The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis.

Suppressors of cytokine signaling (SOCS) negatively regulate the immune response, primarily by interfering with the JAK/STAT pathway. We have developed a small peptide corresponding to the kinase inhibitory region (KIR) sequence of SOCS-1, SOCS1-KIR, which inhibits kinase activity by binding to the activation loop of tyrosine kinases such as JAK2 and TYK2. Treatment of SJL/J mice with SOCS1-KIR beginning 12 days post-immunization with myelin basic protein (MBP) resulted in minimal symptoms of EAE, while most control treated mice developed paraplegia. SOCS1-KIR treatment suppressed interleukin-17A (IL-17A) production by MBP-specific lymphocytes, as well as MBP-induced lymphocyte proliferation. When treated with IL-23, a key cytokine in the terminal differentiation of IL-17-producing cells, MBP-sensitized cells produced IL-17A and IFNγ; SOCS1-KIR was able to inhibit the production of these cytokines. SOCS1-KIR also blocked IL-23 and IL-17A activation of STAT3. There is a deficiency of SOCS-1 and SOCS-3 mRNA expression in CD4(+) T cells that infiltrate the CNS, reflecting a deficiency in regulation. Consistent with therapeutic efficacy, SOCS1-KIR reversed the cellular infiltration of the CNS that is associated with EAE. We have shown here that a SOCS-1 like effect can be obtained with a small functional region of the SOCS-1 protein that is easily produced.

Enhancement of antiviral immunity by small molecule antagonist of suppressor of cytokine signaling.

Suppressors of cytokine signaling (SOCSs) are negative regulators of both innate and adaptive immunity via inhibition of signaling by cytokines such as type I and type II IFNs. We have developed a small peptide antagonist of SOCS-1 that corresponds to the activation loop of JAK2. SOCS-1 inhibits both type I and type II IFN activities by binding to the kinase activation loop via the kinase inhibitory region of the SOCS. The antagonist, pJAK2(1001-1013), inhibited the replication of vaccinia virus and encephalomyocarditis virus in cell culture, suggesting that it possesses broad antiviral activity. In addition, pJAK2(1001-1013) protected mice against lethal vaccinia and encephalomyocarditis virus infection. pJAK2(1001-1013) increased the intracellular level of the constitutive IFN-beta, which may play a role in the antagonist antiviral effect at the cellular level. Ab neutralization suggests that constitutive IFN-beta may act intracellularly, consistent with recent findings on IFN-gamma intracellular signaling. pJAK2(1001-1013) also synergizes with IFNs as per IFN-gamma mimetic to exert a multiplicative antiviral effect at the level of transcription, the cell, and protection of mice against lethal viral infection. pJAK2(1001-1013) binds to the kinase inhibitory region of both SOCS-1 and SOCS-3 and blocks their inhibitory effects on the IFN-gamma activation site promoter. In addition to a direct antiviral effect and synergism with IFN, the SOCS antagonist also exhibits adjuvant effects on humoral and cellular immunity as well as an enhancement of polyinosinic-polycytidylic acid activation of TLR3. The SOCS antagonist thus presents a novel and effective approach to enhancement of host defense against viruses.

HSV-1-induced SOCS-1 expression in keratinocytes: use of a SOCS-1 antagonist to block a novel mechanism of viral immune evasion.

Keratinocytes are important for the acute phase of HSV-1 infection and subsequent persistence in sensory nervous tissue. In this study, we showed that keratinocytes (HEL-30) were refractory to IFN-gamma induction of an antiviral state to HSV-1 infection, while IFN-gamma did induce an antiviral state in fibroblasts (L929). This led us to examine the possible role of suppressor of cytokine signaling-1 (SOCS-1) in this refractiveness. RT-PCR analysis of SOCS-1 mRNA expression in HSV-1-infected cells showed a 4-fold increase for keratinocytes while having a negligible effect on fibroblasts. A similar pattern was observed at the level of SOCS-1 protein induction. Activation of STAT1alpha in keratinocytes was inhibited by HSV-1 infection. A direct effect of HSV-1 on the SOCS-1 promoter was shown in a luciferase reporter gene assay. We have developed a small peptide antagonist of SOCS-1, pJAK2(1001-1013), that had both an antiviral effect in keratinocytes against HSV-1 as well as a synergistic effect on IFN-gamma induction of an antiviral state. HSV-1 ICP0 mutant was inhibited by IFN-gamma in HEL-30 cells and was less effective than wild-type virus in induction of SOCS-1 promoter. We conclude that SOCS-1 plays an important role in the inhibition of the antiviral effect of IFN-gamma in keratinocytes infected with HSV-1. The use of SOCS-1 antagonist to abrogate this refractiveness could have a transformational effect on therapy against viral infections.

Interferon gamma is recognised by importin alpha/beta: enhanced nuclear localising and transactivation activities of an interferon gamma mimetic.

Interferon (IFN) gamma's ability to localise in the nucleus and function in gene activation has been known for some time, although the role of the conventional nuclear transporting importin molecules is unclear. Here, we demonstrate for the first time the direct recognition of IFNgamma and an IFNgamma mimetic peptide by IMPalpha and the IMPalpha/beta heterodimer, where the IFNgamma mimetic shows higher affinity. Significantly, this correlates well both with in vivo ability to target green fluorescent protein to the nucleus in transfected cells as determined by quantitative confocal laser scanning microscopy, as well as GAS promoter activity of a luciferase reporter. This has important implications for IFNgamma's anti-viral action, and the potential use of the IFNgamma mimetic in antiviral therapies.

Peptide mimetics of gamma interferon possess antiviral properties against vaccinia virus and other viruses in the presence of poxvirus B8R protein.

We have developed peptide mimetics of gamma interferon (IFN-gamma) that play a direct role in the activation and nuclear translocation of STAT1alpha transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-gamma receptor but rather bind to the cytoplasmic domain of the receptor chain 1, IFNGR-1, and thereby initiate the cellular signaling. Thus, we hypothesized that these mimetics would bypass the poxvirus virulence factor B8R protein that binds to intact IFN-gamma and prevents its interaction with the receptor. Human and murine IFN-gamma mimetic peptides were introduced into an adenoviral vector for intracellular expression. Murine IFN-gamma mimetic peptide was also expressed via chemical synthesis with an attached lipophilic group for penetration of cell plasma membrane. In contrast to intact human IFN-gamma, the mimetics did not bind poxvirus B8R protein, a homolog of the IFN-gamma receptor extracellular domain. Expression of B8R protein in WISH cells did not block the antiviral effect of the mimetics against encephalomyocarditis or vesicular stomatitis virus, while the antiviral activity of human IFN-gamma was neutralized. Consistent with the antiviral activity, the upregulation of MHC class I molecules on WISH cells by the IFN-gamma mimetics was not affected by B8R protein, while IFN-gamma-induced upregulation was blocked. Finally, the mimetics, but not IFN-gamma, inhibited vaccinia virus replication in African green monkey kidney BSC-40 cells. The data presented demonstrate that small peptide mimetics of IFN-gamma can avoid the B8R virulence factor for poxviruses and, thus, are potential candidates for antivirals against smallpox virus.