In Vivo Accumulation of Cyclin A and Cellular Replication Factors in Autonomous Parvovirus Minute...

  10.1128/JVI.75.9.4394-4398.2001.

2001, 75(9):4394. DOI:J. Virol. Tarig Bashir, Jean Rommelaere and Celina Cziepluch Replication Bodies

Mice-AssociatedParvovirus Minute Virus of AutonomousCellular Replication Factors in

In Vivo Accumulation of Cyclin A and

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JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.9.4394–4398.2001

May 2001, p. 4394–4398 Vol. 75, No. 9

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

In Vivo Accumulation of Cyclin A and Cellular ReplicationFactors in Autonomous Parvovirus Minute Virus of

Mice-Associated Replication BodiesTARIG BASHIR, JEAN ROMMELAERE, AND CELINA CZIEPLUCH*

Applied Tumor Virology Unit F0100 and Institut National de la Sante et de la Recherche Medicale U 375,Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany

Received 14 December 2000/Accepted 6 February 2001

Autonomous parvovirus minute virus of mice (MVM) DNA replication is strictly dependent on cellularfactors expressed during the S phase of the cell cycle. Here we report that MVM DNA replication proceeds inspecific nuclear structures termed autonomous parvovirus-associated replication bodies, where components ofthe basic cellular replication machinery accumulate. The presence of DNA polymerases a and d in these bodiessuggests that MVM utilizes partially preformed cellular replication complexes for its replication. The recruit-ment of cyclin A points to a role for this cell cycle factor in MVM DNA replication beyond its involvement inactivating the conversion of virion single-stranded DNA to the duplex replicative form.

Autonomous parvoviruses and other members of the Parvo-viridae family are unique among animal viruses in having linearsingle-stranded DNA genomes. The termini of their approxi-mately 5,000-nucleotide genomes contain palindromic se-quences which fold into stable hairpin structures and serve asprimers for viral DNA synthesis. The replicative cycle, whichtakes place in the nucleus, is initiated by the synthesis of thecDNA strand leading to the formation of double-stranded rep-licative-form DNA. This reaction, also known as conversion, isexclusively dependent on cellular factors. In addition, the sub-sequent amplification reactions require the activity of the vi-rus-encoded major nonstructural protein NS1 (7).

Several cellular factors which are essential for DNA repli-cation of the prototype autonomous parvovirus minute virus ofmice (MVM) have been identified through cell fractionationand complementation assays in vitro replication systems (5, 8,20). However, very little is known about the subnuclear orga-nization of MVM DNA replication in vivo. In contrast toviruses with double-stranded DNA genomes that replicate inclose proximity to preformed nuclear structures known as pro-myelocytic leukemia (PML) bodies (for review see reference18), autonomous parvovirus H-1 was shown to induce charac-teristic nuclear structures, termed H-1 parvovirus-associatedreplication (PAR) bodies, which are unrelated to PML bodies(9). Similar structures were also characterized in Aleutianmink disease virus-infected cells (21, 22). In this study, weaimed to determine whether MVM also establishes PAR body-like structures in the nuclei of infected cells and to analyze thesubnuclear distribution of cellular factors assumed to be in-volved in MVM DNA replication in vivo. To this end, A9mouse cells were infected with MVMp at a multiplicity ofinfection of 10 PFU per cell. At 15 h postinfection, cells werelabeled for 20 min with bromodeoxyuridine (BrdU) at a con-

centration of 10 mM and then immediately fixed in 1% form-aldehyde for 10 min at room temperature. BrdU incorporationinto replicated viral DNA was detected with a BrdU-specificantibody (Becton Dickinson) without prior denaturation, thusexcluding the detection of chromosomal DNA replication (21).Simultaneously, NS1 was detected with the NS1-specific SP8antibody (11). Analysis by confocal microscopy (LSM510 UV;Zeiss, Jena, Germany) revealed the accumulation of NS1 inspecific nuclear bodies (Fig. 1a) which were also found to bethe sites of ongoing viral DNA replication, as indicated byBrdU incorporation (Fig. 1b and c). At this time postinfection,no signs of virus-induced cytotoxicity were visible (Fig. 1d).From these data, we concluded that MVM DNA replicationproceeds in specific nuclear structures similar to the ones pre-viously described for other autonomous parvoviruses (9, 21,22). We would therefore like to propose the more general termautonomous PAR (APAR) bodies for these virus-inducedstructures.

