Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase Richard W. P. Smith 1, * and Heinz-Peter Nasheuer 1,2 1 Abteilung Biochemie, Institut fu ¨ r Molekulare Biotechnologie, Jena, Germany; 2 National University of Ireland, Galway, Department of Biochemistry, Galway, Ireland Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase a-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA repli- cation of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase a-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex. Keywords: DNA replication; initiation; DNA polymerase a-primase; species specificity; polyomavirus. Polyomavirus DNA replication has served as a model system to study eukaryotic DNA replication [1,2]. JC virus (JCV) belongs to the polyomavirus family and is the causative agent of progressive multifocal leukoencephalo- pathy in immunocompromised humans (reviewed in [3–7]). JCV exhibits a highly restricted host range and this species specificity appears to be governed by host encoded DNA replication factors as hamster glial cells, which support viral early gene transcription, nevertheless fail to replicate JCV DNA [8]. JCV is closely related to simian virus 40 (SV40) and to mouse polyomavirus (PyV), both of which show clear species specificities as lytic infection is limited to primate and mouse cells, respectively [9]. The species specificities of both SV40 and PyV are regulated at the level of initiation of DNA replication [10], a process that has been extensively studied both in vivo and in vitro owing to the development of cell-free DNA replication systems [2,11–16]. Polyomavirus DNA replication is carried out by the host cell machinery supplemented with a single essential viral protein, large T antigen (TAg), which recognizes and partially unwinds the viral replication origin, recruits host proteins such as replication protein A (RPA) and DNA polymerase a-primase, and functions as the replicative helicase [2,17,18]. Species specificity of both SV40 and PyV DNA replication can be reproduced in vitro using DNA carrying the viral core origin and purified replication enzymes [14,19–21]. For both SV40 and PyV it has been clearly demonstrated that the host factor responsible for species specificity is DNA polymerase a-primase, which initiates DNA replication in all eukaryotes [19,22–27]. DNA polymerase a-primase consists of four subunits with apparent molecular masses of 180, 68, 58 and 48 kDa of which the largest and smallest subunits are a DNA polymerase and a primase, respectively [28–30]. SV40 DNA replication in vitro was recently shown to require a functional interaction between the SV40 TAg and the C-terminus of the p180 subunit of human DNA poly- merase a-primase [26]. The genome of JCV is 69% homologous to that of SV40 and expresses an analogous set of proteins [31]. The core origins of replication of the two viruses are also conserved to such an extent that SV40 TAg, which is 72% identical to JCV TAg, can efficiently support JCV DNA replication in vivo and in vitro [32,33]. The high level of conservation between these two primate specific viruses coupled with the fact that JCV DNA replication is inhibited in a nonpermis- sive host in vivo would imply that the restricted host range of JCV is due to a requirement for human DNA polymerase a-primase, as is the case with SV40 [8]. Previously we reported the establishment of a cell-free system for JCV DNA replication [32]. With this system we were able to reproduce many features of JCV DNA replication found in vivo, such as sequence requirements at the origin of replication and the requirement for JCV or SV40 (but not PyV) TAg for efficient replication. Therefore, we applied this system to the question of host specificity regulation and found that this differs from that of SV40 in that it is not determined at the level of initiation of DNA replication by DNA polymerase a-primase in vitro. This appears to be due to differences in the interaction of the JCV and SV40 large T antigens with the initiation complex. Correspondence to H. P. Nasheuer, National University of Ireland, Galway, Department of Biochemistry, Cell Cycle Control Laboratory, Galway, Ireland. Fax: + 353 91 512 504, Tel.: + 353 91 512 409, E-mail: h.nasheuer@nuigalway.ie Abbreviations: JCV, JC virus; SV40, simian virus 40; PyV, mouse polyomavirus; TAg, large T antigen; RPA, replication protein A. *Present address: Institute of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, Scotland UK. (Received 30 October 2002, revised 4 March 2003, accepted 17 March 2003) Eur. J. Biochem. 