The E8^E2C Protein, a Negative Regulator of Viral Transcription and Replication, Is Required for Extrachromosomal Maintenance of Human Papillomavirus Type 31 in Keratinocytes

Abstract

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 E29s 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 E29s 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.