Nuclear Translocation of IFN-γ Is an Intrinsic Requirement for Its Biologic Activity and Can Be Driven by a Heterologous Nuclear Localization Sequence

Subramaniam, Prem S.; Green, Marino M.; Larkin, Joseph; Torres, Barbara A.; Johnson, Howard M.

doi:10.1089/107999001753289569

Cited by 43 publications

(47 citation statements)

References 39 publications

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“…The mimetic data support the conclusion that the versatility of the C terminus of IFN-␥, involving both its ␣-helix and its NLS, is important for IFN-␥ mimetic-mediated STAT1␣ nuclear translocation and associated IFN-␥-like biological activities. Details of the discussion on the mechanism of intracellular trafficking of IFN-␥ and the IFN-␥ mimetics are presented elsewhere (14,(21)(22)(23).…”

Section: Discussionmentioning

confidence: 99%

Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

Ahmed

Burkhart

Subramaniam

et al. 2005

J Virol

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We have developed peptide mimetics of gamma interferon (IFN-␥) that play a direct role in the activation and nuclear translocation of STAT1␣ transcription factor. These mimetics do not act through recognition by the extracellular domain of IFN-␥ 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-␥ and prevents its interaction with the receptor. Human and murine IFN-␥ mimetic peptides were introduced into an adenoviral vector for intracellular expression. There are several studies using mostly nucleoside analogs to test their efficacy against acute exposure to smallpox and other poxviruses (5,6,17). The most promising of these analogs appears to be cidofovir (5). However, undesirable toxic effects, particularly those involving kidneys, have been observed in animal studies (17). Perhaps this is because these drugs do not appear to be virus specific. Thus, there is a timely need for other approaches to the development of poxvirus therapeutic agents. Vaccinia virus, which is used worldwide to vaccinate against smallpox infections, is a prototype of the poxvirus family. These viruses are particularly effective in neutralizing host innate antiviral defense mechanisms, such as the interferon (IFN) system. A major reason for this is the production by these viruses of soluble secreted proteins that bind to and prevent alpha/beta IFN (IFN-␣/␤) and IFN-␥ from binding to their respective receptors on the cell membrane (16). An important virulence factor of poxviruses is the B8R protein, which is a homolog of the extracellular domain of the IFN-␥ receptor and can therefore bind to intact IFN-␥ and prevent its interaction with the receptor (24).We have discovered, characterized, and synthesized small peptide agonists/mimetics of IFN-␥. These peptides, consisting of IFN-␥ C terminus and C terminus analogs, HuIFN-␥(95-134) (from amino acid 95 to 134) for humans and MuIFN-␥(95-133) (from amino acid 95 to 133) for mice, possess potent antiviral activity against vesicular stomatitis virus (VSV) (25)(26)(27)(28). VSV is commonly used in the detection and quantitation of IFN activity. The structural motif required for the recognition of the extracellular domain of the IFN-␥ receptor chain IFNGR-1 and that involved in intracellular signaling are contained in two separate domains on the N terminus and C terminus, respectively, of the IFN-␥ molecule (28, 31). The peptide mimetics act intracellularly to activate the Jak/STAT signaling apparatus and, thus, do not recognize the IFN-␥ receptor extracellular domain (28). Therefore, smallpox and vaccinia virus virulence factor B8R should not interact with the IFN-␥ mimetics and, thus, should not block mimetic antiviral activity. The mimetics were delivered into the cell via penetration by attachment of a lipophilic residue to chemically synthesized peptides, as described earlier for the deliver...

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Section: Discussionmentioning

confidence: 99%

Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

Ahmed

Burkhart

Subramaniam

et al. 2005

J Virol

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“…Microinjection of antibodies raised against the murine NLS containing peptide resulted in loss of STAT1α nuclear translocation in cells treated extracellularly with IFNγ (Subramaniam et al, 1999). Replacement of the NLS in the C-terminus of IFNγ with the NLS from SV40 T antigen resulted in restoration of biological activity of IFNγ (Subramaniam et al, 2001a). This suggests a requirement for an interaction between the NLS-containing region of IFNγ and IFNGR1, which is supported by the observations that IFNGR1-/-cells do not respond to murine intracellular IFNγ or an agonist peptide (Thiam et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

