INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Minute virus of mice (MVM), an autonomous parvovirus, is a small nonenveloped spherical particle with a single-stranded linear DNA as a genome. The 5.1-kb viral DNA codes for two structural (VP) and at least four nonstructural (NS) proteins, of which only the large (83-kDa), mainly nuclear phosphoprotein NS1 is required for progeny virus production in all cell types (for reviews, see references 19 and 33). This multifunctional protein is involved in many processes during the virus cycle. It controls promoter activities (42), causes alteration of the cell physiology (1, 7, 39) and morphology (7, 12), and is the initiator protein for viral DNA replication (17, 18, 36). Thus, it was suggested that the multiple, very diverging NS1 activities are regulated by posttranslational modifications.

After conversion of the single-stranded, linear genome to a covalently closed circular DNA in absence of viral proteins (3), DNA amplification involves the formation of monomeric and concatemeric duplex DNA intermediates that are produced by an unidirectional, single-strand semiconservative DNA replication (for a review, see reference 23). This modified rolling hairpin replication closely resembles the rolling circle replication (RCR) mechanism described for single-stranded plasmids, bacteriophages, and geminiviruses (for a review, see reference 30). In fact, it was shown that plasmids containing left- or right-end MVM origins were suitable substrates for NS1-initiated RCR, in the presence of cellular extracts and deoxynucleoside triphosphates (dNTPs) (22).

Initiation of RCR occurs by site- and strand-specific nicking of origin sequences by the replicator protein NS1 generating the free 3'-hydroxyls necessary for DNA polymerase activities. During this reaction, NS1 becomes covalently attached to the newly generated 5' end and remains connected to replication intermediates as well as virion DNA in vitro (17, 18) and in vivo (20, 21). Site- and strand-specific nicking of both the left- and right-end MVM origins has been shown to require cellular accessory proteins under physiological conditions. While at the left origin the newly described parvovirus initiation factor PIF assists NS1 for nickase activity (9-11), members of the high-mobility-group (HMG) protein family serve for activation of the viral protein at the right-end origin (16, 24).

The minimal origin sequences at the left-end telomere have been mapped and consist of approximately 50 bp within the Y-shaped terminal structure. The left-end origin comprises the binding sites for PIF and NS1, an A/T-rich spacer, and the NS1 nick site (10, 15, 22). Within the terminal hairpin structure, there is a mismatched "bubble" sequence between the binding sites for PIF and NS1, in which a triplet 5'-GAA-3' on one strand opposes a dinucleotide 5'-GA-3'. These tri- and dinucleotide sequences distinguish the otherwise identical origins present in the junction of head-to-head dimer replication intermediates and determine which of the origins serves as a substrate for NS1-dependent nicking. Under physiological conditions, nicking of NS1 in concert with PIF occurred only at origins containing the dinucleotide bubble sequences, whereas a trinucleotide between the NS1 and PIF binding sites abolished this reaction (9). This asymmetric initiation of replication is thought to preserve the flip orientation within the left-end palindrome of MVM virion DNA (for details, see reference 23).

Characterization of conserved motifs among replicator proteins has shown similarities of NS1 with proteins involved in RCR of single-stranded plasmids and bacteriophages (29). Thus, two hallmarks of endonucleases, a metal coordination site (amino acids 126-WHCHVLIGG-134) and a consensus active-site tyrosine (210-YFLTK), could be identified in MVM NS1 (36). A third NS1 motif required for nicking, 399-GPASTGKSIIAQAI-411, was identified as a nucleotide-binding site and is part of the ATPase domain responsible for energy supply during DNA-unwinding reactions (34, 36). In addition, this motif is involved in the control of NS1 self-assembly (38), a prerequisite for both site-specific DNA binding (15) and helicase function (36, 41). Due to its intrinsic helicase activity, NS1 should be able to achieve the double-stranded viral origin unwinding necessary for site-specific nicking without the help of exogenous cellular helicases as recently demonstrated for the adeno-associated virus (AAV) Rep protein (5).

NS1-initiated RCR is dependent on phosphorylation of the viral polypeptide (37). This is likely to be due, at least in part, to the regulation of NS1 helicase function by protein kinase C (PKC) (26), yet the role of phosphorylation in the initial nicking reaction remained elusive. This question was worth investigating, given the complex evolution of the NS1 phosphorylation pattern in the course of a viral infection (13) and the differences between the biochemical profiles of native and dephosphorylated NS1 (34), both arguing for control of the different NS1 functions by distinct phosphorylation and dephosphorylation events. To investigate the role of phosphorylation in NS1 regulation in the initiation of viral DNA replication, i.e., DNA nicking at the left-end origin, we analyzed previously described PKC phosphorylation site mutants that mimic partially phosphorylated or dephosphorylated NS1 (12). Purified wild-type and mutant NS1 polypeptides derived from recombinant vaccinia virus expression in HeLa cells were characterized for the ability to drive site- and strand-specific nicking of the MVM left-end origin under both physiological and nonstringent conditions, as well as for their intrinsic site-specific interactions with the origin and enzymatic (helicase and ATPase) activities. The data presented indicate that T403, a PKC phosphorylation site targeted in vivo (12), alters the affinity of NS1 for its DNA recognition motif, an activity required not only for origin recognition but also for trans activation of the P38 promoter. Furthermore, specific activation of NS1 for viral DNA replication occurs by atypical PKC phosphorylation at T435 and S473, regulating nicking on the level of origin unwinding.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Viruses and cells. Recombinant vaccinia viruses were propagated in monolayer cultures of BSC-40 cells and purified over a sucrose cushion (31), except for the release of the virus from cells by three cycles of freezing and thawing instead of sonication. The vaccinia virus vTF7-3 has been described by Fuerst and coworkers (27); the procedures for construction of wild-type and mutant His₆-tagged NS1 as well as PKC have been described previously (12, 26, 36). BSC-40 cells were grown as monolayer cultures in Dulbecco's modified Eagle medium containing 10% fetal calf serum. HeLa-S3 cells were grown in suspension using Spinner bottles in Joklik's medium containing 5% fetal calf serum.

