The infected cell protein 0 encoded by bovine herpesvirus 1 (bICP0) induces degradation of interferon response factor 3 and, consequently, inhibits beta interferon promoter activity - PubMed (original) (raw)
The infected cell protein 0 encoded by bovine herpesvirus 1 (bICP0) induces degradation of interferon response factor 3 and, consequently, inhibits beta interferon promoter activity
Kazima Saira et al. J Virol. 2007 Apr.
Abstract
The ICP0 protein (bICP0) encoded by bovine herpesvirus 1 is the major viral regulatory protein because it stimulates all viral promoters and, consequently, productive infection. Like other ICP0 analogues encoded by Alphaherpesvirinae subfamily members, bICP0 contains a zinc RING finger near its amino terminus that is necessary for activating transcription, regulating subcellular localization, and inhibiting interferon-dependent transcription. In this study, we discovered that sequences near the C terminus, and the zinc RING finger, are necessary for inhibiting the human beta interferon (IFN-beta) promoter. In contrast to herpes simplex virus type 1-encoded ICP0, bICP0 reduces interferon response factor 3 (IRF3), but not IRF7, protein levels in transiently transfected cells. The zinc RING finger and sequences near the C terminus are necessary for bICP0-induced degradation of IRF3. A proteasome inhibitor, lactacystin, interfered with bICP0-induced degradation of IRF3, suggesting that bICP0, directly or indirectly, targets IRF3 for proteasome-dependent degradation. IRF3, but not IRF7, is not readily detectable in the nuclei of productively infected bovine cells during the late stages of infection. In the context of productive infection, IRF3 and IRF7 are detected in the nucleus at early times after infection. At late times after infection, IRF7, but not IRF3, is still detectable in the nuclei of infected cells. Collectively, these studies suggest that the ability of bICP0 to reduce IRF3 protein levels is important with respect to disarming the IFN response during productive infection.
Figures
FIG. 1.
Schematic of bICP0 mutants used to localize the domains necessary for inhibiting IFN-dependent transcription. (A) Construction and identification of the transposon insertion mutants were previously described (50). The transposon insertion sites were first mapped by restriction endonuclease digestion, and then precise insertion sites were identified by DNA sequencing. The mutants were designated A to O, and the numbers in parentheses denote the amino acid number that was disrupted by transposon insertion. The positions of the zinc RING finger, acidic domain, consensus NLS (KRRR), ATG, and bICP0 stop codon are shown. The transposon mutants that had an effect on activating the thymidine kinase promoter are underlined. (B) Schematic of the 13G/51A and bICP0 deletion mutants. Two amino acid substitutions were inserted into conserved C's of the zinc RING finger of bICP0. The ΔbICP0 mutant construct was prepared by digestion of the wt construct with SalI and XhoI, which deleted sequences from amino acids 357 to 676. The details of the 13G/51A and ΔbICP0 mutants were described previously . The ΔNcoI mutant was previously described (50). Except for mutants B and L, the remainder of transposon mutants and deletion mutants express similar levels of bICP0 protein (25, 50).
FIG. 2.
Identification of bICP0 domains that are necessary to inhibit activation of the human IFN-β promoter. 293 cells (1 × 105) were cotransfected with the IFN-β CAT reporter plasmid (1.0 μg DNA), 1.0 μg of IRF7 (A) or 1.0 μg or IRF3 (B) expression plasmid, and the designated bICP0 expression plasmids (1.0 μg DNA). An empty vector (pcDNA3.1) was used as a control. Cell extracts were collected after 40 h of transfection and analyzed for CAT expression as described in Material and Methods. The value for the IFN-β promoter and IRF3 or IRF7 in the presence of the blank expression vector was set at 100%. Data represent the means from at least three experiments. Error bars show the standard errors for triplicate transfections. *, P < 0.05.
