Host-Specific Replication of BK Virus DNA in Mouse Cell Extracts Is Independently Controlled by DNA Polymerase α-Primase and Inhibitory Activities^▿

DNA.

pUC18-based plasmid DNAs with complete viral origins included pOriBKV (termed B-B-B, for BKV core region flanked by BKV sequence) (see Fig. 7) containing a fragment of the archetype BKV Dik strain (28), pOriSV40 (SV S strain) (42), and plasmids containing BKV-mPyV chimeric origins (P-B-B, B-B-P, and P-B-P, for the BKV [B] core region flanked by BKV and mPyV [P] sequences) (28). The pUC18 plasmid without an insert served as a negative control (pOri−) for cell-free DNA replication. All DNAs for replication assays were purified with Midiprep kits (Qiagen, Germany).

Protein purification.

Human recombinant topoisomerase I (48) and RPA (36, 40) were expressed and purified, and their concentrations and activities were determined as previously described (3, 27). Expression and purification of human and mouse Pol-prim as well as different hybrids were performed as previously described (50). Briefly, High Five (Invitrogen) insect cells (3.0 × 10⁹) were infected with 10 PFU of each recombinant baculovirus per cell and incubated at 27°C for 48 h. Pol-prim complexes were purified by phosphocellulose chromatography, followed by immunoaffinity chromatography (49). Immobilized monoclonal antibody SJK 287-38 was used to purify recombinant Pol-prim complexes containing mouse p180. SJK 237-71 coupled to Sepharose 4B was used to purify recombinant Pol-prim containing human p180. DNA polymerase assays and DNA primase assays were performed as previously described (4, 17, 34).

The BKV TAg was expressed and purified as previously described (28). Briefly, High Five insect cells (Invitrogen) were infected with the recombinant baculovirus (10 PFU per cell). Infected cells were harvested at 48 h, and BKV TAg was purified by immobilized metal affinity chromatography (IMAC) using Talon resin (Clontech). The purity and amount BKV TAg were determined by SDS-PAGE and Coomassie brilliant blue staining.

SV40 TAg was purified from extracts of insect High Five cells infected with recombinant baculoviruses by immunoaffinity chromatography using monoclonal antibody PAb101 coupled to protein A Sepharose (8, 37).

ELISAs.

Enzyme-linked immunosorbent assays (ELISAs) were carried out under the buffer conditions used for in vitro DNA replication, as described previously (12, 42). In brief, plastic 96-well microtiter plates (Immuno Maxi Sorb; Nunc) were coated overnight with excess (1 μg) recombinant human or mouse Pol-prim. Unbound protein was washed with phosphate-buffered saline (PBS). Wells were blocked for 2 h with 0.3 ml of 3% bovine serum albumin (BSA) (Sigma) in PBS. After the plates were washed, the indicated amounts (see Fig. 2) of BKV TAg (in 50 μl) were added, and plates were incubated for 1 h at room temperature and then washed with PBS. Polyclonal antibody against SV40 TAg that recognizes BKV TAg, kindly provided by W. Deppert, was added; the plates were incubated overnight at 4°C, washed, and then visualized by incubation with anti-rabbit immunoglobulin serum conjugated with horseradish peroxidase (Jackson Laboratories, Inc.) in the presence of 3% BSA for 1 h, followed by reaction with ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid); Sigma]. The reaction was allowed to proceed for 20 min, and then plates were quantified spectrophotometrically at 410 nm.

DNA synthesis on φX174 ssDNA.

The DNA synthesis on φX174 ssDNA was carried out according to Smith et al. (46). Briefly, the reaction mixture contained the following: 1 μg of φX174 ssDNA (NEB); 20 mM Tris-acetate (pH 7.3); 5 mM magnesium acetate; 20 mM potassium acetate; 1 mM dithiothreitol (DTT); 0.1 mg/ml BSA; 1 mM ATP; 0.1 mM each CTP, GTP, UTP, dATP, dCTP, dGTP, and dTTP; and 0.1 mM [α³²P]dCTP (3,000 Ci/mmol; Perkin-Elmer). Comparisons between human and mouse Pol-prim were made by titration, and the amount of enzyme complexes was varied from 0.5 to 2.0 units (3.04 U/μg for human Pol-prim, 2.88 U/μg for mouse Pol-prim, and 1 U to 100 pmol of deoxynucleoside monophosphate [dNMP] incorporation in a 1-h reaction) of each complex per assay. The incorporation of dCMP was determined by acid precipitation of DNA and followed by scintillation counting.

