Journal of General Virology

Inhibition of transcription-regulating properties of nonstructural protein 1 (NS1) of the parvovirus minute virus of mice by a dominant-negative mutant form of NS1

Laurent Deleu, Aurora Pujol,† Jürg P. F. Nüesch and Jean Rommelaere

Applied Tumor Virology Programme, Abteilung F0100 and Institut National de la Santé et de la Recherche Médicale U 375, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany

Nonstructural protein 1 (NS1) of minute virus of mice is involved in viral DNA replication, transcriptional regulation and cytotoxic action in the host cell. Viral DNA replication is dependent on the ability of NS1 to form a homo-oligomer. To investigate whether oligomerization is required for NS1 transcriptional activities, a functionally impaired mutant derivative of NS1 that was able to interact with the wild-type (wt) protein and inhibit its activity in a dominant-negative manner was designed. This mutant provided evidence that transactivation of the parvoviral P38 promoter and transinhibition of a heterologous promoter by NS1 were both affected by the co-expression of the wt and the dominant-negative mutant form of NS1. These results indicate that additional functions of NS1, involved in promoter regulation, require oligomer formation.

Autonomous parvoviruses are small non-enveloped viruses that infect a variety of animal species, including humans. Their 5 kb genome is a linear single-stranded DNA molecule flanked by palindromic sequences. The coding sequences of the prototype strain of minute virus of mice (MVMp) used in the present study is divided into two overlapping transcription units (Pintel et al., 1983) that encode two capsid proteins (VP) and at least four non-structural (NS) proteins (Cotmore & Tattersall, 1987). Of these latter polypeptides, NS1, an 83 kDa nuclear phosphoprotein, is the only polypeptide essential for a productive infection in all cell types (Cater & Pintel, 1992; Naeger et al., 1990). NS1 is endowed with intrinsic ATPase, site-specific endonuclease, helicase and DNA-binding activities (Baldauf et al., 1997; Christensen et al., 1997; Cotmore et al., 1995; Cotmore & Tattersall, 1998; Nüesch et al., 1995; Wilson et al., 1991). These various functions are involved in viral DNA replication and transcription, which allows NS1 to serve both as an initiator of viral DNA amplification and as a regulator of viral gene expression. NS1 strongly induces the viral P38 promoter (Rhode & Richard, 1987) and activates or inhibits gene expression from heterologous promoters (Faisst et al., 1993; Legendre & Rommelaere, 1992; Vanacker et al., 1993). Although the molecular mechanisms involved in these various effects are still elusive, genetic analysis has shown that the transcription-regulating domains of NS1 are confined to the amino- and carboxy-terminal portions of the protein and can be dissociated from its replicative function (Legendre & Rommelaere, 1992).

To be fully functional, a number of proteins involved in DNA metabolism require self-association. This is the case of most helicases (West, 1996), many replication and transcription factors (Baler et al., 1993; Lazazzera et al., 1996; Mastrangelo et al., 1991), and multifunctional proteins, such as the large tumour antigen of simian virus 40 (SV40) (San Martin et al., 1997). Transcription factors forming oligomers can be inactivated in a dominant-negative manner by co-expressing a non-functional derivative capable of associating with the wild-type (wt) protein and forming inactive oligomers (Smith & Birrer, 1996). Recently, it was demonstrated that NS1 is able to form homo-oligomers in an ATP-dependent manner (Nüesch & Tattersall, 1993). This self-association is required for NS1 to fulfill its helicase function and to support viral DNA replication (Pujol et al., 1997). In order to study promoter regulation by NS1, we used this observation to design an approach involving a dominant-negative mutant. We engineered a mutant form of NS1 that is non-functional with regard to transcriptional activities and unable to bind to its DNA recognition motif, but can still oligomerize with full-length NS1. Should such a mutant act in a dominant-negative manner, this would argue for a role of self-association in the transcription-regulating functions of NS1. A putative dominant-negative mutant NS1 derivative, DT, which is unable to bind to its DNA cognate motif (deletion of aa 95–254) and lacks the acidic activation domain (deletion of 67 aa from the C terminus), but contains the oligomerization domain, was designed. A control mutant, DOT, which is identical to the DT mutant but impaired for oligomerization due to a deletion within the domain required for self-association (VETTVT-X₉-IQT) was also constructed (Fig. 1 A) (Pujol et al., 1997). All constructs were engineered by PCR using the wt NS1 DNA sequence and cloned into the cytomegalovirus (CMV)-driven expression vector pX (Pujol et al., 1997). The two different mutant derivatives were expressed at similar levels, as shown by immuno-blotting of extracts prepared from A9 cells transfected with a construct harbouring NS1 under the control of the bacteriophage T7 promoter and infected with a recombinant vaccinia virus, vTF7-3, expressing the bacteriophage T7 RNA polymerase (Fig. 1 B) (Nüesch et al., 1992). By two-hybrid analysis, the O deletion of aa 277–309 has been shown to prevent NS1 from homo-oligomerization (Pujol et al., 1997).