270, 2030–2037 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03579.x Materials and methods Proteins SV40 TAg, JCV TAg and the DNA polymerase a-primase complexes (p180-p68-p58-p48) were purified from baculovirus infected insect cells as described previously [22,32,34,35]. Human and murine DNA polymerase a-primase were immunopurified using the monoclonal antibodies SJK237-71 and SJK287-38, respectively [36]. Human RPA was bacterially expressed and purified as outlined previously [37,38]. Human topoisomerase I was expressed in insect cells and purified as described by Søe et al. [39] and was a generous gift of K. Søe (IMB-Jena, Germany). The monoclonal antibodies SJK237-71 and SJK287-38, specific for DNA polymerase a-primase, were purified by affinity chromatography [40]. Protein concen- tration was determined according to Bradford [41] using a commercial reagent with BSA as a standard (Biorad, Munich). DNA polymerase a and DNA primase assays were performed as previously described [34,42,43]. Preparation of S100 extracts and replication of SV40 and JCV in vitro S100 extracts were prepared from logarithmically growing FM3A cells as previously described [27,34]. Cells were harvested by centrifugation, then washed twice with phos- phate buffered saline (NaCl/P i ) and once with hypotonic buffer. The cells were resuspended in hypotonic buffer, incubatedfor10minonice,andbrokenby12strokesina Dounce homogenizer. The extracts were centrifuged at 4 °C and 11 000 g. The supernatant was then adjusted to 100 m M NaCl and clarified by a second centrifugation at 100 000 g (S100 extract). Depletion of DNA polymerase a-primase from S100 extracts was performed essentially as previously described [22,27,32]. The replication of SV40 and JCV DNA in vitro was performed as previously described [22,27,32]. Briefly, the assay contained 0.6 lg SV40 or JCV TAg, 250 ng of pUC- HS or pJC389 or pJC433 DNA (carrying the replication origin of SV40 or JCV, respectively [21,32]), and 200 lg S100 in 30 m M Hepes/NaOH (pH 7.8), 1 m M dithiothreitol, 7m M magnesium acetate, 1 m M EGTA (pH 7.8), 4 m M ATP, 0.3 m M CTP, GTP, and UTP, 0.1 m M dATP and dGTP, 0.05 m M dCTP and dTTP, 40 m M creatine phos- phate, and 80 lgÆmL )1 creatine kinase, and 5 lCi each of [a 32 P]dCTP and [a 32 P]dTTP (3000 CiÆmmol )1 , Amersham- Biosciences). DNA polymerase a-primase was added as indicated. The incorporation of radioactive dNMP was measured by acid-precipitation of DNA and scintillation counting. The total radioactivity was measured after spotting 5 lL of a 200-fold dilution of the replication assay onto GF52 filters (Schleicher & Schu ¨ ll, Dassel, Germany). EcoRI and DpnI digestion of product DNA was carried out as described by Kautz et al. [23]. Initiation of replication on JCV DNA Initiation reactions were performed essentially as previously described [22,32,44,45]. Briefly, the JCV initiation assay (40 lL) was assembled on ice and contained 0.25 lg pJC389 (carrying the JCV replication origin), 0.6 lgJCV TAg, and 0.5 lgRPA,in30m M Hepes/KOH (pH 7.8), 7m M magnesium acetate, 1 m M EGTA, 1 m M dithiothre- itol, 0.2 m M UTP, 0.2 m M GTP, 0.01 m M CTP, 4 m M ATP, 40 m M creatine phosphate, 1 lg creatine kinase, 0.3 lg topoisomerase I, 0.25 mgÆmL )1 heat treated BSA, and 20 lCi of [a- 32 P]CTP (3000 CiÆmmol )1 , NEN Life Science, Brussels). Recombinant DNA polymerase a-primase was added as indicated in the figure legends. After incubation for 2 h at 37 °C one-eighth of the reaction mixture was spotted onto DE81 paper to estimate the amount of incorporated nucleotides [46]. For size analysis the reaction products were precipitated with 0.8 M LiCl, 10 lg of sonicated salmon sperm DNA (Sigma), 10 m M MgCl 2 and 120 lL of ethanol for 1 h on dry ice, washed twice with 75% ethanol/water, dried, redissolved in 45% formamide/5 m M EDTA/0.05% (w/v) xylene cyanol FF/0.05% (w/v) bromphenol blue at 65 °C for 30 min, heated for 3 min at 95 °C, and electro- phoresed in denaturing 20% polyacrylamide gels for 3–4 h at 600 V as described previously [22]. The reaction products were visualized by autoradiography and quantified with a phosphoimager (Amersham Biosciences). JCV and SV40 monopolymerase systems These assays were adapted from Ishimi et al.