In vitro DNA replication assays.

Replication of DNAs in vitro was assayed as previously described (28) with slight modifications. Briefly, the reaction mixture (40 μl) contained the following: 20 mM HEPES, pH 7.8; 7 mM Mg acetate [MgAc]; 1 mM DTT; 4 mM ATP, 200 μM each CTP, UTP, and GTP; 50 μM dCTP; 100 μM each dATP, dTTP, and dGTP; 40 mM creatine phosphate (pH 7.8); 40 μg/ml creatine kinase; 5 μCi of [α³²P]dCTP (3,000 Ci/mmol, Perkin-Elmer); 0.25 μg of test plasmid DNA; FM3A cell extract (100 μg of total protein); 0.2 μg of purified BKV TAg; and purified recombinant human Pol-prim in the indicated amounts (see Fig. 4). After incubation for 60 min at 37°C, reaction products were precipitated with cold 10% (wt/vol) trichloroacetic acid (TCA) containing 2.5% (wt/vol) sodium pyrophosphate, spotted on glass fiber filters (GF/C; Whatman), washed with 1 M HCl, and analyzed by scintillation counting.

Monopolymerase DNA replication assay.

The SV40 and BKV monopolymerase replication assay contained 0.5 μg of pOriBKV DNA (28) or 0.5 μg of pOriSV40 (42); 50 ng of topoisomerase I; 100 ng of Pol-prim; 1 μg of RPA in 30 mM HEPES, pH 7.8; 7 mM MgAc; 0.1 mM EGTA; 0.5 mM 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 BSA; and 5 μCi of [α³²P]dCTP (3,000 Ci/mmol, Perkin-Elmer) in 40 μl. Purified BKV or SV40 TAg (0.2 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) TCA containing 2.5% (wt/vol) sodium pyrophosphate, spotted on glass fiber filters (GF/C; Whatman), washed with 1 M HCl, and analyzed by scintillation counting.

Initiation of replication of BKV and SV40 DNA.

Initiation reactions were performed as previously described (29, 31, 46) with slight modifications. The initiation reaction mixtures contained 0.25 μg of origin-containing DNA, 1.6 μg of TAg, 1 μg of RPA, 30 mM HEPES-KOH (pH 7.8), 7 mM MgAc, 1 mM EGTA, 1 mM DTT, 0.2 mM UTP, 0.2 mM GTP, 0.01 mM CTP, 4 mM ATP, 40 mM creatine phosphate, 1 μg of creatine kinase, 0.3 μg of topoisomerase I, 0.2 mg/ml of BSA, and 10 μCi of [α-³²P]CTP (3,000 Ci/mmol; Perkin-Elmer). Recombinant Pol-prim was added as indicated in the figure legends. Half of the reaction mixture was spotted on DE 81 paper (Whatman), precipitated with cold 10% (wt/vol) TCA containing 2.5% (wt/vol) sodium pyrophosphate, washed with 1 M HCl, and analyzed by scintillation counting. The remaining reaction products were precipitated with ethanol in the presence of 0.8 M LiCl-0.5 mg/ml DNA, redissolved in 45% formamide-5 mM EDTA-0.09% xylene cyanol FF at 65°C for 30 min, heated for 3 min at 95°C, and separated by denaturing 20% polyacrylamide gel electrophoresis for 3 to 4 h at 600 to 800 V as previously described (42). The reaction products were visualized by autoradiography using an FLA-5100 imager (Fuji, Europe).

FM3A extract.

FM3A cell extracts were prepared as previously described (28). Briefly, logarithmically growing FM3A cells in RPMI medium supplemented with 10% fetal bovine serum (FBS) were collected by centrifugation and washed with phosphate-buffered saline and then with hypotonic buffer. Cells were homogenized and NaCl adjusted to 100 mM. Extracts were clarified by centrifugation at 20,000 × g for 30 min and stored at −80°C.