NS1 mutants. Mutants Y210F and Y197F, the former harboring an amino acid substitution for the linkage tyrosine and the latter being impaired in site-specific interaction with the cognate DNA recognition motif, were described in detail by Nüesch and coworkers (36). The PKC phosphorylation site mutant S473A was described by Dettwiler et al. (26); construction and characterization of the additional PKC consensus phosphorylation site mutants (S283A, T363A, T403A, T435A, and T463A) were described by Corbau and coworkers (12).

Plasmids and DNA substrates. Plasmids containing the active and inactive left-end origins (pL1-2TC and pL1-2GAA, respectively) have been described by Cotmore and Tattersall (22). Double-stranded minimal origins were obtained as 95-bp EcoRI fragments and radiolabeled by fill-in reactions using Sequenase (Amersham/Pharmacia). Oligonucleotides Ori-ssNickTC and Ori-ssNickGAA, generating duplex PIF and NS1 binding sites while extruding the consensus nicking sequence as a single-strand bubble (Fig. 7A), were synthesized by MWG-Biotech AG (Ebersberg, Germany), heated for 10 min at 100°C, and then slowly cooled to allow annealing of complementary sequences. Radioactive labeling was performed by fill-in reactions of the 5' overhang using Sequenase.

Production and purification of recombinant proteins by means of vaccinia virus expression. PKC and NS1 polypeptides were produced from recombinant vaccinia viruses in suspension cultures of HeLa-S3 cells (34), using vTF7-3 together with the appropriate recombinant vaccinia viruses (15 PFU of each per cell) containing the NS1 or PKC genes, respectively, under the control of the T7 promoter. Infected cultures were harvested 18 h postinfection, whole (PKC) or nuclear (NS1) extracts were prepared, and His-tagged recombinant proteins were purified using Ni²⁺-NTA agarose (Qiagen) columns (36). Protein preparations were analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining and tested for distinct biochemical properties.

Nicking assays. NS1-mediated site-specific nicking and the resultant covalent attachment of NS1 to the 5' end of the nicked product were analyzed as described previously (9, 36). Approximately 100 ng of purified NS1 (as determined by SDS-PAGE and Coomassie blue staining) and 50 ng of baculovirus-produced PIF heterodimer p76-p96 (J. Christensen, S. F. Cotmore, and P. Tattersall, submitted for publication) were incubated with 100 ng of HaeIII-cleaved plasmid pUC19 as nonspecific competitor DNA, 100 mM NaCl to mimic physiological conditions, 1 ng of ³²P-labeled substrate DNA, and 3 mM ATP in 20 mM HEPES-KOH (pH 7.5)-5 mM MgCl₂-5 mM KCl-1 mM dithiothreitol (DTT) for 1 h at 37°C. Substrates consisted of the purified 95-bp EcoRI fragments of pL1-2TC or pL1-2GAA, Ori-NickTC/GAA, or self-annealed partially single-stranded oligonucleotides and were 3' end labeled by fill-in reactions using Sequenase and [-³²P]dATP. For nicking assays under nonstringent conditions (without requirement for cofactors), reactions were performed in the absence of NaCl, competitor DNA, and PIF. When indicated, ATP was replaced with the same amount of the nonhydrolyzable -S-ATP analogue to measure NS1 nickase activity in absence of external energy sources. Reactions were stopped by adding 0.1% SDS and 2.5 mM EDTA and analyzed after heat denaturation for 5 min at 100°C by 7% PAGE in the presence of 0.1% SDS. In addition, immunoprecipitations were performed using the NS1-specific antiserum NS_N (36), and the immune complexes were analyzed after proteinase K digestion and phenol-chloroform extraction by 7% PAGE in the presence of SDS.

Site-specific binding of NS1 to the MVM 3' origin of replication. Site-specific binding assays using NS1 and the MVM left-end origin were performed as described elsewhere (15). Briefly, plasmid pL1-2TC, which contains the minimal active left-end replication origin (22), was digested with restriction enzymes Sau3AI and NarI, and the DNA fragments were 3' end labeled by filling in with Sequenase, [-³²P]dGTP, and unlabeled dATP, dCTP, and dTTP. Binding assays were carried out in 100 µl of 20 mM Tris-HCl (pH 8.0)-10% glycerol-1% NP-40-5 mM DTT-100 mM NaCl supplemented with labeled, restriction-digested pL1-2TC DNA, 500 ng of oligod(I-C), 0.5 mM -S-ATP, and 50 ng of purified NS1. After interactions were allowed to take place for 30 min on ice, 2 µl of antiserum NS_N was added and incubation was continued for another hour. Immune complexes were precipitated with protein A-Sepharose, deproteinized, and analyzed by nondenaturing 7% PAGE in the presence of 0.1% SDS.

