TopBP1 Regulates Human Papillomavirus Type 16 E2 Interaction with Chromatin

Abstract

Human papillomavirus type 16 (HPV16) E2 regulates transcription from and replication of the viral genome, in association with viral and cellular factors. HPV16 E2 interacts functionally with TopBP1, a cellular protein essential for the initiation of cellular, and potentially viral, DNA replication. This report demonstrates that the absence of TopBP1 results in the redistribution of HPV16 E2 into an alternative cellular protein complex, resulting in enhanced affinity for chromatin. This redistribution does not significantly alter the ability of HPV16 E2 to either activate or repress transcription. We also show colocalization of both proteins on chromatin at late stages of mitosis, suggesting that TopBP1 could be the mitotic chromatin receptor for HPV16 E2. The possible significance of the results for the regulation of the viral life cycle is discussed.All human papillomaviruses (HPVs) encode an E2 protein which regulates the replication of and transcription from the viral genome (5). We have previously identified TopBP1 as a functional cellular interacting partner for HPV type 16 (HPV16) E2 (hereafter called E2) which increases its transcriptional and DNA replication activity (2, 3). TopBP1 is a BRCT repeat-containing protein that can regulate many aspects of nucleic acid metabolism, including transcription, replication, and DNA damage and repair processes (6,10,12,14,(19)(20)(21)(22). As both E2 and TopBP1 are chromatin-associated proteins, we investigated whether TopBP1 may be a chromatin receptor for E2 and a regulator of E2 function.TopBP1 is not essential for E2 transcriptional activity. 293T cells (chosen due to their high transfectability and ease of TopBP1 knockdown as demonstrated below) were cultured and used in transcription assays as previously described (18). TopBP1 was depleted or mock depleted using pSUPERTopBP1 or pSUPER plasmids (8). From the results shown in Fig. 1A, it is clear that removal of TopBP1 has little effect on the ability of E2 to activate transcription from the thymidine kinase promoter or to repress transcription from the HPV18 promoter. Thirty micrograms of protein from each lysate from the assays whose results are shown in Fig. 1A was Western blotted and probed, as described previously (3), for TopBP1 and E2 (Fig. 1B). Good depletion of TopBP1 can be observed in samples transfected with the pSUPER-TopBP1 plasmid as opposed to the control plasmid. In samples where TopBP1 is depleted, the levels of E2 are markedly elevated. Using the same protein preparation technique, we demonstrated that this increased level of E2 was not due to enhanced stability of the E2 protein (not shown). We therefore investigated the ability of TopBP1 to alter the subcellular localization, and therefore potentially the solubilization, of E2.Depletion of TopBP1 alters the subcellular localization of E2. 293T cells transiently transfected with E2 and TopBP1 depletion plasmids were subjected to cytoplasmic/nuclear fractionation as depicted in Fig. 1C (17). Figure 1D shows that depletion of TopBP1 results in E...

Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2

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Interaction of the Papillomavirus E8∧E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression

“…Binding of E2 to Brd4, ChlR1, and Mklp2 has been implicated in mediating the plasmid segregation function of E2 (4,36,47,82). Transcriptional activation by E2 depends upon binding to several cellular proteins, such as Brd4, cNAP1, Gps2, p300, and TopBP1 (7,16,27,39,49). E2 has also been shown to repress the HPV E6/E7 promoter from promoter-proximal binding sites (70).…”

Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

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