Influenza A virus infection down regulates JAK1.
To investigate the regulation of JAK1 during influenza A virus infection, JAK1 protein abundance was quantified using Western blotting. We infected cells with three different strain of influenza viruses: A/mallard/Huadong/S/2005 (H5N1), A/chicken/Jiangsu/WJ-14/2015 (H7N9) and A/chicken/Taixing/10/2010 (H9N2). Following all subtypes of IAV infection, the protein level of JAK1 was strongly decreased in both A549 cells, 293T cells and MDCK cells (Fig. 1a). IAV infection-induced JAK1 downregulation was time- and virus dose-dependent, and the protein level of JAK1 was decreased during the replication of IAV, as assayed by the expression of viral NP in infected cells (Fig. 1b and 1c). These data demonstrate that IAV infection significantly downregulates the protein level of JAK1.
IAV infection induces the ubiquitination and proteasome degradation of JAK1.
We determined the stage of JAK1 downregulation during IAV infection. First, the mRNA level of JAK1 in IAV infected cells and control cells were detected using real-time quantitative PCR. IAV infection had no effect on the mRNA level of JAK1 (Fig. 2a), suggesting that the inhibition of JAK1 expression occurred at the post-transcriptional stage. Then we treated IAV infected cells and control cells with the translation inhibitor cycloheximide (CHX) and then detected the protein stability of JAK1. The result showed that IAV infection accelerated JAK1 protein degradation (Fig. 2b). As the ubiquitin proteasome system was reported to mediate the degradation of JAK1[16], we determined if IAV infection induces JAK1 ubiquitination . To detect the ubiquitination of JAK1, Flag-tagged JAK1 and HA-tagged Ub were transiently overexpressed in 293T cells, and the ubiquitination of JAK1 was assessed using immunoprecipitation and Western blotting. IAV infection increased the ubiquitination level of JAK1, indicating that JAK1 ubiquitination is induced by IAV infection (Fig. 2c). Proteasome-dependent and lysosome-dependent protein degradation pathway are two distinct protein degradation mechanisms. We sought to investigate the JAK1 degradation pathway induced by IAV infection. Following infection with IAV, cells were treated with MG-132 (proteasome inhibitor) or NH4Cl (lysosome inhibitor). The degradation of JAK1 induced by IAV was repressed by MG-132 but not by NH4Cl, suggesting that proteasome-dependent pathway mediated the degradation of JAK1 during IAV infection (Fig. 2d).
IAV infection induces cell less responsive to type I and type II IFNs.
Jak1 and the downstream transcription factors are required for the cellular responses to IFNs. Given the ability of IAV to reduce the protein level of JAK1, we sought to gain insight into the influence of IAV in IFNs mediated signaling pathway. We first investigated the activation of transcription factor STAT1 in response to type I and type II IFNs in the presence or absence of IAV infection. Treatment with recombinant human IFN-α or IFN-γ induced STAT1 activation, eliciting high levels of pSTAT1 (Fig. 3a and 3b). However, IAV infection strongly inhibited STAT1 phosphorylation after stimulation with IFN-α or IFN-γ, and at the same time the protein level of JAK1 was significantly downregulated (Fig. 3a and 3b). IFNs treatment can induce the expression of antiviral ISGs through JAK/STAT pathway, and ISGs play key rule in and immune regulation and controlling virus replication[6, 17]. We then determined whether IAV infection induced JAK1 degradation regulates the expression of ISGs. The transcription profiling of ISGs in response to IFN-α and IFN-γ was analyzed using qPCR. IAV infection substantially inhibited IFN-α induced expression of ISG56 and Mx1, as well as IFN-γ triggered Tap-1 and Lmp-2 (Fig. 3c and 3d). Collectively, these results indicate that IAV infection induces JAK1 degradation and decreases cellular sensitivity to IFNs.
Rescuing JAK1 expression inhibits IAV replication.
To further demonstrate the association of JAK1 degradation and IFNs response attenuation during IAV infection, 293T cells were transfected with JAK1-His or control vector. Most of the overexpressed JAK1 was degraded during IAV infection, and JAK1 overexpression downregulated the expression of IAV protein NS1(Fig. 4a) and NP (data not shown), indicating that JAK1 degradation induced by IAV infection was beneficial to virus replication. JAK1 overexpression partially restored the activation of STAT1 in response to IFN-α and IFN-γ in the presence of IAV infection (Fig. 4b and 4c). The mRNA level of ISGs were also increased by JAK1 overexpression (Fig. 4d and 4e). Transient expression of JAK1 cannot fully restore the phosphorylation of STAT1 as well as the expression of ISGs. That may be because IAV infection can also inhibit the IFNs response by other ways, such as by decreasing the protein level of IFN receptors in several cell types[11]. Collectively, overexpression of JAK1 can partly restore cellular response to type I and type II IFNs and display anti influenza activity, suggesting that JAK1 degradation play a critical role in attenuating the antiviral activity of IFNs during IAV infection.
IAV infection induced SOCS1 mediate the degradation of JAK1
SOCSs family proteins play an important role in negatively regulation JAK/STAT signaling pathway[18, 19]. Previous studies have revealed that IAV infection upregulates SOCS1 and SOCS3, and SOCS1/3 antagonist peptide can protect mice against lethal influenza infection[20]. IAV infection induced SOCS1 can inhibit the phosphorylation of STAT1[12]. SOCS1 and SOCS3 also target JAK1 for degradation[21, 22]. Therefore, we investigated whether SOCS1 and SOCS3 are involved in the degradation of JAK1 during IAV infection. The protein level of SOCS1 was significantly increased during IAV infection, and the protein level of SOCS3 was not changed (Fig. 5a). IAV infection also upregulated the mRNA level of SOCS1 (Fig. 5b). To further verify the correlation of SOCS1 protein expression and JAK1 degradation, SOCS1 protein level in 293T cells were knocked down using siRNA (Fig. 5c). In SOCS1 knockdown cells, the protein level of JAK1 was notably higher than that of control cells (Fig. 5c), indicating that SOCS1 directly mediated JAK1 degradation during IAV infection. Importantly, knocked down of SOCS1 inhibited the expression of virus protein NP upon IAV infection (Fig. 5c). These results indicate that IAV infection induced SOCS1 is a negative regulator of cellular antiviral activity, and SOCS1 plays an important role in downregulating the protein level of JAK1. To determine if SOCS1 mediates JAK1 ubiquitination and degradation, 293T cells were transfected with siRNA targeting SOCS1, then transfected with Flag-tagged JAK1 and HA-tagged ubiquitin, and then infected with IAV. SOCS1 knockdown attenuated the ubiquitination of JAK1 during IAV infection (Fig. 5d). Collectively, these results indicate that SOCS1 mediates the ubiquitination and degradation of JAK1 during IAV infection.
Knock down of SOCS1 rescues IFNs response
To further ascertain the importance of SOCS1 in inducing JAK1 degradation during IAV infection, SOCS1 were knocked down using siRNA before testing the cellular response to IFNs SOCS1 knockdown rescued the protein level of JAK1 as well as the phosphorylation of STAT1 induced by type I and type II IFNs (Fig. 6a and 6b). To further investigate the association between SOCS1 and IFNs response inhibition, the expression level of ISGs were detected by real-time PCR; SOCS1 knockdown enhanced the expression of ISGs induced by IFNs (Fig. 6c and 6d), indicating that SOCS1 knock down can enhance the IFNs induced ISGs expression during IAV infection. Collectively, these results illustrate that SOCS1 expression induced by IAV infection can downregulate the protein level of JAK1, making cells less responsive to IFNs.