Lupberger J. Viral manipulation of STAT3: Evade, exploit, and injure. PLoS Pathog. 2018;14 doi: 10.1371/journal.ppat.1006839. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Chang Z., Wang Y., Zhou X., Long J.E. STAT3 roles in viral infection: antiviral or proviral? Future Virol. 2018;13:557–574. doi: 10.2217/fvl-2018-0033. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Ampomah P.B., Moraes L.A., Lukman H.M., Lim L.H.K. Formyl peptide receptor 2 is regulated by RNA mimics and viruses through an IFN-beta-STAT3-dependent pathway. FASEB J. 2018;32:1468–1478. doi: 10.1096/fj.201700584RR. [DOI] [PubMed] [Google Scholar]
39.Liu S., Liu S., Yu Z., Zhou W., Zheng M., Gu R., Hong J., Yang Z., Chi X., Guo G., et al. STAT3 regulates antiviral immunity by suppressing excessive interferon signaling. Cell Rep. 2023;42 doi: 10.1016/j.celrep.2023.112806. [DOI] [PubMed] [Google Scholar]
40.Adorni M.P., Papotti B., Borghi M.O., Raschi E., Zimetti F., Bernini F., Meroni P.L., Ronda N. Effect of the JAK/STAT Inhibitor Tofacitinib on Macrophage Cholesterol Metabolism. Int. J. Mol. Sci. 2023;24 doi: 10.3390/ijms241612571. [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Kinoshita S., Ogawa W., Okamoto Y., Takashima M., Inoue H., Matsuki Y., Watanabe E., Hiramatsu R., Kasuga M. Role of hepatic STAT3 in the regulation of lipid metabolism. Kobe J. Med. Sci. 2008;54:E200–E208. [PubMed] [Google Scholar]
42.Chen Y.Y., Ge J.Y., Zhu S.Y., Shao Z.M., Yu K.D. Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis. Nat. Commun. 2022;13:791. doi: 10.1038/s41467-022-28452-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.White Z., Sun Z., Sauge E., Cox D., Donen G., Pechkovsky D., Straub V., Francis G.A., Bernatchez P. Limb-girdle muscular dystrophy type 2B causes HDL-C abnormalities in patients and statin-resistant muscle wasting in dysferlin-deficient mice. Skelet. Muscle. 2022;12:25. doi: 10.1186/s13395-022-00308-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Verma D.K., Gupta D., Lal S.K. Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus. Viruses. 2018;10 doi: 10.3390/v10110650. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Wang J., Sun J., Hu J., Wang C., Prinz R.A., Peng D., Liu X., Xu X. A77 1726, the active metabolite of the anti-rheumatoid arthritis drug leflunomide, inhibits influenza A virus replication in vitro and in vivo by inhibiting the activity of Janus kinases. FASEB J. 2020;34:10132–10145. doi: 10.1096/fj.201902793RR. [DOI] [PubMed] [Google Scholar]
46.Bajimaya S., Frankl T., Hayashi T., Takimoto T. Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses. Virology. 2017;510:234–241. doi: 10.1016/j.virol.2017.07.024. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Kawaguchi A., Hirohama M., Harada Y., Osari S., Nagata K. Influenza Virus Induces Cholesterol-Enriched Endocytic Recycling Compartments for Budozone Formation via Cell Cycle-Independent Centrosome Maturation. PLoS Pathog. 2015;11 doi: 10.1371/journal.ppat.1005284. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Hillmer E.J., Zhang H., Li H.S., Watowich S.S. STAT3 signaling in immunity. Cytokine Growth Factor Rev. 2016;31:1–15. doi: 10.1016/j.cytogfr.2016.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Olivetta E., Percario Z., Fiorucci G., Mattia G., Schiavoni I., Dennis C., Jäger J., Harris M., Romeo G., Affabris E., Federico M. HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation. J. Immunol. 2003;170:1716–1727. doi: 10.4049/jimmunol.170.4.1716. [DOI] [PubMed] [Google Scholar]
50.Gao Q., Yang Y., Luo Y., Chen X., Gong T., Wu D., Feng Y., Zheng X., Wang H., Zhang G., et al. African Swine Fever Virus Envelope Glycoprotein CD2v Interacts with Host CSF2RA to Regulate the JAK2-STAT3 Pathway and Inhibit Apoptosis to Facilitate Virus Replication. J. Virol. 2023;97 doi: 10.1128/jvi.01889-22. [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Hui K.P., Li H.S., Cheung M.C., Chan R.W., Yuen K.M., Mok C.K., Nicholls J.M., Peiris J.S.M., Chan M.C.W. Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3. Sci. Rep. 2016;6 doi: 10.1038/srep28593. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Wang J., Prinz R.A., Liu X., Xu X. In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2. Viruses. 2020;12 doi: 10.3390/v12101141. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.McCartney E.M., Helbig K.J., Narayana S.K., Eyre N.S., Aloia A.L., Beard M.R. Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus. Hepatology. 2013;58:1558–1568. doi: 10.1002/hep.26496. [DOI] [PubMed] [Google Scholar]
