Stimulation of BK Virus DNA Replication by NFI Family Transcription Factors

Plasmids.

Test replication templates pUC-wt-BKV and pUC-Δen-BKV were generated by the insertion of PCR fragments of the intact BKV NCCR (positions 5031 to 282) and the NCCR without the enhancer (positions 5031 to 32) of the archetype Dik strain (GenBank accession number AB211369) into the XmaI/PstI sites of pUC18. pUC-6mtNFIs-BKV was generated by the ligation of a synthesized mutant BKV NCCR (GenScript) into the XmaI/PstI sites of pUC18. pUC-5mtNFIsW1-BKV, pUC-5mtNFIsW2-BKV, pUC-5mtNFIsW3-BKV, and pUC-5mtNFIsW6-BKV were derived from pUC-6mtNFIs-BKV by using the QuikChange site-directed mutagenesis kit (Stratagene). To construct test templates pUC-BKV-1fNFI, pUC-BKV-1rNFI, pUC-BKV-2rNFI, and pUC-BKV-4rNFI, the synthetic oligonucleotides 5′-CACATGGAATGTAGCCAAAACTGCA-3′ and 5′-GTTTTGGCTACATTCCATGTGTGCA-3′ (LNF1 consensus sites are underlined) were annealed, self-ligated, and inserted into the PstI site of pUC-Δen-BKV. Competition template pBC-wt-BKV was generated by the insertion of PCR fragments of the intact NCCR (positions 5031 to 282), enhancer (positions 33 to 282), and core origin (positions 5103 to 32) of BKV (Dik) into the XmaI/PstI, NotI/PstI, and NotI/XhoI sites of pBC-Sk(+). pBC-BKV-A89G, pBC-BKV-A143G, and pBC-BKV-A141T were mutated by using QuikChange site-directed mutagenesis.

The expression vector for BKV Tag, pCMV-BKTAg-Flag, lacking a simian virus 40 (SV40) origin, was described previously (46). NFI expression vectors pCH-NFIA, pCH-NFIB, pCH-NFIC, pCH-NFIX, and pCH-empty were kindly provided by Richard Gronostajski (18). pCH-hNFIC/CTF1 was generated by the insertion of the human NFIC/CTF1 cDNA (amplified by PCR from pCMV-CTF-1ΔUTR, kindly provided by Nicolas Mermod) (49), using the primers 5′-AGCTGGGCCCATGGATGAGTTCCAC-3′ and 5′-TTGCGCTAGCCTATCCCAGATACCAGGAC-3′, into the NheI/ApaI sites of the pCH-empty vector. The Pol-primase subunits were cloned into the expression vector pCMV with a T7 epitope tag at their N termini. The expression plasmids were kindly provided by E. Sock and M. Wegner (University of Erlangen—Nürnberg).

Bacterial expression vectors for truncated BKV Tag (pGEX3X-BKTHD, pGEX3X-BKTHDHR, and pGEX3X-BKTHR) were generated by the insertion of PCR-amplified fragments into the EcoRI/BamHI sites of pGEX3X. Primers used for PCR were BKTHD (5′-ATAGGATCCCAGGCTTAAAGGAGCATGATTTTAAC-3′ and 5′-CGGCCAATTCTTAATCAAGAATACATTTCCCCATG-3′), BKTHDHR (5′-ATAGGATCCCAGGCTTAAAGGAGCATGATTTTAAC-3′ and 5′-ACGCGAATTCTTATTTTGGGGGTGGTGTTTTAG-3′), and BKTHR (5′-ATATGGATCCCAATTACAAGAGAAGAGGATTCAG-3′ and 5′-ACGCGAATTCTTATTTTGGGGGTGGTGTTTTAG-3′).

Cell cultures and antibodies.

HK-2 human proximal tubular kidney cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone), 4 mM l-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Lonza). HEK293 cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose) (Invitrogen) supplemented with 10% fetal bovine serum (HyClone), 4 mM l-glutamine, 100 μg/ml penicillin, and 100 μg/ml streptomycin (Lonza). RPTECs (renal primary tubular cells) were cultured in renal epithelial growth medium with supplements (Lonza). All cells were grown at 37°C with 5% CO₂ in a humidified incubator.

