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. 2014 Apr 2;6(4):1502-24.
doi: 10.3390/v6041502.

The carboxy terminal region of the human cytomegalovirus immediate early 1 (IE1) protein disrupts type II inteferon signaling

Bindu Raghavan  1 , Charles H Cook  2 , Joanne Trgovcich  3
Affiliations

The carboxy terminal region of the human cytomegalovirus immediate early 1 (IE1) protein disrupts type II inteferon signaling

Bindu Raghavan et al. Viruses. .

Abstract

Interferons (IFNs) activate the first lines of defense against viruses, and promote innate and adaptive immune responses to viruses. We report that the immediate early 1 (IE1) protein of human cytomegalovirus (HCMV) disrupts signaling by IFNγ. The carboxyl-terminal region of IE1 is required for this function. We found no defect in the initial events in IFNγ signaling or in nuclear accumulation of signal transducer and activator of transcription 1 (STAT1) in IE1-expressing cells. Moreover, we did not observe an association between disruption of IFNγ signaling and nuclear domain 10 (ND10) disruption. However, there is reduced binding of STAT1 homodimers to target gamma activated sequence (GAS) elements in the presence of IE1. Co-immunoprecipitation studies failed to support a direct interaction between IE1 and STAT1, although these studies revealed that the C-terminal region of IE1 was required for interaction with STAT2. Together, these results indicate that IE1 disrupts IFNγ signaling by interfering with signaling events in the nucleus through a novel mechanism.