Helicase assay. Helicase assays were carried out as described elsewhere (36). M13-VAR used as substrate was prepared by annealing the M13rev primer (Amersham) to M13 single-stranded DNA followed by extension for 5 min at room temperature in the presence of T7 polymerase and dNTPs, including [-³²P]dATP. ³²P-labeled fragments of various lengths were obtained by addition of dideoxy-GTP and further incubation for 20 min. Purified NS1 was incubated with 20 ng of substrate for 40 min in the presence of 3 mM ATP. The reactions were stopped by addition of SDS and EDTA, and the products were analyzed by 7% nondenaturing PAGE in the presence of 0.1% SDS.

ATPase assay. As previously described (34), NS1 used for ATPase assays was further purified by centrifugation through a 1.5-ml glycerol gradient (15 to 40%) banding NS1 in the middle of the gradient. ATPase activities were measured in the individual fractions matched for their NS1 contents. Titrations between 2 to 50 ng of NS1 protein were performed in 20 mM Tris-HCl (pH 7.5)-100 mM NaCl-5 mM MgCl₂-5 mM DTT-0.01% NP-40-0.1 µg of M13 single-stranded DNA with 30 µM ATP and 0.5 µCi of [-³²P]ATP (3,000 Ci/mmol; Amersham) for 20 min at room temperature. The reactions were terminated by addition of 100 µl of 7.5% (wt/vol) acid-washed charcoal in 50 mM HCl-5 mM H₃PO₄, and free phosphate was separated from unreacted charcoal-bound ATP by centrifugation. A 50-µl sample of the ³²P_i-containing supernatant was analyzed by scintillation counting.

Phosphopeptide analysis of wild-type and mutant NS1 phosphorylated by atypical PKC. Purified dephosphorylated NS1 (500 ng) was subjected to in vitro phosphorylation using 50 ng of recombinant PKC. Reactions were performed with 30 µCi of [-³²P]ATP in the presence of 1 µg of L--phosphatidyl-L-serine per ml for 40 min at 37°C as described elsewhere (26, 34). The reaction was stopped by adding SDS and EDTA and heating at 70°C for 30 min. ³²P-labeled NS1 was purified by SDS-PAGE, blotted on a polyvinylidene difluoride membrane, and digested with chymotrypsin (Boehringer Mannheim) for 18 h at 37°C. Phosphopeptides were recovered and analyzed by two-dimensional thin-layer chromatography and electrophoresis as described elsewhere (34).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

PKC activates dephosphorylated NS1 for site- and strand-specific nicking at the left-end origin. Previous investigations have shown that NS1-dependent RCR initiated at the left-end MVM origin is regulated by PKC phosphorylation under in vitro conditions (37). This dependency of NS1 replicative functions on phosphorylation is consistent with the altered biochemical profile of dephosphorylated NS1^O compared with the native NS1^P protein (34). NS1 is thought to drive RCR both by initiating this reaction through site- and strand-specific nicking of the viral origin (which produces a free 3'-hydroxyl serving as a primer for DNA polymerases) and by unwinding DNA in front of the replication fork at the subsequent stage of strand displacement synthesis. This prompted us to first determine whether NS1 phosphorylation plays a role in the initiation (nicking) step of MVM DNA replication. To this end, we performed nicking reactions under nearly physiological salt conditions, using purified native or dephosphorylated NS1, together with recombinant parvovirus initiation factor PIF (11) and the cofactor ATP. As shown in Fig. 1, native NS1^P was able to recognize the left-end origin and to drive the site- and strand-specific endonuclease reaction resulting in covalent attachment of NS1 to the 5' end of the nicked DNA, while its dephosphorylated counterpart NS1^O was strongly impaired in this reaction. Moreover, supplementing the reaction with the activated recombinant lambda isoform of PKC was able to restore the nicking function of NS1^O, demonstrating that the capacity of NS1 for initiating MVM DNA replication is controlled by PKC phosphorylation.

View larger version (23K): [in this window] [in a new window]   FIG. 1.   Nicking of the MVM left-end origin by NS1. (A) Diagram of substrate and denatured products of the nicking reaction. Labeled 3' ends are marked by asterisks; the gray area delineates the minimal left-end origin. PIF (hatched section) and NS1 (cross-hatched section) binding sites, which are separated by two bases (TC/GA) corresponding to the doublet bubble sequence, as well as the nick site (arrowhead) are indicated. The circled NS1 depicts the covalently linked NS1 at the 5' end of the nicked strand. The dashed line indicates the predicted unlabeled 42-nucleotide (nt) single-stranded product DNA. (B) NS1 nickase activity was determined using a 32P-end-labeled fragment containing the left-end origin of replication as a substrate in the presence of physiological concentrations of NaCl, competitor DNA, and the cellular cofactor PIF. Reaction products were heat denatured in the presence of 0.1% SDS and analyzed for cleavage and covalent attachment of NS1 by 7% PAGE in the presence of SDS. Input, migration of free substrate and uncleaved upper strand, Nicked, the 3'-end-labeled reaction products identified due to a mobility shift caused by the covalently attached NS1 proteins; Y210F, NS1 linkage tyrosine mutant serving as a negative control; NS1P, native purified NS1 derived from vaccinia virus expression in HeLa cells; NS1O, dephosphorylated NS1; NS1O + PKC, nicking reaction driven by NS1O in the presence of activated recombinant PKC.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Effect of mutagenesis at consensus PKC phosphorylation sites on NS1 nicking activity. (A) Domain structure of NS1. The consensus PKC phosphorylation sites chosen for mutagenesis are indicated by their amino acid numbers. The spotted bar represents the amino acid 256-672 fragment of NS1 (designated NS1-He) containing the majority of NS1phosphorylation sites (12). The common N terminus of NS1 and NS2 is shown as a dotted box. The NTP-binding site, oligomerization region, nuclear localization signal (NLS), and nicking motifs (metal coordination site and linkage tyrosine) are indicated. (B) NS1-dependent nicking reactions were performed under nearly physiological salt concentrations, in the presence of ATP, competitor DNA, and PIF. The 3'-end-labeled 95-bp EcoRI fragment of pL1-2TC served as a substrate. Y210F (the linkage tyrosine mutant) and Y197F (a mutant deficient for site-specific recognition of the origin) served as negative controls. Nicking and covalent attachment of NS1 were analyzed by 7% PAGE in the presence of SDS either directly after heat denaturation (B) or after immunoprecipitation with NSN, deproteinization, and heat denaturation (C). Migrations of substrate DNA, containing residual substrate and unnicked positive strand (Input; 95 nucleotides), and nicked product (53 nucleotides) are indicated. Note that the input DNA is not quantitatively immunoprecipitated and/or is only partially removed by the washing procedure. wt, wild type.