54.Niu Y., Si Y., Li Y., Chi X., Li X., Liu X., Li D., Cheng M., Fan J., Si S., Yang W. A novel small-molecule inhibitor of hepatitis C virus replication acts by suppressing signal transducer and activator of transcription 3. J. Antimicrob. Chemother. 2015;70:2013–2023. doi: 10.1093/jac/dkv077. [DOI] [PubMed] [Google Scholar]
55.Lupberger J., Duong F.H., Fofana I., Zona L., Xiao F., Thumann C., Durand S.C., Pessaux P., Zeisel M.B., Heim M.H., Baumert T.F. Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha. Hepatology. 2013;58:1225–1235. doi: 10.1002/hep.26404. [DOI] [PubMed] [Google Scholar]
56.Wang W.B., Levy D.E., Lee C.K. STAT3 negatively regulates type I IFN-mediated antiviral response. J. Immunol. 2011;187:2578–2585. doi: 10.4049/jimmunol.1004128. [DOI] [PubMed] [Google Scholar]
57.Wang H., Yuan M., Wang S., Zhang L., Zhang R., Zou X., Wang X., Chen D., Wu Z. STAT3 Regulates the Type I IFN-Mediated Antiviral Response by Interfering with the Nuclear Entry of STAT1. Int. J. Mol. Sci. 2019;20 doi: 10.3390/ijms20194870. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Shen W., Shao W., Wang Q., Wang B., Zhao G., Gu A., Jiang Z., Hu H. Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice. J. Biomed. Sci. 2023;30:44. doi: 10.1186/s12929-023-00933-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Chen K., Zhao Z., Wang G., Zou J., Yu X., Zhang D., Zeng G., Tang C. Interleukin-5 promotes ATP-binding cassette transporter A1 expression through miR-211/JAK2/STAT3 pathways in THP-1-dervied macrophages. Acta Biochim. Biophys. Sin. 2020;52:832–841. doi: 10.1093/abbs/gmaa071. [DOI] [PubMed] [Google Scholar]
60.Kleinfelter L.M., Jangra R.K., Jae L.T., Herbert A.S., Mittler E., Stiles K.M., Wirchnianski A.S., Kielian M., Brummelkamp T.R., Dye J.M., Chandran K. Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion. mBio. 2015;6 doi: 10.1128/mBio.00801-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Watanabe T., Kawakami E., Shoemaker J.E., Lopes T.J., Matsuoka Y., Tomita Y., Kozuka-Hata H., Gorai T., Kuwahara T., Takeda E., et al. Influenza virus-host interactome screen as a platform for antiviral drug development. Cell Host Microbe. 2014;16:795–805. doi: 10.1016/j.chom.2014.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Han J., Perez J.T., Chen C., Li Y., Benitez A., Kandasamy M., Lee Y., Andrade J., tenOever B., Manicassamy B. Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication. Cell Rep. 2018;23:596–607. doi: 10.1016/j.celrep.2018.03.045. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Mecate-Zambrano A., Sukumar S., Seebohm G., Ciminski K., Schreiber A., Anhlan D., Greune L., Wixler L., Grothe S., Stein N.C., et al. Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly. PLoS Pathog. 2020;16 doi: 10.1371/journal.ppat.1008775. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Wang S., Zhao Z., Bi Y., Sun L., Liu X., Liu W. Tyrosine 132 phosphorylation of influenza A virus M1 protein is crucial for virus replication by controlling the nuclear import of M1. J. Virol. 2013;87:6182–6191. doi: 10.1128/JVI.03024-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Eierhoff T., Hrincius E.R., Rescher U., Ludwig S., Ehrhardt C. The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells. PLoS Pathog. 2010;6 doi: 10.1371/journal.ppat.1001099. [DOI] [PMC free article] [PubMed] [Google Scholar]
66.Yao D., Bao L., Li F., Liu B., Wu X., Hu Z., Xu J., Wang W., Zhang X. H1N1 influenza virus dose dependent induction of dysregulated innate immune responses and STAT1/3 activation are associated with pulmonary immunopathological damage. Virulence. 2022;13:1558–1572. doi: 10.1080/21505594.2022.2120951. [DOI] [PMC free article] [PubMed] [Google Scholar]
67.He L., Zhao G., Zhong L., Liu Q., Duan Z., Gu M., Wang X., Liu X., Liu X. Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China. Arch. Virol. 2013;158:2531–2541. doi: 10.1007/s00705-013-1771-y. [DOI] [PubMed] [Google Scholar]
68.Gao R., Gu M., Liu K., Li Q., Li J., Shi L., Li X., Wang X., Hu J., Liu X., et al. T160A mutation-induced deglycosylation at site 158 in hemagglutinin is a critical determinant of the dual receptor binding properties of clade 2.3.4.4 H5NX subtype avian influenza viruses. Vet. Microbiol. 2018;217:158–166. doi: 10.1016/j.vetmic.2018.03.018. [DOI] [PubMed] [Google Scholar]
69.Hu J., Hu Z., Song Q., Gu M., Liu X., Wang X., Hu S., Chen C., Liu H., Liu W., et al. The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice. J. Virol. 2013;87:2660–2672. doi: 10.1128/JVI.02891-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
70.Zheng Z.Y., Tian L., Bu W., Fan C., Gao X., Wang H., Liao Y.H., Li Y., Lewis M.T., Edwards D., et al. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation. Cell Rep. 2015;12:511–524. doi: 10.1016/j.celrep.2015.06.044. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1 and S2 and Table S1

