Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45 - PubMed (original) (raw)

Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45

Narayanan Sathish et al. J Biol Chem. 2011.

Abstract

IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two β-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.

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Figures

FIGURE 1.

KSHV ORF45 binds to a specific region on IRF-7 encompassing amino acids 283–466. The full-length IRF-7 and a series of truncation mutants across the entire length of IRF-7 cloned into the pACT2 vector (prey) were individually co-transformed along with the full-length ORF45 in the pAS2-1 vector (bait) into yeast strain Y190. Yeast transformants positive for the prey-bait interaction were selected on plates lacking leucine, tryptophan, and histidine titrated against increasing concentrations (10–50 m

) of 3-AT and subsequently assayed for β-galactosidase activity (standard colony filter assay), the findings of which are represented on the right of each prey construct. Dark blue rectangles indicate the IRF-7 prey plasmids that interact with KSHV ORF45, whereas the white open rectangles indicate IRF-7 constructs that lost their ability to interact with the latter. The numbers on the side of the rectangles indicate the corresponding amino acid position. The region in IRF-7 identified as being sufficient for its interaction with KSHV ORF45 is enclosed within the red lines. The upper panel depicts a schematic representation of the domain structure of IRF-7 with the N-terminal DBD followed by the CAD and the VAD. The C-terminal half of IRF-7 is constituted by the ID and SRD. The numbers below this representation indicate the corresponding amino acid positions of the respective domains.

FIGURE 2.

The entire ID of IRF-7 is required for binding with KSHV ORF45. A, the full-length and a series of small deletion mutants (ID1–ID10) of IRF-7 cloned into the pACT2 vector (prey) were individually co-transformed along with the full-length ORF45 in the pAS2-1 vector (bait) into yeast strain Y190. Yeast transformants positive for the prey-bait interaction were selected on plates lacking leucine, tryptophan, and histidine titrated against increasing concentrations (10–50 m

) of 3-AT and subsequently assayed for β-galactosidase activity (standard colony filter assay), the findings of represented on the right of each prey construct. B, subconfluent monolayers of HEK 293T cells were co-transfected with either the pCMV-2-Flag-IRF-7 (full-length) or the respective pCMV-2-Flag-IRF-7 deletion mutants (ID1–ID10) along with the ORF45 expressing plasmid (pCR3.1-ORF45). Expression levels of the IRF-7 full-length/deletion mutants in whole cell extracts (WCE) as detected by a Western blot (WB) employing anti-FLAG antibody is shown in the upper panel. The cell extracts were immunoprecipitated (IP) with an anti-FLAG antibody and the immunoprecipitates were resolved on SDS-PAGE gels and subjected to a Western blot with an anti-ORF45 antibody to detect levels of co-precipitating ORF45 (lower panel).

FIGURE 3.

Deletion of the IRF-7 ID results in a constitutive and a hyperactive form of IRF-7. Reporter plasmids containing the firefly luciferase gene under control of the human IFNA1 promoter were co-transfected into subconfluent monolayers of HEK 293T cells along with either the wild-type (WT) IRF-7 or the ID-deleted form of IRF-7 (Δ283–466). pRL-TK reporter plasmid encoding the Renilla luciferase was included as an internal control. At 8 h post-transfection, cells were either left uninfected (black bars) or infected with Sendai virus (gray bars). Cells were lysed and the luciferase (Luc) activities measured at 24 h post-infection and represented relative to the Renilla luciferase internal control gene.

FIGURE 4.

Any small deletions in the IRF-7 ID results in a complete loss of IRF-7 transactivation. Reporter plasmids containing the firefly luciferase gene under control of the human IFNA1 promoter were co-transfected into subconfluent monolayers of HEK 293T cells along with either the wild-type (WT) IRF-7 or the respective small deletion mutants of IRF-7 across the ID region. pRL-TK reporter plasmid encoding the Renilla luciferase was included as an internal control. At 8 h post-transfection, cells were either left uninfected (black bars) or infected with Sendai virus (gray bars). Cells were lysed and the luciferase (Luc) activities were measured at 24 h post-infection and are represented relative to the Renilla luciferase internal control gene.

FIGURE 5.