The role of IFNγ nuclear localization sequence in intracellular function

Ahmed

Burkhart

Mujtaba

et al. 2003

Journal of Cell Science

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IntroductionThe current view is that interferon γ (IFNγ) activates cells via interaction with the extracellular domain of the receptor complex (reviewed in Stark et al., 1998). This in turn results in the activation of the receptor-associated tyrosine kinases JAK1 and JAK2, leading to phosphorylation and dimerization of the transcription factor STAT1α, which dissociates from the receptor cytoplasmic domain and undergoes nuclear translocation. This current view ascribes no further role to IFNγ or the receptor chains IFNGR1 and IFNGR2 in IFNγ signaling. The above scenario ignores several events associated with IFNγ signaling. First, both IFNγ and one of the receptor chains, IFNGR1, undergo internalization or endocytosis and nuclear translocation Subramaniam et al., 2000). Second, at least three separate studies showed that intracellular IFNγ possessed biological activity that was qualitatively not unlike that of extracellular IFNγ. In one, microinjection of human IFNγ into mouse macrophage cells induced Ia expression (Smith et al., 1990). Extracellularly added human IFNγ did not have an effect, since it does not recognize the extracellular domain of the mouse IFNγ receptor complex. Another study involved internalizing human IFNγ into mouse macrophages via a liposomal delivery system, resulting in induction of an antiproliferative effect (Killion et al., 1994).Finally, intracellular expression of the human IFNγ gene in mouse fibroblasts in a non-secretory form resulted in induction of antiviral activity (Sanceau et al., 1987).Recently, we have determined the structural basis for nuclear translocation of murine IFNγ by identification of a polycationic nuclear localization sequence in its C-terminus (Subramaniam et al., 1999). Further, internalized IFNγ binds to the cytoplasmic domain of the IFNGR1 receptor chain . Immunoprecipitation experiments showed that a cytoplasmic complex of IFNγ-IFNGR1-STAT1α complexed to the nuclear importin α protein, NPI-1, occurs in an NLSdependent fashion . Thus, the internalization of IFNγ and nuclear transport of IFNγ and IFNGR1 appear to be a mechanism for nuclear import of the IFNγ transcription factor STAT1α.In the present study, we have expressed a non-secretable form of human IFNγ and found it to be biologically active. In order to demonstrate that the NLS of IFNγ was key to the intracellular events described above, positively charged amino acids in the NLS were replaced with alanines, such that the NLS sequence 128 KTGKRKR 134 was mutated to 128 ATGAAAA 134 . Non-secreted forms of IFNγ or its NLSmutated version were tested in murine and human cells for their ability to induce IFNγ activity, to activate STAT1α and to carry out nuclear translocation of STAT1α. The data show that

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“…Replacement of the IFN-␥(95-133) NLS region with the NLS of the SV40 large T antigen resulted in a similar antiviral activity against EMC virus challenge in tissue culture. This suggests that the NLS in IFN-␥ and IFN-␥(95-133) is classic and functions through the components of the Ran/importin pathway utilized by the SV40 T antigen NLS for transportation into the nucleus for signal transduction events triggered by receptor-ligand binding (15,17,19). Also shown here, the concentrations of IFN-␥(95-133) that had antiviral effects in tissue culture were not toxic to cells.…”

Section: Discussionmentioning

confidence: 55%

“…The C terminus of murine IFN-␥, consisting of residues 95 to 133, binds to the membrane-proximal region of the cytoplasmic domain of the receptor and thus can independently activate IFN-␥ signal transduction mechanisms while bypassing the need of binding the N terminus of the IFN-␥ receptor, which is at the extracellular side of the membrane (16,19,21,22). This is consistent with the data shown here that IFN-␥(95-133) peptide has antiviral activity in tissue culture and in mice in the presence of the poxvirus B8R protein.…”

