Phosphoproteomics to Characterize Host Response During Influenza A Virus Infection of Human Macrophages

Abstract

Influenza A viruses cause infections in the human respiratory tract and give rise to annual seasonal outbreaks, as well as more rarely dreaded pandemics. Influenza A viruses become quickly resistant to the virus-directed antiviral treatments, which are the current main treatment options. A promising alternative approach is to target host cell factors that are exploited by influenza viruses. To this end, we characterized the phosphoproteome of influenza A virus infected primary human macrophages to elucidate the intracellular signaling pathways and critical host factors activated upon influenza infection. We identified 1675 phosphoproteins, 4004 phosphopeptides and 4146 nonredundant phosphosites. The phosphorylation of 1113 proteins (66%) was regulated upon infection, highlighting the importance of such global phosphoproteomic profiling in primary cells. Notably, 285 of the identified phosphorylation sites have not been previously described in publicly available phosphorylation databases, despite many published large-scale phosphoproteome studies using human and mouse cell lines. Systematic bioinformatics analysis of the phosphoproteome data indicated that the phosphorylation of proteins involved in the ubiquitin/proteasome pathway (such as TRIM22 and TRIM25) and antiviral responses (such as MAVS) changed in infected macrophages. Proteins known to play roles in small GTPase-, mitogen-activated protein kinase-, and cyclin-dependent kinase- signaling were also regulated by phosphorylation upon infection. In particular, the influenza infection had a major influence on the phosphorylation profiles of a large number of cyclin-dependent kinase substrates. Functional studies using cyclin-dependent kinase inhibitors showed that the cyclin-dependent kinase activity is required for efficient viral replication and for activation of the host antiviral responses. In addition, we show that cyclin-dependent kinase inhibitors protect IAV-infected mice from death. In conclusion, we provide the first comprehensive phosphoproteome characterization of influenza A virus infection in primary human macrophages, and provide evidence that cyclin-dependent kinases represent potential therapeutic targets for more effective treatment of influenza infections.

Fig. 1.

Phosphoproteome analysis of influenza A virus-infected primary human macrophages. A, Macrophages were infected with influenza A virus for the times indicated. After this, total cell lysates were prepared and analyzed with Western blotting with anti-NP and anti-NS1, anti-phospho(Ser396)-IRF3, anti-IRF3 and anti-IκBα antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) detection was used to confirm equal sample loading. B, The workflow for the phosphoproteomics experiment. C, Phosphoprotein and -peptide identification results when pooling the identifications of both biological replicates. D, Classification of the uniquely phosphorylated proteins based on their molecular type. Ingenuity Pathway Analysis (IPA^®) assigned a specific molecular type to 502 of the 1113 proteins identified as uniquely phosphorylated upon IAV infection.

Fig. 2.

The phosphorylation profiles change upon influenza A virus infection for host proteins known to be involved in different stages of viral infection. In total 180 proteins of the 1113 uniquely phosphorylated proteins were directly associated with the biological function “viral infection” according to IPA^®, and 177 of these had annotated subcellular locations. These 177 proteins are shown and their molecular type is indicated by specific shapes. The purple nodes are proteins, which have unique phosphorylation sites in both uninfected (control) and infected conditions. The red nodes are proteins, which have unique phosphorylation sites only after infection. The green nodes are proteins that have unique phosphorylation sites only in the control sample. Proteins associated with small GTPase signaling (connected with magenta colored edges), replication of influenza A virus (connected with orange colored edges), and endocytosis (connected with blue colored edges) are highlighted with edges connected to the respective biological function. Phosphoproteins with novel phosphorylation sites are highlighted in bold text coding and yellow colored outlining.

Fig. 3.

Cyclin-dependent kinases are activated upon influenza A virus infection, and their specific inhibitors rescue primary macrophages from virus-induced cell death. A, The uniquely phosphorylated substrate proteins of CDK1, CDK2, CDK7, and CDK9 are shown with their gene names. The substrates for CDK1 and CDK2 are classified according to their biological process (note that one protein can belong to more than one class). Black colored coded proteins have unique phosphorylations in control and IAV samples, red colored coded proteins have unique phosphorylations only after IAV infection, and blue colored proteins have unique phosphorylations only in the untreated, control sample. The bold text coded proteins were identified to have novel phosphosites. B, Mock- or influenza A virus (MOI 3) infected primary macrophages were treated with increasing concentrations of various CDK inhibitors (SNS-032, flavopiridol, dinaciclib, roscovitine, and pablociclib). Cell viability was measured with the CTG assay at 24 h post-infection.