Antibodies used for Western blotting included anti-NFI (sc-870; Santa Cruz), anti-p53 (Cell Signaling), anti-c-Jun (sc-1694; Santa Cruz), anti-Sp1 (sc-59; Santa Cruz), anti-Ets1 (sc-111; Santa Cruz), anti-NF-κB (sc-372; Santa Cruz), anti-CREB (sc-58; Santa Cruz), anti-Smad3 (sc-8332; Santa Cruz), M2 anti-Flag (Sigma), anti-β-actin (sc-47778; Santa Cruz), anti-hemagglutinin (HA) (Roche), and anti-T7 (Novagen) antibodies. Polyclonal rabbit antiserum against recombinant human primase p58-p48 expressed in Escherichia coli cells was prepared and purified as previously described (87).

DNA replication assays.

DNA replication assays with transfected cells were performed as previously described (46, 84), with transfection procedures optimized by luciferase/β-galactosidase reporter assays. The in vitro monopolymerase assays were performed as previously described (46, 85), with the following slight modifications: the assay mixture included 0.5 μg of BKV template DNA, 50 ng topoisomerase I, 1 μg replication protein A (RPA), and Pol-primase (as indicated in the figure legends) in a solution containing 30 mM HEPES (pH 7.8); 7 mM magnesium acetate; 0.1 mM EGTA; 0.5 mM dithiothreitol (DTT); 200 μM each UTP, GTP, and CTP; 4 mM ATP; 100 μM each dATP, dGTP, and dTTP; 10 μM dCTP; 40 mM creatine phosphate; 1 μg creatine kinase; 0.1 mg/ml heat-treated bovine serum albumin (BSA); and 5 μCi [α-³²P]dCTP (3,000 Ci/mmol; Perkin-Elmer) in 40 μl. The amount of added NFI protein is indicated in the figure legends. Purified BKV Tag (0.4 μg) was added to start the reaction, and after incubation for 60 min at 37°C, the reaction products were precipitated with cold 10% (wt/vol) trichloroacetic acid (TCA) containing 2.5% (wt/vol) sodium pyrophosphate, spotted onto glass fiber filters (GF/C; Whatman), washed with 1 M HCl, and analyzed by scintillation counting.

Nuclear extracts.

HEK293 cells from 10 liters of a suspension culture were purchased from The National Cell Culture Center (NCCC) (Minneapolis, MN). Cell pellets were washed in 5 packed cell volumes (PCV) of a solution containing ice-cold hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride [PMSF], 0.5 mM DTT) and centrifuged in a Beckman GH-3.7 rotor at 3,000 rpm for 5 min at 4°C. Collected cells were resuspended in 3 PCV of ice-cold hypotonic buffer and allowed to swell on ice for 10 min; swollen cells were transferred into a chilled glass Dounce tissue grinder, homogenized with 10 to 15 strokes using a type B pestle, and centrifuged in a Beckman GH-3.7 rotor at 3,500 rpm for 15 min at 4°C; the supernatants were removed; the pellets were resuspended in 1 packed nuclear volume (PNV) of low-salt buffer (20 mM HEPES [pH 7.9], 25% glycerol, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT); and 0.183 PNV of 5 M NaCl was added dropwise to the resuspended nuclei while mixing gently by swirling, placed onto a rotating platform for 30 min at 4°C, and centrifuged at 16,000 rpm (37,000 × g) in a Sorvall SA-600 rotor for 1 h at 4°C. The supernatants were collected and dialyzed in dialysis buffer (20 mM HEPES [pH 7.9], 20% glycerol, 5 mM NaCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT). The protein concentration of the extracts was adjusted to 1.4 μg/μl, determined by a Bradford protein assay (9a). Small-scale preparations of HK-2 cell, HeLa cell, RPTEC, and HEK293 cell nuclear extracts for Western blotting were made by using nuclear extract kits (Active Motif).

Competitive EMSAs.

Competitive electrophoretic mobility shift assays (EMSAs) were performed according to previously described procedures (11), with the following modifications: 4 pmol competitor oligonucleotides, 2 μg of HeLa cell nuclear extracts (sc-2120; Santa Cruz) or 0.25 μg of purified human NFIC (hNFIC)/CTF1 (Abcam), and 50 ng of poly(dI · dC) were incubated in 1× HEPES binding buffer (25 mM HEPES [pH 7.5], 6 mM MgCl₂, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 0.5 mM PMSF, 5% glycerol) for 10 min at room temperature (RT); 20 fmol biotin-labeled probe for the NFI site was then added to the reaction mixture and incubated for 20 min at room temperature. For antibody supershift EMSAs, 1 μl of NFI antibody (sc-5567; Santa Cruz) was added after the binding reaction, and the mixture was incubated for another 10 min at room temperature. The reaction products were fractionated in 5% nondenaturing polyacrylamide gels (prerun in 0.5× Tris-borate-EDTA [TBE] at 100 V for 30 min) in 0.5× TBE at 100 V until the bromophenol blue dye reached the bottom of the gel. The products were transferred onto nylon membranes in 0.5× TBE at 65 V for 30 min, UV cross-linked for 15 min, and visualized by chemiluminescent nucleic acid detection (Pierce).

