Identification of an arginine-rich motif in human papillomavirus type 1 E1^E4 protein necessary for E4-mediated inhibition of cellular DNA Synthesis In Vitro and in Cells

Effects of the HPV E4 protein has on cellular replication

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replicationlicensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated
keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA
replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is
accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to
activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells
into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replicationlicensing
factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA
replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing
loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and
suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement
of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We
hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal
keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the
infected cell in favor of viral genome amplification.

Archived with thanks to Journal of Virology