Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.     

SOCS-3 is tyrosine phosphorylated in response to interleukin-2 and suppresses STAT5 phosphorylation and lymphocyte proliferation.

Members of the recently discovered SOCS/CIS/SSI family have been proposed as regulators of cytokine signaling, and while targets and mechanisms have been suggested for some family members, the precise role of these proteins remains to be defined. To date no SOCS proteins have been specifically implicated in interleukin-2 (IL-2) signaling in T cells. Here we report SOCS-3 expression in response to IL-2 in both T-cell lines and human peripheral blood lymphocytes. SOCS-3 protein was detectable as early as 30 min following IL-2 stimulation, while CIS was seen only at low levels after 2 h. Unlike CIS, SOCS-3 was rapidly tyrosine phosphorylated in response to IL-2. Tyrosine phosphorylation of SOCS-3 was observed upon coexpression with Jak1 and Jak2 but only weakly with Jak3. In these experiments, SOCS-3 associated with Jak1 and inhibited Jak1 phosphorylation, and this inhibition was markedly enhanced by the presence of IL-2 receptor beta chain (IL-2Rbeta). Moreover, following IL-2 stimulation of T cells, SOCS-3 was able to interact with the IL-2 receptor complex, and in particular tyrosine phosphorylated Jak1 and IL-2Rbeta. Additionally, in lymphocytes expressing SOCS-3 but not CIS, IL-2-induced tyrosine phosphorylation of STAT5b was markedly reduced, while there was only a weak effect on IL-3-mediated STAT5b tyrosine phosphorylation. Finally, proliferation induced by both IL-2- and IL-3 was significantly inhibited in the presence of SOCS-3. The findings suggest that when SOCS-3 is rapidly induced by IL-2 in T cells, it acts to inhibit IL-2 responses in a classical negative feedback loop.     

IFN-gamma suppresses STAT6 phosphorylation by inhibiting its recruitment to the IL-4 receptor

Polarized Th1 cells show a stable phenotype: they become insensitive to IL-4 stimulation and lose the potential to produce IL-4. Previously, we reported that IFN-gamma played a critical role in stabilizing Th1 phenotype. However, the mechanism by which IFN-gamma stabilizes Th1 phenotype is not clear. In this study, we compared STAT6 phosphorylation in wild-type (WT) and IFN-gamma receptor knockout (IFNGR(-/-)) Th1 cells. We found a striking diminution of STAT6 phosphorylation in differentiated WT Th1 cells, but not in differentiated IFNGR(-/-) Th1 cells. The impairment of STAT6 phosphorylation in differentiated WT Th1 cells was not due to a lack of IL-4R expression or phosphorylation. Jak1 and Jak3 expression and phosphorylation were comparable in both cell types. No differential expression of suppressor of cytokine signaling 1 (SOCS1), SOCS3, or SOCS5 was observed in the two cell types. In addition, Src homology 2-containing phosphatase mutation did not affect IL-4-induced STAT6 phosphorylation in differentiated Th1 cells derived from viable motheaten (me(v)/me(v)) mice. These results led us to focus on a novel mechanism. By using a pulldown assay, we observed that STAT6 in WT Th1 cells bound less effectively to the phosphorylated IL-4R/GST fusion protein than that in IFNGR(-/-) Th1 cells. Our results suggest that IFN-gamma may suppress phosphorylation of STAT6 by inhibiting its recruitment to the IL-4R.     

Model-driven experimental analysis of the function of SHP-2 in IL-6-induced Jak/STAT signaling

Misregulated interleukin-6 (IL-6)-induced Jak/STAT signaling contributes to many diseases. Under non-pathological conditions Jak/STAT signaling is tightly regulated by a complex network of regulators. One of these regulators is the protein tyrosine phosphatase SH2-containing phosphatase 2 (SHP2). Although SHP2 is known to be a negative regulator of IL-6-induced Jak/STAT signaling, its exact molecular function is not entirely understood. To elucidate the function of SHP2 in IL-6 signaling we followed a systems biology approach, in which modeling, stepwise model refinement, and experimental analysis are closely linked. We come up with an identifiable model of early Jak/STAT signaling that describes the data and proves to be predictive. The model-based analysis implies that (1) the stepwise association of IL-6 with gp80 and gp130 and (2) STAT3 dimerization at the receptor are essential for the dynamics of early pathway activation, and (3) phosphorylation of SHP2 is nonlinear. Furthermore, the modeling results indicate that SHP2 does not act as a feedback inhibitor in an early phase of IL-6-induced Jak/STAT signaling. However, experimental data reveal that SHP2 exhibits a basal repressory function.     