Fig. 4.

The effect of cyclin-dependent kinase inhibitors on virus replication and antiviral and chemokine response in influenza A virus-infected human macrophages. A, Primary human macrophages differentiated from three donors were pretreated with CDK inhibitors SNS-032 (0.3 μm) or flavopiridol (0.3 μm) for 1 h after which they were left uninfected or infected with IAV for 6 h. After this, the cells from the separate donors were collected and pooled, and total cellular RNA was prepared. The mRNA expression of IAV M2 and NP genes was analyzed with quantitative RT-PCR. Data is presented as mean and S.D. of n = 3 independent measurements. The results were compared with the infected and untreated sample. B, Primary human macrophages differentiated from three donors were pretreated with CDK inhibitors of SNS-032 (0.3 μm) or flavopiridol (0.3 μm) 1 h before infection. The protein abundances of influenza A virus NS1 and NP proteins 18 h post-infection were measured with Western blotting, using GAPDH as a loading control. C, Primary human macrophages differentiated from three donors were pretreated with CDK inhibitors of SNS-032 (0.3 μm) or flavopiridol (0.3 μm) 1 h before infection. The expression of IFN-β, IL-29, CXCL10, and CXCL11 mRNAs in IAV infected macrophages was analyzed with quantitative RT-PCR. Data is presented as mean and S.D. of n = 3 independent measurements. The results were compared with the infected and untreated sample. D, Macrophages were pretreated with SNS-032 (0.3 μm) for 1 h after which they were left uninfected or infected with IAV for 6 h. After this, the cell lysates were prepared and analyzed with Western blotting with anti-phospho-IRF3 and anti-IκBα antibodies. Comparable data were obtained from two different experiments.

Fig. 5.

Cyclin-dependent kinase inhibitors block influenza A virus-induced apoptosis and inflammasome activation. A, Macrophages were pretreated with SNS-032 (0.3 μm) or flavopiridol (0.3 μm) for 1 h after which they were left uninfected or infected with IAV for 18 h. Caspase 3/7, caspase 8, and caspase 9 activity was measured as described under Experimental Procedures. The changes in activity were calculated and compared with the mock/untreated sample (= 1). Data is presented as mean and S.D. of n = 3 biological replicates. The results were compared with the infected and untreated sample. B, Macrophages were pretreated with SNS-032 (0.3 μm) or flavopiridol (0.3 μm) for 1 h after which they were left uninfected or infected with IAV for 18 h. After this, total cell lysates were prepared and expression of cleaved caspase 3 (Asp175) and Bcl-xL expression was analyzed with Western blotting. C, Macrophages from three different donors were pretreated with SNS-032 (0.3 μm) or flavopiridol (0.3 μm) after which they were infected with IAV for 18 h. After this, the cell culture supernatants were collected and IL-18 secretion was analyzed with a Luminex assay. Data is presented as mean and S.D. of n = 3 independent measurements. The results were compared with the infected and untreated sample. D, The activity of caspase 1 was measured in 18 h infected or noninfected macrophages, nontreated or treated with SNS-032 at 0.3 μm at 18 h post-infected. Data is presented as mean and S.D. of n = 3 biological replicates. The results were compared with the infected and untreated sample.

Fig. 6.

SNS-032 treatment rescues influenza A virus-infected mice. Mortality and morbidity of SNS-032 treated and DMSO treated mice after lethal influenza infection. At day 0 the mice were infected with 0.5 LD₅₀ of mouse-adapted PR8 or mock infected. Six mice per group were repeatedly treated intraperitoneally with SNS-032 at 1 day prior and 1, 3, 5, 7, and 9 days post infection. A, Kaplan-Meier survival curves of the infected mice show perfect protection gained by SNS-032 treatment (log-rank test, p < 0.01). B, Relative body weight curves for the two different groups of infected mice. SNS-032 treated mice lost significantly less body weight than the DMSO treated mice at days 4–7 (two-way ANOVA, Bonferroni's multiple comparison adjustment, p < 0.0001). At 9 days post infection more than half of the DMSO treated mice had died and hence from day 9 on, the body weight of the surviving DMSO treated mice was not included in the figure. C, Cytokine levels in mouse lung homogenates at day 5 were determined using mouse cytokine array panel A kit. Lung homogenates from four mice per group were combined before measuring the relative intensity of the luminescence for the different cytokines. Representative results from two experiments are shown.