Replication protein A is not a species-specific factor in cell-free BKV DNA replication.

The monopolymerase replication assay was then used to analyze the species specificity of BKV DNA replication in vitro with DNA containing the BKV origin of replication (28). BKV TAg supports DNA replication in vitro and shows specificity for the origin DNA in this assay as well as species specificity since the addition of mouse but not human cell extracts inhibits BKV DNA replication (28). However, whether components of the replication machinery regulate host-specific inhibition of BKV DNA replication remained unknown. Therefore, the question arose as to whether mouse RPA, a host replication factor essential for the initiation of viral DNA replication, would inhibit BKV DNA replication or whether mouse RPA would be able to substitute for human RPA in BKV DNA replication. Preliminary data suggested that RPA is not responsible for inhibition since during the purification of IA from mouse FM3A extracts, RPA and IA were discovered in different purification fractions (data not shown). However, to prove that RPA is not identical with IA, mouse RPA, which was partially purified by ssDNA-cellulose chromatography, was tested in BKV and SV40 DNA replication assays. Mouse RPA is fully active in both systems. Mouse RPA is neither the IA nor the species-specific factor of BKV DNA replication (Fig. 1).

BKV large T antigen physically and functionally interacts with both human and mouse DNA polymerase α-primase in similar ways.

SV40 TAg has been shown to interact physically with purified Pol-prim and independently with all four Pol-prim subunits (11, 46, 57). In contrast, physical interactions of purified Pol-prim with BKV TAg have not been investigated. Therefore, we tested whether the physical interactions of BKV TAg with the Pol-prim complex are species specific by a modified ELISA (12). The results revealed binding of BKV T TAg to human and mouse Pol-prim complexes with similar efficiencies (Fig. 2). Therefore, BKV large TAg showed no species specificity in physical interactions with Pol-prim on this level. In parallel, SV40 TAg binding to Pol-prim complexes was analyzed, and consistent with previous data, no host specificity was observed (42; also data not shown). The results revealed that human and mouse Pol-prims physically bind to BKV TAg with similarly efficiencies. This raises the question of whether the initiation complex containing mouse Pol-prim and BKV TAg would be functional or could inhibit BKV DNA replication. The latter occurrence was previously seen when peptides containing TAg-binding sites of human Pol-prim or RPA were added to the DNA replication assays prior to the start of the reaction (10, 52).

Pol-prim has been shown to be the major host factor responsible for species-specific replication of mPyV and SV40 DNAs in human and mouse cell extracts, respectively (8, 23, 46, 50). The activity of human and mouse Pol-prim was tested in assays with ssDNA as a template (Fig. 3 and data not shown). Pol-prim is able to perform DNA synthesis on ssDNA substrates alone or in the presence of other initiation proteins, e.g., RPA and large TAg. As previously described, RPA inhibits Pol-prim synthesis on ssDNA, but this inhibition is reversed by viral TAg, suggesting that all three proteins together form an active synthesis complex (Fig. 3 and data not shown). This assay provides the evidence for the functional activity of the purified proteins and their ability to cooperate in DNA synthesis. We investigated whether BKV TAg stimulates Pol-prim synthesis on an ssDNA substrate in a species-specific manner. Both SV40 and BKV TAg stimulated mouse and human Pol-prim complexes on φX174 DNA in the presence or absence of RPA (Fig. 3 and data not shown). Consistent with previous SV40 studies, these experiments revealed that BKV TAg behaves in the same way as SV40 TAg by efficiently stimulating the Pol-prim activity but not in a species-specific manner.

Mouse DNA polymerase α-primase does not support BKV replication in vitro.