Predicted structure of the IRF-7 IAD segment. A, sequence alignment of the IRF-3 and IRF-7 IAD. Primary amino acid sequence alignment of the IRF-3 and IRF-7 IAD. The predicted secondary structures of IRF-7 are shown below the alignment with H representing the α-helices and S representing the β sheets. Hydrophobic amino acids in the IRF-7 sequences are indicated in red. The numbers alongside the sequences represent the respective amino acid positions. B, predicted structure of the IRF-7 IAD segment. The x-ray crystal structure of IRF-3 (PDB 1J2F) was obtained from the Protein Data Bank at the Research Collaboratory for Structural Bioinformatics. The C-terminal section of the B chain (residues 189–422) served as the structure template. The sequence of IRF-7 (residues 281–503) was aligned to the IRF-3 structure by sequence similarity, and the coordinates of homologous amino acids were transferred from IRF-3 to IRF-7 using the Homology module of Insight II (Accelrys). The coordinates of unassigned residues of IRF-7 were estimated by standard interpolation methods with the final predicted structure energy minimized using the Discover module of Insight II. The region spanning the H3-H4-H5 α-helices (within the boxed square) is illustrated in C as a space-filling model clearly showing the physical interaction of the three helices along with the increased hydrophobicity of this region. The hydrophobicity scale index (25) is shown alongside the right panel with hydrophobicity increasing from top to the bottom of the scale.

FIGURE 6.

Interaction patterns of IRF-7 exons with the IRF-7 ID. A, the different IRF-7 exons (prey) cloned into the pACT2 vector were tested for their abilities to interact with the bait IRF-7 ID (aa 283–466) by a standard yeast two-hybrid (Y2H) assay. Yeast transformants positive for the prey-bait interaction were first selected on plates lacking leucine, tryptophan, and histidine (incorporated with 50 m

3-AT, taken as the cut-off point) and subsequently assayed for β-galactosidase activity by a standard colony filter assay. The upper panel shows the growth patterns of prey-bait transformed yeast colonies on plates lacking histidine (with 50 m

3-AT) as an indicator of HIS3 activity. The lower panel exhibits the β-galactosidase assay findings on these transformed yeast colonies as an indicator of LacZ activity. B, schematic representation of the interaction patterns of the different IRF-7 exons with the IRF-7 ID. The different IRF-7 exons (1–10) are shown schematically (upper panel) with the numbers indicating the corresponding amino acid positions of the respective exons. Findings of the above Y2H assay revealed the interaction of both the N-terminal (exons 1/2 and 3) and C-terminal (exon 10) exons (indicated by dark blue lines) with the IRF-7 ID (enclosed within the red bordered rectangle). The lower panel represents a schematic representation of the different domains of IRF-7, including the DBD, CAD, VAD, ID, and SRD. Extrapolation from the IRF-7 exon interaction pattern is also indicated in the lower panel, wherein both the N-terminal DBD and C-terminal SRD (indicated by blue lines underneath) interact with the IRF-7 ID (indicated within the red bordered rectangle).

FIGURE 7.

Deletions of the hydrophobic amino acid-rich IRF-7 ID region spanning H3–H4 α-helices results in increased transcription of the IFNA1 promoter. A, a series of progressive deletions in the ID of IRF-7 (designated as mutants ID10–ID20) across the entire length of the ORF45-binding region was generated and cloned into a pCMV-FLAG vector. Reporter plasmids containing the firefly luciferase gene under control of the human IFNA1 promoter were co-transfected into subconfluent monolayers of HEK 293T cells along with either the wild-type (WT) IRF-7 or the progressive deletion mutants (ID10–ID20). pRL-TK reporter plasmid encoding the Renilla luciferase (Ren) was included as an internal control. At 8 h post-transfection, cells were either left uninfected or infected with Sendai virus. Cells were lysed and the luciferase (Luc) activities measured at 24 h post-infection and represented relative to the Ren internal control gene. The mean luciferase activity with respect to transcription of the IFNA1 promoter either in the absence or presence of virus is indicated on the left. The red bordered circles represent localization of the H3 and H4 α-helices in the IRF-7 ID. The upper panel depicts a schematic representation of the domain structure of IRF-7 with the DBD, CAD, VAD, ID, and SRD. B, the expression of these mutants in HEK 293T cells was analyzed by a Western blot (WB) using an anti-FLAG antibody. This was performed to check for both the presence of expression and comparable levels of expression of these mutant plasmids.

FIGURE 8.

Deletions of the hydrophobic amino acid-rich IRF-7 ID region spanning H3–H4 α-helices results in nuclear translocation of IRF-7. A set of progressive IRF-7 ID deletion mutants (ID10–ID20) as well as the wild type IRF-7 cloned into an EGFP-tagged vector were individually transfected into HEK 293T cells grown on coverslips. The transfected cells were challenged with Sendai virus 16 h post-transfection. Twelve hours post-infection, both the uninfected (−) and virus-infected (+) cells were washed with PBS and subsequently fixed. The coverslips were mounted onto slides and examined under a confocal microscope (Nikon) for the intracellular localization patterns of the transfected IRF-7 expressing plasmids.

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Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45 - PubMed (original) (raw)