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. 2020 Mar 13;12(3):311.
doi: 10.3390/v12030311.

Suppression of a Subset of Interferon-Induced Genes by Human Papillomavirus Type 16 E7 via a Cyclin Dependent Kinase 8-Dependent Mechanism

Sadie Rice  1 , Seong-Man Kim  1 , Cynthia Rodriguez  1 , William Songock  1 , Gaurav Raikhy  1 , Rebecca Lopez  1 , Lauren Henderson  1 , Arjun Yusufji  1   2 , Jason Bodily  1
Affiliations

Suppression of a Subset of Interferon-Induced Genes by Human Papillomavirus Type 16 E7 via a Cyclin Dependent Kinase 8-Dependent Mechanism

Sadie Rice et al. Viruses. .

Abstract

Persistent infection by human papillomaviruses (HPVs), small, double-stranded DNA viruses that infect keratinocytes of the squamous epithelia, can lead to the development of cervical and other cancers. The viral oncoprotein E7 contributes to viral persistence in part by regulating host gene expression through binding host transcriptional regulators, although mechanisms responsible for E7-mediated transcriptional regulation are incompletely understood. Type I IFN signaling promotes the expression of anti-viral genes, called interferon-stimulated genes (ISGs), through the phosphorylation and activation of STAT1. In this study, we have observed that the CR3 domain of E7 contributes to the episomal maintenance of viral genomes. Transcriptome analysis revealed that E7 transcriptionally suppresses a subset of ISGs but not through regulation of STAT1 activation. Instead, we discovered that E7 associates with Mediator kinase CDK8 and this is correlated with the recruitment of CDK8 to ISG promoters and reduced ISG expression. E7 fails to suppress ISGs in the absence of CDK8, indicating that CDK8 function contributes to the suppression of ISGs by E7. Altogether, E7/CDK8 association may be a novel mechanism by which E7 inhibits innate immune signaling.

Keywords: IFN signaling; Mediator kinase CDK8; STAT1; interferon-stimulated genes; oncoprotein E7; papillomaviruses; transcription.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of E6/E7 and E6/E7 F57A cell lines. Western blot analysis of protein levels of E7 (top panel), p53 (middle panel), pRb (bottom panel), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human foreskin keratinocytes (HFKs), pLXSN E6/E7, and pLXSN E6/E7 F57A cells grown in monolayer culture. Three HFK donors (A–C) were used.
Figure 2
Figure 2
Levels of interferon-stimulated genes (ISG)s. RT-qPCR analysis for ISGs (IFIT1, IFI27, IFI44L, XAF1, IFI6, and MX2) in HFKs, pLXSN E6/E7, and pLXSN E6/E7 F57A cells grown in monolayer culture. Values were normalized to the cyclophilin A housekeeping gene, with HFK set to 1.
Figure 3
Figure 3
Effect of Ruxolitinib on ISG levels. RT-qPCR analysis of IFIT1 (a) or IFI27 (b) transcript levels in HFKs, pLXSN E6/E7, and pLXSN E6/E7 F57A cells treated with DMSO (control) or 10 μM Ruxolitinib (Rux) for 24 h under monolayer culture conditions. Transcripts were normalized to cyclophilin A housekeeping gene, with DMSO-treated pLXSN E6/E7 samples set to 1.
Figure 4
Figure 4
STAT1 levels and modification. (a) Western blot analysis of protein levels of pY701 (top panel), pS727 (middle panel), total STAT1 (bottom panel), and GAPDH in HFKs, pLXSN E6/E7, and pLXSN E6/E7 F57A cells that were treated with vehicle (IFNβ−) or 50 U/mL IFNβ (IFNβ+) for 2 h under monolayer culture conditions. Protein from uninfected human foreskin fibroblasts (HFF) treated with IFNβ was a positive control for STAT1 activation. (b) RT-qPCR analysis of STAT1 transcripts in untreated HFKs, pLXSN E6/E7, and pLXSN E6/E7 F57A cells. Transcripts were normalized to cyclophilin A housekeeping gene with pLXSN E6/E7 values set to 1.
Figure 5
Figure 5
Interaction of E7 and CDK8. (a) Expression vectors encoding HA-tagged E7s from human papillomavirus (HPV) types 16, 11, 18, and the indicated mutants (16E7) were transfected into U2OS cells. Thirty-six hours later, total cell lysates were harvested. CDK8-containing complexes were immunoprecipitated with anti-CDK8 antibodies. Anti-HA antibody was used to detect HA-E7 by Western blotting (IB) of the immunoprecipitates (top panel). Immunoblot of total HA-E7, CDK8, and GAPDH present in the transfected U2OS cell lysates (middle panels and bottom panel) are also shown. (b) Immunoprecipitation was performed using total lysates from HPV16+ cells from two HFK donors with either anti-E7 antibodies (top panel), anti-CDK8 antibodies (bottom panel) or IgG (negative control). CDK8 was detected in both the anti-E7 immunoprecipitate and in the input by immunoblotting (top panel). E7 was detected in both the anti-CDK8 immunoprecipitate and in the input by immunoblotting (bottom panel).
Figure 6
Figure 6
Effect of CDK8 knockdown on ISG levels. (a) RT-qPCR analysis of IFI44L and XAF1 transcript levels in non-target (NT) or CDK8 knockdown (KD) HFK, pLXSN E6/E7, and pLXSN E6/E7 F57A cells. Transcripts were normalized to cyclophilin A housekeeping gene with NTC values for each cell line set to 1. (b) Western blot analysis of CDK8 and GAPDH protein levels in HFK, pLXSN E6/E7, and pLXSN E6/E7 F57A cells from three HFK donor backgrounds.
Figure 7
Figure 7
Chromatin immunoprecipitation of CDK8 from ISG promoters. ChIP was performed with IgG (control) or CDK8-specific antibodies using chromatin from HFK, pLXSN E6/E7, and pLXSN E6/E7 F57A cells. Eluted DNA from ChIP was used in a qPCR reaction with primers specific for the IFI27 and XAF1 promoters. Relative enrichment of CDK8 was determined by normalizing the CDK8 signal to IgG, with IgG samples set to 1.
Figure 8
Figure 8
Model for the transcriptional suppression of ISGs by E7. (a) Factors necessary for transcription, such as Mediator and Pol II, are present at the promoters of ISGs in HFKs, and transcription occurs. (b) Wild type E7 associates with and recruits CDK8 to ISG promoters and this results in gene suppression, possibly due to loss of Pol II recruitment. (c) In cells containing F57A mutant E7, CDK8 is not efficiently recruited to ISG promoters due to reduced association between E7 F57A and CDK8. ISGs remain activated and expressed.
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