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, 88 (15), 8545-55

E1-mediated Recruitment of a UAF1-USP Deubiquitinase Complex Facilitates Human Papillomavirus DNA Replication

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E1-mediated Recruitment of a UAF1-USP Deubiquitinase Complex Facilitates Human Papillomavirus DNA Replication

Michaël Lehoux et al. J Virol.

Abstract

The human papillomavirus (HPV) E1 helicase promotes viral DNA replication through its DNA unwinding activity and association with host factors. The E1 proteins from anogenital HPV types interact with the cellular WD repeat-containing factor UAF1 (formerly known as p80). Specific amino acid substitutions in E1 that impair this interaction inhibit maintenance of the viral episome in immortalized keratinocytes and reduce viral DNA replication by up to 70% in transient assays. In this study, we determined by affinity purification of UAF1 that it interacts with three deubiquitinating enzymes in C33A cervical carcinoma cells: USP1, a nuclear protein, and the two cytoplasmic enzymes USP12 and USP46. Coimmunoprecipitation experiments indicated that E1 assembles into a ternary complex with UAF1 and any one of these three USPs. Moreover, expression of E1 leads to a redistribution of USP12 and USP46 from the cytoplasm to the nucleus. Chromatin immunoprecipitation studies further revealed that E1 recruits these threes USPs to the viral origin in association with UAF1. The function of USP1, USP12, and USP46 in viral DNA replication was investigated by overproduction of catalytically inactive versions of these enzymes in transient assays. All three dominant negative USPs reduced HPV31 DNA replication by up to 60%, an effect that was specific, as it was not observed in assays performed with a truncated E1 lacking the UAF1-binding domain or with bovine papillomavirus 1 E1, which does not bind UAF1. These results highlight the importance of the USP1, USP12, and USP46 deubiquitinating enzymes in anogenital HPV DNA replication.

Importance: Human papillomaviruses are small DNA tumor viruses that induce benign and malignant lesions of the skin and mucosa. HPV types that infect the anogenital tract are the etiological agents of cervical cancer, the majority of anal cancers, and a growing proportion of head-and-neck cancers. Replication of the HPV genome requires the viral protein E1, a DNA helicase that also interacts with host factors to promote viral DNA synthesis. We previously reported that the E1 helicase from anogenital HPV types associates with the WD40 repeat-containing protein UAF1. Here, we show that UAF1 bridges the interaction of E1 with three deubiquitinating enzymes, USP1, USP12, and USP46. We further show that these deubiquitinases are recruited by E1/UAF1 to the viral origin of DNA replication and that overexpression of catalytically inactive versions of these enzymes reduces viral DNA replication. These results highlight the need for an E1-associated deubiquitinase activity in anogenital HPV genome replication.