[11].The monopolymerase assay (40 lL) was assembled on ice and contained 0.5 lg pUC-HS (carrying the SV40 replication origin) or 0.5 lg pJC433 (carrying the JCV replication origin [32]), 1 lg SV40 or JCV T antigen, 1.4 ng topoiso- merase I and 0.5 lgRPA,in30m M Hepes/KOH (pH 7.8), 7m M magnesium acetate, 0.1 m M EGTA, 1 m M dithio- threitol, 0.2 m M UTP, 0.2 m M GTP, 0.2 m M CTP, 4 m M ATP, 20 m M dATP, 20 m M dGTP, 2 m M dCTP, 2 m M dTTP, 40 m M creatine phosphate, 1 lg creatine kinase, 0.25 mgÆmL )1 heat treated BSA, and 5 lCi [a- 32 P]dCTP and 5 lCi [a- 32 P]dTTP (each 3000 CiÆmmol )1 , Amersham- Biosciences). Recombinant DNA polymerase a-primase was added as indicated. After incubation for 90 min at 37 °C, one-quarter of the reaction mixture was used to determine the level of incorporation by spotting onto DE81 paper, washing with 0.5 M NaHCO 3 and liquid scintillation counting [46]. Results Replication of JCV DNA in crude cell extracts with recombinant human and murine DNA polymerase a-primase Previously we reported the establishment of in vitro systems for the replication of JCV DNA, either using crude cell extracts or purified proteins only [32]. Both these systems were dependent upon recombinant JCV TAg and the presence of a JCV origin of DNA replication. Here we applied these systems to study the dependence of JCV DNA replication on human replication proteins. Figure 1 repre- sents a comparison between the SV40 (panel A) and JCV (panel B) cell-free DNA replication systems. Figure 1B shows a DNA replication assay using mouse FM3A S100 crude cell extracts depleted of DNA polymerase a-primase supplemented with JCV TAg, a plasmid carrying the JCV Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2031 replication origin (pJC389) and either recombinant human or murine DNA polymerase a-primase expressed using the baculovirus system (Fig. 1B, columns 1–7). The replication activity of JCV TAg in mouse cell extracts is not dependent on the sequence of the plasmid as the incorporation of radioactive dNMPs was the same whether the plasmid pJC389 or pJC433 was used in the cell-free replication assay (data not shown). In parallel, we show an SV40 DNA replication assay using the same cell extracts but with SV40 TAg and a plasmid (pUC-HS) that carries the SV40 replication origin (Fig. 1A, columns 1–7 [26,27]). As we showed previously, SV40 DNA replication is absolutely dependent upon human DNA polymerase a-primase (Fig. 1A, columns 1–5 [26,27]) and more specifically, upon the human p180 subunit as the hybrid DNA polymerase a-primase complex consisting of the murine p180 subunit together with the human p68, p58 and p48 subunits (MH 3 ) is inactive (Fig. 1A, columns 6–7 [26,27]). In contrast, replication of JCV DNA does not show the same strict requirement for human DNA polymerase a-primase (Fig. 1B, columns 1–7). Both murine DNA polymerase a-primase and the hybrid complex, MH 3 , show significant activity in the DNA replication assay. In both the SV40 and JCV assays, incorporation of nucleotides is dependent upon an intact origin of replication; either plasmids carrying the noncognate PyV origin or a disabled JCV origin are inactive (Fig. 1A, column 8 and Fig. 1B, columns 8 and 9). Background values determined without added DNA polymerase a-primase were higher in the JCV system, presumably due to residual endogenous murine enzyme in the cell extracts due to incomplete depletion. However, as this background incorporation did not repre- sent full rounds of replication in either system (Fig. 2A,B, columns 1–2) other relevant values (Fig. 1A,B, lanes 2–7) were corrected for it. We further characterized the products of the replication reactions by digesting the resulting DNA with the restriction enzyme DpnI, which digests only fully methylated DNA. The plasmid DNA used as template in our assay was purified from Escherichia coli and is therefore fully methy- lated. However, one or more full rounds of replication will result in hemimethylated or unmethylated products and will consequently lead to DpnI resistance which is indeed observed after replication of JCV DNA either with human or with murine DNA polymerase a-primase (Fig. 2B, lanes 4 and 6). The lack of species specificity we observed was reproducible with various independently expressed and purified batches of JCV TAg (data not shown) and with various batches of the template DNAs pJC389 and pJC433 ([32]; Figs 1 and 2). As expected murine DNA polymerase a-primase did not support SV40 DNA replication (Fig. 2A, lanes 5 and 6). JCV DNA replication with purified proteins We note that in the cell-free system JCV DNA replication is markedly less efficient when driven by murine compared with human DNA polymerase a-primase. In order to Fig. 1. DNA replication assays in murine FM3A cell extracts depleted of DNA polymerase a-primase using the SV40 (A) and JCV (B) systems. (A) The SV40 system makes use of SV40 TAg and pUC-HS template DNA containing the SV40 origin of DNA