Persistent infections with human papillomavirus type 16 (HPV16), HPV18, or HPV31 are necessary for the development of cervical cancer, implying that HPVs have evolved immunoevasive mechanisms. Recent global transcriptome analyses indicated that these HPV types downregulate the constitutive expression of interferon (IFN)-stimulated genes (ISGs), but the underlying mechanism is not well understood. Comparative analyses of ISG transcription in keratinocytes with complete HPV16, -18, and -31 genomes revealed that antiviral genes (IFIT1 and MX1), genes involved in IFN signaling (STAT1), proapoptotic genes (TRAIL and XAF1), and pathogen recognition receptors (TLR3, RIG-I, and MDA5) are inhibited to similar extents by HPV16, -18, and -31. The lower expression of pathogen receptors in HPV-positive cells correlated with a greatly impaired induction of IFN-β and also of IFN-λ1, -2, and -3 upon receptor stimulation. IFN-κ is constitutively expressed in normal keratinocytes and is strongly repressed by HPV16, -18, and -31. ISGs downregulated in HPV-positive cells can be reactivated by IFN-κ expression. The viral E6 and E7 oncogenes are sufficient for IFN-κ repression, with E6 being mainly responsible. E6 inhibits IFN-κ transcription independently from binding to PDZ proteins. IFN-κ expression can be activated in only one cell line by E6AP knockdown but can be activated in all tested HPV-positive cells by addition of a DNA methyltransferase inhibitor, suggesting that HPVs modulate DNA methylation. Taken together, these results suggest that carcinogenic HPVs target IFN-κ by different pathways in keratinocytes to inhibit both antiviral ISGs and pathogen recognition receptors, which in turn reduces the expression of inducible IFNs.

The viral E2 protein is a major regulator of papillomavirus DNA replication. An important way to influence viral replication is through modulation of the activity of the E2 protein. This could occur through the action of truncated E2 proteins, called E2 repressors, whose role in the replication cycle of human papillomaviruses (HPVs) has not been determined. In this study, using cell lines that contain episomal copies of the “high-risk” HPV type 31 (HPV31), we have identified viral transcripts with a splice from nucleotide (nt) 1296 to 3295. These transcripts are similar to RNAs from other animal and human papillomaviruses and have the potential to fuse a small open reading frame (E8) to the C terminus of E2, resulting in an E8 ̂E2C fusion protein. E8 ̂E2C transcripts were present throughout the complete replication cycle of HPV31. A genetic analysis of E8 ̂E2C in the context of the HPV31 genome revealed that mutation of the single ATG of the E8 gene, introduction of a stop codon downstream of the ATG, or disruption of the splice donor site at nt 1296 led to a dramatic 30- to 40-fold increase in the transient DNA replication levels in both normal and immortalized human keratinocytes. High-level expression of E8 ̂E2C from heterologous vectors was found to inhibit E1-E2-dependent DNA replication of an HPV31 origin of replication construct as well as to interfere with E2's ability to transactivate reporter gene constructs. In addition, HPV31 E8 ̂E2C strongly repressed the basal activity of the major viral early promoter P97 independent of E2. E8 ̂E2C may therefore exert its negative effect on viral DNA replication through modulating E2's ability to enhance E1-dependent DNA replication as well as by regulating viral gene expression. Surprisingly, HPV31 genomes that were unable to express E8 ̂E2C could not be maintained extrachromosomally in human keratinocytes in long-term assays despite high transient DNA replication levels. This suggests that the E8 ̂E2C protein may play a role in copy number control as well as in the stable maintenance of HPV episomes.

The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes. We show that HPV42, so far considered a nononcogenic, “low-risk” HPV, recapitulates the molecular hallmarks of oncogenic, “high-risk” HPVs. Using machine learning, we find that HPV-driven transformation elicits a germ cell–like transcriptional program conserved throughout all HPV-driven cancers (DPA, cervical carcinoma, and head and neck cancer). We further show that this germ cell–like transcriptional program, even when reduced to the top two genes (CDKN2A and SYCP2), serves as a fingerprint of oncogenic HPVs with implications for early detection, diagnosis, and therapy of all HPV-driven cancers. Significance: We identify HPV42 as a uniform driver of DPA and add a new member to the short list of tumorigenic viruses in humans. We discover that all oncogenic HPVs evoke a germ cell–like transcriptional program with important implications for detecting, diagnosing, and treating all HPV-driven cancers. See related commentary by Starrett et al., p. 17. This article is highlighted in the In This Issue feature, p. 1

Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8−), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8− and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16.

High-risk human papillomaviruses (hr-HPV) establish persistent infections in keratinocytes, which can lead to cancer of the anogenital tract. Interferons (IFNs) are a family of secreted cytokines that induce IFN-stimulated genes (ISGs), many of which display antiviral activities. Transcriptome studies have indicated that established hr-HPV-positive cell lines display a reduced expression of ISGs, which correlates with decreased levels of interferon kappa (IFN-κ), a type I IFN constitutively expressed in keratinocytes. Prior studies have also suggested that IFN-β has anti-hr-HPV activity but the underlying mechanisms are not well understood. The downregulation of IFN-κ by hr-HPV raises the possibility that IFN-κ has anti-HPV activity. Using doxycycline-inducible IFN-κ expression in CIN612-9E cells, which maintain extrachromosomally replicating HPV31 genomes, we demonstrated that IFN-κ inhibits the growth of these cells and reduces viral transcription and replication. Interestingly, the initiation of viral early transcription was already inhibited at 4 to 6 h after IFN-κ expression. This was also observed with recombinant IFN-β, suggesting a common mechanism of IFNs. Transcriptome sequencing (RNA-seq) analysis identified 1,367 IFN-κ-regulated genes, of which 221 were modulated >2-fold. The majority of those (71%) matched known ISGs, confirming that IFN-κ acts as abona fidetype I IFN in hr-HPV-positive keratinocytes. RNA interference (RNAi) and cotransfection experiments indicated that the inhibition of viral transcription is mainly due to the induction of Sp100 proteins by IFN-κ. Consistent with published data showing that Sp100 acts as a restriction factor for HPV18 infection, our results suggest that hr-HPV target IFN-κ to prevent Sp100 expression and identify Sp100 as an ISG with anti-HPV activity.IMPORTANCEHigh-risk HPV can establish persistent infections which may progress to anogenital cancers. hr-HPV interfere with the expression of interferon (IFN)-stimulated genes (ISGs), which is due to reduced levels of IFN-κ, an IFN that is constitutively expressed in human keratinocytes. This study reveals that IFN-κ rapidly inhibits HPV transcription and that this is due to the induction of Sp100 proteins. Thus, Sp100 represents an ISG for hr-HPV.

Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins. Human papillomaviruses (HPV) have been shown to cause ano-genital and oropharyngeal cancers and have been also implicated in non-melanoma skin cancer. HPV have a two-stage replication cycle: in undifferentiated keratinocytes only a low level of genome replication without virus production can be observed whereas in differentiated keratinocytes high-level genome replication and virus production takes place. Previous studies have suggested that some HPV encode an E8^E2C protein that limits genome replication in undifferentiated cells. We now demonstrate that E8^E2C proteins from phylogenetically diverse HPV types interact with NCoR/SMRT corepressor complexes to limit viral transcription and genome replication. While NCoR/SMRT complexes are known to mediate the transcription repression functions of a wide variety of host transcription factors, this is the first evidence that NCoR/SMRT proteins also are involved in the repression of the replication of viral origins.