In vitro pulldown assays.

BKV Tag with an N-terminal Flag epitope was expressed by using the Bac-to-Bac baculovirus system (Invitrogen) as follows: 1.5 × 10⁷ infected Hi-Five insect cells were harvested at 48 h postinfection (p.i.) and lysed in 1 ml of 0.5% NP-40 lysis buffer (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 5 mM KCl, 1.0 mM MgCl₂, 0.5% NP-40, 10% glycerol, 1× PhosSTOP phosphatase inhibitors [Roche], 1× complete protease inhibitor cocktail [Roche]) by incubation on a rotating platform at 4°C for 30 min and homogenization with a glass Dounce tissue grinder. Lysates were cleared by centrifugation at 20,000 × g in a Sorvall SA-600 rotor for 30 min at 4°C. Supernatants were incubated with 60 μl of an anti-Flag M2 affinity gel (Sigma) by rotation at 4°C for 2 h and washed three times with 1 ml of ice-cold phosphate-buffered saline (PBS); each of the gel suspensions was then incubated with extracts of infected and uninfected Hi-Five cells and then incubated with 1 ml of HEK293 cell nuclear extracts (1.4 μg/μl) plus 1× complete protease inhibitor cocktail (Roche) by rotation at 4°C for 12 h. The gel was washed three times with 1 ml of ice-cold PBS and boiled for 5 min with 50 μl of 1× SDS sample buffer.

Glutathione S-transferase (GST)-truncated BKV Tag proteins were expressed in E. coli Rosetta 2 cells (Novagen) cultured in LB medium on a shaking platform at 225 rpm at 25°C. Expression was induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when the A₆₀₀ reached 0.4 to 0.5. After induction, E. coli cells were cultured at 25°C with shaking at 225 rpm for 20 h and then sonicated (twice for 300 s each with a 20% duty cycle at maximum power) in L1 buffer (50 mM Tris-Cl [pH 8.0], 250 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM PMSF, 1× complete protease inhibitor cocktail [Roche]). NP-40 (0.1%) was added to the lysates after sonication and incubated on ice for 10 min. After centrifugation at 20,000 × g in a Sorvall SA-600 rotor for 30 min at 4°C, supernatants were incubated with glutathione beads (GE Healthcare) at 4°C for 2 h, followed by washing 2 times with 20 bed volumes of L1 buffer. A small portion (1/20) of the beads for each fusion protein was boiled with SDS sample buffer, and the quantity of bound fusion proteins was determined by SDS-PAGE and Coomassie blue staining. Beads with approximately equal amounts of each fusion protein (∼50 μg) were incubated with 800 μl of HEK293 cell nuclear extract (1.4 μg/μl) supplemented with 1× complete protease inhibitor cocktail (Roche) by rotation at 4°C for 12 h. The beads were washed 5 times with 1 ml of L1 buffer and then boiled for 5 min with 100 μl 1× SDS sample buffer. All samples were fractionated by SDS-PAGE and analyzed by Coomassie blue staining, followed by Western blotting.

Coimmunoprecipitation assays.

HEK293 cells (approximately 6 × 10⁶ cells) were transfected with expression vectors by using LipofectAMINE and Plus reagent (Invitrogen) in 100-mm-diameter plates as previously described (46). At 48 h, the cells were lysed with 750 μl of a solution containing 1% Triton lysis buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1% Triton X-100, 30 μM ethidium bromide, 1 mM PMSF, complete protease inhibitor cocktail [Roche], 1× PhosSTOP phosphatase inhibitors [Roche]) by rotation for 1 h at 4°C, and lysates were cleared by centrifugation at 20,000 × g in a Sorvall SA-600 rotor for 30 min at 4°C. A sample (10 μl) was taken for the protein input control, and the remaining supernatants were incubated with 50 μl of an anti-Flag M2 affinity gel (Sigma) or 50 μl of an anti-HA affinity matrix (Roche) by rotation at 4°C for 2 h, followed by four washes with 1 ml of 1% Triton lysis buffer. Following the last wash, pelleted beads were suspended in 40 μl of 1× SDS sample buffer and boiled for 5 min. Samples of 20 μl of the supernatant were analyzed by SDS-PAGE and Western blotting.