replication. (B) The JCV system uses JCV TAg and pJC389 DNA with the JCV origin. The cell extracts were supplemented with 0.5 and 1.0 DNA polymerase units of the indicated DNA polymerase a-primase complexes (H 4 , human heterotetramer; M 4 , murine heterotetramer; MH 3 , murine p180 with human p68, p58 and p48). Enzyme activities were determined beforehand with a DNA polymerase assay on activated calf thymus DNA. (A) and (B) column 1, TAg omitted; column 8, pJC389I-/II-, containing a disabled JCV replication origin [32], was used as template for human DNA polymerase a-primase with SV40 (A) and JCV TAg (B), respectively. (B) column 9, pUC-Py1, containing the PyV replication origin [21], used with human DNA polymerase a-primase and JCV TAg. Values in panels (A) and (B), columns 2–7 were corrected for background incorporation determined in either system without the addition of DNA polymerase a-primase. Standard deviations from two to four experiments are indicated as error bars. The experiments in (A) and (B) were performed in parallel. 2032 R. W. P. Smith and H. P. Nasheuer (Eur. J. Biochem. 270) Ó FEBS 2003 characterize further these processes we made use of purified systems. Firstly we examined the efficiency of primer formation at the origin of replication during the initiation step of JCV DNA replication. Figure 3 shows that this is efficiently carried out by both the human and murine DNA polymerase a-primase complexes although quantification of the reaction products shows the murine complex to be approximately 30% less efficient than the human complex, especially in the synthesis of products greater than four nucleotides in length. Murine DNA polymerase a-primase is absolutely inactive in initiating SV40 DNA replication in an assay consisting of purified enzymes [26,27]. We then reproduced the absence of JCV species specifi- city using the monopolymerase DNA replication system, which makes use of purified enzymes only and allows measurement of deoxyribonucleotide incorporation after coupled initiation and elongation by DNA polymerase a-primase [11]. Figure 4 shows that SV40 DNA replication in this system is clearly dependent upon DNA polymerase a-primase being of human origin (panel A) but that this is not the case for JCV (panel B). This shows that the ability of murine DNA polymerase a-primase to replicate JCV DNA is not dependent upon factors other than the replication proteins used in this assay. However, overall deoxynucleo- tide incorporation is less efficient than by human DNA polymerase a-primase as shown above with the cell-free assay (Fig. 1), which suggests that steps in JCV DNA replication subsequent to primer formation may be slightly inhibited by the murine enzyme complex. SV40 TAg confers species specificity to JCV origin dependent DNA replication It has been reported that SV40 TAg is capable of supporting JCV DNA replication both in vivo and in vitro [32,33,47]. Therefore, we asked whether substitution of SV40 TAg for JCV TAg would render JCV DNA replication species- specific with regard to the nature of the DNA polymerase a-primase complex catalysing the reaction, as is the case Fig. 2. DNA synthesis products of the SV40 and JCV DNA replication systems. Murine FM3A cell extracts depleted of DNA poly- merase a-primase were supplemented with 1.0 DNApolymeraseunitsofH4orM4ornot supplemented (–Pol). One-quarter of the DNA synthesis products were analysed for complete DNA replication by digestion with EcoRI and DpnI (even numbered lanes). In parallel, the products were linearized with EcoRI (odd numbered lanes). The positions of linearized template DNAs are indicated by arrows. Fig. 3. Autoradiogram of an in vitro JCV DNA replication initiation assay with 0.2 and 0.4 units of primase of either human (H4) or murine (M4) DNA polymerase a-primase complexes. Specific primase activities were determined beforehand with a primase assay on poly (dT). Lanes 1 and 2, control reaction with DNA polymerase a-primase lacking TAg or vice versa; lanes 3 and 4, 0.2 U and 0.4 U of human; lanes 5 and 6, 0.2 U and 0.4 U of murine DNA polymerase a-primase. The approximate sizes of the reaction products are indicated on the right in nucleotides (nt). Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2033 with SV40 DNA replication. Figure 5 shows that murine DNA polymerase a-primase is indeed incapable of replica- ting JCV DNA when the replication complex contains SV40 TAg (columns 2–3). The reciprocal experiment to investigate whether JCV TAg would relieve the species specificity of SV40 DNA replication is not feasible as JCV TAg is incapable of supporting SV40 DNA replication [32,33]. Discussion It has been firmly established that the species specificity of lytic infection by the polyomaviruses SV40 and PyV is determined at the level of DNA replication both in vivo and in vitro by the nature of the host DNA polymerase a-primase complex [2,19,21–27]. Here we report that the closely related JC virus does not show such a strict specificity in its DNA replication in vitro. Although murine DNA polymerase a-primase is approximately 50% less efficient than is its human counterpart in the replication of JCV DNA in our assays (Figs 1–5), nucleotide incorpor- ation by this complex is significantly above the values determined in the SV40 DNA replication systems (Figs 1,2 and 4 [21,26,27]). In the purified initiation system of JCV DNA replication, murine DNA polymerase a-primase can catalyse primer formation at the JCV origin (Fig. 3), a reaction the murine complex does not support in the SV40 system [22,26,27]. Importantly, we show that nucleotide incorporation in the JCV system by murine DNA polymerase a-primase is dependent upon the JCV DNA replication origin (Fig. 1B) and results in DpnI-resistant products (Fig. 2), indicating that it is due to bona fide DNA replication and not a consequence of Ôfilling inÕ of gaps or other short patch repair events. The fact that we observe a complete round of plasmid replication in murine cell extracts indicates that other essential replication proteins, such as DNA poly- merases d and e, proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), topoisomerase I and DNA ligase are not responsible for JCV species specificity in vitro. Our murine FM3A cell extracts contain relatively low levels of endogenous RPA and are therefore supplemented with human RPA. However, if this is left out we nevertheless observe significant, albeit overall less efficient, incorporation by both murine and human DNA polymerase a-primase (data not shown) indicating that RPA also is not a species- specific factor. In apparent contradiction of our results, Feigenbaum et al. [8] showed that cultured nonpermissive hamster glial cells were unable to replicate transfected JCV DNA. Their observation and our data could be reconciled if the murine but not the hamster cellular DNA replication machinery were permissive for JCV DNA replication. We consider this unlikely. A more likely reason for the discrepancy between Feigenbaum’s data and our own is the difference in the ÔstateÕ of the DNA in the two assays. Transfected DNA will become associated with histones to form chromatin in the cell nucleus whereas our in vitro assays are carried out with naked plasmid DNA. Nucleosomes may interfere with the initiation of replication and evidence exists that the binding of transcription factors to sites in the regulatory regions adjacent to the replication origin of the SV40 chromosome may play a role in relieving such nucleosome repression [48– 52]. In vivo JCV DNA replication shows a greater depend- ency on such flanking regions than does SV40 [33]. It could be that JCV species specificity is governed by a host factor such as nuclear factor I (NF-I) required to facilitate initiation of replication in vivo but not in vitro, perhaps by alleviating nucleosome repression or by assisting in origin unwinding under conditions of altered template DNA superhelicity [50]. The findings that JCV DNA replication is stimulated by NF-I in vivo but not in vitro support this explanation [50]. This view is consistent with the knowledge that introducing an SV40 sequence into the JCV genome Fig. 4. SV40 and JCV DNA replication using the monopolymerase system. The SV40 system contains SV40 TAg and DNA with the SV40 replication origin (A); the JCV system con- tains JCV TAg and DNA with the JCV rep- lication origin (B). DNA polymerase (0.25 and 0.5 U) of the indicated DNA polymerase a-primase complexes were added (H4, human, columns 2–3; M4, murine, columns 4–5; –Pol, none, panels (A) and (B), column 1). Enzyme activities were determined beforehand with a DNA polymerase assay on activated calf thymus DNA. Standard deviations from three experimentsareindicatedaserrorbars.The experiments in (A) and (B) were performed in parallel. 2034 R. W. P. Smith and H. P. Nasheuer (Eur. J. Biochem. 