Section: Discussionmentioning

confidence: 99%

“…The IFN-␥ N terminus binds to the extracellular domain of the receptor, while the C terminus of murine IFN-␥, consisting of residues 95 to 133 [IFN-␥(95-133)], binds instead to the membrane-proximal region of the cytoplasmic domain of the receptor (17,19,21,22). Contained within this C-terminal region of IFN-␥ is a required polycationic sequence, 126 RKRKRSK 132 , which is similar to the prototypical nuclear localization sequence (NLS) of simian virus 40 (SV40) T antigen (15).…”

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The Gamma Interferon (IFN-γ) Mimetic Peptide IFN-γ(95-133) Prevents Encephalomyocarditis Virus Infection both in Tissue Culture and in Mice

Mujtaba

Patel

et al. 2006

Clin Vaccine Immunol

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We have demonstrated previously that the C-terminal gamma interferon (IFN-␥Gamma interferon (IFN-␥) plays a critical role in normal immune functions. Its many biological effects include, but are not limited to, the induction of a number of antiviral proteins, up-regulation of major histocompatibility complex antigen expression, and involvement in B-cell maturation and immunoglobulin isotype switching (4, 6, 7). Among viral infections in which a protective role for IFN-␥ is clearly demonstrated are infection with hepatitis B virus, herpes simplex virus, lymphocytic choriomeningitis virus, and some poxviruses (1, 2, 8). These activities are induced as the IFN-␥ molecules interact with a single class of cell surface receptor consisting of a ligand-binding subunit ␣ chain and an associated cofactor protein necessary for efficient signal transduction (15,22).We have shown previously that murine IFN-␥ binds to a soluble form of its receptor via both the N terminus and the C terminus (21, 22). The IFN-␥ N terminus binds to the extracellular domain of the receptor, while the C terminus of murine IFN-␥, consisting of residues 95 to 133 [IFN-␥(95-133)], binds instead to the membrane-proximal region of the cytoplasmic domain of the receptor (17,19,21,22). Contained within this C-terminal region of IFN-␥ is a required polycationic sequence, 126 RKRKRSK 132 , which is similar to the prototypical nuclear localization sequence (NLS) of simian virus 40 (SV40) T antigen (15). We have shown previously that the C-terminal IFN-␥ peptide IFN-␥(95-133) possesses IFN-␥ agonist activity, such as induction of major histocompatibility complex class II molecules (2, 21, 22) without toxicity, on macrophages. Furthermore, this mimetic peptide possesses antiviral activity similar to that of IFN-␥ in tissue culture against vaccinia virus, vesicular stomatitis virus, and encephalomyocarditis (EMC) virus (1). Vaccinia virus, a member of the poxvirus family, encodes the B8R protein, which is an important virulence factor of poxviruses in that it neutralizes host innate antiviral defense mechanisms by binding to 20). The therapeutic activity of in the presence of B8R protein has not been determined in vivo in any viral infection models thus far. EMC virus is a rodent picornavirus belonging to the genus Cardiovirus, and it has an extremely wide range of hosts. It infects pigs, rodents, cattle, elephants, raccoons, marsupials, and primates such as baboons, monkeys, chimpanzees, and humans (5,11,25). Instances of human infection with EMC virus have manifested as generalized febrile illness, but the virus has also been isolated from patients with more-severe illnesses, such as encephalitis, meningitis, and cardiomyopathy (9, 11). In mice, EMC virus infection is lethal (10,14,25). In this study we characterized the antiviral effects of the IFN-␥ mimetic peptide IFN-␥(95-133) with and without the NLS region, as well as a peptide with a substitution of the SV40 T antigen NLS )SV40], and evaluated the therapeutic activity of the IFN-␥ mimetic peptide in...

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Nuclear Translocation of IFN-γ Is an Intrinsic Requirement for Its Biologic Activity and Can Be Driven by a Heterologous Nuclear Localization Sequence

Cited by 43 publications

References 39 publications

Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

Peptide Mimetics of Gamma Interferon Possess Antiviral Properties against Vaccinia Virus and Other Viruses in the Presence of Poxvirus B8R Protein

The role of IFNγ nuclear localization sequence in intracellular function

The Gamma Interferon (IFN-γ) Mimetic Peptide IFN-γ(95-133) Prevents Encephalomyocarditis Virus Infection both in Tissue Culture and in Mice

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