Adenosine acts through A2 receptors to inhibit IL-2-induced tyrosine phosphorylation of STAT5 in T lymphocytes: role of cyclic adenosine 3',5'-monophosphate and phosphatases

Adenosine is a purine nucleoside with immunosuppressive activity that acts through cell surface receptors (A(1), A(2a), A(2b), A(3)) on responsive cells such as T lymphocytes. IL-2 is a major T cell growth and survival factor that is responsible for inducing Jak1, Jak3, and STAT5 phosphorylation, as well as causing STAT5 to translocate to the nucleus and bind regulatory elements in the genome. In this study, we show that adenosine suppressed IL-2-dependent proliferation of CTLL-2 T cells by inhibiting STAT5a/b tyrosine phosphorylation that is associated with IL-2R signaling without affecting IL-2-induced phosphorylation of Jak1 or Jak3. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reversed by the protein tyrosine phosphatase inhibitors sodium orthovanadate and bpV(phen). Adenosine dramatically increased Src homology region 2 domain-containing phosphatase-2 (SHP-2) tyrosine phosphorylation and its association with STAT5 in IL-2-stimulated CTLL-2 T cells, implicating SHP-2 in adenosine-induced STAT5a/b dephosphorylation. The inhibitory effect of adenosine on IL-2-induced STAT5a/b tyrosine phosphorylation was reproduced by A(2) receptor agonists and was blocked by selective A(2a) and A(2b) receptor antagonists, indicating that adenosine was mediating its effect through A(2) receptors. Inhibition of STAT5a/b phosphorylation was reproduced with cell-permeable 8-bromo-cAMP or forskolin-induced activation of adenylyl cyclase, and blocked by the cAMP/protein kinase A inhibitor Rp-cAMP. Forskolin and 8-bromo-cAMP also induced SHP-2 tyrosine phosphorylation. Collectively, these findings suggest that adenosine acts through A(2) receptors and associated cAMP/protein kinase A-dependent signaling pathways to activate SHP-2 and cause STAT5 dephosphorylation that results in reduced IL-2R signaling in T cells.     

Interleukin-6 increases rat metalloproteinase-13 gene expression through Janus kinase-2-mediated inhibition of serine/threonine phosphatase-2A

Interleukin-6 (IL-6) increases metalloproteinase-13 (MMP-13) gene expression by increasing phosphorylated c-Jun and by inhibiting serine/threonine phosphatase-2A (PP2A) activity. We investigated the mechanisms by which IL-6 induces c-Jun phosphorylation and PP2A inactivation in Rat-1 fibroblasts. We show that IL-6 increased MMP-13 mRNA, phosphorylated c-Jun, and activator protein 1 (AP1) binding activity without increasing c-Jun-N-terminal kinase (JNK) activity. These effects did not seem to be mediated by ERK, p38 MAP kinase, phosphatidylinositol-3-kinase, calmoduline-dependent protein kinase, protein kinase C (PKC) or protein kinase A since inhibition with specific inhibitors did not abrogate these effects. IL-6 increases PP2A catalytic subunit tyrosine phosphorylation. Inhibition of the tyrosine kinase Jak2, with the specific inhibitor AG490, abrogated this effect. Likewise, this Jak2 inhibitor blocked the effects of IL-6 on c-Jun phosphorylation, AP1 binding activity and metalloproteinase-13 gene expression. We conclude that IL-6 increases MMP-13 gene expression by activation of Jak2, resulting in tyrosine phosphorylation of the catalytic subunit of PP2A, which in turn decreases PP2A activity and prolongs c-Jun phosphorylation.     

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.