Synthetic NFI sites proximal to the core origin stimulate BKV DNA replication in HK-2 cells.

Six putative NFI sites occur in the BKV archetype enhancer (Dik strain) (Fig. 1), and EMSAs were employed to evaluate NFI binding to these sites. As a positive control for NFI protein-oligonucleotide complexes, an EMSA was performed with a biotin-labeled oligonucleotide with the sequence of the NFI site in the adenovirus inverted terminal repeat (ITR) (45). As expected, a shift of the migration of the oligonucleotide caused by NFI binding was observed in the absence of a competitor (Fig. 2A, lanes 1 and 6), and the addition of a 200× excess of the unlabeled oligonucleotide fully competed for NFI binding (Fig. 2A, lanes 2 and 7); however, an oligonucleotide with a point mutation in the NFI site did not alter the band shift (Fig. 2A, lane 3). The addition of an anti-NFI antibody to the binding reaction mixture caused a supershift (Fig. 2A, lane 4, dashed arrow), confirming that the band shift was caused by NFI. An excess of BKV enhancer DNA also competed for NFI binding (Fig. 2A, lane 5), confirming that the BKV archetype enhancer contains functional NFI sites. Oligonucleotides with sequences of the six NFI binding sites in the BKV archetype enhancer (Fig. 1 and 3A) competed for NFI binding with different efficiencies (Fig. 2A, lanes 8 to 13), suggesting that these sites have different affinities for NFI proteins. Oligonucleotides with sequences of NFI sites closer to the BKV core-ori competed more efficiently than did those with distal sites.

Sequences corresponding to the first NFI site in the P block (Fig. 1) in different orientations and copy numbers were substituted for enhancer sequences (Fig. 2B) and assayed for Tag-dependent DNA replication in HK-2 cells (Fig. 2C, lanes 2 to 5). The addition of synthetic NFI sites to templates lacking the enhancer stimulated their replication, with the extent correlating with the number of sites (Fig. 2C, lanes 6 to 8) but not their orientations (Fig. 2C, lanes 3 and 4). The replication of templates containing multiple synthetic NFI sites was always weaker than the replication of templates with the archetype enhancer (Fig. 2C, lanes 1, 5, 7, and 8), suggesting that other elements also stimulate BKV DNA replication in these cells or that a particular spatial configuration of NFI sites is required.

Mutation of the BKV NCCR NFI sites reduces NFI binding and DNA replication.

Point mutations were introduced into the consensus sequence “TGGN_5∼7GCCAA” of each of the six NFI sites in the BKV enhancer, with the design of the mutations confirmed with MatInspector (72) to ensure that no new transcription factor binding sites were created (Fig. 3A). Oligonucleotides with mutated NFI sites were demonstrably defective for NFI binding in competitive EMSAs (Fig. 3B). A 200-fold excess of the BKV enhancer DNA with mutations in all six NFI sites (6mtNFIs BK enhancer) only slightly reduced the NFI binding to the adenovirus ITR oligonucleotide (Fig. 3C, compare lanes 1 and 3), whereas the wild-type (WT) enhancer sequence completely abolished NFI binding (Fig. 3C, lane 2). These results indicate that the mutation of the six identified NFI sites greatly reduces NFI binding to the BKV archetype enhancer.

Assays of the replication of BKV templates containing mutations of all six NFI sites (pUC-6mtNFIs-BKV) compared with the replication of the wild-type template (pUC-wt-BKV) in HK-2 cells transfected with each template separately indicated that both templates replicated with similar efficiencies (data not shown). However, the use of a competitive assay in which a competitor (3.4-kb) template, pBC-wt-BKV, containing the archetype NCCR was cotransfected into HK-2 cells together with test mutant templates (2.6 kb) (Fig. 4A) and a Tag expression vector (pCMV-BKT-Flag), revealed that the level of replication of the test templates with the enhancer containing six mutant NFI sites (pUC-6mtNFIs-BKV) was greatly reduced (Fig. 4B, compare lane 3 with lane 1), as was also observed for a test template without the enhancer (Fig. 4B, lane 2). Test templates with mutations in Ets1 binding sites in the enhancer (pUC-mtEts1s-BKV) replicated with an efficiency similar to that of the wild-type template (Fig. 4B, compare lane 5 with lane 1), indicating that the effect of the mutations on DNA replication in the competitive assay is attributable to the NFI site.

