Expression of E8^E2 Is Required for Wart Formation by Mouse Papillomavirus 1 In Vivo

Abstract

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells. IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

Functions of Papillomavirus E8^E2 Proteins in Tissue Culture and In Vivo

“…The general increase in viral transcription upon E8^E2 inactivation suggests that E8^E2 might regulate all viral promoters [ 23 , 41 ]. In line with this, analyses of reporter constructs have revealed that HR-HPV major early promoters, the HPV6 E7 promoter, the HPV16 E8 promoter, and MmuPV1 promoters, can be efficiently repressed by E8^E2 [ 27 , 34 , 43 , 49 , 50 ]. E8^E2 is an efficient repressor of E6 and E7 transcription, and this is relevant for cellular immortalization by HPV49 as well as the growth of HPV18-positive HeLa cells [ 19 , 23 , 50 ].…”

“…HPV1, 5, 8, 11, 16, 18, 31, and 49 E8^E2 ko genomes replicate to much higher levels than wild-type (wt) genomes in short-term assays in immortalized human keratinocytes (HPV16, 31), normal human keratinocytes (HPV1, 8, 16, 31, 49), or the U2OS osteosarcoma cell line (HPV5, 11, 18) [ 8 , 14 , 20 , 23 , 27 , 39 , 40 , 41 , 42 ]. Furthermore, MmuPV1 E8^E2 ko genomes display greatly increased viral gene expression in cultured murine keratinocytes similar to HPV E8^E2 ko in human keratinocytes suggesting that E8^E2 function is conserved among human and animal PVs [ 43 ]. Evolutionary conservation, viral mutants and complementation assays strongly indicate that the E8^E2 fusion protein is responsible for the phenotypes of PV E8^E2 mutants: (1) Disruption of the E8 exon splice donor site, the E8 ATG, or translation termination linker (TTL) mutants in E8 result in increased genome replication [ 14 ], arguing that the spliced E8^E2 product is responsible.…”

“…It is thus possible that E8^E2 is not expressed in sufficient amounts in domestic rabbit keratinocytes, which would explain the lack of a phenotype in vivo. In contrast, studies with MmuPV1 revealed that the knock-out of E8^E2 increases early and late viral gene transcription in murine keratinocytes similar to HPV E8^E2 ko in human keratinocytes [ 43 ]. Remarkably, MmuPV1 E8^E2 ko genomes neither induced tail warts nor were able to maintain long-term gene expression in the vaginal tract of immunodeficient nude mice ( Figure 2 ) [ 43 ].…”

“…In contrast, studies with MmuPV1 revealed that the knock-out of E8^E2 increases early and late viral gene transcription in murine keratinocytes similar to HPV E8^E2 ko in human keratinocytes [ 43 ]. Remarkably, MmuPV1 E8^E2 ko genomes neither induced tail warts nor were able to maintain long-term gene expression in the vaginal tract of immunodeficient nude mice ( Figure 2 ) [ 43 ]. This suggests that the primary function of MmuPV1 E8^E2 in vivo is not the limitation of viral gene expression to avoid immune detection but hints at an important requirement for E8^E2 for tumor formation in the absence of cellular immunity.…”

“…In contrast to E2, transcriptional repression by E8^E2 acts over a distance of more than 1 kbp ( Figure 3 ) [ 34 , 51 ]. Furthermore, E8^E2 inhibits the E1/E2-dependent replication of the viral origin ( Figure 3 ) [ 27 , 39 , 41 , 42 , 43 ]. Thus, increased replication of E8^E2 ko genomes is most likely due to a combined effect of increased viral transcription and de-repressed E1/E2-dependent replication.…”