These findings lead to the question of whether mouse or human Pol-prim can replace each other or inhibit the DNA synthesis. Mouse Pol-prim was added to the BKV monopolymerase assay instead of human Pol-prim, but the mouse enzyme complex could not substitute for the human Pol-prim complex (Fig. 4, compare bars 2 to 4 with bars 5 to 7). In comparison to the human enzyme complex, the activity of mouse Pol-prim in the assay with pBKVori plasmid was about 4- to 5-fold lower than that of human Pol-prim (Fig. 4 and 5 A). This lack of replication activity of the mouse Pol-prim cannot be explained by an inactivation process during expression or purification of the mouse enzyme complex since the same fraction was active in mPyV DNA replication in vitro, had activity comparable to that of the human Pol-prim in DNA synthesis on the ssDNA template, and physically and functionally interacted with BKV TAg (Fig. 2 and 3).

Although the mouse enzyme complex itself was inactive in BKV TAg-dependent DNA replication (Fig. 4, bars 2 to 4), in the presence of the human Pol-prim the mouse Pol-prim did not inhibit BKV DNA synthesis but even slightly stimulated the incorporation of radioactive dNMPs carried out by human Pol-prim (Fig. 4, compare bar 7 with bars 8 and 9). These results suggest that Pol-prim in the mouse replication extracts is not responsible for the inhibition of BKV DNA replication in mouse cell extracts. However, the enzyme complex is a species-specific factor as mouse Pol-prim is not able to efficiently replicate BKV DNA in the presence of BKV TAg, whereas human Pol-prim in the experiments made in parallel is fully active.

BKV DNA replication requires the p180 and p68 subunits of human DNA polymerase α-primase for optimal activity.

It is known that SV40 DNA replication requires only the presence of the large p180 DNA polymerase subunit of human origin in a chimeric Pol-prim complex with the three small subunits of mouse origin to replicate SV40 DNA in mouse extracts in the presence of SV40 TAg (50). In contrast, Pol-prim with the small p48 primase subunit of mouse origin and the three other subunits of human origin is sufficient to allow mPyV DNA replication in human cell extracts in the presence of PyV TAg (8, 23). Since Pol-prim is an important species-specific factor for BKV replication as well, we studied how different mouse-human hybrid Pol-prim complexes containing subunits of both human and mouse origin support BKV DNA replication.

To this end, we expressed in insect cells and then purified six hybrid Pol-prim complexes (MHHH [MH3], HMMM [HM3], HHMM [H2M2], HHHM [H3M], HHMH [H2MH], and HMHH [HMH2]) to near homogeneity. In these designations, H represents a human subunit, and M indicates a subunit of mouse origin; the subunits are noted in order, p180, p68, p58, and p48. BKV and SV40 monopolymerase DNA replication experiments were performed in parallel (Fig. 5) to compare the BKV DNA replication system to the well-studied SV40 system. Mouse Pol-prim is not active in the monopolymerase systems in either the BKV or SV40 system, and the incorporation of radioactive dNMPs on plasmid DNA containing a viral replication origin was close to background level (Fig. 5, M4). Figure 5A shows that the MH3 complex, which consists of mouse p180 and three smaller subunits of human origin, showed some activity, but it was less than 30% of the BKV DNA replication activity with human Pol-prim (H4) (Fig. 5A, compare the three MH3 columns with the H4 columns). These findings suggest a major role of p180 in modulating the species specificity of BKV DNA replication. The importance of the human origin of the p180 and the p68 subunits is underlined by the findings that all hybrid Pol-prim complexes containing the two largest subunits from human (H2M2, H2MH, and H3M) efficiently replicate BKV DNA and reach the level of DNA replication activity of recombinant human Pol-prim having all four subunits of human origin. These results also show that all subunits of Pol-prim contribute to species specificity of replication of BKV origin-containing DNA in the presence of BKV TAg. In contrast to these findings, SV40 DNA replication in vitro with these chimeric Pol-prim complexes reveals a dependence on p180's being of human origin (Fig. 5B) (50). Moreover, the chimeric Pol-prim complexes do not reach the full activity of the recombinant human Pol-prim in the SV40 DNA replication system, whereas H2M2, H2MH, and H3M showed equivalent DNA replication activity in comparison with H4 in the BKV system (compare Fig. 5A and B). These findings suggest that species specificity requirements of BKV DNA replication are less restrictive than those of SV40 DNA replication using the same biochemical assay system.