Determination of NS1 activities regulated through PKC phosphorylation sites. (i) Affinity of NS1 to its cognate recognition motif. Origin recognition is an essential feature of replicator proteins. To investigate whether the interaction of NS1 with its cognate recognition motif [ACCA]_2-3 is modulated through phosphorylation, we performed site-specific DNA binding assays as previously described (15) using plasmid pL1-2TC containing the active MVM left-end origin as a substrate. Purified wild-type and mutant NS1 polypeptides were incubated with ³²P-end-labeled pL1-2TC Sau3A/NarI fragments in the presence of -S-ATP. NS1-DNA complexes were immunoprecipitated with the antiserum NS_N, digested with proteinase K, and analyzed by nondenaturing PAGE in the presence of 0.1% SDS. The linkage tyrosine mutant Y210F served as a negative control. As shown in Fig. 3, all PKC site mutants tested were able to interact specifically with the DNA fragment containing the left-end origin of replication, yet the phosphorylation mutants differed in affinity to the [ACCA]_2-3 element, as apparent from the significantly reduced (T403A) or enhanced (S473A and in particular T363A) extent of binding in comparison with wild-type NS1. Moreover, none of the mutants under investigation was deficient for homo-oligomerization (12), a prerequisite for site-specific DNA binding (15). Thus, in agreement with the previously reported difference between native and dephosphorylated NS1 with regard to DNA binding (34), the affinity of NS1 for its cognate DNA recognition motif may indeed constitute one of the levels at which NS1 functioning is regulated by phosphorylation. However, the significant capacity of all four nicking-deficient NS1 mutants for DNA binding suggested that other steps in the nicking process might be modulated by phosphorylation.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Site-specific binding of wild-type and mutant NS1 to the left-end origin. The ability of wild-type (wt) and mutant NS1 proteins to interact site specifically with the left-end origin was analyzed using 3'-end-labeled, Sau3AI/NarI-digested pL1-2TC as substrate (Input). The labeled substrate was incubated to interaction with NS1 in the presence of nonhydrolyzable -S-ATP and competitor oligo(dI-dC). NS1-bound fragments were immunoprecipitated with NSN, deproteinized, and analyzed by 7% PAGE in the presence of SDS. Y210F served as a negative control. The migration of the origin-containing fragment or1 is indicated.

(ii)Double-stranded origin nicking. To compare the intrinsic nicking activities of wild-type and mutant NS1 proteins, low-stringency conditions were used to avoid the interference of above-mentioned variations in their affinities to the cognate DNA recognition motif. It has indeed been shown that under these non-physiological conditions, NS1 is able to nick the origin siteand strandspecifically, independently of accessory factors and in the absence of its ability to interact site specifically with the ACCA motif. This ATP-dependent reaction takes place in the absence of the cofactor PIF and fails to distinguish between the active and inactive left-end origins of replication. However, the efficiency of the PIF-independent reaction is at least 50-fold less than that of a reaction carried out in the presence of this cellular cofactor (36; J. P. F. Nüesch, unpublished observations). As described above, the 3'-end-labeled EcoRI fragment of pL1-2TC containing the left-end origin was incubated with wild-type or mutant purified NS1 proteins, in the presence of ATP but in the absence of salt and competitor DNA. NS1-attached DNA was then immunoprecipitated with NS_N antiserum in the presence of SDS and analyzed by SDS-PAGE after deproteinization and heat denaturation. The specificity of the reaction was ascertained by using NS1:Y210F (the linkage tyrosine mutant) as a negative control or by substituting nonhydrolyzable analogue -S-ATP for ATP. As shown in Fig. 4, mutants T363A, T435A, and S473A were still unable to nick the origin under relaxed conditions, whereas T403A was competent for this reaction, although to a lesser extent than the wild-type protein. These data point to T363, T435, and/or S473 as potential regulatory sites for the intrinsic nicking activity of NS1. On the other hand, the failure of T403A to nick the origin under physiological conditions (Fig. 2B) indicates that its deficiency in site-specific recognition of the ACCA motif (Fig. 3) might account for its lack of nickase activity in the presence of competitor DNA and physiological salt concentrations. Moreover, it confirms that NS1 is targeting the origin of replication and suggests that the cooperation with PIF (Christensen et al., submitted) occurs in a ternary complex with the origin DNA rather than in solution. It is worth noting that this mutant is also defective in trans activation of the P38 promoter driving the capsid genes (12), which shares with origin nicking the dependence on NS1 association with its cognate motif (8).