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Figures

Figure 1
Figure 1
Diminished interferon-induced gene expression in human fibroblasts expressing IE1. MRC-5 fibroblasts were nucleofected with plasmid pIE622 harboring the UL123 gene specifying IE1, or the pFIN2 empty vector. At 24 hours post-nucleofection, the cells were exposed to 100 U/mL of IFNγ or were left untreated. At 6 hours after treatment, total RNA was isolated and subjected to Real Time RT-PCR analysis as described in Materials and Methods. The fold increase in CIITA (A) (p value = 0.053) or IRF1 (B) (p value 0.017) transcript levels in IFN treated cells relative to untreated cells was determined by the ΔΔCt method. Data shown are the average of three independent experiments.
Figure 2
Figure 2
The carboxy-terminal region of IE1 is required for disruption of IFNγ signaling. (A) Schematic of the IE1 protein and truncation mutants generated in this study. Top line: a schematic of the IE1 protein indicating the amino acid positions comprising the coding exons (below) and the positions of the known functional domains (above). The coding exons are shown in white boxes. The functional domains of exon 4 are shown in shaded boxes and include the leucine zipper (L), the zinc finger (ZF) and the acidic domain (AD). A schematic of the five different IE1 N-terminal truncation mutants generated in this study are shown below. The sixth mutant is a C-terminal truncation mutant. The designation of the plasmids harboring the truncation mutants is based on the deletion of relevant exons or functional domains shown on the right, and the deleted amino acid residues are shown in parentheses. The gene sequences were inserted in frame to sequences specifying a FLAG epitope (depicted as a flag). (B) Interferon Stimulated Gene (ISG) induction in fibroblasts expressing truncated IE1 proteins. The indicated plasmids were nucleoporated into MRC5 cells and 24 hours after nucleofection the cells were treated with 100 U/mL IFNγ. At 6 hours after treatment total RNA was isolated. Real Time RT-PCR analysis was carried out for CIITA and 18S rRNA (endogenous control). The fold increase in CIITA transcript levels in IFN treated cells relative to untreated cells was determined by the ΔΔCt method. Shown is the average of two independent experiments. A film image of immunoblot analysis is shown below. In replicate cultures of the first experiment, cells were treated as above and solubilized. Equal amounts of protein from each lysate were subjected to electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to nitrocellulose sheets and reacted with the indicated antibodies. Anti-FLAG antibody was used to detect the presence of FLAG tagged IE1 proteins and antibody to GAPDH was used to evaluate protein loading.
Figure 3
Figure 3
Disruption of ND10 structures in cells expressing IE1 and the C-terminal truncation mutant. Immunofluorescence images of IE1 and Promyelocytic leukemia protein (PML) in fibroblast cells. MRC5 cells were nucleofected with the indicated constructs. At 24 hours after nucleofection cells were fixed in methanol and reacted with mouse monoclonal anti-IE1 antibody (Mab810) followed by anti-mouse alexafluor 488 as secondary antibody. The cells were then exposed to rhodamine conjugated anti PML antibody (PGM3). IE1 reactivity is visualized in green (left column), PML reactivity is visualized in red (middle column) and merged images are shown in the right column.
Figure 4
Figure 4
IE1 does not alter the state or abundance of key signaling molecules of the IFNγ pathway. Film images of electrophoretically separated cell lysates reacted with antibodies to IFNγ pathway signaling components. MRC5 cells were nucleofected with plasmid harboring full length IE1 (FL), the Δ345–491 C-terminal truncation IE1 (ΔAD) or empty vector (FIN2). At 24 hours after nucleofection, cells were exposed to IFNγ for 30 minutes or left untreated. Cells were solublized and equal amounts of protein from each lysate were subjected to electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to nitrocellulose sheets and reacted with the indicated antibodies. Anti-FLAG antibody was used to detect the presence of FLAG tagged IE1 proteins and antibody to GAPDH was used to evaluate protein loading.
Figure 5
Figure 5
Nuclear translocation of STAT1 is not altered by IE1. (A) Immunofluorescence images of IE1 and STAT1 in cells with and without IFNγ treatment. MRC5 cells were nucleofected with the full length IE1 (FL) or empty vector (FIN2). After 24 hours cells were exposed to IFNγ for 30 minutes or left untreated. Cells were fixed in methanol and reacted with mouse monoclonal anti-IE1 antibody (Mab810) and rabbit polyclonal anti‑STAT1 antibody followed by anti-mouse alexafluor 543 and anti-rabbit alexafluor 488 as secondary antibodies. STAT1 reactivity is visualized in green (left column), IE1 reactivity is visualized in red (middle column) and merged images are shown in the right column. (B) Film images of electrophoretically separated cell lysates reacted with antibodies to STAT1 and GAPDH (loading control). MRC5 cells were nucleofected with plasmid harboring full length IE1 (FL), the Δ345–491 C-terminal truncation IE1 (ΔAD) or empty vector (FIN2). At 72 hours after nucleofection, cells were exposed to IFNγ for 30 minutes or left untreated. Cells were harvested and nuclear and cytoplasmic fractions were separated. Equal amounts of protein from the nuclear and cytoplasmic lysates were subjected to electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to nitrocellulose sheets and reacted with the indicated antibodies.
Figure 6
Figure 6
Reduced binding of STAT1 to GAS elements in the presence of IE1. Film image of an Electrophoretic Mobility Shift Assay shows a reduction in the amount of IRF1 GAS element probe that is shifted upon mixture with nuclear extracts from MRC5 cells ectopically expressing IE1 (A) and HCMV infected cells (B). In A, MRC5 cells were nucleofected with plasmid harboring full length IE1 (FL), the Δ345–491 C-terminal truncation IE1 (ΔAD) or empty vector (FIN2). At 24 hours after nucleofection, cells were exposed to 100 U/mL of IFNγ or left untreated. At 30 minutes after treatment, cells were solubilized and nuclear extracts were isolated as described in Materials and Methods. Nuclear extracts were mixed with the GAS element of the IRF1 gene with or without addition of unlabeled probe and with or without anti-STAT1 antibody. Resulting complexes were resolved by electrophoresis on a 4.5% native polyacrylamide gel. In a second experiment (B), MRC5 cells were exposed to 1 PFU per cell of the AD169 strain of HCMV or left uninfected. At 12 hours after infection, cells were solubilized and processed as described above. The black arrow indicates the shifted bands and the grey arrow indicates the supershifted bands.
Figure 7
Figure 7
IE1 does not interact with STAT1 by co-immunoprecipitation. Film images of electrophoretically separated cell lysates and proteins isolated by immunoprecipation. (A) MRC5 cells were exposed to HCMV AD169 at 3 PFU per cell or left untreated. At 12 hours after infection cells were treated with 100 U/mL of IFNγ for 30 minutes. The cells were harvested and solubilized. 350 µg of total protein from each sample was reacted with anti‑IE1 antibody (mouse monoclonal Mab810). The isolated proteins along with the 10 µg total lysates were resolved by denaturing PAGE and transferred to a nitrocellulose membrane. The membrane was reacted with antibodies for STAT1, IE1 and STAT2 and the antibody reactive bands were visualized by chemiluminescence. The left panel shows films exposed for 10 seconds and the right panel shows films exposed for 5 minutes. The arrow indicates the position of the isolated, full length IE1. (B) MRC5 cells were nucleofected with full length IE1 (FL), the Δ345–491 C-terminal truncation IE1 (ΔAD) or empty vector (FIN2). 48 hours after nucleofection cells were exposed to 100 U/mL of IFNγ for 30 minutes. The preparation of cell lysates, immunoprecipitations, PAGE and western hybridization and detection were carried out as above. The left panel shows films exposed for 10 seconds and the right panel shows films exposed for 5 minutes. The short and long arrows indicate the position of the Δ345–491 IE1 in the total lysate and immunoprecipitates respectively.
Figure 8
Figure 8
Phosphorylated STAT2 does not accumulate in the nucleus of IFNγ-treated MRC-5 cells. Film images of electrophoretically separated nuclear lysates reacted with antibodies to p-STAT2 and Lamin (loading control). MRC5 cells were nucleofected with full length IE1 (FL), the Δ345–491 C-terminal truncation IE1 (ΔAD) or empty vector (FIN2). At 72 hours after nucleofection, cells were exposed to IFNγ or IFNβ for 30 minutes or left untreated. Cells were harvested and nuclear and cytoplasmic fractions were separated. Equal amounts of protein from the nuclear lysates were subjected to electrophoresis in a denaturing polyacrylamide gel. Proteins were transferred to nitrocellulose sheets and reacted with the indicated antibodies.
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