mmc1.pdf^{(301.2KB, pdf)}

Data Availability Statement

•
All data reported in this paper will be shared by the lead contact upon request.
•
This paper does not report original code.
•
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
•
The authors declare that the data supporting the findings of this study are available within the article and from the corresponding author upon request.

[bib1] 1.Paules C., Subbarao K. Influenza. Lancet. 2017;390:697–708. doi: 10.1016/S0140-6736(17)30129-0. [DOI] [PubMed] [Google Scholar]

[bib2] 2.Nguyen J.L., Yang W., Ito K., Matte T.D., Shaman J., Kinney P.L. Seasonal Influenza Infections and Cardiovascular Disease Mortality. JAMA Cardiol. 2016;1:274–281. doi: 10.1001/jamacardio.2016.0433. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib3] 3.Allard R., Leclerc P., Tremblay C., Tannenbaum T.N. Diabetes and the severity of pandemic influenza A (H1N1) infection. Diabetes Care. 2010;33:1491–1493. doi: 10.2337/dc09-2215. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib4] 4.Smith M., Honce R., Schultz-Cherry S. Metabolic Syndrome and Viral Pathogenesis: Lessons from Influenza and Coronaviruses. J. Virol. 2020;94 doi: 10.1128/JVI.00665-20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib5] 5.Louie J.K., Acosta M., Samuel M.C., Schechter R., Vugia D.J., Harriman K., Matyas B.T., California Pandemic H1N1 Working Group A novel risk factor for a novel virus: obesity and 2009 pandemic influenza A (H1N1) Clin. Infect. Dis. 2011;52:301–312. doi: 10.1093/cid/ciq152. [DOI] [PubMed] [Google Scholar]

[bib6] 6.Hancková M., Betakova T. Pandemics of the 21st Century: The Risk Factor for Obese People. Viruses. 2021;14:25. doi: 10.3390/v14010025. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib7] 7.Schultz B.M., Paduro C.A., Salazar G.A., Salazar-Echegarai F.J., Sebastian V.P., Riedel C.A., Kalergis A.M., Alvarez-Lobos M., Bueno S.M. A Potential Role of Salmonella Infection in the Onset of Inflammatory Bowel Diseases. Front. Immunol. 2017;8:191. doi: 10.3389/fimmu.2017.00191. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib8] 8.Griffiths W.J., Wang Y. Cholesterol metabolism: from lipidomics to immunology. J. Lipid Res. 2022;63 doi: 10.1016/j.jlr.2021.100165. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib9] 9.Reboldi A., Dang E. Cholesterol metabolism in innate and adaptive response. F1000Res. 2018;7 doi: 10.12688/f1000research.15500.1. F1000 Faculty Rev-1647. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib10] 10.Louie A.Y., Tingling J., Dray E., Hussain J., McKim D.B., Swanson K.S., Steelman A.J. Dietary Cholesterol Causes Inflammatory Imbalance and Exacerbates Morbidity in Mice Infected with Influenza A Virus. J. Immunol. 2022;208:2523–2539. doi: 10.4049/jimmunol.2100927. [DOI] [PubMed] [Google Scholar]