View larger version (40K): [in this window] [in a new window]   FIG. 4.   NS1-driven cleavage of the left-end origin in the absence of site-specific DNA binding. NS1-dependent nicking was investigated at low-salt conditions in absence of both PIF and nonspecific competitor DNA, allowing site-specific nicking to occur in the presence of ATP due to the intrinsic affinity of NS1 for DNA (38). The 3'-end-labeled 95-bp EcoRI fragment of pL1-2TC was used as a substrate. The NS1 mutant Y210F served as a negative control. In the sample labeled -S-ATP, the analogue was substituted for ATP. Site-specific nicking and covalent attachment of NS1 were analyzed by 7% PAGE in the presence of SDS after immunoprecipitation with NSN, deproteinization and heat denaturation. Migration of the input size DNA and nicked product are indicated. wt, wild type.

(iii) DNA unwinding. Cleavage and trans esterification reactions by replicator proteins are thought to occur at the single-stranded DNA level and in the absence of ATP consumption (5, 6). Therefore, partial unwinding of the MVM origin may be a prerequisite for NS1 nicking. Besides its involvement in nicking, NS1 may contribute to origin unwinding through its helicase activity. Thus, the deficiency of T363A, T435A, and S473A for nicking could be a result from their inability to unwind and/or cleave the origin. The former possibility would be consistent with recent reports showing that the processive helicase activity of NS1 is strongly dependent on phosphorylation (34) and is regulated by atypical PKCs (26). This prompted us to measure the helicase activity of our PKC phosphorylation site mutants in standard assays using M13-VAR (a circular single-stranded DNA with annealed ³²P-labeled fragments of various lengths) as a template. This substrate was incubated with increasing amounts (3 to 300 ng/reaction) of wild-type and mutant NS1 proteins in the presence of ATP, and the unwinding of radiolabeled fragments was determined by nondenaturing PAGE in the presence of 0.1% SDS. S473A, previously shown to be deficient for helicase activity (26), served as a negative control. As illustrated in Fig. 5, T363A and T435A were severely impaired in DNA unwinding, with a residual activity at least 100-fold lower than that of the wild-type protein. This suggested that the incompetence of these mutants to nick the MVM origin (Fig. 2B and 4) might be, at least in part, due to their defect in origin unwinding (see below). In contrast, the amino acid substitution T403A, located within the nucleotide-binding site, had little effect on the processive helicase activity of NS1. This result indicated that the above-mentioned impairment of mutant T403A in site-specific DNA binding (Fig. 3) resulted from the loss of a distinct function and not from a general inactivation of the protein. In addition, it confirmed that the nicking-negative phenotype of T403A (Fig. 2B) could be attributed for the most part to its defect in origin recognition rather than subsequent enzymatic reactions (Fig. 4).

View larger version (84K): [in this window] [in a new window]   FIG. 5.   Helicase activity of wild-type and mutant NS1. Unwinding activity of serial dilutions of wild-type and mutant NS1 proteins (200, 20, and 2 ng/sample) was investigated in standard helicase assays using M13-VAR as a substrate in the presence of 2 mM ATP. The reaction products were analyzed by 7% PAGE in the presence of 0.1% SDS. Lanes 1 and 2, native (NAT) and heat-denatured (DEN) input substrate; lane 3, NS1:S473A serving as a negative control; lanes 4 to 6, wild-type NS1 (NS1 wt); lanes 7 to 9, NS1:T363A; lanes 10 to 12, NS1:T435A; lanes 13 to 15, NS1:T403A.

(iv) ATPase activity. The loss of helicase function upon dephosphorylation of NS1 was suggested to be at least in part the result of a reduced ATPase activity (34). Therefore, we determined the ATPase activities of wild-type NS1 and the helicase-negative mutants T363A, T435A, and S473A. To ensure the absence of endogenous cellular ATPases in the assay, we further fractionated the Ni²⁺ affinity-purified NS1 proteins through glycerol gradients and analyzed consecutive fractions according to their NS1 concentration as previously reported (34). K405R, an NTP-binding site mutant, served as a negative control. The results of multiple independent experiments are summarized in Fig. 6, showing the relative activities of mutants versus wild-type NS1 (100%). All of the NS1 phosphorylation site mutants under investigation had significantly reduced ATPase activity compared to wild-type NS1. It should be stated, however, that this reduction was limited (50 to 85% residual activity) and unlikely to account on its own for the drastic impairment of these mutants in the nicking and helicase functions (Fig. 2B, 4, and 5).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Effect of mutagenesis at consensus PCK phosphorylation sites on ATPase activity of NS1. Release of 32Pi was determined by scintillation counting after incubation of [-32P]ATP with wild-type (wt) or mutant NS1 proteins. Average values from multiple assays (each using different NS1 fractions from glycerol gradients) are shown with their standard deviation bars. The NS1 mutant K405R served as a negative control. Data are expressed relative to the ATPase activity of native wild-type NS1.