270) Ó FEBS 2003 can extend the host range of JCV replication in vivo [52]. Alternatively, rodent, but not human, chromatin might contain a nondiffusable factor that inhibits JCV TAg- dependent DNA replication or the phosphorylation of JCV TAg is different in human and mouse cells interfering with the replication activity in vivo but not with the purified baculovirus-expressed protein. This explanation is consis- tent with findings that specific residues of SV TAg must be phosphorylated whereas other may not be [17]. In summary, the available evidence strongly suggests that, although DNA replication is the species-specific process common to JCV, SV40 and PyV, different host factors in each case ultimately determine the restriction of virus propagation to a particular host. For SV40 and PyV these are, respectively, the p180 and p48 subunits of DNA polymerase a-primase in initiation of DNA replication [22–24,26,27,53]. For JCV a different level of control appears to be in operation although we cannot rule out that the lower initiation efficiency of murine DNA polymerase a-primase is at least in part involved when compounded by other factors not present in our assays. This view is supported by the recent finding that the JC virus receptor is widely distributed on cells which contrasts with the cellular restriction of virus propagation [54]. Factors involved in this regulation might be cellular proteins such as puralpha, p53, or alternative splicing products of viral proteins [51,55,56]. Substitution of SV40 TAg for that of JCV inhibits the formation of an active initiation complex with murine DNA polymerase a-primase (Fig. 5), mimicking the molecular basis of SV40 host specificity. We recently showed that species specificity of SV40 DNA replication is probably the consequence of a failure of SV40 TAg to undergo a productive functional interaction with C-terminal elements of the murine p180 subunit of DNA polymerase a-primase [26,53]. Our results imply that JCV TAg is not, or is to a much lesser extent, inhibited in these interactions. SV40 and JCV TAg share 72% sequence identity with most nonhomology towards the C-termini of the proteins [31]. The cell-free in vitro JCV DNA replication system would be useful in determining which regions of SV40 TAg are responsible for its species–specific interactions with host DNA polymerase a, for instance by studying the activity of chimeric TAg polypeptides derived in part from SV40 and in part from JCV sequences. Acknowledgements We thank J. Fuchs and A. Schneider for technical assistance. This work was financially supported by the Deutsche Forschungsgemeinschaft (Na190/12 and Na190/13-1) and the EC (CT970125). The IMB is a Gottfried-Wilhelm-Leibniz-Institut and financially supported by the federal government and the Land Thu ¨ ringen. References 1. Challberg, M. & Kelly, T.J. (1989) Animal virus DNA replication. Annu. Rev. Biochem. 58, 671–717. 2. Smith, R.W.P. & Nasheuer, H.P. (2000) Control of papovaviral DNA replication. In Recent Research Developments in Virology,2 (Pandalai, S.G., ed.), pp. 67–92. Transworld Research Network, Trivandrum, India. 3. Berger, J.R. & Concha, M. (1995) Progressive multifocal leu- koencephalopathy: The evolution of a disease once considered rare. J. Neurovirol. 1, 5–18. 4. Frisque, R.J. & White, F.A. III (1992) The molecular biology of JC virus, causative agent of progressive multifocal leukoence- phalopathy. In Molecular Neurovirology (Ross, R., ed.), pp. 25– 158. Humana Press, Cliffton, NJ, USA. 5. Gallia, G.L., Houff, S.A., Major, E.O. & Khalili, K. (1997) Review: JC virus infection of lymphocytes – revisited. J. Infect Dis. 176, 1603–1609. 6. Major, E.O. & Ault, G.S. (1995) Progressive multifocal leuko- encephalopathy: clinical and laboratory observations on a viral induced demyelinating disease in the immunodeficient patient. Curr. Opin. Neurol. 8, 184–190. 7. Walker, D.L. & Frisque, R.J. (1986) The biology and molecular biology of JC virus. In The Papovaviridae, the Polyomaviruses (Salzman, N., ed.), pp. 327–377. Plenum Press, New York, USA. 8. Feigenbaum, L., Khalili, K., Major, E. & Khoury, G. (1987) Regulation of the host range of human papovavirus JCV. Proc. NatlAcad.Sci.USA84, 3695–3698. 9. Tooze, J. (1981) Molecular biology of tumor virus, part 2. DNA Tumor Viruses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. Fig. 5. Cell-free DNA replication assays in murine FM3A cell extracts depleted of DNA polymerase a-primase. TAgs (SV40 & JCV) and DNA polymerase a-primase complexes (human & murine) were tested in parallel experiments for their ability to support DNA replication of a plasmid carrying the JCV origin. Columns 2 and 3, SV40 Tag with human and murine DNA polymerase a-primase, respectively; columns 4 and 5, JCV Tag with human and murine DNA polymerase a-pri- mase, respectively. DNA polymerase (0.5 U) of the indicated DNA polymerase a-primase complexes were added. Values were corrected for background incorporation in the absence of added DNA poly- merase a-primase (see Fig. 1). Column 1, control with H4 and plasmid DNA but lacking TAg. Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2035 10. Bennett, E.R., Naujokas, M. & Hassell, J.A. (1989) Requirements for species-specific papovavirus DNA replication. J. Virol. 63, 5371–5385. 11. Ishimi, Y., Claude, A., Bullock, P. & Hurwitz, J. (1988) Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J. Biol. Chem. 263, 19723–19733. 12. Li, J.J. & Kelly, T.J. (1984) Simian virus 40 DNA replication in vitro. Proc. Natl Acad. Sci. USA 81, 6973–6977. 13. Li, J.J. & Kelly, T.J. (1985) Simian virus 40 DNA replication in vitro: Specificity of initiation and evidence for bidirectional replication. Mol. Cell. Biol. 5, 1238–1246. 14. Murakami, Y., Eki, T., Yamada, M., Prives, C. & Hurwitz, J. (1986) Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication. Proc. Natl Acad. Sci. USA 83, 6347–6351. 15. Waga, S., Bauer, G. & Stillman, B. (1994) Reconstitution of complete SV40 DNA replication with purified replication factors. J. Biol. Chem. 269, 10923–10934. 16. Waga, S. & Stillman, B. (1998) The DNA replication fork in eukaryotic cells. Annu.Rev.Biochem.67, 721–751. 17. Fanning, E. & Knippers, R. (1992) Structure and function of simian virus 40 large T antigen. Annu.Rev.Biochem.61, 55–85. 18. Hassell, J.A. & Brinton, B.T. (1996) SV40 and polyomavirus DNA replication. In DNA Replication in Eukaryotic Cells (DePamphilis, M.L., ed.), pp. 639–677. Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY, USA. 19. Eki, T., Enomoto, T., Masutani, C., Miyajima, A., Takada, R., Murakami, Y., Ohno, T., Hanaoka, F. & Ui, M. (1991) Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication. J. Virol. 65, 4874–4881. 20.Murakami,Y.,Wobbe,C.R.,Weissbach,L.,Dean,F.B.& Hurowitz,J.(1986)RoleofDNApolymerasea and DNA pri- mase in simian virus 40 DNA replication in vitro. Proc. Natl Acad. Sci. USA 83, 2869–2873. 21. Schneider, C.K., Weißhart, L.A., Guarino, I. & Dornreiter & Fanning, E. (1994) Species specific functional interactions of DNA polymerase a-primase with SV40 T antigen require SV40 origin DNA. Mol. Cell. Biol. 14, 3176–3185. 22. Bru ¨ ckner, A., Stadlbauer, F., Guarino, L.A., Brunahl, A., Schneider, C., Rehfuess, C., Prives, C., Fanning, E. & Nasheuer, H P. (1995) The mouse DNA polymerase a-primase subunit p48 mediates species-specific replication of polyoma virus DNA in vitro. Mol. Cell. Biol. 15, 1716–1724. 23. Kautz, A., Schneider, A., Weisshart, K., Geiger, C. & Nasheuer, H.P. (2001) Different regions of primase subunit p48 control mouse polyomavirus and simian virus 40 DNA replication in vitro. J. Virol. 75, 1751–1760. 24. Kautz, A., Weisshart, K., Schneider, A., Grosse, F. & Nasheuer, H.P. (2001) Amino acids 257–288 of mouse p48 control the cooperation of polyomavirus large T antigen, replication protein A, and DNA polymerase a-primase to synthesize DNA in vitro. J. Virol. 75, 8569–8578. 25. Nasheuer, H.P., Smith, R.W.P., Bauerschmidt, C., Grosse, F. & Weisshart, K. (2002) Initiation of eukaryotic DNA replication – regulation and mechanisms. Prog. Nucl. Acids Res. Mol. Biol. 72, 41–94. 26. Smith,R.W.,Steffen,C.,Grosse,F.&Nasheuer,H.P.(2002) Species specificity of simian virus 40 DNA replication in vitro requires multiple functions of human DNA polymerase a. J. Biol. Chem. 277, 20541–20548. 27. Stadlbauer, F., Voitenleitner, C., Bru ¨ ckner, A., Fanning, E. & Nasheuer, H P. (1996) Species-specific replication of simian virus 40 DNA in vitro requires the p180 subunit of human DNA polymerase a-primase. Mol. Cell. Biol. 16, 94–104. 28. Hu ¨ bscher, U., Nasheuer, H.P. & Syva ¨ oja, J. (2000) Eukaryotic DNA polymerases, a growing family. Trends Biochem. Sci. 25, 143–147. 29. Hu ¨ bscher, U., Maga, G. & Spadari, S. (2002) Eukaryotic DNA polymerases. Annu. Rev. Biochem. 71, 133–163. 30. Shcherbakova, P.V., Bebenek, K. & Kunkel, T.A. (2003) Func- tions of eukaryotic DNA polymerases. Sci. SAGE KE 2003, re3 (26 February 2003), http://sageke.sciencemag.org/cgi/content/full/ sageke;2003/8/re3 31. Frisque, R.J., Bream, G.L. & Cannell, M.T. (1984) Human polyomavirus JC virus genome. J. Virol. 51, 458–469. 32. Nesper,J.,Smith,R.W.P.,Kautz,A.R.,Sock,E.,Wegner,M., Grummt, F. & Nasheuer, H.P. (1997) A cell-free replication system for human polyomavirus JC DNA. J. Virol. 71, 7421–7428. 33. Sock, E., Wegner, M., Fortunato, E.A. & Grummt, F. (1993) Large T-antigen and sequences within the regulatory region of JC virus both contribute to the features of JC virus DNA replication. Virology 197, 537–548. 