E2 Proteins of High Risk Human Papillomaviruses Down-Modulate STING and IFN-κ Transcription in Keratinocytes

<div><p>In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence<b>.</b> To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.</p></div

STING and IFN-κ down-regulation in HPV positive clinical specimens.

<p>(A) HPV E2s transcription level was significantly increased in HPV positive normal and LSIL when compared to HPV negative normal cases (<i>P</i><0.05). (B) STING transcription level was significantly decreased in LSIL with HPV positive and HPV E2 positive cases (<i>P</i><0.05). Normal cases with HPV positive also showed low STING transcription level when compared to normal HPV negative cases (<i>P</i><0.05). (C) IFN-κ transcription level was significantly down-regulated in normal HPV positive cases and HPV positive LSIL cases when compared to normal HPV negative cases (<i>P</i><0.05). (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

HPV E2s expression in recombinant adenovirus HPV E2-transduced human primary keratinocytes.

<p>(A) GFP signals of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD under fluorescence microscope on live cells (10X). (B) GFP protein expression was detected by western blotting analysis. GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD showed the expected protein sizes of ∼32 kD, 62 kD, 64 kD, 50 kD, and 45 kD, respectively and are indicated by asterisks. The β-actin protein was used as loading control. (C) The mRNA expression levels of recombinant adenoviruses GFP, GFP-HPV16 E2, GFP-HPV18 E2, GFP-HPV18 E2TAD, and GFP-HPV18 E2DBD are shown as CT values (mean±SD). (D) The suppressive effect of HPV E2 on HPV18 E6/E7 transcription in HeLa cells is shown as relative transcripts levels compared to cells not expressing E2 using real-time PCR. (*<i>P</i>≤0.05).</p

Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein.

<p>Top 10 canonical pathways modulated in HPK expressing HPV16 E2 protein.</p

Genome-wide profiling in HPK transduced by HPV16 E2.

<p>Microarray was performed 48-off of <i>P</i><0.05, 1.5-fold change. (A) Gene Ontology (GO) was used to define biological categories and functions. (B) Hierarchical clustering was constructed from immune associated genes as defined by GO. Cluster 1 and 2 were up-regulated cluster genes and cluster 3 and 4 were down-regulated cluster genes. (C) Signature genes altered in HPK expressing HPV16 E2, immune associated genes were analyzed by GO. Chromatin remodeling factors, transcription factors, and transcription co-factors were analysed by Animal Transcription Factor Database (AnimalTFDB), red numbers represent up-regulated genes; Green numbers represent down-regulated genes.</p

STING and IFN-κ transcripts were down-regulated by HPV E2s.

<p>(A) STING transcripts level analyzed by RT-PCR was significantly down-regulated by HPV16 E2, HPV18 E2 and the HPV18 E2 TAD in HPK and B) IFN-κ transcription level was significantly down-regulated by HPV16 E2, HPV18 E2 and HPV18 TAD in HPK. C) STING transcripts level was modulated by E2 in NUH49 as shown in (A). D) IFN-κ transcripts level was modulated by E2 in ITB3 and NUH49 as in (B). (A–D) HPV18 E2TAD significantly down-regulated STING and IFN-κ genes transcription when compared to HPV18 E2DBD. (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

Transcriptional reduction of IFN-κ in STING-silenced HPK and poly I:C stimulated HPK.

<p>(A) STING and IFN-κ transcription level in HPK cells after transfection with siSTING and siIFNK for 48 h. IFN-κ transcription level in STING-knockdown HPK was reduced by ∼20% (<i>P</i> = 0.06). STING transcription level in IFN-κ-knockdown HPK was not changed. (B) ISGs transcription levels were down-modulated in IFN-κ-knockdown HPK. (C) TAD of HPV18 E2 abrogated STING transcription in Poly I:C induced HPK cells. Cells transduced with GFP as control and GFP-E218TAD were stimulated with poly I:C (10 µg/ml) for 3, 6, and 12 h. STING transcription level was measured at each time point. (D) IFN-κ transcription level was abrogated by HPV18 E2TAD when examined at each time point of IFN-κ induction by poly I:C (E) ISGs were determined after stimulation of HPK expressing HPV18 E2TAD with poly I:C (10 µg/ml) for 3 h. (*<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001).</p

Transcription network- associated genes modulated by HPV16E2 in HPK.

<p>Transcription network- associated genes modulated by HPV16E2 in HPK.</p

Top 10 modulated pathways in HPK expressing HPV16 E2 protein.

<p>Top 10 modulated pathways in HPK expressing HPV16 E2 protein.</p