Cleavage and trans esterification of partially single-strand templates do not require ATP consumption and are independent of NS1 helicase activity. Regulation of simian virus 40 (SV40) DNA replication has been shown to involve DNA-unwinding activity and the phosphorylation of large T antigen (LT). Given the similarities of NS1 and SV40 LT, the question arose of whether the helicase-negative phenotype of the NS1 phosphorylation mutants T363A, T435A, and S473A was responsible for their inability to initiate parvovirus DNA replication. To test this hypothesis, we determined whether these mutants became competent for nicking under conditions where the nick site was kept in a denatured (i.e., single-stranded) configuration. Partially single-stranded nicking substrates Ori-ssNickTC and Ori-ssNickGAA (corresponding to active- and inactive left-end origins, respectively) were designed such that the PIF and NS1 binding sites were present as double-stranded DNA, while the nicking consensus sequence was extruded in a loop structure (Fig. 7A). In a first step, we determined the cofactor requirements for the cleavage of ssNick substrates by wild-type NS1 under physiological conditions (100 mM NaCl, competitor DNA) in comparison to previously characterized templates containing the nick site in the duplex configuration (dsNick) (9). The linkage tyrosine mutant Y210F served as a negative control. As shown in Fig. 7B, NS1 requires both ATP and the cellular accessory protein PIF to achieve nicking and covalent attachment at a dsNick origin. No nicking occurred when ATP was replaced by nonhydrolyzable -S-ATP, PIF was omitted from the reaction, or the inactive (G) origin (triplet-bubble sequence) was used as a template. In contrast, nicking at the partially single-stranded template occurred independent of the presence or absence of PIF, did not require hydrolyzable ATP, and did not distinguish between the active TC origin and the inactive GAA origin. NS1 requires ATP binding for oligomerization (38) and consequently site-specific DNA binding (15); thus, we were not able to perform the nicking reactions in absence of trinucleotides. Given the consumption of ATP for DNA unwinding (36), these results demonstrate that the ssNick origin can be cleaved in the absence of further denaturation and that the NS1 cleavage and trans esterification reactions do not require external energy sources, as recently reported for the related Rep68 protein of AAV (5, 6). Furthermore, our data show that unwinding of the dsNick origin is a prerequisite for its subsequent nicking by NS1. In addition, the dispensability of PIF for the cleavage of ssNick origins suggests that through its cooperative binding with NS1 (Christensen et al., submitted), PIF directs NS1-dependent unwinding to the nick site, thereby controlling the asymmetry of the resolution of head-to-head dimers (18) by activating the appropriate origin for replication initiation (22).

View larger version (56K): [in this window] [in a new window]   FIG. 7.   Effect of extrusion of the nick site in a single-stranded loop on its NS1-driven cleavage. (A) Schematic representation of the partially single-stranded Ori-ssNickTC/GAA substrates. Binding sites for PIF and NS1 (boxes), the consensus nick site (arrow), and the TC/GAA bubble sequence are indicated. (B) Determination of cofactor requirements for the NS1-dependent cleavage of the genuine (dsNickOri; lanes 1 to 5) and partially single-stranded (ssNickOri; lanes 6 to 14) left-end origins. Reactions were carried out in the presence of physiological salt concentration and competitor DNA, i.e., under conditions involving the site-specific interaction of NS1 with its cognate DNA recognition motif. ATP or nonhydrolyzable -S-ATP, the cofactor PIF, and either wild-type (wt) or mutant (Y210F; negative control) NS1 were added as indicated. Substrates T and G correspond to the dinucleotide (TC) and trinucleotide (GAA) versions of the bubble and to the active and inactive forms of the origin, respectively.

View larger version (52K): [in this window] [in a new window]   FIG. 8.   Capacity of NS1 phosphorylation site mutants for specific cleavage of the partially single-stranded left-end origin. The 3'-end-labeled Ori-ssNickTC substrate was incubated with NS1 in the presence of physiological salt concentration, competitor DNA, PIF, and nonhydrolyzable -S-ATP. Mutants Y210F and Y197F served as negative controls; wild-type (wt) NS1 served as a positive control. Nicking and covalent attachment of NS1 were analyzed by 7% PAGE in the presence of SDS, either after heat denaturation (A) or after immunoprecipitation with NSN, deproteinization, and heat denaturation (B) Migrations of input DNA and nicked product are indicated.

Determination of atypical PKC phosphorylation sites in NS1. The loss of nicking activity observed upon dephosphorylation of NS1 could be restored by addition of recombinant atypical PKC (Fig. 1B), suggesting that in contrast to RCR, no additional kinases are required to activate the polypeptide for the nicking reaction (37). This led us to speculate that the nicking and/or helicase deficiency of above-mentioned consensus phosphorylation site mutants might be attributed to their lack of phosphorylation by atypical PKCs at these sites. To substantiate this possibility, we determined whether besides the previously determined PKC phosphorylation site S473 (26), the NS1 residues T363 and T435 were targets for atypical PKCs. To test this possibility, in vitro kinase assays were performed using recombinant PKC and dephosphorylated wild-type or mutant NS1 proteins as substrates. ³²P-labeled NS1 was purified by SDS-PAGE, digested with chymotrypsin, and analyzed for its phosphopeptide pattern by two-dimensional electrophoresis-chromatography. As illustrated in Fig. 9, T435A and S473A lacked each a distinct phosphopeptide, strongly suggesting that the respective threonine and serine residues serve as targets for PKC. In contrast, T363A (Fig. 9) and T403A (data not shown) showed no consistent difference in their chymotryptic phosphopeptide maps compared to wild-type NS1, indicating that these consensus PKC phosphorylation sites in NS1 are not target sites for PKC. Altogether, these results indicated that T435 and S473, which have been shown to be targets for phosphorylation in vivo (12, 26), serve as sites for regulation of DNA-unwinding functions by atypical PKC.

