HPV18 Persistence Impairs Basal and DNA Ligand–Mediated IFN-β and IFN-λ1 Production through Transcriptional Repression of Multiple Downstream Effectors of Pattern Recognition Receptor Signaling

Abstract

Although it is clear that high-risk human papillomaviruses (HPVs) can selectively infect keratinocytes and persist in the host, it still remains to be unequivocally determined whether they can escape antiviral innate immunity by interfering with pattern recognition receptor (PRR) signaling. In this study, we have assessed the innate immune response in monolayer and organotypic raft cultures of NIKS cells harboring multiple copies of episomal HPV18 (NIKSmcHPV18), which fully recapitulates the persistent state of infection. We show for the first time, to our knowledge, that NIKSmcHPV18, as well as HeLa cells (a cervical carcinoma-derived cell line harboring integrated HPV18 DNA), display marked downregulation of several PRRs, as well as other PRR downstream effectors, such as the adaptor protein stimulator of IFN genes and the transcription factors IRF1 and 7. Importantly, we provide evidence that downregulation of stimulator of IFN genes, cyclic GMP-AMP synthase, and retinoic acid-inducible gene I mRNA levels occurs at the transcriptional level through a novel epigenetic silencing mechanism, as documented by the accumulation of repressive heterochromatin markers seen at the promoter region of these genes. Furthermore, stimulation of NIKSmcHPV18 cells with salmon sperm DNA or poly(deoxyadenylic-deoxythymidylic) acid, two potent inducers of PRR signaling, only partially restored PRR protein expression. Accordingly, the production of IFN-β and IFN-λ was significantly reduced in comparison with the parental NIKS cells, indicating that HPV18 exerts its immunosuppressive activity through downregulation of PRR signaling. Altogether, our findings indicate that high-risk human papillomaviruses have evolved broad-spectrum mechanisms that allow simultaneous depletion of multiple effectors of the innate immunity network, thereby creating an unreactive cellular milieu suitable for viral persistence.

Human Papillomavirus E7 Oncoprotein Subverts Host Innate Immunity via SUV39H1-Mediated Epigenetic Silencing of Immune Sensor Genes

“…We recently reported that downregulation of RIG-I, cGAS, and STING mRNA levels in hrHPV-harboring cells occurs at the transcriptional level through a novel epigenetic silencing mechanism, as shown by the presence of repressive heterochromatin marks at the promoter region of these genes (25). In the present study, we expand on those findings and show that in hrHPV-transformed cells the increase in the repressive H3K9me2 and H3K9me3 marks is achieved through transcriptional induction of the H3K9-specific methyltransferase SUV39H1 (37).…”

“…HeLa and HEK293 cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich) and CaSki cells in RPMI (Thermo Fisher Scientific), both supplemented with 10% fetal bovine serum (Sigma-Aldrich). NIKSmcHPV18 cells, stably harboring a high viral load of HPV18 episomal genomes, were obtained and cultured as previously described (25).…”

“…In recent studies, we demonstrated that in NIKSmcHPV18 keratinocytes carrying episomal HPV18, as well as in HeLa cells harboring an integrated HPV18 genome, induction of both beta interferon (IFN-␤) and IFN-1 by DNA ligands is significantly impaired compared to that of parental cells (25). Furthermore, we found that downregulation of stimulator of IFN genes (STING), cyclic GMP-AMP synthase (cGAS), and retinoic acid-inducible gene I (RIG-I) mRNA levels occurs at the transcriptional level through a novel epigenetic silencing mechanism based on the accumulation of repressive heterochromatin marks, especially H3Lys9me2 (H3K9me2), at the promoter region of these genes (25). The incorporation of histone marks in chromatin represents a dynamic balance between enzymes depositing the mark (writers) and other enzymes removing it (erasers) (26).…”

The APOBEC3 genes and their role in cancer: insights from human papillomavirus

“…Antiviral responses can be triggered through the sensing of foreign DNA in endosomes by a subset of Toll-like receptors (TLRs), or in the cytoplasm by the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) pathway (Lebre et al 2007, Suspène et al 2017. Both pathways result in the induction of type-1 interferons (IFNs), which in turn induce a host of interferon-stimulated genes (ISGs, including several A3s) with a broad range of antiviral activities, and both are inhibited by HPV, suggesting a role in sensing the virus (Hasan et al 2007, Albertini et al 2018. Human keratinocytes express several TLRs, among which TLR9 is activated by DNA-containing unmethylated CpG motifs including a region from the HPV16 E6 gene (Hasan et al 2007, Lebre et al 2007).…”

“…Both cGAS and the retinoic acid-inducible gene I (RIG-I, a sensor of viral RNA) have been implicated in keratinocyte responses to HPV infection, and RIG-I is required for induction of A3A expression by cytoplasmic DNA in the monocytic leukaemia cell line, THP-1 (Suspène et al 2017, Albertini et al 2018, Chiang et al 2018. Until recently, it was thought that sensing of viral DNA was limited to the cytoplasm or endosome; however, sensors of nuclear viral DNA (IFI16, recently reported to restrict HPV18 replication Lo Cigno et al 2015 and IFIX) have been described that also act together with cGAS to induce IFN responses (reviewed in Diner et al 2015), thus providing another mechanism by which A3 activity could be induced in HPV-infected cells.…”