Adeno-Associated Virus Type 2 Rep Protein Inhibits Human Papillomavirus Type 16 E2 Recruitment of the Transcriptional Coactivator p300

Abstract

Infection by human adeno-associated virus type 2 (AAV2) is a possible protective factor in the development of cervical carcinomas associated with human papillomaviruses (HPV). The replicative proteins of AAV2 (Rep) have been implicated in the inhibition of papillomavirus replication and transforming activities, although the molecular events underlying these effects are poorly understood. We observed that each of the four forms of AAV2 Rep inhibited the E1-and E2-driven replication of oncogenic HPV type 16 (HPV16). Rep40, corresponding to the C-terminal domain of all Rep proteins, inhibited both HPV DNA replication and HPV16 E2-mediated transactivation. Rep40 specifically bound the N-terminal transactivation domain of HPV16 E2 both in vitro and in vivo. This interaction was found to specifically disrupt the binding of E2 to the cellular transcriptional coactivator p300. Accordingly, the inhibitory effect of Rep on HPV16 E2 transactivation was rescued by the overexpression of p300. These data indicate a novel role of Rep in the down-regulation of papillomaviruses through inhibition of complex formation between the HPV16 E2 transcriptional activator and its cellular coactivator, p300.

Interaction between the HPV E7 oncoprotein and the transcriptional coactivator p300

“…U2-OS cells were transiently transfected with a CAT reporter construct containing six E2-binding sites, together with plasmids expressing E2, p300 and E7. As can be seen in Figure 6a, p300 stimulates E2-dependent transcriptional activity, and this is in agreement with previous studies (Lee et al, 2000;Marcello et al, 2000).…”

“…The HPV E2 protein has also been shown to interact with p300/CBP (Lee et al, 2000;Marcello et al, 2000). E2 protein can function as transcriptional repressor or activator, depending upon the levels of E2 expression (Choe et al, 1989;Bouvard et al, 1994;Stubenrauch et al, 1998).…”

“…At 48 h post-transfection, cells were harvested and assayed for luciferase activity. The results show the mean of three independent experiments and standard deviations are also shown b HPV E7 interaction with p300 A Bernat et al Recent reports (Lee et al, 2000;Marcello et al, 2000) have shown a strong synergistic transcriptional activity between the HPV-E2 protein and p300. This is very relevant to the context in which E7 would usually be expressed; therefore, we chose to assess the effects of E7 upon p300 coactivation function.…”

The Human Papillomavirus E6 protein and its contribution to malignant progression

“…Finally, it should also be borne in mind that the E6/p300 interaction, and its possible contribution towards survival of the transformed cell, could be a by-product of the regulation of viral gene expression. Thus, the HPV major transcriptional activator, E2, also interacts with p300/CBP (Lee et al, 2000a;Marcello et al, 2000). This appears to involve a cellular protein, AMF-1/Gps2, which in turn enhances p300 activity but which, intriguingly, is also a target for E6 mediated degradation (Peng et al, 2000;Degenhardt and Silverstein, 2001).…”

Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation

“…35 S-labeled proteins were synthesized in vitro using the TNT quick coupled transcriptiontranslation system (Promega) according to the manufacturer's procedures, using pcDNA-MGC2663, pcDNA-MGC2663/242, pcDNA-ORF50, or pcDNA-ORF50/548 as a template. Recombinant proteins immobilized on beads were pretreated with 0.2 U of DNase I and 0.2 g of RNase A per l for 0.5 h at 25°C in pretreating buffer (50 mM Tris-HCl [pH 8.0], 5 mM MgCl 2 , 2.5 mM CaCl 2 , 100 mM NaCl, 5% glycerol, 1 mM dithiothreitol) as described previously (22). The beads were washed twice with binding buffer (20 mM Tris-Cl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol) and resuspended in the same buffer before the labeled proteins were added, followed by incubation for 1 h at 4°C.…”