Fig. 1. Schematic representation and expression of the wt and mutant forms of NS1. (A) Hatched boxes indicate the positions of NS1 DNA-binding, oligomerization and C-terminal acidic activation domains. The regions of NS1 that are deleted in the mutant derivatives are indicated by open spaces. The vertical line shows the position of the NLS. Numbers correspond to amino acid residues. (B) Protein extracts from A9 cells transfected with the indicated constructs were subjected to immunoblotting using an NS1-specific antiserum.

NS1 exhibits a mainly nuclear localization during natural MVM infection (Cotmore et al., 1986) and when produced independently of other parvoviral proteins or viral DNA in transient expression systems (Nüesch et al., 1992; Nüesch & Tattersall, 1993). Previously, it has been shown that mutant NS1 polypeptides with an impaired nuclear localization signal (NLS) display nuclear localization when co-expressed with wt NS1. Plasmids expressing wt NS1, FLAG-NS1DT or FLAG-NS1DOT, under the control of the CMV early promoter were transfected separately into A9 cells. At 36 h post-transfection, proteins were detected by double-immunofluorescence using an M2 monoclonal antibody (Kodak), which recognizes the FLAG epitope, together with the polyclonal antisera SP8 (Brockhaus et al., 1996), which recognizes the C terminus of NS1 absent in the DT and DOT proteins. Immunofluorescence staining was analysed by confocal laser microscopy. As presented in Fig. 2(A), wt NS was found mainly in the nucleus of A9 cells, as described previously (Nüesch et al., 1992). As expected from the lack of the NLS in the mutant forms (Fig. 1), the DT derivative only displayed cytoplasmic localization, while the DOT mutant exhibited uniform distribution between the cytoplasm and the nucleus (Fig. 2 C). The migration of DOT into the nucleus is most likely due to both the small size of this mutant (55 kDa) and its inability to form higher order oligomers, which may allow the polypeptide to enter the nucleus independently of an NLS. When A9 cells were co-transfected with pX-NS1_wt and pX-FLAG-NS1DT at a ratio of 1:1, the DT mutant co-localized with wt NS1 in the nuclear compartment (Fig. 2 D). In contrast, A9 cells co-transfected with DOT- and wt NS1-expressing vectors showed no co-localization of mutant DOT with wt NS1 (Fig. 2 E). These findings clearly indicate that the ability of DT to interact with NS1 is due to the self-association domain, as DOT, which lacks this domain, fails to associate with wt NS1.

Fig. 2. Subcellular localization of wt and mutant forms of NS1 expressed separately or in combination. The subcellular localization of NS1 polypeptides was determined by double immunofluorescence staining at 36 h post-transfection of A9 cells with plasmids expressing wt NS1 (A), NS1 DT (B) or NS1 DOT (C), wt NS1 and DT (D), or wt NS and DOT (E). In co-transfection experiments (D, E), wt and mutant plasmids were transfected at a 1:1 ratio. Representative confocal optical sections through transfected cells are shown. (i) Expression of NS1 DT or NS1 DOT, as detected with M2 monoclonal antibodies and revealed with Texas red-conjugated goat anti-mouse antibodies (red). (ii) Expression of wt NS1, as detected with SP8 antiserum and revealed with FITC-conjugated goat anti-rabbit antibodies (green). (iii) Merged images of (i) and (ii) indicate when proteins co-localize (yellow). Pictures were obtained with a Zeiss 310 confocal laser scanning microscope, with readings at 488 and 534 nm wavelengths for FITC and Texas red fluorescence, respectively. Images were obtained with a Mitsubishi sublimation printer. An oil immersion X63 Plan Apochromat objective was used.

