Control mechanisms of productive papillomavirus replication by the viral E8^E2 protein

• Applicant: Professor Dr. Frank Stubenrauch
• Subject Area: Virology
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407440854

Infections with high risk human papillomaviruses (HPV) can result in intraepithelial lesions that can progress to invasive cancers. HPV have linked their replication cycle to the differentiation state of the infected keratinocyte. HPV establish a non-productive, persistent infection in keratinocytes in the basal layer of the epithelium. In suprabasal layers, infected cells start their differentiation program, but also re-enter the cell cycle and activate DNA replication. Then, productive replication is initiated which is characterized by the amplification of viral genomes and activation of the viral late promoter which drives expression of the viral E4 protein and the viral capsid proteins. Despite intensive research, the mechanisms responsible for genome amplification and late promoter induction in differentiated cells are not very well understood.The conserved HPV E8^E2 transcription factor is a potent repressor of viral replication and gene expression in undifferentiated cells, but its evolutionary advantage for PV is not understood. Results obtained in the previous funding period using the Mus musculus PV1 (MmuPV1)-mouse model have revealed that the MmuPV1 E8^E2 protein is also a potent repressor of viral gene expression and, identical to HPV, uses cellular NCoR/SMRT corepressor complexes for repression. Surprisingly, E8^E2 knock-out (ko) or NCoR/SMRT-binding deficient genomes are unable to induce tail warts or manifest infections of the genital tract of T-cell deficient Foxn1nu/nu mice. E8^E2 mt genomes greatly enhance the fraction of undifferentiated keratinocytes expressing the late viral E4 protein and this results in a G2 cell cycle arrest. This most likely interferes with the division and, thus, expansion of MmuPV1 E8^E2 mt infected keratinocytes in the basal layer in infected mice and therefore wart formation. Consistent with the idea that E8^E2 mainly represses productive replication, we also find that changes of the cellular transcriptome induced by a HPV16 E8^E2 ko mt genome in organotypic cultures correlate with the extent of viral late gene expression. Novel antibodies against the HPV16 E2 protein reveal that E8^E2 inhibits the formation of nuclear viral E2 foci which, most likely, represent viral replication centers and that such cells express E4 protein. This strongly suggests that the main function of E8^E2 is to prevent E2 protein accumulation in undifferentiated cells which would result in viral genome amplification, late promoter induction and E4 expression. Based on these results, we plan to identify host transcriptome changes induced by productive PV replication and address the functional relevance of selected genes for productive replication. Furthermore, we plan to explore the mechanisms by which E8^E2 controls E2 accumulation and thus productive replication. In summary, this project aims to identify mechanisms controlling productive PV replication which may help to find conserved targets for anti-PV therapy.

• DFG Programme: Research Grants