34. Stadlbauer, F., Brueckner, A., Rehfuess, C., Eckerskorn, C., Lottspeich, F., Fo ¨ rster,V.,Tseng,B.Y.&Nasheuer,H P.(1994) DNA replication in vitro by recombinant DNA polymerase a-primase. Eur. J. Biochem. 222, 781–793. 35. Stigger, E., Dean, F.B., Hurwitz, J. & Lee, S.H. (1994) Recon- stitution of functional human single-stranded DNA-binding pro- tein from individual subunits expressed by recombinant baculoviruses. Proc. Natl Acad. Sci. USA 91, 579–583. 36. Tanaka, S., Hu, S Z., Wang, T.S F. & Korn, D. (1982) Pre- paration and preliminary characterization of monoclonal anti- bodies against human DNA polymerase a. J. Biol. Chem. 257, 8386–8390. 37. Henricksen, L.A., Umbricht, C.B. & Wold, M.S. (1994) Recombinant replication protein A: expression, complex formation, and functional characterization. J. Biol. Chem. 269, 11121–11132. 38. Nasheuer, H P., von Winkler, D., Schneider, C., Dornreiter, I., Gilbert, I. & Fanning, E. (1992) Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. Chromosoma. 102, S52–S59. 39. Søe,K.,Dianov,G.,Nasheuer,H.P.,Bohr,V.A.,Grosse,F.& Stevnsner, T. (2001) A human topoisomerase I cleavage complex is recognized by an additional human topisomerase I molecule in vitro. Nucleic Acids Res. 29, 3195–3203. 40. Harlow,E.&Lane,D.P.(1988)Antibodies: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 41. Bradford, M. (1976) A rapid and sensitive methode for the quantitation of microgram quantities of protein utilicing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 42. Nasheuer, H P. & Grosse, F. (1987) Immunoaffinity-purified DNA polymerase a displays novel properties. Biochemistry 26, 8458–8466. 43. Nasheuer, H P. & Grosse, F. (1988) DNA polymerase a-primase from calf thymus. Determination of the polypeptide responsible for primase activity. J. Biol. Chem. 263, 8981–8988. 44. Matsumoto, T., Eki, T. & Hurwitz, J. (1990) Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. Proc. Natl Acad. Sci. USA 87, 9712–9716. 45. Weisshart, K., Fo ¨ rster, H., Kremmer, E., Schlott, B., Grosse, F. & Nasheuer, H.P. (2000) Protein–protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. J. Biol. Chem. 275, 17328–17337. 46. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 2036 R. W. P. Smith and H. P. Nasheuer (Eur. J. Biochem. 270) Ó FEBS 2003 47. Lynch, K.J. & Frisque, R.J. (1990) Identification of critical ele- ments within the JC virus DNA replication origin. J. Virol. 64, 5812–5822. 48. Cheng, L.Z., Workman, J.L., Kingston, R.E. & Kelly, T.J. (1992) Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16. Proc. Natl Acad. Sci. USA 89, 589–593. 49. Cheng, L. & Kelly, T.J. (1989) Transcriptional activator nuclear factor I stimulates the replication of SV40 minichromosomes in vivo and in vitro. Cell. 59, 541–551. 50. Sock, E., Wegner, M. & Grummt, F. (1991) DNA replication of human polyomavirus JC is stimulated by NF-1 in vivo. Virology 182, 298–308. 51. Staib, C., Pesch, J., Gerwig, R., Gerber, J.K., Brehm, U., Stangl, A. & Grummt, F. (1996) p53 inhibits JC virus DNA replication in vivo and interacts with JC virus large T-antigen. Virology. 219, 237–246. 52. Vacante, D.A., Traub, R. & Major, E.O. (1989) Extension of JC virus host range to monkey cells by insertion of a simian virus 40 enhancer into the JC virus regulatory region. Virology 170, 353– 361. 53. Smith, R. & Nasheuer, H. (2002) Control of complex formation of DNA polymerase alpha-primase and cell-free DNA replication by the C-terminal amino acids of the largest subunit p180. FEBS Lett. 527, 143–146. 54. Suzuki, S., Sawa, H., Komagome, R., Orba, Y., Yamada, M., Okada, Y., Ishida, Y., Nishihara, H., Tanaka, S. & Nagashima, K. (2001) Broad distribution of the JC virus receptor contrasts with a marked cellular restriction of virus replication. Virology 286, 100–112. 55. Daniel, D.C., Wortman, M.J., Schiller, R.J., Liu, H., Gan, L., Mellen, J.S., Chang, C.F., Gallia, G.L., Rappaport, J., Khalili, K. & Johnson, E.M. (2001) Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Puralpha on DNA replication initiated at the JC virus origin. J. Gen. Virol. 82, 1543–1553. 56. Prins, C. & Frisque, R.J. (2001) JC virus T¢ proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells. J. Neurovirol. 7, 250–264. Ó FEBS 2003 Initiation of DNA replication (Eur. J. Biochem. 270) 2037 . Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase a-primase Richard W. P. Smith 1, * and Heinz-Peter Nasheuer 1,2 1 Abteilung. polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase a-primase of either human or murine origin indicating that the mechanism of its strict