ABSTRACT
The E2 protein encoded by human papillomaviruses (HPV) is a sequence-specific DNA-binding protein that recruits viral and cellular proteins. Bromodomain-containing protein 4 (BRD4) is a highly conserved interactor for E2 proteins that has been linked to E2's functions as transcription modulator, activator of viral replication and segregation factor for viral genomes. In addition to BRD4, a short form of BRD4 (BRD4S) is expressed from the BRD4 gene which lacks the C-terminal domain of BRD4. E2 proteins interact with the C-terminal motif (CTM) of BRD4, but a recent study suggested that the phospho-dependent interaction domain (PDID) and the basic interaction domain (BID) in BRD4 also bind to E2. These domains are also present in BRD4S. We now find that HPV31 E2 interacts with the isolated PDID domain in living cells and also with BRD4S which is present in detectable amounts in HPV-positive cell lines and is recruited into HPV31 E1 and E2 induced replication foci. Overexpression and knockdown experiments surprisingly indicate that BRD4S inhibits activities of E2. In line with that, the specific knockdown of BRD4S in the HPV31-positive CIN612-9E cell line induces mainly late viral transcripts. This occurs only in undifferentiated but not differentiated cells in which the productive viral replication cycle is induced. These data suggest that the BRD4S-E2 interaction is important to prevent HPV late gene expression in undifferentiated keratinocytes which may contribute to immune evasion and HPV persistence.ImportanceHuman papillomaviruses (HPV) have coevolved with their host by using cellular factors like bromodomain-containing protein 4 (BRD4) to control viral processes such as genome maintenance, gene expression and replication. We here show that, in addition to the C-terminal motif in BRD4, the phospho-dependent interaction domain in BRD4 interacts with E2 proteins which enable the recruitment of BRD4S, the short isoform of BRD4, to E2. Knock-down and overexpression of BRD4S reveals that BRD4S is a negative regulator of E2 activities. Importantly, the knockdown of BRD4S induces mainly L1 transcripts in undifferentiated CIN612-9E cells, which maintain replicating HPV31 genomes. Our study reveals an inhibitory role of BRD4S on HPV transcription, which may serve as an immune escape mechanism by the suppression of L1 transcripts and thus contribute to the establishment of persistent HPV infections.
ABSTRACT
Orf virus (ORFV) represents a suitable vector for the generation of efficient, prophylactic antiviral vaccines against different pathogens. The present study investigated for the first time the therapeutic application of ORFV vector-based vaccines against tumors induced by cottontail rabbit papillomavirus (CRPV). ORFV-CRPV recombinants were constructed expressing the early CRPV gene E1, E2, E7, or LE6. In two independent experiments we used in total 23 rabbits which were immunized with a mixture of the four ORFV-CRPV recombinants or empty ORFV vector as a control 5 weeks after the appearance of skin tumors. For the determination of the therapeutic efficacy, the subsequent growth of the tumors was recorded. In the first experiment, we could demonstrate that three immunizations of rabbits with high tumor burden with the combined four ORFV-CRPV recombinants resulted in significant growth retardation of the tumors compared to the control. A second experiment was performed to test the therapeutic effect of 5 doses of the combined vaccine in rabbits with a lower tumor burden than in nonimmunized rabbits. Tumor growth was significantly reduced after immunization, and one vaccinated rabbit even displayed complete tumor regression until the end of the observation period at 26 weeks. Results of delayed-type hypersensitivity (DTH) skin tests suggest the induction of a cellular immune response mediated by the ORFV-CRPV vaccine. The data presented show for the first time a therapeutic potential of the ORFV vector platform and encourage further studies for the development of a therapeutic vaccine against virus-induced tumors.IMPORTANCE Viral vectors are widely used for the development of therapeutic vaccines for the treatment of tumors. In our study we have used Orf virus (ORFV) strain D1701-V for the generation of recombinant vaccines expressing cottontail rabbit papillomavirus (CRPV) early proteins E1, E2, LE6, and E7. The therapeutic efficacy of the ORFV-CRPV vaccines was evaluated in two independent experiments using the outbred CRPV rabbit model. In both experiments the immunization achieved significant suppression of tumor growth. In total, 84.6% of all outbred animals benefited from the ORFV-CRPV vaccination, showing reduction in tumor size and significant tumor growth inhibition, including one animal with complete tumor regression without recurrence.
