Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA

<div><p>Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.</p></div

DNA damage after UV irradiation and etoposide in the presence of LT or OBD. A

<p>: Representative images of comet assays from uninduced MEF cells or OBD expressing cells after 40 J/m² UV, or 100 µM etoposide (E). <b>B</b>: Top Panel: CASP calculated tail moments (TM) from analysis of comet assays of uninduced or LT expressing MEFs immediately after UV at 4, 40 or 400 J/m². Data are shown for a representative experiment, where at least 100 comets were quantitated for each cell line. Bottom Panel: Uninduced or OBD-expressing MEFs were untreated or exposed to etoposide (E), 0 and 100 µM, for 45 or 60 minutes. CASP calculated tail moments (TM) from analysis of alkaline comet assays are shown. <b>C</b>: Quantification of DNA damage with or without TiQA (30 mM) pretreatment in uninduced or OBD expressing MEFs after exposure to 40 J/m² UV Irradiation. CASP calculated tail moment (TM) from analysis of comet assays are shown for a representative experiment, where at least 100 comets were quantitated for each condition. <b>D, E</b>: LT/OBD Does Not Affect Formation of UV Photolesions. <b>D</b>: MEFs inducibly expressing LT or uninduced controls (CON) were exposed to 0, 4, 40 and 400 J/m² UV irradiation and then stained immediately with antibody against CPD to measure the degree of damage by FACS analysis. <b>E</b>: As in D, except stained immediately with anti-64PP antibody.</p

The binding of OBD/LT to RPA is necessary to sensitize cells to damage. A:

<p>Binding of RPA: Uninduced MEFs (IND −) or MEFs inducibly expressing wild type LT (IND +) for 48 h were harvested and then immunoprecipitated with anti-T or anti-RPA70. The immunoprecipitates and whole cell extracts were blotted with antibody against endogenous RPA14, RPA32, RPA70 and LT. Extracts with only agarose beads and no antibody were used as control +(C). <b>B</b>: 293T HEK cells were cotransfected with control CMV vector (−) or CMV vectors expressing wild type LT; S306P/V358A; E343K/E344K; HA-OBD and K308E (Top Panel) and wild type LT, E320A, K308E and PGRD (Bottom Panel) as well as GFP-tagged RPA. Cells were harvested 48 hours post transfection and then immunoprecipitated with anti-T or anti-HA serum (OBD). The immunoprecipitates and whole cell extracts were blotted with antibody against GFP to show RPA70, against LT and against HA to show OBD. <b>C</b>: RPA binding defective mutants K308E and E320A fail to sensitize cells to UV. MEFs inducibly expressing wild type, E320A, or K308E LT were untreated or exposed to UV light (40 J/m²). Morphologies of the cells are shown 16 hours after stress treatment. <b>D</b>: CASP calculated tail moments (TM) from analysis of comet assays from uninduced MEFs, wild type (WT) or K308E LT expressing cells that were untreated or treated with UV (40 J/m²) and immediately analyzed for comets. <b>E</b>: LT interferes with double-stranded DNA break repair: DR-U2OS cells maintained under growing conditions (10% FCS) were cotransfected with I-SceI plasmid and empty vector, LT or K308E. Populations of GFP-positive cells arising from homologous recombination were determined by flow cytometry 48 hours post-transfection. Percentages of GFP positive cells arising from HR as measured by flow cytometry are shown as: Con-0.0%, LT- 0.0%, K308E- 0.0%, Sce1- 3.9%, Sce1 + LT- 1.3%, Sce1+ K308E- 3.6%. A representative of four experiments is shown.</p

Overexpression of RPA protects against sensitization to DNA damage by LT, while knockdown of RPA mimics LT phenotype. A