[bib11] 11.Gao P., Ji M., Liu X., Chen X., Liu H., Li S., Jia B., Li C., Ren L., Zhao X., et al. Apolipoprotein E mediates cell resistance to influenza virus infection. Sci. Adv. 2022;8 doi: 10.1126/sciadv.abm6668. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib12] 12.Hu B., Höfer C.T., Thiele C., Veit M. Cholesterol Binding to the Transmembrane Region of a Group 2 Hemagglutinin (HA) of Influenza Virus Is Essential for Virus Replication, Affecting both Virus Assembly and HA Fusion Activity. J. Virol. 2019;93 doi: 10.1128/JVI.00555-19. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib13] 13.Li Y.J., Chen C.Y., Yang J.H., Chiu Y.F. Modulating cholesterol-rich lipid rafts to disrupt influenza A virus infection. Front. Immunol. 2022;13 doi: 10.3389/fimmu.2022.982264. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib14] 14.Ripa I., Andreu S., Lopez-Guerrero J.A., Bello-Morales R. Membrane Rafts: Portals for Viral Entry. Front. Microbiol. 2021;12 doi: 10.3389/fmicb.2021.631274. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib15] 15.Tang B., Sun E.Z., Zhang Z.L., Liu S.L., Liu J., Kusumi A., Hu Z.H., Zeng T., Kang Y.F., Tang H.W., Pang D.W. Sphingomyelin-Sequestered Cholesterol Domain Recruits Formin-Binding Protein 17 for Constricting Clathrin-Coated Pits in Influenza Virus Entry. J. Virol. 2022;96 doi: 10.1128/jvi.01813-21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib16] 16.Li G., Su B., Fu P., Bai Y., Ding G., Li D., Wang J., Yang G., Chu B. NPC1-regulated dynamic of clathrin-coated pits is essential for viral entry. Sci. China Life Sci. 2022;65:341–361. doi: 10.1007/s11427-021-1929-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib17] 17.Motsa B.B., Stahelin R.V. Lipid-protein interactions in virus assembly and budding from the host cell plasma membrane. Biochem. Soc. Trans. 2021;49:1633–1641. doi: 10.1042/BST20200854. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib18] 18.Kleinehr J., Wilden J.J., Boergeling Y., Ludwig S., Hrincius E.R. Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets. Viruses. 2021;13 doi: 10.3390/v13102068. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib19] 19.Bukrinsky M.I., Mukhamedova N., Sviridov D. Lipid rafts and pathogens: the art of deception and exploitation. J. Lipid Res. 2020;61:601–610. doi: 10.1194/jlr.TR119000391. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib20] 20.Duan Y., Gong K., Xu S., Zhang F., Meng X., Han J. Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics. Signal Transduct. Targeted Ther. 2022;7:265. doi: 10.1038/s41392-022-01125-5. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib21] 21.Sun X., Whittaker G.R. Role for influenza virus envelope cholesterol in virus entry and infection. J. Virol. 2003;77:12543–12551. doi: 10.1128/JVI.77.23.12543-12551.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib22] 22.Barman S., Nayak D.P. Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells. J. Virol. 2007;81:12169–12178. doi: 10.1128/JVI.00835-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib23] 23.Elkins M.R., Williams J.K., Gelenter M.D., Dai P., Kwon B., Sergeyev I.V., Pentelute B.L., Hong M. Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR. Proc. Natl. Acad. Sci. USA. 2017;114:12946–12951. doi: 10.1073/pnas.1715127114. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib24] 24.Teo Q.W., Wong H.H., Heunis T., Stancheva V., Hachim A., Lv H., Siu L., Ho J., Lan Y., Mok C.K.P., et al. Usp25-Erlin1/2 activity limits cholesterol flux to restrict virus infection. Dev. Cell. 2023;58:2495–2509.e6. doi: 10.1016/j.devcel.2023.08.013. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib25] 25.de Vries M., Herrmann A., Veit M. A cholesterol consensus motif is required for efficient intracellular transport and raft association of a group 2 HA from influenza virus. Biochem. J. 2015;465:305–314. doi: 10.1042/BJ20141114. [DOI] [PubMed] [Google Scholar]