The parvoviral P38 promoter is activated in the presence of wt NS1. Transcriptional activation mediated by NS1 is dependent on site-specific interaction of NS1 with its cognate DNA-binding motif, which is located within the tar region of the promoter (Christensen et al., 1995), as well as NS1 interaction with the transcription factor SP1 (Krady & Ward, 1995; Lorson et al., 1998). In order to investigate whether oligomerization of NS1 is indeed a prerequisite for its ability to transactivate the P38 promoter, we attempted to inhibit the activity of wt NS1 by co-expression with DT that was C-terminally fused to the SV40 large tumour antigen NLS allowing migration to the nucleus as confirmed by immunofluorescence (data not shown). The plasmid P38-Luc, which contains the luciferase gene under the control of the MVMp P38 promoter, was used as a reporter to reveal the transactivation capacity of NS1 in transient expression assays. A9 mouse fibroblasts were co-transfected with 50 ng of P38-Luc and various amounts of pX, pX-NS1, pX-DT-NLS or pX-DOT-NLS. Luciferase activity was measured 36 h post-transfection. To exclude any effect of the pX plasmid on P38 promoter function, luciferase activities from extracts of cells transfected with P38-Luc in the presence or absence of 400 ng of pX were compared. Since no differences were found between the two conditions (data not shown), the total amount of the effector plasmid was maintained at 400 ng per transfection. As illustrated in Fig. 3(A), wt NS1 was able to transactivate the P38 promoter up to 100-fold above that of the background level detected with the pX control vector. In contrast, no significant transactivation was observed with NS1-DT-NLS or NS1-DOT-NLS. Using co-expression of either mutant polypeptide with wt NS1, we determined whether interaction with DT would impair NS1 capacity for transactivation. Mouse A9 cells were co-transfected with P38-Luc (reporter), pX-NS1_wt (transactivator) and the potential transinhibitor pX-DT-NLS (or pX-DOT-NLS as a negative control) in wt versus mutant ratios of 1:1, 1:3 or 1:6. As illustrated in Fig. 3(B), P38 transactivation by wt NS1 was reduced in cells co-expressing DT-NLS in a dose-dependent manner. In contrast, the oligomerization-negative DOT-NLS protein failed to suppress wt NS1-induced transactivation, even when supplied in a sixfold excess over pX-NS1_wt. The reduction of wt NS1-induced P38 activity in the presence of DT, but not DOT, under conditions in which target cells accumulate similar amount of both types of mutant proteins (Fig. 2), argues for oligomerization as the mechanism by which the DT form exerts its dominant-negative effect. This interpretation is supported by recent work that shows that NS1 directly binds, in an ATP-dependent manner, to a promoter P38 region that mediates the NS1 transactivation response in cis (Christensen et al., 1995). The ATP-dependence of NS1-specific binding to DNA can be circumvented in the presence of antibodies that are likely to cross-link NS1 molecules. These data, together with the footprinting analysis of NS1–DNA interactions (Christensen et al., 1995), point to the fact that NS1 homo-oligomerization is involved in specific DNA binding and ensuing promoter transactivation. This is in keeping with the proposed mode of action whereby the dominant-negative DT mutant forms inactive oligomers with wt NS1.