To determine whether a specific NFI site is responsible for the stimulatory effect on DNA replication, pUC-6mtNFIs-BKV templates with single NFI sites reverted to the WT were assayed for replication. Wild-type NFI sites 1 and 2 (closer to core-ori) appeared to be more stimulatory for BKV DNA replication than NFI sites 3 and 6 (Fig. 4C, lanes 3 to 7).

The requirement of a competitor template to observe the stimulatory effect of NFI sites on BKV DNA replication might be explained by the recruitment of factors required for DNA replication that are made limiting by use of the competitor template. The observation that NFI sites closer to the core-ori are more stimulatory for replication than are sites distal to the core-ori suggests that NFI may target components of the initiation complex, such as Tag, Pol-primase, replication protein A (RPA), or topoisomerase I. Evidence favoring this notion is provided by the experiments described below.

NFI interacts with BKV Tag.

We tested whether NFI interacts with BKV Tag by antibody pulldown assays. Full-length Flag-tagged BKV Tag (BKT-Flag) was mixed with HEK293 nuclear extracts, protein complexes were collected by using Flag antibody resin, and associated candidate proteins were detected with specific antibodies to each one (Fig. 5A). Initially, a pan-NFI antibody recognizing all NFI isotypes was used in the pulldown assays; other antibodies also detected the association of BKV Tag with p53, Sp1, and c-Jun but not Ets1, CREB, NF-κB p65, or Smad3 (Fig. 5A). Domains of BKV Tag that interact with these transcription factors were determined with GST-tagged truncated BKV Tags (Fig. 5B): the BKV Tag helicase/ATPase domain (HD) pulled down NFI, p53, c-Jun strongly, and Sp1 weakly but not CREB or NF-κB (Fig. 5C, lanes 4 and 5), indicating that NFI, p53, and c-Jun interact with the helicase/ATPase domain, while Sp1 may also interact with other Tag domains in addition to the HD. The latter observations are consistent with previous reports that BKV Tag and SV40 Tag complex with p53 (80), the SV40 Tag origin binding domain (OBD) interacts with Sp1 (39), and SV40 interacts with c-Jun (7). None of the transcription factors tested appeared to interact with the BKV Tag C-terminal region (Fig. 5C, lane 3). Truncated BKV Tags did not pull down factors not observed to complex with full-length BKV Tag, including Ets1, NF-κB, CREB, and Smad3 (Fig. 5A and C and data not shown).

Isotype-specific antibodies directed against NFIA, NFIB, and NFIC were used to attempt to distinguish isotype-specific interactions with BKV Tag, but only NFIA was detected at a high level in the HEK293 cell extracts and was pulled down by Tag (data not shown). To determine other isotypes of NFI that interact with BKV Tag, HA-tagged NFI isotypes and Flag-tagged BKV Tag were overexpressed in HEK293 cells, and their interactions with Tag were assessed by co-IP assays using either anti-HA or anti-Flag antibodies (Fig. 5D). Four HA-tagged NFI isotypes were expressed at similar levels (Fig. 5D); however, NFIB strongly upregulated the expression of Tag driven by the CMV promoter (Fig. 5D, lane 3), leading to a higher level of coprecipitation of Tag with NFIB than with other NFI isotypes. In contrast, HA-NFIA only weakly coprecipitated with Tag (Fig. 5D, lane 2), which might be due to a high level of endogenous NFIA competing against the transiently expressed NFIA in an interaction with Tag (Fig. 5D, lane 2, and data not shown). In control assays, no interaction between Tag and the HA-tagged Gal4 DNA binding domain (Gal4dbd) was detected, even though Tag was expressed at an extremely high level (Fig. 5D, lane 6), and no interaction of Tag with endogenous NF-κB p65 was detected (Fig. 5D). These results are in agreement with data from in vitro pulldown assays (Fig. 5A and C), indicating a specific interaction of BKV Tag with HA-tagged NFI isotypes.

Increased Tag expression partially rescues the replication deficiency of BKV templates with mutant NFI sites.