View larger version (86K): [in this window] [in a new window]   FIG. 9.   Characterization of atypical PKC phosphorylation of NS1 by comparative phosphopeptide analysis. Wild-type (wt) NS1 and indicated phosphorylation site mutants were first dephosphorylated, then incubated with activated recombinant PKC in the presence of [-32P]ATP, purified by SDS-PAGE, and digested with chymotrypsin. The resulting phosphopeptides were analyzed by two-dimensional electrophoresis-chromatography. Arrows point to phosphopeptides that are characteristically missing in corresponding mutant NS1 proteins. Since S473A is a poor substrate for PKC in vitro, the corresponding autoradiograph was overexposed to visualize the presence of the residual PKC phosphorylation sites in NS1.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Viral DNA replication initiated at the MVM left-end origin has been shown to be regulated by NS1 phosphorylation through members of the PKC family (26, 37). This regulation was further investigated in the present work by analyzing the initial step, i.e., the site- and strand-specific nicking of the origin, which generates the free 3'-hydroxyl necessary for DNA polymerase activity. Two NS1 residues (T435 and S473) previously shown to become phosphorylated in vivo (12, 26) were found to be targets for atypical PKC in vitro. The substitution of alanines for either of these residues dramatically impaired NS1 nickase activity. Yet the NS1 mutants T435A and S473A were not deficient for site-specific interaction with the NS1 cognate recognition motif or for their intrinsic trans esterification activity, since they were capable of cleaving the origin under physiological conditions in a strand- and site-specific fashion, provided that the nick site was extruded in a single-stranded loop. Altogether, these data showed that T435A and S473A did not undergo an overall inactivation caused by their amino acid substitutions and supported the assumption that the distinct phenotypes of these mutants were due to their lack of phosphorylation at the target residues. However, a phosphorylation-independent correlation cannot be excluded at present. Apart from this restriction, both mutants pointed to DNA unwinding as a regulated NS1 function under control of atypical PKC phosphorylation. In addition, by dissecting the individual steps of the NS1-driven nicking process, we demonstrated that ATP hydrolysis was required as a source of energy for partial unwinding of the origin but was dispensable for subsequent DNA cleavage and trans esterification reactions. Indeed, in keeping with recent reports concerning the related AAV Rep68 protein (5, 6), NS1 was found to nick and become covalently attached to origin DNA in the absence of ATP consumption, provided that the consensus nick site was contained within a single-strand structure. The present work shows that unwinding of the MVM left-end origin is a prerequisite for its sequence-specific cleavage by NS1. Moreover, the identification of two replication-deficient phosphorylation mutants, which have selectively lost helicase activity while keeping other known replicative functions (origin recognition and binding, strand and site-specific DNA cleavage, trans esterification, ATPase [this work], and oligomerization [12]), argues for origin unwinding as a step of MVM DNA replication that is regulated through phosphorylation of NS1 by atypical PKC.

Tight regulation of the initiation of viral DNA replication is not a feature unique to parvoviruses and can be best exemplified by the coupling of the onset of SV40 DNA replication with the entry of host cells into S phase (45). This could be attributed to the activation of SV40 LT, which shows striking functional and structural similarities to the parvoviral NS1 protein (2, 34), and unwinds the origin of replication as a result of the phosphorylation of residue T124 by cyclin A/cdk2 (a hallmark of S phase). This regulation represents merely one facet of the complex dependence of SV40 LT on phosphorylation, which involves both up- and down-modulating effects, several LT target residues, and cellular protein kinases (for a review, see reference 45). The present study, demonstrating that the ability of NS1 to initiate MVM DNA replication is also regulated by phosphorylation at the level of origin unwinding, further substantiates the resemblance between the two multifunctional viral proteins. In parvovirus, like SV40, DNA replication starts at the time of host cell entry into S phase (19). However, it is noteworthy that in contrast with SV40 LT, the capacity of NS1 for processing the origin is not the primary factor restricting parvovirus DNA replication to S phase of the cell cycle. The first step of parvovirus DNA replication consists of the so-called conversion of the viral single-stranded genome to a duplex DNA, which is necessary for transcription of the viral genes including NS1. It has recently been shown that, like the initiation of SV40 DNA replication, parvovirus DNA conversion is tightly coupled with the G₁/S transition due to its requirement for cyclin A/cdk2 (4), yet this dependence involves the cellular replication machinery independently of any viral cofactor. Thus, NS1 appears only at a later stage, to allow amplification of double-stranded replication forms after initiation of replication at the origins located at either end of the viral DNA (for a review see reference 23). Therefore, the control of NS1 origin-unwinding activity by phosphorylation appears to serve a purpose other than the coordination of viral DNA replication with S phase of the cell. The assignment of the protein kinases responsible for the activation of NS1 replicative functions to the PKC family (37) leads us to speculate that this regulation may contribute to the responsiveness of parvovirus replication to the differentiation (28) and transformation (14) status of the host cell, given the involvement of PKC in these processes (for reviews, see reference 32 and 40).

