Interferon-beta treatment increases human papillomavirus early gene transcription and viral plasmid genome replication by activating interferon regulatory factor (IRF)-1

Abstract

Interferons (IFNs) have been used to treat mucosal lesions caused by human papillomavirus (HPV) infection, such as intraepithelial precursor lesions to cancer of the uterine cervix, genital warts or recurrent respiratory papillomatosis, to potentially reduce or eliminate replicating HPV plasmid genomes. Mucosal HPVs have evolved mechanisms that impede IFN-beta synthesis and downregulate genes induced by IFN. Here we show that these HPV types directly subvert a cellular transcriptional response to IFN-beta as a potential boost in infection. Treatment with low levels of human IFN-beta induced initial amplification of HPV-16 and HPV-11 plasmid genomes and increased HPV-16 or HPV-31 DNA copy numbers up to 6-fold in HPV-immortalized keratinocytes. IFN treatment also increased early gene transcription from the major early gene promoters in HPV-16, HPV-31 and HPV-11. Furthermore, mutagenesis of the viral genomes and ectopic interferon regulatory factor (IRF) expression in transfection experiments using IRF-1(-/-), IRF-2(-/-) and dual knockout cell lines determined that these responses are due to the activation of IRF-1 interaction with a conserved interferon response element demonstrated in several mucosal HPV early gene promoters. Our results provide a molecular explanation for the varying clinical outcomes of IFN therapy of papillomatoses and define an assay for the modulation of the HPV gene program by IFNs as well as other cytokines and signaling molecules in infection and therapy.

Fig. 1.

IRF-1 and IRF-2 bind to a conserved IRE 5′ to the HPV-16 E6–E7 promoter. ( A ) Synthetic oligonucleotides (shown in upper case while adjacent sequences are shown as lower case) spanning a putative IRE in the HPV-16 P97 promoter and 10 other HPVs (boxed area). Nucleotide substitutions (shaded) disrupting IRF binding were introduced into a conserved IRE motif in HPV-16, HPV-11 and interferon-stimulated response element (ISRE) (ISG15 promoter) control sequence. Adjacent tataa boxes are underlined. ( B ) Specific cellular complexes formed with the HPV oligonucleotide probes in electrophoretic mobility shift assays. The composite cellular IRF complex consists of IRF-1 and IRF-2 while papillomavirus regulatory factor binding to HPV-16 had been described previously ( 55 ). Recombinant IRF proteins (rIRF-1 and rIRF-2) formed specific complexes with defined cellular ISRE, from the IFN-stimulated gene-15 (ISG15) and guanylate binding protein (GBP) promoters, as well as the HPV-16 wt IRE. Polyclonal antisera specific for each IRF protein was used to supershift these complexes. ( C ) Chromatin immunoprecipitations were performed with extracts derived from SCC13 cells transfected with HPV-16 wt plasmids. Antiserum, specific for histone 2A (αH2A), was included as a positive control in this composite of two independent experiments. HA refers to the hemagglutinin antisera control. Relative immunoprecipitation activity was expressed as a ratio of HPV-16 immunoprecipitation signal to the truncated competitor PCR product, measured by scanning densitometry.

Fig. 2.

IFN-mediated activation of the HPV-16 P97 promoter is dependent on the IRF pathway in keratinocytes. ( A ) Mobility shift assays were used to detect the binding of known IFN-inducible regulatory proteins in IFN-treated keratinocyte extracts to the HPV-16 IRE and control sequences in vitro . The ISRE probe (from the ISG15 promoter) detects the tripartite interferon-stimulated gene factor-3 complex and IRF protein binding while the consensus SIE probe detects Stat dimer formation. The GTIIC wt probe (from SV40) detects IFN-unresponsive TEF-1 binding as a negative control. ( B ) Primary HFK cells were first starved in low-serum media prior to treatment with IFN-β, RNA harvest and analysis of IRF-1/IRF-2 messenger RNA by RNase protection. Scanning densitometry determined normalized promoter activities relative to the GAPDH internal control. The 28S message was monitored as an additional internal control. ( C ) The HPV-16 promoter, P97-cat, IRF-responsive (IRF-2 cat) and Stat-responsive (IRF-1 cat) constructs were used in these assays. ( D ) Primary keratinocytes were transiently transfected with the HPV-16 P97-cat, IRF and Stat-responsive targets prior to IFN-β treatment and extract preparation for chloramphenicol acetyl transferase assays. The adenovirus E2 promoter construct (AdE2- cat ) served as an IFN-unresponsive negative control in these assays. Normalized activities represent an average of two to four independent experiments. ( E ) Transfections in HPK-I cells, which harbor integrated HPV-16, were similarly treated in parallel.

Fig. 3.

IFNs activate HPV-16, HPV-11 and HPV-31 E6–E7 transcription from replicating viral genomes. Nucleotide substitutions in the HPV IRE that block IRF-1 binding were introduced into the ( A ) whole HPV-16 and ( B ) HPV-11 genomes, transiently transfected into HeLa cells and then treated with IFN-β prior to RNA harvest and analysis by RNase protection. ( C ) Baseline activities of constructs containing the indicated IRE and/or E2 DNA-binding domain (DBD) mutations were compared. Normalized, promoter activities (relative to the SV40 internal transfection control) were determined by scanning densitometry. ( D ) Clonal HPV-16 W12-E cells were treated with IFN-β prior to total RNA harvest and analysis by RNase protection assay. The normalized ratio of P97 transcription to GAPDH (as an internal control) was determined by scanning densitometry in a representative experiment. ( E ) Clonal CIN612 cells were treated with IFN-γ and similarly analyzed.

Fig. 4.

IFN-mediated activation of the HPV-16 P97 promoter is dependent on the IRF-1 pathway in vivo . ( A ) IRF or Stat-responsive chloramphenicol acetyl transferase plasmids were transiently transfected into wt, 3T3, fibroblasts, expressing both IRF-1 and IRF-2, and then treated with IFN-β (10 U/ml) as described. Shaded bars indicate mock-induced target activities, whereas solid bars represent IFN-treated cultures. Normalized activities represent an average of three to six independent experiments. The IFN-unresponsive HPV-16 IRE mut II and AdE2- cat constructs were included as negative controls. ( B ) The IRF-1 knockout cells (IRF-1 ^−/− ), ( C ) IRF-2 knockout cells (IRF-2 ^−/− ) and ( D ) IRF-1 and IRF-2 double knockout cells (IRF-1/2 dko) were similarly transfected and treated in parallel.

Fig. 5.

Treatment with low levels of IFN-β can increase mucosal HPV copy numbers in transient transfections and stably replicating keratinocytes. ( A ) An increasing dose of IFN-β induced replication from HPV-16 and HPV-11 wt plasmids transiently transfected into SCC13 cells. Mutation of the conserved IRE (IRE mut II—as illustrated in Figure 1A ), which disrupts IRF binding to these constructs, served as a negative control. ( B ) Stably replicating HPV-16 wt cervical keratinocyte cells (HCK) were treated with an optimal IFN-β dose (10 U/ml) in time-course inductions. ( C ) HPV-16 wt, ( D ) HPV-16 IRE mut and ( E ) HPV-31 wt cell lines derived from primary HFK were similarly treated. Changes in copy numbers were quantified by scanning densitometry.