The interaction of BKV Tag with NFI provides a possible basis for the stimulatory effect upon replication by NFI sites in the enhancer. Initially, BKV Tag was expressed at a low level in the competitive DNA replication assays (Fig. 4B); to investigate if BKV Tag is the only limiting factor in these assays, increasing amounts of a pCMV-BKV Tag expression vector were introduced into HK-2 cells, and the levels of DNA replication of wild-type and mutant (NFI site) templates were compared (Fig. 6). BKV Tag expressed from 400 ng of pCMV-BKTAg saturated replication (Fig. 6, lane 7 to 9), but this amount, and even the addition of 900 ng pCMV-BKTAg, only partially rescued the replication of the template with the mutant NFI sites (pUC-6mtNFIs-BKV), compared with the robust replication of the wild-type template (pUC-wt-BKV) (Fig. 6, compare lane 9 with land 7 and compare lane 12 with lane 10). Furthermore, templates with a deleted enhancer (pUC-Δen-BKV) replicated poorly, even when BKV Tag was highly expressed (Fig. 6, lanes 8 and 11). These observations indicate that high levels of BKV Tag alone cannot correct the replication deficiency of templates with mutant NFI sites and suggest that BKV Tag is not the sole limiting factor in these replication assays.

NFIC/CTF1 stimulates BKV DNA replication in vitro when DNA polymerase-α primase is limiting.

BKV Tag interacts with different NFI isotypes in co-IP assays (Fig. 5D), but their importance for BKV replication is difficult to test in vivo due to the concurrent expression of multiple endogenous NFI isotypes. The NFIC/CTF1 isotype stimulates the initiation of adenovirus DNA replication by recruiting adenovirus DNA polymerase to the origin of replication (9, 19), and the proline-rich transactivation domain of the NFIC/CTF1 isotype stimulates SV40 DNA replication when tethered to the SV40 origin (61). These observations prompted us to assess whether NFIC/CTF1 can stimulate BKV DNA replication in the monopolymerase replication system that contains Pol-primase, RPA, and topoisomerase I (46, 85) in the absence of other cellular factors.

When Pol-primase was made limiting in the monopolymerase system, the DNA replication of the wild-type BKV template was stimulated strongly by NFIC/CTF1 in a concentration-dependent manner (Fig. 7, lanes 4 to 6). In contrast, with high levels of Pol-primase, the addition of NFIC/CTF1 had no effect on the replication of the wild-type BKV template (Fig. 7, lanes 1 to 3). Furthermore, no stimulation was observed with the NFI binding-site mutant BKV template regardless of the Pol-primase levels (Fig. 7, lanes 7 to 12). These findings indicate that NFIC/CTF1 stimulates the initiation of BKV DNA replication in vitro only when Pol-primase is limiting and suggest that Pol-primase is a limiting factor targeted by NFI to stimulate BKV DNA replication in vivo in the competitive replication assays.

NFIC/CTF1 interacts with the cellular DNA polymerase-α primase p58 subunit.

To further investigate the stimulation of replication by NFI, we studied whether it and Pol-primase proteins interact. Pol-primase consists of four subunits: two smaller subunits, p48 and p58, constitute the primase, and two larger subunits, p180 and p68, constitute the DNA polymerase-α catalytic and regulatory subunit p68, respectively (50, 51, 65, 82). HA-tagged human NFIC/CTF1 was ectopically expressed with each of four T7-tagged human Pol-primase subunits in HEK293 cells (Fig. 8A), and their interaction was examined by co-IP assays. The expression of p180 and p68 subunits was much more efficient than the expression of the primase subunits (as detected with the anti-T7 antibody) (Fig. 8A, lanes 3 to 6), but no interaction between NFIC/CTF1 and either the p180 or p68 subunits was detected (data not shown). Because the p58 expression level was low (Fig. 8A, lane 4), a more sensitive antibody against p58 was used, instead of the anti-T7 antibody, for the detection of p58 (Fig. 8B). In this assay, p58 was found to coprecipitate with NFIC/CTF1 (Fig. 8B, lane 1), and p58 co-IP was not detected in three control experiments, in which only p58 (Fig. 8B, lane 2), only NFIC/CTF1 (Fig. 8B, lane 3), or neither (Fig. 8B, lane 4) was expressed. No other significant distinct band was detected in the coprecipitated fraction. Unfortunately, despite several attempts, the level of expression of p48 was extremely low. An interaction between p48 and NFIC/CTF1 was not detected (Fig. 8A, lane 3, and data not shown), but we cannot exclude that p48 might bind to NFI.