[bib26] 26.Thaa B., Levental I., Herrmann A., Veit M. Intrinsic membrane association of the cytoplasmic tail of influenza virus M2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation. Biochem. J. 2011;437:389–397. doi: 10.1042/BJ20110706. [DOI] [PubMed] [Google Scholar]

[bib27] 27.Vandermeer M.L., Thomas A.R., Kamimoto L., Reingold A., Gershman K., Meek J., Farley M.M., Ryan P., Lynfield R., Baumbach J., et al. Association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study. J. Infect. Dis. 2012;205:13–19. doi: 10.1093/infdis/jir695. [DOI] [PubMed] [Google Scholar]

[bib28] 28.Fedson D.S. Pandemic influenza: a potential role for statins in treatment and prophylaxis. Clin. Infect. Dis. 2006;43:199–205. doi: 10.1086/505116. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib29] 29.Episcopio D., Aminov S., Benjamin S., Germain G., Datan E., Landazuri J., Lockshin R.A., Zakeri Z. Atorvastatin restricts the ability of influenza virus to generate lipid droplets and severely suppresses the replication of the virus. FASEB J. 2019;33:9516–9525. doi: 10.1096/fj.201900428RR. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib30] 30.Budd A., Alleva L., Alsharifi M., Koskinen A., Smythe V., Mullbacher A., Wood J., Clark I. Increased survival after gemfibrozil treatment of severe mouse influenza. Antimicrob. Agents Chemother. 2007;51:2965–2968. doi: 10.1128/AAC.00219-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib31] 31.Luo J., Yang H., Song B.L. Mechanisms and regulation of cholesterol homeostasis. Nat. Rev. Mol. Cell Biol. 2020;21:225–245. doi: 10.1038/s41580-019-0190-7. [DOI] [PubMed] [Google Scholar]

[bib32] 32.Cai D., Wang J., Gao B., Li J., Wu F., Zou J.X., Xu J., Jiang Y., Zou H., Huang Z., et al. RORγ is a targetable master regulator of cholesterol biosynthesis in a cancer subtype. Nat. Commun. 2019;10:4621. doi: 10.1038/s41467-019-12529-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib33] 33.Branche E., Wang Y.T., Viramontes K.M., Valls Cuevas J.M., Xie J., Ana-Sosa-Batiz F., Shafee N., Duttke S.H., McMillan R.E., Clark A.E., et al. SREBP2-dependent lipid gene transcription enhances the infection of human dendritic cells by Zika virus. Nat. Commun. 2022;13:5341. doi: 10.1038/s41467-022-33041-1. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib34] 34.Yuan S., Chu H., Chan J.F., Ye Z.W., Wen L., Yan B., Lai P.M., Tee K.M., Huang J., Chen D., et al. SREBP-dependent lipidomic reprogramming as a broad-spectrum antiviral target. Nat. Commun. 2019;10:120. doi: 10.1038/s41467-018-08015-x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib35] 35.Schneider W.M., Luna J.M., Hoffmann H.H., Sanchez-Rivera F.J., Leal A.A., Ashbrook A.W., Le Pen J., Ricardo-Lax I., Michailidis E., Peace A., et al. Genome-Scale Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks. Cell. 2021;184:120–132.e14. doi: 10.1016/j.cell.2020.12.006. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib36] 36.Roca Suarez A.A., Van Renne N., Baumert T.F., Lupberger J. Viral manipulation of STAT3: Evade, exploit, and injure. PLoS Pathog. 2018;14 doi: 10.1371/journal.ppat.1006839. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib37] 37.Chang Z., Wang Y., Zhou X., Long J.E. STAT3 roles in viral infection: antiviral or proviral? Future Virol. 2018;13:557–574. doi: 10.2217/fvl-2018-0033. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib38] 38.Ampomah P.B., Moraes L.A., Lukman H.M., Lim L.H.K. Formyl peptide receptor 2 is regulated by RNA mimics and viruses through an IFN-beta-STAT3-dependent pathway. FASEB J. 2018;32:1468–1478. doi: 10.1096/fj.201700584RR. [DOI] [PubMed] [Google Scholar]

