Interactions of human papillomavirus type 11 helicase *E1 with host transcription and replication machinery

Human papillomaviruses (HPVs) infect undifferentiated keratinocytes. Viral protein E2 activates viral transcription in undifferentiated keratinocytes, but as keratinocyte differentiation progresses, E2 associates with the viral helicase, E1, at the viral origin to activate vegetative DNA replication. It is not understood how E2's function switches from transcription to replication during keratinocyte differentiation. To investigate the mechanisms surrounding the initiation of viral replication, the interactions of viral proteins E1 and E2 with host transcription and replication factors were investigated.;To study E1's recruitment to the viral replication origin, the interaction between E2 and host transcription factor TATA binding protein (TBP) was considered. In the viral genome, the placement of a TBP binding site adjacent to the E2 binding site indicated that TBP could influence HPV replication. In vitro replication assays using TBP and its carboxy-tenninal version, TBPc, demonstrated that TBP potently inhibits viral replication and that increasing E1 cannot overcome TBP-induced inhibition, indicating that TBP is a non-competitive inhibitor of E1 binding. Though fluorescence anisotropy studies demonstrated a direct interaction between E2 and TBP or TBPc, this could not fully account for the inhibition of replication. Rather, fluorescence anisotropy studies indicate that TBP sterically inhibits E2:DNA binding. It is proposed that host transcription factors regulate HPV DNA replication as well as viral transcription.;After E1:E2 binding at the origin, DNA replication depends on the recruitment of polymerase a (pol-prim). E1 interacts with the p68 subunit of pol-prim, displacing E2, to initiate DNA replication. Here, we show with fluorescence anisotropy studies that p68 aids E1 binding to DNA. Fluorescence anisotropy also demonstrates that p68 and pol-prim bind and bend DNA. To confirm this bending phenomenon, an empirical ruler based on fluorescent resonance energy transfer (FRET) and human TBP-induced DNA bending was created. In concert with spectroscopic and crystallographic results, our results yielded an estimated TBP-induced DNA bend of 107°. FRET experiments using p68 confirmed that p68 bends DNA, and fluorescence anisotropy experiments indicate p68's affinity for A-rich tracts. It is proposed that p68-induced bending may be necessary for replication initiation for a variety of small DNA viruses that depend on pol-prim machinery.