<p>: Stable, MEF cell lines that inducibly express OBD were used to obtain cells overexpressing RPA using GFP-tagged RPA70 ([RPA]^O). Morphology of uninduced cells (UN) and cells expressing OBD is shown 16 h after exposure to UV light (40 J/m²). <b>B</b>: Cell extracts of MEFs inducibly expressing OBD or expressing OBD as well as GFP-RPA were tested by western blot for endogenous (ENDO) and GFP-RPA (GFP) with an anti-RPA70 antibody. <b>C</b>: CASP calculated tail moments (TM) from analysis of comet assays of uninduced or OBD-expressing (WT) cells with or without exogenous GFP-RPA70 overexpression either without UV treatment or immediately after UV (40 J/m²). Data are shown for a representative experiment, where at least 100 comets were quantitated for each cell line. <b>D</b>. Morphology of cells in which RPA has been knocked down. Stable, MEF cell lines that inducibly express OBD were used to obtain cells in which RPA has been transiently knocked down using siRNA directed towards RPA70. Morphology of uninduced cells (UN) and cells expressing OBD is shown 16 h after exposure to 100 µM etoposide (E). Scrambled siRNA (siScr) expressing uninduced (UN) and OBD expressing MEFs was used as negative control. <b>E</b>. Uninduced MEFs (UN) or MEFs expressing OBD with transient knockdown of RPA70 (siRPA70) or without (CON) (siScr) were exposed to 40 J/m² UV light. Cell extracts harvested 1 h post treatment were resolved by SDS PAGE and tested by western blotting for PARP-1, phospho-JNK1/2 (pJNK), phospho-p38 (pp38), total JNK1/2 (JNK), endogenous RPA70, total p38 and OBD.</p

LT inhibits localization of repair proteins into nuclear foci after UV exposure. A

<p>: Uninduced MEFs or MEFs expressing either LT or K308E were exposed to 40 J/m² UV. After one hour, they were stained with antibody to RPA70 (FITC), RAD51 (TRITC). Individual fluorescence images and the merged DAPI images that stain nuclei are shown. <b>B</b>: Uninduced MEFs or MEFs expressing either LT or K308E were exposed to 40 J/m² UV. After one hour, they were stained with antibody to RPA70 (FITC), RAD9 (TRITC). ). Individual fluorescence images and the merged DAPI images that stain nuclei are shown.</p

Sensitization by LT triggers apoptotic changes. A

<p>: Uninduced MEFs that were not expressing LT and LT-expressing MEFs were unexposed or exposed to increasing amounts of UV (4, 40 or 400 J/m²). After one hour, cell extracts were harvested, separated by SDS PAGE, and blotted with antibodies against BAD, BclXL, Bim, LT, with p38 as a loading control. <b>B</b>: OBD affects localization of Bim after UV exposure. Uninduced MEFs (UN) not expressing or MEFs expressing OBD were untreated or exposed to 40 J/m² UV light. After one hour, they were stained (TRITC) with antibody to Bim and DAPI. Individual fluorescence images are shown. <b>C: Top Panel</b>: Morphologies of cells in which Bim has been knocked down. Stable MEF cell lines that inducibly express OBD were used to obtain cells in which Bim was stably knocked down using shRNA directed towards Bim. Morphologies of uninduced cells (UN) and cells expressing OBD are shown 16 hours after exposure to 40 J/m² UV light. Scrambled shRNA (shScr) containing uninduced (UN) MEFs and OBD expressing MEFs without shRNA were used as controls. <b>Bottom Panel</b>: Expression in uninduced MEFs (UN) or MEFs expressing OBD with shRNA targeting Bim. Cell extracts harvested 1 h post treatment were resolved by SDS PAGE and tested by western blotting for Bim, OBD with actin as the loading control. Scrambled shRNA (shScr) MEFs and MEFs expressing OBD were used as negative controls.</p