Fig. 3. Effect of NS1, NS1 DT and NS1 DOT on reporter gene expression driven by either the P38 promoter (A, B) or the CMV early promoter (C, D). (A, B) Cultures of 1.5x10⁵ A9 cells were co-transfected with 50 ng of the reporter plasmid P38-Luc and indicated amounts of effector plasmid pX-NS1wt, pX-DT-NLS or pX-DOT-NLS. Effector plasmids were given either separately (A) or in combination (B). The DNA inoculum was adjusted to 400 ng with the control plasmid pX. Transfected cultures were incubated for 48h and assayed for luciferase activity. The relative light units (RLU) measured for the P38 promoter-driven reporter plasmid co-transfected with each effector are shown (A) and the fold induction of the P38 promoter is shown for the different effectors (B). (C, D) The CMV early promoter-driven reporter construct pCMV SEAP was used to measure the transinhibiting effects of wt or mutant NS1-expressing plasmids that were inoculated either separately (C) or in combination (D). All transfection mixtures were adjusted to 600 ng of effector plasmid with the empty vector pX. SEAP activity was measured with the Phospha-Light kit (Tropix), according to the manufacturer's instructions. The RLU measured for the CMV promoter-driven reporter plasmid co-transfected with each effector are shown (C) and the fold inhibition of the CMV promoter is shown for the different effectors (D). Data are average values from three independent transfections, each carried out in triplicate.

Besides regulating the viral P38 promoter, NS1 is also able to suppress transcription from a number of cellular and heterologous viral promoters (Legendre & Rommelaere, 1992; Rhode & Richard, 1987). At present, it is not clear whether this inhibition results from a specific repression by NS1 or a mere squelching of transcription factors that interact with the N- or C-terminal domains of the polypeptides (Legendre & Rommelaere, 1992). The dominant-negative mutant approach was used to investigate whether homo-oligomerization is involved in the transinhibiting activity of NS1. The early promoter of CMV was chosen as a target for NS1-dependent transinhibition. A fixed amount (50 ng) of an expression vector expressing secreted alkaline phosphatase (SEAP) from the CMV promoter (pCMV SEAP) (Tropix) was co-transfected with increasing amounts of plasmids expressing either wt NS1 or the mutant derivatives DT-NLS or DOT-NLS. The total amount of the effector plasmid was maintained at 400 ng per transfection for the same reason as that described for the P38 promoter. As illustrated in Fig. 3(C), wt NS1 suppressed CMV promoter activity by more than fourfold, whereas both of the deletion mutants were deficient in this respect. When plasmids expressing wt NS1 and DT were co-transfected, the mutant polypeptide exerted a dominant-negative effect on the wt protein, in that it prevented the latter from transrepressing the CMV early promoter (Fig. 3 D). In contrast, the oligomerization-deficient NS1 DOT mutant form was ineffective in suppressing the transinhibitory activity of wt NS1. These results indicate that NS1-mediated transinhibition of the CMV promoter is achieved by a multimeric form of the polypeptide requiring functionally active subunits.

In conclusion, the NS1 DT mutant protein proved able to negatively interfere with the capacity of wt NS1 for both activation and repression of target promoters in a way that is dependent on the self-association of the NS1 polypeptide. These data indicate that the transcription-regulating function of NS1 is likely to require NS1 homo-oligomerization. Further investigations are necessary to determine whether homo-oligomerization controls the interaction of NS1 with the transcription machinery and/or other transcription factors.

L. Deleu and A. Pujol contributed equally to this work. We are grateful to Dr Johanna Bridges for the confocal laser scanning microscopy pictures and to Dr Romuald Corbau for the kind gift of P38-Luc plasmid. We acknowledge the help of Dr Jean-Claude Jauniaux and Celina Cziepluch in the establishment of the yeast two-hybrid system. We also thank Dr Jean-Marc Vanacker, Nathalie Salome, François Fuks and Jan Cornelis for helpful discussions. This work was supported by the Commission of the European Communities, the Deutsche Krebshilfe–Dr Mildred Scheel Stiftung für Krebsforschung and the German–Israeli Foundation for Scientific Research and Development. A.P. and L.D. are fellows of the Commission of the European Community.

† Present address: Human Molecular Genetics Laboratory, IGBMC, 1 rue Laurent Fries, BP 163, 67404 Illkirch Cedex, France

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