[bib39] 39.Liu S., Liu S., Yu Z., Zhou W., Zheng M., Gu R., Hong J., Yang Z., Chi X., Guo G., et al. STAT3 regulates antiviral immunity by suppressing excessive interferon signaling. Cell Rep. 2023;42 doi: 10.1016/j.celrep.2023.112806. [DOI] [PubMed] [Google Scholar]

[bib40] 40.Adorni M.P., Papotti B., Borghi M.O., Raschi E., Zimetti F., Bernini F., Meroni P.L., Ronda N. Effect of the JAK/STAT Inhibitor Tofacitinib on Macrophage Cholesterol Metabolism. Int. J. Mol. Sci. 2023;24 doi: 10.3390/ijms241612571. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib41] 41.Kinoshita S., Ogawa W., Okamoto Y., Takashima M., Inoue H., Matsuki Y., Watanabe E., Hiramatsu R., Kasuga M. Role of hepatic STAT3 in the regulation of lipid metabolism. Kobe J. Med. Sci. 2008;54:E200–E208. [PubMed] [Google Scholar]

[bib42] 42.Chen Y.Y., Ge J.Y., Zhu S.Y., Shao Z.M., Yu K.D. Copy number amplification of ENSA promotes the progression of triple-negative breast cancer via cholesterol biosynthesis. Nat. Commun. 2022;13:791. doi: 10.1038/s41467-022-28452-z. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib43] 43.White Z., Sun Z., Sauge E., Cox D., Donen G., Pechkovsky D., Straub V., Francis G.A., Bernatchez P. Limb-girdle muscular dystrophy type 2B causes HDL-C abnormalities in patients and statin-resistant muscle wasting in dysferlin-deficient mice. Skelet. Muscle. 2022;12:25. doi: 10.1186/s13395-022-00308-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib44] 44.Verma D.K., Gupta D., Lal S.K. Host Lipid Rafts Play a Major Role in Binding and Endocytosis of Influenza A Virus. Viruses. 2018;10 doi: 10.3390/v10110650. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib45] 45.Wang J., Sun J., Hu J., Wang C., Prinz R.A., Peng D., Liu X., Xu X. A77 1726, the active metabolite of the anti-rheumatoid arthritis drug leflunomide, inhibits influenza A virus replication in vitro and in vivo by inhibiting the activity of Janus kinases. FASEB J. 2020;34:10132–10145. doi: 10.1096/fj.201902793RR. [DOI] [PubMed] [Google Scholar]

[bib46] 46.Bajimaya S., Frankl T., Hayashi T., Takimoto T. Cholesterol is required for stability and infectivity of influenza A and respiratory syncytial viruses. Virology. 2017;510:234–241. doi: 10.1016/j.virol.2017.07.024. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib47] 47.Kawaguchi A., Hirohama M., Harada Y., Osari S., Nagata K. Influenza Virus Induces Cholesterol-Enriched Endocytic Recycling Compartments for Budozone Formation via Cell Cycle-Independent Centrosome Maturation. PLoS Pathog. 2015;11 doi: 10.1371/journal.ppat.1005284. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib48] 48.Hillmer E.J., Zhang H., Li H.S., Watowich S.S. STAT3 signaling in immunity. Cytokine Growth Factor Rev. 2016;31:1–15. doi: 10.1016/j.cytogfr.2016.05.001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib49] 49.Olivetta E., Percario Z., Fiorucci G., Mattia G., Schiavoni I., Dennis C., Jäger J., Harris M., Romeo G., Affabris E., Federico M. HIV-1 Nef induces the release of inflammatory factors from human monocyte/macrophages: involvement of Nef endocytotic signals and NF-kappa B activation. J. Immunol. 2003;170:1716–1727. doi: 10.4049/jimmunol.170.4.1716. [DOI] [PubMed] [Google Scholar]