Polyoma large T sensitizes cells to DNA damaging agents. A

<p>: Secondary mouse embryo fibroblasts (MEFs) uninfected or infected were untreated (negative control) or exposed to UV light (4, 40, or 400 J/m²) or treated by addition of etoposide (100 µM) to the medium at 18 hours post infection. Phase contrast pictures were taken sixteen hours later. <b>B</b>: Mouse embryo fibroblasts (MEFs) uninduced (UN) or induced to express LT by the removal of doxycyclin for 48 hours were untreated, exposed to a 40 J/m² dose of UV light or treated by addition of etoposide (100 µM) to the medium. Phase contrast pictures were taken sixteen hours later. <b>C: Top Panel</b>: MEFs expressing LT (48 hours post induction) or infected secondary MEFs (18 hours post infection) were stained with antibody to LT (FITC). Individual fluorescence images and the merged DAPI images that stain nuclei are shown. <b>C: Bottom Panel</b>: Cell extracts from uninduced MEFs or MEFs expressing LT (48 hour post-induction) and infected secondary MEFs (18 hours post infection) or uninfected MEFs were harvested, separated by SDS PAGE, and blotted with antibodies against LT or actin (loading control). <b>D</b>: Mouse embryo fibroblasts (MEFs) uninduced (UN) or induced to express OBD by the removal of doxycyclin for 48 hours were untreated (negative control), exposed to a 40 J/m² dose of UV light or treated by addition of etoposide (100 µM) to the medium. Phase contrast pictures were taken sixteen hours later. <b>E</b>: Polyoma large T enhances stress responses to UV: Uninduced MEFs or those expressing LT were untreated or exposed to UV light (4, 40, or 400 J/m²), 48 hours post induction. (For LT-induced MEFs, no cells remain after 400 J/m².) After one hour, cell extracts were harvested, separated by SDS PAGE, and blotted with antibodies against phospho JNK (pJNK1/2), total JNK, phospho p38 (pp38), total p38 or LT. <b>F</b>: DAPI staining of nuclear chromatin from uninduced (UN) or OBD-expressing cells 6 h after UV (0 and 40 J/m²) or etoposide treatment (100 µM) (E) in the presence or absence of overnight pretreatment with PARP inhibitor TiQA (30 mM). Panel 3 (OBD+ UV40) and panel 7 (OBD+ E) show apparent hallmarks of apoptosis with densely stained nuclear granular bodies within fragmented nuclei, highly condensed and fragmented chromatin. Panel 6 and panel 10 show lightly and evenly stained nuclei indicating that TiQA protects cells from apoptotic induction by UV irradiation and etoposide treatment. <b>G</b>: OBD enhances DNA laddering: Low molecular weight DNA was extracted from uninduced or OBD expressing MEFs after 40 J/m² UV or 100 µM etoposide (E) treatment. Serum starved NIH 3T3 positive controls (S) cells undergoing apoptosis exhibit DNA laddering. Lane M represents DNA size markers. <b>H</b>: Cell extracts as in E were tested by western blotting for LT and PARP-1, with p38 as a loading control.</p

Neither DNA binding nor transcriptional activation are required for enhancement of DNA damage. A

<p>: Site-specific (S306P) and non-specific (S306P/V358A) DNA binding mutant of LT sensitize cells to UV. Uninduced (UN) MEFs or MEFs inducibly expressing wild type (WT) and S306P T or S306P,V358A T were untreated or exposed to UV light (40 J/m²). Morphology of uninduced cells (UN) and cells expressing OBD is shown 16 h after exposure to UV light (40 J/m²). <b>B</b>: Top Panel: CASP calculated tail moments (TM) from analysis of comet assays from uninduced MEFs, wild type (WT) or S306P,V358A (S/V) expressing cells that were untreated or treated with UV (40 J/m²). Data are shown for a representative experiment, where at least 100 comets were quantitated for each cell line. Bottom Panel: CASP calculated tail moment (TM) from analysis of comet assays from uninduced MEFs, wild type (WT) or S306P expressing cells that were untreated or treated with UV (40 J/m²). Data are shown for a representative experiment, where at least 100 comets were quantitated for each cell line. <b>C</b>: NIH 3T3 cells maintained under growing conditions (10% CS) were cotransfected with Gal4TK-Luc reporter and Gal4-CREB (CREB) and WT OBD or mutant P402R/G403D (PGRD). Cells were harvested 48 hours post-transfection and assayed for luciferase activity. Assays were done as previously described (30). <b>D</b>: CASP calculated tail moment (TM) from analysis of comet assays from uninduced MEFs, wild type (WT) or PGRD LT expressing cells that were untreated or treated with UV (40 J/m²) and immediately analyzed for comets. Data are shown for a representative experiment, where at least 100 comets were quantitated for each cell line.</p