Downregulation of Toll-Like Receptor 9 Expression by Beta Human Papillomavirus 38 and Implications for Cell Cycle Control

Abstract

Innate immunity is the first line of host defense against infections. Many oncogenic viruses can deregulate several immune-related pathways to guarantee the persistence of the infection. Here, we show that the cutaneous human papillomavirus 38 (HPV38) E6 and E7 oncoproteins suppress the expression of the double-stranded DNA sensor Toll-like receptor 9 (TLR9) in human foreskin keratinocytes (HFK), a key mediator of the antiviral innate immune host response. In particular, HPV38 E7 induces TLR9 mRNA downregulation by promoting accumulation of ⌬Np73␣, an antagonist of p53 and p73. Inhibition of ⌬Np73␣ expression by antisense oligonucleotide in HPV38 E6/E7 HFK strongly rescues mRNA levels of TLR9, highlighting a key role of ⌬Np73␣ in this event. Chromatin immunoprecipitation experiments showed that ⌬Np73␣ is part of a negative transcriptional regulatory complex with IB kinase beta (IKK␤) that binds to a NF-B responsive element within the TLR9 promoter. In addition, the Polycomb protein enhancer of zeste homolog 2 (EZH2), responsible for gene expression silencing, is also recruited into the complex, leading to histone 3 trimethylation at lysine 27 (H3K27me3) in the same region of the TLR9 promoter. Ectopic expression of TLR9 in HPV38 E6/E7 cells resulted in an accumulation of the cell cycle inhibitors p21 WAF1 and p27 Kip1 , decreased CDK2-associated kinase activity, and inhibition of cellular proliferation. In summary, our data show that HPV38, similarly to other viruses with well-known oncogenic activity, can downregulate TLR9 expression. In addition, they highlight a new role for TLR9 in cell cycle regulation. IMPORTANCEThe mucosal high-risk HPV types have been clearly associated with human carcinogenesis. Emerging lines of evidence suggest the involvement of certain cutaneous HPV types in development of skin squamous cell carcinoma, although this association is still under debate. Oncogenic viruses have evolved different strategies to hijack the host immune system in order to guarantee the persistence of the infection. Their capability to evade the immune system is as important as their ability to promote cellular transformation. Therefore, understanding the viral mechanisms involved in viral persistence is a valid tool to evaluate their potential role in human carcinogenesis. Here, we show that E6 and E7 oncoproteins from the cutaneous HPV38 downregulate the expression of the double-stranded DNA sensor TLR9 of innate immunity. We also present evidence that the HPV38-mediated downregulation of TLR9 expression, in addition to its potential impact on the innate immune response, is linked to cell cycle deregulation. In addition to the well-characterized mucosal high-risk human papillomaviruses (HPV), a subgroup of cutaneous HPV types belonging to the genus beta of the HPV phylogenetic tree appears to be associated with human carcinogenesis (1-3). These HPV types are suspected to be involved together with UV radiation in the development of nonmelanoma skin cancer (4, 5). Beta HPV types were originally isolat...

UV Radiation Activates Toll-Like Receptor 9 Expression in Primary Human Keratinocytes, an Event Inhibited by Human Papillomavirus 38 E6 and E7 Oncoproteins

“…Therefore, it is plausible that inhibition of TLR9 signaling may also be necessary to deregulate additional events and to generate the ideal conditions for completion of the viral life cycle. In a previous study, we showed that overexpression of TLR9 in HPV38-immortalized keratinocytes results in accumulation of the cell cycle inhibitor p21 WAF1/Cip1 and in decreased cellular proliferation (3). The inhibitory effect of TLR9 on cell cycle progression has also been observed in head and neck cancer cell lines, in which TLR9 expression resulted in a reduction of cellular proliferation and in inhibition of the cells' ability to grow in an anchorage-independent manner (6).…”

“…Cells were lysed and sonicated as described previously (3). The TLR9 promoter (wild type or mutated) was used as a template to amplify the NF-B RE region near site C. PCR amplification was performed using a biotinylated forward primer and a nonbiotinylated reverse primer (listed in Table 2).…”

“…Previous studies have shown that the basal levels of TLR9 expression are downregulated by various viruses, such as the alpha human papillomavirus type 16 (HPV16), the beta HPV38, Epstein-Barr virus, and Merkel cell polyomavirus (1)(2)(3)(4)(5). The blocking of TLR9 function by several oncogenic viruses is a crucial event related to host immune evasion and guarantees the persistence of the infection.…”

Rationale For Immune-Based Therapies in Merkel Polyomavirus-Positive and -Negative Merkel Cell Carcinomas

“…MCPyV-LT and -sT have been shown to inhibit TLR9 expression in an epithelial and MCC cell line in vitro which may reduce inflammatory responses (Figure 2: process 'D') [66]. Several other oncogenic viruses (including HPV, EBV and HBV) have also been shown to alter TLR9 expression, suggesting that this is a common strategy to limit immune a ctivation [67].…”

Manipulation of the innate immune response by human papillomaviruses

“…In addition, no changes are seen in TLR9 levels in HPV18-positive cells while TLR3 levels are reduced (Reiser et al, 2011). Moreover, HPV38 down-regulates TLR9 levels through the action of the transcriptional regular ΔNp73 (Pacini et al, 2015). It is therefore difficult to make general conclusions about TLRs and HPV infections as effects may be type specific.…”