[bib50] 50.Gao Q., Yang Y., Luo Y., Chen X., Gong T., Wu D., Feng Y., Zheng X., Wang H., Zhang G., et al. African Swine Fever Virus Envelope Glycoprotein CD2v Interacts with Host CSF2RA to Regulate the JAK2-STAT3 Pathway and Inhibit Apoptosis to Facilitate Virus Replication. J. Virol. 2023;97 doi: 10.1128/jvi.01889-22. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib51] 51.Hui K.P., Li H.S., Cheung M.C., Chan R.W., Yuen K.M., Mok C.K., Nicholls J.M., Peiris J.S.M., Chan M.C.W. Highly pathogenic avian influenza H5N1 virus delays apoptotic responses via activation of STAT3. Sci. Rep. 2016;6 doi: 10.1038/srep28593. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib52] 52.Wang J., Prinz R.A., Liu X., Xu X. In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2. Viruses. 2020;12 doi: 10.3390/v12101141. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib53] 53.McCartney E.M., Helbig K.J., Narayana S.K., Eyre N.S., Aloia A.L., Beard M.R. Signal transducer and activator of transcription 3 is a proviral host factor for hepatitis C virus. Hepatology. 2013;58:1558–1568. doi: 10.1002/hep.26496. [DOI] [PubMed] [Google Scholar]

[bib54] 54.Niu Y., Si Y., Li Y., Chi X., Li X., Liu X., Li D., Cheng M., Fan J., Si S., Yang W. A novel small-molecule inhibitor of hepatitis C virus replication acts by suppressing signal transducer and activator of transcription 3. J. Antimicrob. Chemother. 2015;70:2013–2023. doi: 10.1093/jac/dkv077. [DOI] [PubMed] [Google Scholar]

[bib55] 55.Lupberger J., Duong F.H., Fofana I., Zona L., Xiao F., Thumann C., Durand S.C., Pessaux P., Zeisel M.B., Heim M.H., Baumert T.F. Epidermal growth factor receptor signaling impairs the antiviral activity of interferon-alpha. Hepatology. 2013;58:1225–1235. doi: 10.1002/hep.26404. [DOI] [PubMed] [Google Scholar]

[bib56] 56.Wang W.B., Levy D.E., Lee C.K. STAT3 negatively regulates type I IFN-mediated antiviral response. J. Immunol. 2011;187:2578–2585. doi: 10.4049/jimmunol.1004128. [DOI] [PubMed] [Google Scholar]

[bib57] 57.Wang H., Yuan M., Wang S., Zhang L., Zhang R., Zou X., Wang X., Chen D., Wu Z. STAT3 Regulates the Type I IFN-Mediated Antiviral Response by Interfering with the Nuclear Entry of STAT1. Int. J. Mol. Sci. 2019;20 doi: 10.3390/ijms20194870. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib58] 58.Shen W., Shao W., Wang Q., Wang B., Zhao G., Gu A., Jiang Z., Hu H. Dietary diosgenin transcriptionally down-regulated intestinal NPC1L1 expression to prevent cholesterol gallstone formation in mice. J. Biomed. Sci. 2023;30:44. doi: 10.1186/s12929-023-00933-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib59] 59.Chen K., Zhao Z., Wang G., Zou J., Yu X., Zhang D., Zeng G., Tang C. Interleukin-5 promotes ATP-binding cassette transporter A1 expression through miR-211/JAK2/STAT3 pathways in THP-1-dervied macrophages. Acta Biochim. Biophys. Sin. 2020;52:832–841. doi: 10.1093/abbs/gmaa071. [DOI] [PubMed] [Google Scholar]