Figures

FIG 1
FIG 1
UAF1 interacts with USP1, USP12, and USP46 in C33A cervical carcinoma cells. (A) C33A stable cell lines expressing 3F-UAF1 (UAF1) or 3F alone (Control) were submitted to anti-Flag immunoprecipitation. Purified protein complexes were separated by SDS-PAGE and stained with silver nitrate; gel slices were then excised and their protein content was identified by LC-MS/MS. Major identified proteins are indicated on the right, along with their respective spectral counts, Mascot scores, and peptide coverage. USP12 and USP46, two proteins with high sequence identity, were often codetected, and their results are indicated together. (B) Co-IP of 3F-UAF1 with GFP-tagged USP1, USP12, or USP46 or with USP14 as a negative control. Cells transfected with the USP1 expression plasmid were treated for 24 h with 10 μM lactacystin prior to cell lysis. Results for input cell extracts (IN) are shown at the bottom.
FIG 2
FIG 2
The WD40 repeat region of UAF1 is required for interaction with USP12 and USP46. (A) Schematic representation of truncated UAF1 proteins. The WD40 repeat region of UAF1 is indicated by a black box; and the two SUMO-like domains (SLD1 and SLD2) are indicated by gray boxes. The results obtained with USP12 and USP46 are summarized on the right (++, strong binding; +, weak binding; −, no binding). Also summarized are the results that we previously obtained with HPV E1 (21). (B) Coimmunoprecipitation mapping assay. The indicated 3F-UAF1 truncated proteins were expressed in C33A cells together with RFP-USP12 or RFP-USP46 and immunoprecipitated using an anti-Flag antibody. The presence of RFP-USPs in the immunoprecipitates was determined by Western blotting with an anti-RFP antibody. The results for input cell extracts (IN) are shown at the bottom. (C) Colony formation assay. C33A cells were transfected with expression vectors encoding the indicated UAF1 proteins and grown for approximately 3 weeks in G418-containing medium. Colonies were fixed with methanol and stained with methylene blue.
FIG 3
FIG 3
E1 assembles as a ternary complex with UAF1 and associated USPs. (A) Coimmunoprecipitation of YFP-E1 with 3F-UAF1 and RFP-USP1, RFP-USP12, or RFP-USP46. YFP-E1, as either the wild-type (WT) protein or the UAF1-binding-defective VE protein as a control, was immunoprecipitated at 48 h posttransfection using an anti-GFP antibody from cells transfected with the indicated expression vectors. Immunoprecipitates were analyzed by Western blotting with anti-RFP (for USPs), anti-Flag (for UAF1), or anti-GFP (for E1) antibodies. (B and C) Fluorescence confocal microscopy images showing the subcellular localization of the indicated RFP-USPs expressed either alone (B) or together with 3F-UAF1 and GFP-E1 (C). The UAF1-binding-defective E1 VE protein was used as a negative control. Nuclei were stained with DAPI.
FIG 4
FIG 4
USP1, USP12, and USP46 are recruited to the HPV origin by E1 and UAF1. (A) ChIP assays were performed in C33A cells cotransfected with expression vectors for 3F-E1 (either the WT or OBD mutant protein, as indicated), RFP-E2, GFP-USP (USP1, USP12, USP46, or USP14), or GFP alone (−), together with an origin (ori)-containing plasmid and a Renilla luciferase (Rluc)-containing plasmid as an internal control. GFP-USP fusion proteins were immunoprecipitated with a GFP antibody or an HA antibody as a specificity control. The results of the ori enrichment levels determined by qPCR are shown after normalization to the amount of input DNA using the internal control (RLuc). Each value is the average of three replicates, with the standard deviations presented as error bars. (B) The same as for panel A but using the mutant E1 VE protein that is defective for binding UAF1. (C) The same as for panel A but using an anti-Flag antibody to immunoprecipitate the indicated E1 proteins.
FIG 5
FIG 5
Depletion of USP1, USP12, USP46, or UAF1 by shRNAs impairs cellular proliferation. (A) Colony formation assay. C33A cells were transfected with the indicated shRNA expression vectors and selected for approximately 3 weeks in puromycin-containing medium. Drug-resistant colonies were fixed with methanol and stained with methylene blue. (B) Cell cycle analysis. C33A cells transiently expressing the indicated shRNA were trypsinized at 72 h posttransfection, and their DNA was stained with Hoechst and analyzed by flow cytometry.
FIG 6
FIG 6
Catalytically inactive USP1, USP12, and USP46 do not inhibit cellular proliferation. (A) Amino acid sequence alignment of a short region of USP1, USP12, and USP46 surrounding the catalytic cysteine (highlighted). (B) Anti-RFP Western blot showing the expression of the different RFP-USP catalytically inactive (ci) enzymes. β-Tubulin (Tub.) was used as a loading control. (C) Colony formation assay. C33A cells were transfected with the indicated RFP-USPci expression plasmids and then selected for approximately 3 weeks in G418-containing medium. Colonies were fixed with methanol and stained with methylene blue. (D) Cell cycle analysis. C33A cells transiently expressing the indicated GFP-USPci enzymes were trypsinized at 48 h posttransfection, and their DNA was stained with Hoechst and analyzed by flow cytometry.
FIG 7
FIG 7
Specific inhibition of HPV DNA replication by catalytically inactive USP1, USP12, and USP46. (A) HPV DNA replication levels in cells expressing increasing amounts of RFP-tagged USP1ci, USP12ci, and USP46ci (8.75, 37.7, and 75 ng of the RFP-USPci expression vector). DNA replication activities are reported as a percentage of the signal obtained with cells cotransfected with the empty RFP vector as a control (−). Each value is the average of at least three independent experiments, each performed in triplicate, with the standard deviations presented as error bars. (B) The same as for panel A but using a firefly luciferase reporter plasmid lacking the viral origin. (C) Effect of the RFP-USPci enzymes on BPV1 DNA replication. The experiment was performed as described for panel A but using a BPV1 DNA replication assay.
FIG 8
FIG 8
Inhibition of HPV DNA replication by catalytically inactive USP1, USP12, and USP46 requires the UAF1-binding site in E1. (A) Schematic representation of the HPV31 E1 wild-type protein (E1 WT) and of the truncated E1 derivative (E1Δ) lacking the N-terminal 40 amino-acid-long UAF1-binding site (hatched box). The relative DNA replication activity of these two proteins is indicated on the right. (B to D) Effect of overproducing RFP-tagged USP1ci (B), RFP-USP12ci (C), and RFP-USP46ci (D) on HPV DNA replication catalyzed by either the E1 wild-type protein or E1Δ. Increasing amounts of RFP-USPci expression vectors (8.75, 37.7, and 75 ng) were used. DNA replication activities are reported as a percentage of the signal obtained with cells cotransfected with the empty RFP vector as a control (−). Each value is the average of three independent experiments, each performed in triplicate, with the standard deviations presented as error bars.

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