The role of NS1 in the parvoviral life cycle is not limited to DNA replication, as it also includes the regulation of viral gene expression (notably the trans activation of the P38 promoter controlling capsid gene expression [42]) and the induction of cellular disturbances which ultimately lead to cell lysis and release of progeny particles (1, 7, 12, 39, 44). In keeping with its multiple functions, NS1 expression occurs early and persists during virus replication (13, 43). Yet it would a priori be an advantage to parvoviruses if the various NS1 functions were not all activated concomitantly, allowing, for instance, viral DNA amplification to occur before capsids sequester progeny genomes or NS1 cytotoxicity negatively interferes with virus replication. Given that NS1 is phosphorylated at multiple sites and has a different biochemical profile depending on its phosphorylation state (34), we proposed that phosphorylation might contribute to priming NS1 for distinct tasks necessary for viral DNA replication. Moreover, the NS1 phosphorylation pattern shows consistent changes during the viral life cycle (13) and might thus direct NS1 functions in a temporally ordered fashion, allowing the viral product to drive progeny particle formation before inducing cell killing (12, 13, 34). The present study further supports the role of phosphorylation in NS1 regulation, indicating that a dissociation of NS1 activities can indeed be achieved through phosphorylation. More particularly, our data strongly suggest that the phosphorylation of specific residues (T435 and S473) is required to activate NS1 in regard to its ability to unwind DNA and initiate viral DNA replication. The T435A and S473A mutants are still capable of site-specific DNA binding (this work) and promoter trans activation (12). Therefore, the balance of NS1 replicative and transcriptional activities may be tipped toward the latter under conditions in which NS1 is not phosphorylated on T435 and S473 residues, as mimicked by site-directed mutagenesis in the present study. Another example of functional dissociation is given by the NS1 phosphorylation site mutant T363A which proved to have an especially high affinity for its cognate DNA recognition motif while being inactive for both nicking (this work) and trans activation (12). It may be speculated that this state would prime NS1 for joining (and putatively organizing) recently described subnuclear structures called APAR bodies, which develop into replication factories (25), through NS1 binding to its multiple cognate motifs within parvoviral DNA. It remains to be determined, however, whether phosphorylation at above-mentioned NS1 residues does indeed occur in a sequential order during virus infection, to commit NS1 into distinct tasks in the course of time.

The NS1-driven site-specific nicking reactions require cellular cofactors, namely, the HMG1/2 proteins at the right-end origin (24) and the transcription factor PIF at the left-end origin (9, 10; Christensen et al., submitted). Furthermore, PIF is thought to define the asymmetry of the NS1-dependent replication and resolution of head-to-head dimeric replication intermediates, thereby allowing the left-end palindrome in virion DNA to keep its flip orientation (9, 22; Christensen et al., submitted). In the presence of PIF, NS1 distinguishes between the two origins that face each other in the dimer bridge and differ only in a single nucleotide, GA versus GAA in the so-called bubble region, by cleaving the GA-containing (active) origin and leaving its GAA-containing (inactive) counterpart intact. This is explained in part by the cooperative binding of PIF and NS1 which stabilize the protein complex on the DNA template in absence of ATP (Christensen et al., submitted). The NS1-PIF complex could facilitate the local origin unwinding that is driven by NS1 in the presence of ATP hydrolysis and allows the exposed nick site to be cleaved. In addition, it might position the NS1 protein during this reaction in order to ensure the site specificity of single-stranded DNA nicking. Using a loop-containing substrate, we have demonstrated the NS1-dependent reaction of site-specific cleavage and trans esterification loses both its requirement for PIF and its distinction between the active and inactive origins, when the nick site is present in a single-stranded structure, while keeping its specificity for the nick site. This result provides direct evidence that a major role of PIF consists in allowing NS1 to unwind origin DNA around the nick site and/or to stabilize this energetically unfavorable structure necessary for cleavage and trans esterification. Whether this local unwinding occurs through NS1 helicase function or merely results from conformational changes induced by ATP hydrolysis remains to be shown. It is worth noting that the requirement for a cellular cofactor such as PIF appears to be superfluous when DNA secondary structures (e.g., a stem-and-loop conformation) are naturally present around the nick site and presumably position the Rep proteins and/or induce torsion within the origin to allow unwinding, as suggested for AAV (5, 6), geminivirus, plasmid, and bacteriophage origins (for a review see reference 30).

Native NS1 is involved not only in the initiation, i.e., nicking reaction of parvovirus DNA replication, but also in the consecutive RCR type of DNA amplification. While necessary and sufficient to make NS1^o competent for origin nicking and covalent attachment under nearly physiological salt conditions, atypical PKC phosphorylation of NS1 is not sufficient to drive RCR (37). Therefore, it appear that at least one additional regulatory component is necessary to fully activate the NS1 replicative functions. The nature of this additional component(s) is not known at present, except for its purification profile and cofactor requirements, pointing to members of the classical and/or novel PKCs (37). Besides complexes with the accessory proteins required for nicking (Christensen et al., submitted), NS1 interacts directly with at least one component of the replication complex in solution, i.e., human single-stranded DNA-binding protein (J. Christensen, unpublished data). The association of NS1 with the replication complex is further suggested by the specificity shown by NS1 regarding the type of DNA polymerase with which it can cooperate to drive RCR (37). It may be speculated that in addition to its role in the processing of the origin, NS1 participates in parvoviral DNA elongation by unwinding DNA in front of the replication fork through its processive helicase function, and that the proper coordination of this activity with the DNA polymerase is ensured by the physical interaction of NS1 with element(s) of the replication complex as previously shown for SV40 LT (45). Further work is required to unravel the regulatory pathway(s) that activate NS1 for its cooperation with the cellular DNA elongation machinery.