FIG. 3.
bICP0 expression correlates with reduced IRF3 protein levels. (A) 293 cells (1 × 105) were transfected with a plasmid expressing IRF3 (2.5 μg) or IRF7 (2.5 μg) and Flag-tagged bICP0 (2.5 μg) expression vector as described for Fig. 2. Whole-cell lysate was collected at 40 h after transfection and IRF3 or IRF7 protein levels measured by Western blot analysis using 100 μg of total cell lysate. (B) 293 cells were transiently transfected with IRF3 alone, IRF3 and bICP0, or IRF3 and mutant O. Total RNA was isolated 40 h after transfection. For RT-PCR, 1 μg of total RNA was used, and 10% of the resulting cDNA was used as the template for PCR. Primers specific for IRF3 and β-actin (control) were used. The β-actin and IRF3 bands were quantified using BioRad Molecular Images. The ratio between IRF3 and β-actin was calculated for each lane. The IRF3 lane was normalized to 1.
FIG. 4.
bICP0 reduces IRF3 levels in transiently transfected cells. (A) 293 cells were transfected with the designated amounts of plasmids (μg DNA). (B) IRF3 protein expression was analyzed in 9.1.3 cells. IRF3 plasmid (2.5 μg) was cotransfected with bICP0 plasmid (2.5 μg). At 40 h after transfection, cell lysate was collected as described in Materials and Methods. (C) 9.1.3 cells were transfected with the designated plasmids (2.5 μg DNA). At 24 h after transfection, the designated cultures were treated with lactacystin (15 μM; Calbiochem catalog no. 426100) or dimethyl sulfoxide, which was used to suspend lactacystin. IRF3 protein levels were detected by Western blot analysis. For each lane, 100 μg protein was used. The level of DNA in each lane was the same because an empty expression vector (pcDNA3.1−) was added to the transfection mixture to make the total DNA equal to 5 μg.
FIG. 5.
Effect of bICP0 expression on IRF3 protein expression and subcellular localization. Equivalent amounts of IRF3 and Flag-tagged bICP0 plasmids (wt, 13G/51A mutant, or the mutant O construct) were transfected into 9.1.3 cells. After 24 h of transfection, immunostaining was performed using the anti-IRF3 and anti-Flag antibodies as described in Materials and Methods. Cultures were then stained with Cy2-conjugated anti-goat IgG antibody (IRF3, green) and Cy5-conjugated anti-mouse IgG antibody (bICP0, red). Stained cells were visualized by confocal microscopy. In the cultures containing lactacystin, cultures were transfected and then treated with 15 μM lactacystin. The images are representative of three different experiments (at least 100 cells were examined for each sample in each experiment).
FIG. 6.
HSV-1-encoded ICP0 does not reduce IRF3 protein levels in transfected cells. IRF3 (0.5 μg DNA) and the Flag-tagged ICP0 construct (0.5 μg DNA) were transfected into 9.1.3 cells. As designated, certain cultures were transfected with an empty vector (pCMV2C; 0.5 μg DNA) and the IRF3 expression construct (0.5 μg DNA). After 24 h of transfection, immunostaining was performed using the anti-IRF3 and anti-Flag antibodies. Cultures were then stained with Cy2-conjugated anti-goat IgG antibody (IRF3, green) and Cy5-conjugated anti-mouse IgG antibody (ICP0, red). Stained cells were visualized by confocal microscopy. The ICP0 panels show just the Flag antibody staining, and the ICP0+IRF3 panels show the merge between ICP0 and IRF3 staining. Images are representative of three different experiments (at least 100 cells were examined for each sample). The lower right panel shows a Western blot study demonstrating that transfection of 9.1.3 cells with increasing concentrations of HSV-1 ICP0 does not reduce IRF3 protein levels. The amount of Flag-ICP0 construct (μg DNA) is shown. For all lanes, 2.5 μg IRF3 was used. As a loading control, β-actin levels were examined.
FIG. 7.
Localization of IRF3 or IRF7 (green) in BHV-1-infected cells. 9.1.3 cells were infected with BHV-1 at an MOI of 1. Cells were then immunostained with the anti-IRF3 antibody or anti-IRF7 antibody at 0, 4, 16, or 24 h postinfection (pi). Images were visualized by confocal microscopy, and the results are representative of three different experiments.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources