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. 2018 Oct 16;10(10):567.
doi: 10.3390/v10100567.

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activity

Matteo Biolatti  1 , Valentina Dell'Oste  2 , Sara Scutera  3 , Francesca Gugliesi  4 , Gloria Griffante  5 , Marco De Andrea  6   7 , Tiziana Musso  8 , Santo Landolfo  9
Affiliations

The Viral Tegument Protein pp65 Impairs Transcriptional Upregulation of IL-1β by Human Cytomegalovirus through Inhibition of NF-kB Activity

Matteo Biolatti et al. Viruses. .

Abstract

Interleukin-1β (IL-1β) is a key effector of the inflammasome complex in response to pathogens and danger signals. Although it is well known that assembly of the inflammasome triggers proteolytic cleavage of the biologically inactive precursor pro-IL-1β into its mature secreted form, the mechanism by which human cytomegalovirus (HCMV) regulates IL-1β production via the inflammasome is still poorly understood. Here, we show that the infection of human foreskin fibroblasts (HFFs) with a mutant HCMV lacking the tegument protein pp65 (v65Stop) results in higher expression levels of mature IL-1β compared to its wild-type counterpart, suggesting that pp65 mediates HCMV immune evasion through downmodulation of IL-1β. Furthermore, we show that enhanced IL-1β production by the v65Stop mutant is due in part to induction of DNA binding and the transcriptional activity of NF-κB. Lastly, we demonstrate that HCMV infection of HFFs triggers a non-canonical IL-1β activation pathway where caspase-8 promotes IL-1β maturation independently of caspase-1. Altogether, our findings provide novel mechanistic insights into the interplay between HCMV and the inflammasome system and raise the possibility of targeting pp65 to treat HCMV infection.

Keywords: caspase-8; human cytomegalovirus (HCMV); inflammasome; interleukin-1β (IL-1β); pp65.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
HCMV (human cytomegalovirus) pp65 inhibits IL-1β (interleukin-1β) response following HCMV infection of HFFs (human foreskin fibroblasts) through NF-κB. (A) HFFs were infected at an MOI of 1 with wild-type or v65Stop HCMV and were analyzed by RT-qPCR. IL-1β mRNA expression levels following HCMV vs. mock infection were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are shown as mean fold changes ± SD (*, p < 0.05; ***, p < 0.001; two-way ANOVA followed by Bonferroni’s post-tests). (B) HFFs were transduced with AdVLacZ (black bar) or AdVpp65 (grey bar) at an MOI of 50. Subsequently, cells were infected with v65Stop HCMV (MOI of 1). At 15 hpi, IL-1β mRNA expression was normalized to that of GAPDH and is shown as a mean ± SD fold change (*, p < 0.05; unpaired t-test). (C) HFFs were infected with wild-type or v65Stop HCMV at an MOI of 1. Supernatants were collected at 6, 24, and 48 hpi and were assessed by ELISA for IL-1β production. Results are shown as a mean ± SD fold change (***, p < 0.001; two-way ANOVA followed by Bonferroni’s post-tests). (D) HFFs were infected with wild-type, wild-type UV, v65Stop, or v65Stop UV at an MOI of 1. Supernatants were collected at 6, 24, and 48 hpi and were assessed by ELISA for IL-1β production. Results are shown as the mean ± SD fold change (*, p < 0.05; ***, p < 0.001; two-way ANOVA followed by Bonferroni’s post-tests). (E) Schematic representation of the IL-1β luciferase promoter plasmid with the sequences containing the two putative NF-κB binding sites indicated as NF-κB#1 and NF-κB#2 (left panel). HFFs were transiently electroporated with luciferase plasmids encoding the wild-type (pIL-1β-WT) or deletion mutant (pIL-1β-NF-κB-KO) IL-1β promoter fragments, and pRL-SV40. Twenty-four hours later, the cells were mock-infected or infected with wild-type or v65Stop HCMV at an MOI of 1. At 24 hpi, firefly and Renilla luciferase activities were measured. The luciferase activity in whole-cell lysates was normalized to Renilla luciferase activity and is expressed as relative light units (RLU) (right panel). Results are shown as the mean ± SD fold change (*, p < 0.05; ***, p < 0.001; one-way ANOVA followed by Bonferroni’s post-tests). (F) HFFs mock-infected or infected with wild-type or v65Stop HCMV at an MOI of 1 were fixed at 24 hpi and were subjected to immunofluorescence analysis. HCMV-IEA (red) and p65/RelA (green) were visualized using primary antibodies, followed by secondary antibody staining, in the presence of 10% HCMV-negative human serum. Nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (blue). The graph (right panel) shows the levels of nuclear NF-κB in infected cells. The data represent the mean fold changes (*, p < 0.05; unpaired t-test). (G) Schematic representation of the probe containing the NF-κB#1 binding site (left panel). HFFs were left untreated (mock) or infected with wild-type or v65Stop (MOI of 1). At 24 hpi, the cells were lysed and the nuclear fraction was analyzed for NF-κB binding activity using the Universal EZ-TFA transcription factor assay colorimetric kit (right panel). The data show the means ± SD (**, p < 0.01; ***, p < 0.001; one-way ANOVA followed by Bonferroni’s post-test).
Figure 2
Figure 2
HCMV primes IL-1β activation in a caspase-8 dependent manner. (A) HFFs were infected with wild-type or v65Stop HCMV at an MOI of 1 for 24 and 48 h and were processed by fluorimetric assay for caspase-1 and caspase-8 activation. Fold changes were calculated after normalization of HCMV vs. mock-infected cells. Data are shown as mean ± SD (*, p < 0.05; **, p < 0.01; one-way ANOVA followed by Bonferroni’s post-tests). (B–D) HFFs were electroporated with pools of siRNA targeting caspase-1 (siCASP1), caspase-8 (siCASP8), or scrambled control siRNA (siCTRL). (B) The efficiency of caspase-1 and caspase-8 depletion was assayed by RT-qPCR (the data are shown as mean fold changes plus SD; *, p < 0.05; ***, p < 0.001; by unpaired t-test). (C,D) siCTRL, siCASP1, and siCASP8 HFFs were infected with wild-type or v65Stop HCMV at an MOI of 1 and were examined at 24 hpi by fluorimetric assay for caspase-1 (C) and caspase-8 (D) activation. Results are shown as the mean ± SD fold change (***, p < 0.001 two-way ANOVA followed by Bonferroni’s post-tests). (E) Cells were infected as described in (C,D). Supernatants were collected at 24 hpi and were analyzed by IL-1β ELISA. Results are shown as the mean ± SD fold change over wild-type siCTRL set as 1.0. A statistically significant difference compared to siCTRL is indicated by asterisks (**, p < 0.05; unpaired t-test). (F) Model depicting the proposed functional role of pp65 modulation of IL-1β activity during HCMV infection.
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