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Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production

View Article: PubMed Central - PubMed

ABSTRACT

Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation.

No MeSH data available.


Related in: MedlinePlus

The suppression of NF-κB nuclear translocation upon neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. After BMMs were pretreated with 0.1 μM MLN4924 or DMSO of equal volume for 30 min, cells were infected with HSV-1 for the indicated periods of time. (a) The subcellular localization of p65 subunit of NF-κB (red) was revealed by indirect immunofluorescence staining with a p65-specific antibody. Nuclei were counterstained for DNA by DAPI (blue). (b) Cell lysates were harvested and subjected to immunoblotting analysis with the indicated antibodies.
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fig5: The suppression of NF-κB nuclear translocation upon neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. After BMMs were pretreated with 0.1 μM MLN4924 or DMSO of equal volume for 30 min, cells were infected with HSV-1 for the indicated periods of time. (a) The subcellular localization of p65 subunit of NF-κB (red) was revealed by indirect immunofluorescence staining with a p65-specific antibody. Nuclei were counterstained for DNA by DAPI (blue). (b) Cell lysates were harvested and subjected to immunoblotting analysis with the indicated antibodies.

Mentions: Our previous data indicate that neddylation inhibition leads to delayed, but not totally blocked, IκBα degradation in response to HSV-1 infection (Figures 1a and 3a). To further examine the extent of impaired NF-κB activation, we checked p65 nuclear translocation in response to HSV-1 infection at later time points with or without MLN4924 pretreatment. As shown in Figure 5a, p65 remained in the nucleus of control BMMs 8–12 h after HSV-1 infection (Figure 5a). p65 equally distributed in the cytoplasm and nucleus of MLN4924-pretreated BMMs 8 h after HSV-1 infection (Figure 5a). After 12 h of HSV-1 infection, p65 showed predominantly nuclear localization even in the presence of MNL4924 (Figure 5a). Thus, neddylation inhibition leads to delayed, but not totally blocked, NF-κB nuclear translocation in response to HSV-1 infection. On the other hand, IRF3 phosphorylation decreased to the basal level in control BMMs 12–24 h after HSV-1 infection, which was unaffected in the presence of MLN4924 (Figure 5b and data not shown). Therefore, neddylation inhibition does not affect IRF3 activation even at the later time points of HSV-1 infection.


Neddylation is required for herpes simplex virus type I (HSV-1)-induced early phase interferon-beta production
The suppression of NF-κB nuclear translocation upon neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. After BMMs were pretreated with 0.1 μM MLN4924 or DMSO of equal volume for 30 min, cells were infected with HSV-1 for the indicated periods of time. (a) The subcellular localization of p65 subunit of NF-κB (red) was revealed by indirect immunofluorescence staining with a p65-specific antibody. Nuclei were counterstained for DNA by DAPI (blue). (b) Cell lysates were harvested and subjected to immunoblotting analysis with the indicated antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5037273&req=5

fig5: The suppression of NF-κB nuclear translocation upon neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. After BMMs were pretreated with 0.1 μM MLN4924 or DMSO of equal volume for 30 min, cells were infected with HSV-1 for the indicated periods of time. (a) The subcellular localization of p65 subunit of NF-κB (red) was revealed by indirect immunofluorescence staining with a p65-specific antibody. Nuclei were counterstained for DNA by DAPI (blue). (b) Cell lysates were harvested and subjected to immunoblotting analysis with the indicated antibodies.
Mentions: Our previous data indicate that neddylation inhibition leads to delayed, but not totally blocked, IκBα degradation in response to HSV-1 infection (Figures 1a and 3a). To further examine the extent of impaired NF-κB activation, we checked p65 nuclear translocation in response to HSV-1 infection at later time points with or without MLN4924 pretreatment. As shown in Figure 5a, p65 remained in the nucleus of control BMMs 8–12 h after HSV-1 infection (Figure 5a). p65 equally distributed in the cytoplasm and nucleus of MLN4924-pretreated BMMs 8 h after HSV-1 infection (Figure 5a). After 12 h of HSV-1 infection, p65 showed predominantly nuclear localization even in the presence of MNL4924 (Figure 5a). Thus, neddylation inhibition leads to delayed, but not totally blocked, NF-κB nuclear translocation in response to HSV-1 infection. On the other hand, IRF3 phosphorylation decreased to the basal level in control BMMs 12–24 h after HSV-1 infection, which was unaffected in the presence of MLN4924 (Figure 5b and data not shown). Therefore, neddylation inhibition does not affect IRF3 activation even at the later time points of HSV-1 infection.

View Article: PubMed Central - PubMed

ABSTRACT

Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation.

No MeSH data available.


Related in: MedlinePlus