[bib60] 60.Kleinfelter L.M., Jangra R.K., Jae L.T., Herbert A.S., Mittler E., Stiles K.M., Wirchnianski A.S., Kielian M., Brummelkamp T.R., Dye J.M., Chandran K. Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion. mBio. 2015;6 doi: 10.1128/mBio.00801-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib61] 61.Watanabe T., Kawakami E., Shoemaker J.E., Lopes T.J., Matsuoka Y., Tomita Y., Kozuka-Hata H., Gorai T., Kuwahara T., Takeda E., et al. Influenza virus-host interactome screen as a platform for antiviral drug development. Cell Host Microbe. 2014;16:795–805. doi: 10.1016/j.chom.2014.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib62] 62.Han J., Perez J.T., Chen C., Li Y., Benitez A., Kandasamy M., Lee Y., Andrade J., tenOever B., Manicassamy B. Genome-wide CRISPR/Cas9 Screen Identifies Host Factors Essential for Influenza Virus Replication. Cell Rep. 2018;23:596–607. doi: 10.1016/j.celrep.2018.03.045. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib63] 63.Mecate-Zambrano A., Sukumar S., Seebohm G., Ciminski K., Schreiber A., Anhlan D., Greune L., Wixler L., Grothe S., Stein N.C., et al. Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly. PLoS Pathog. 2020;16 doi: 10.1371/journal.ppat.1008775. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib64] 64.Wang S., Zhao Z., Bi Y., Sun L., Liu X., Liu W. Tyrosine 132 phosphorylation of influenza A virus M1 protein is crucial for virus replication by controlling the nuclear import of M1. J. Virol. 2013;87:6182–6191. doi: 10.1128/JVI.03024-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib65] 65.Eierhoff T., Hrincius E.R., Rescher U., Ludwig S., Ehrhardt C. The epidermal growth factor receptor (EGFR) promotes uptake of influenza A viruses (IAV) into host cells. PLoS Pathog. 2010;6 doi: 10.1371/journal.ppat.1001099. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib66] 66.Yao D., Bao L., Li F., Liu B., Wu X., Hu Z., Xu J., Wang W., Zhang X. H1N1 influenza virus dose dependent induction of dysregulated innate immune responses and STAT1/3 activation are associated with pulmonary immunopathological damage. Virulence. 2022;13:1558–1572. doi: 10.1080/21505594.2022.2120951. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib67] 67.He L., Zhao G., Zhong L., Liu Q., Duan Z., Gu M., Wang X., Liu X., Liu X. Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China. Arch. Virol. 2013;158:2531–2541. doi: 10.1007/s00705-013-1771-y. [DOI] [PubMed] [Google Scholar]

[bib68] 68.Gao R., Gu M., Liu K., Li Q., Li J., Shi L., Li X., Wang X., Hu J., Liu X., et al. T160A mutation-induced deglycosylation at site 158 in hemagglutinin is a critical determinant of the dual receptor binding properties of clade 2.3.4.4 H5NX subtype avian influenza viruses. Vet. Microbiol. 2018;217:158–166. doi: 10.1016/j.vetmic.2018.03.018. [DOI] [PubMed] [Google Scholar]

[bib69] 69.Hu J., Hu Z., Song Q., Gu M., Liu X., Wang X., Hu S., Chen C., Liu H., Liu W., et al. The PA-gene-mediated lethal dissemination and excessive innate immune response contribute to the high virulence of H5N1 avian influenza virus in mice. J. Virol. 2013;87:2660–2672. doi: 10.1128/JVI.02891-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[bib70] 70.Zheng Z.Y., Tian L., Bu W., Fan C., Gao X., Wang H., Liao Y.H., Li Y., Lewis M.T., Edwards D., et al. Wild-Type N-Ras, Overexpressed in Basal-like Breast Cancer, Promotes Tumor Formation by Inducing IL-8 Secretion via JAK2 Activation. Cell Rep. 2015;12:511–524. doi: 10.1016/j.celrep.2015.06.044. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Influenza A virus infection activates STAT3 to enhance SREBP2 expression, cholesterol biosynthesis, and virus replication

Jingting Zhang

Yunhan Wu

Yujie Wang

Penggang Liu

Kaituo Liu

Jing Sun

Pinghu Zhang

Xiaoquan Wang

Xiufan Liu

Xiulong Xu

Summary

Graphical abstract

Highlights

Introduction

Results

IAV infection induces SREBP2 activation

Figure 1.

SREBP2 activation is required for IAV replication

Figure 2.

IAV infection activates STAT3 to enhance virus replication

Figure 3.

Figure 4.

STAT3 upregulates SREBP2 expression

Figure 5.

STAT3 promotes IAV replication by enhancing cholesterol biosynthesis

Figure 6.

JAK2 activates STAT3 to promote cholesterol biosynthesis

Figure 7.

Discussion

Limitations of the study

STAR★Methods

Key resources table

Resource availability

Lead contact

Materials availability

Data and code availability

Experimental model and study participant details

Cell culture and virus

Method details

EC50 and CC50

Plasmids

CRISPR/Cas9 sgRNA design

SREBP2, STAT3, and JAK2 transfection

Western blot

RT-qPCR analyses

Virus binding assay

Filipin staining

Quantification and statistical analysis

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

EC₅₀ and CC₅₀