Initiation of BKV replication by human and mouse DNA polymerase α-primase.

To study the activity of human-mouse hybrid Pol-prims in early stages of DNA replication, we optimized assays for the initiation of BKV and SV40 DNA replication. The primase activities of human and mouse Pol-prim and of a selection of chimeric Pol-prim complexes (H4, M4, H2M2, and MH3) were assessed using an ssDNA substrate. The ranges of primase activity were consistent with previously published data (50). Calculated from these experiments, amounts of Pol-prim used in the initiation assays with viral origins cloned into plasmids were normalized to the same enzyme activity.

The initiation activities of these hybrid Pol-prim complexes were similar to their activities in DNA replication. These data showed that human primase subunits are not essential in both the SV40 and BKV systems. The hybrid H2M2 complex was highly active in the BKV and SV40 systems. The exchange of the large DNA polymerase p180 subunit in a Pol-prim complex of human origin for one of mouse origin caused a reduction in replication levels in both systems. This shows the importance of the p180 subunit in species specificity of both initiation and overall BKV and SV40 replication in vitro. However, it is important to note that hybrid Pol-prim complexes, similar to the human Pol-prim, are more active in the initiation of BKV DNA replication than in the initiation with SV40 DNA (Fig. 6 A).

In addition, initiation products were analyzed by denaturing electrophoresis (Fig. 6B). In these analyses human Pol-prim and the hybrid complex H2M2 were highly active. In contrast, mouse Pol-prim showed very low activity. However, the mouse enzyme complex revealed some basal activity above the background presented in lane 11. As seen by comparing lanes 3, 7, and 9 with lane 5 of Fig. 6B, human and hybrid Pol-prims form longer RNA products than mouse Pol-prim, suggesting that mouse Pol-prim is able to start primer synthesis during the initiation of BKV DNA replication but cannot efficiently elongate these short primers, leading to a later abortion of the BKV DNA replication in vitro.

Interactions of the inhibitory activity with flanking regions of the BKV core origin.

Previous studies have shown that the core origins of SV40 and mPyV but not their flanking regions are responsible for the species specificity of the viral DNA replication (2). Recent results with BKV DNA replication also suggest that the main species-specific function may associate with the core origin (28). The latter idea is supported by the findings presented here that Pol-prim modulates the species specificity of BKV DNA in vitro. The presence of inhibitory activities (IAs) in mouse cell extracts, which block the BKV DNA replication in these extracts supplemented with human Pol-prim (28), raises the question of whether the IAs also interact with the core origin or whether they require different activities of the BKV origin that associate with the early region and the enhancer regions on the late site adjacent to the core origin. The involvement of the core origin-flanking sites would suggest that multiple independent mechanisms are present in mouse cells to control the species specificity of BKV DNA replication, which is different from the current model of species-specific control of polyomavirus DNA replication. In such a context we expected human Pol-prim to be a species-specific function associated with the core origin, and by supplementing mouse cell extracts with hum Pol-prim, we could investigate whether changing the flanking sequences of BKV to mPyV would make these human Pol-prim-supplemented extracts permissive for BKV TAg-dependent replication of chimeric human-mouse polyomavirus DNA in vitro (Fig. 7).

The addition of increasing amounts of human Pol-prim (minimum requirement of 2 U, equivalent to 660 ng of enzyme complex) in the presence of 0.2 μg of BKV TAg allowed mouse crude replication extracts to efficiently replicate DNA containing BKV-mPyV chimeric origins P-B-B and P-B-P, which have the early flanking region of the wild-type BKV origin replaced by the equivalent polyomavirus sequences. In contrast, both BKV wild-type B-B-B and the chimeric B-B-P origin showed background levels of BKV TAg-dependent DNA replication at any concentration of human Pol-prim. These results indicate that BKV DNA replication is independently regulated by both Pol-prim, representing a core origin species-specific factor, and the newly identified inhibitory activities, which require origin-flanking sequences for their action and specifically sequences located in the early region of the BKV origin of replication. These findings yield new insight into the regulation of species specificity of BKV polyomavirus, which differs from that of other Polyomaviridae such as mPyV and SV40.