MVM DNA replication starts only after host cells enter theS phase of the cell cycle. Recently, it was shown that in vitro theconversion reaction is activated by the cell cycle factor cyclin A,whose production is induced at the G1/S transition (1). CyclinA is involved in the regulation of the S and G2 phases (25) andhas been reported to be required for chromosomal (16) andsimian virus 40 (10, 12) DNA replication. In order to testwhether cyclin A is present in APAR bodies, we performeddouble immunofluorescence labeling and confocal microscop-ical analysis of MVM-infected A9 cells. Cyclin A, which ishomogeneously distributed throughout the nuclei of mock-infected cells and absent from nucleoli (reference 2 and datanot shown), was indeed found to massively accumulate to-gether with NS1 in the APAR bodies of infected cells (Fig. 2ato c). Even if one assumes that the subnuclear location atwhich the conversion reaction takes place predetermines thesite of APAR body formation, a sole role for cyclin A in theconversion reaction is difficult to reconcile with such a strongaccumulation of cyclin A in APAR bodies at 15 h postinfec-tion, when conversion is thought to be complete. This result

* Corresponding author. Mailing address: Applied Tumor Virology,Abteilung F0100 and INSERM U 375, Deutsches Krebsforschung-szentrum, Postfach 101949, 69009 Heidelberg, Germany. Phone: 496221 424977. Fax: 49 6221 424962. E-mail: C.Cziepluch@dkfz-heidelberg.de.

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could indicate that the continuous presence of cyclin A is alsorequired for subsequent NS1-dependent replicative steps. Onecan furthermore speculate that the sequestration of this factoris responsible for the previously described parvovirus-inducedcell cycle arrest (23, 24) because deprivation of cyclin A maylead to a failure of cdk1 activation in the G2 phase, thuspreventing cell cycle progression.

Besides cyclin A, cyclin E is involved in the regulation of theG1/S transition and is known to play a role in chromosomalDNA replication (14, 16). In contrast to cyclin A, however,cyclin E did not accumulate in APAR bodies (Fig. 2d to f).Since the cyclin E-specific antibody best recognized the humanprotein, in these experiments we infected human NBE cellsand verified that MVMp indeed induces the formation ofAPAR bodies in this human cell line, as detected by NS1-specific (Fig. 2d) and BrdU-specific (data not shown) antibod-ies. The failure to detect an accumulation of cyclin E does notexclude the presence of minute amounts, of this factor inAPAR bodies. Our data are, however, in agreement with re-cent in vitro experiments showing no contribution of cyclin Eor cdk2 in the initiation of conversion (1).

Cyclin A forms an active complex with cdk2 at the beginningof the S phase. This complex is assumed to be involved in theregulation of DNA replication, since both cyclin A and cdk2were found to be associated with replication foci in mammaliancells (3) as well as with replicating DNA in the simian virus 40in vitro replication system (13). We therefore attempted todetect cdk2 in APAR bodies. Using several different cdk2-specific antibodies, cdk2 could not be shown to accumulate inAPAR bodies (Fig. 2g to i). The observed accumulation ofcyclin A in APAR bodies (Fig. 2b) would then point to a

structural role for cyclin A in these bodies, independent ofcomplex formation with cdk2. However, it is still possible thatonly catalytic amounts too small to be detected by the methodused here are required for MVM DNA replication in APARbodies.

Parvovirus DNA replication proceeds according to a lead-ing-strand-synthesis mechanism and has been shown to be de-pendent on proliferating cell nuclear antigen (PCNA) in vitro(5). These and other lines of evidence point to DNA polymer-ase d as the enzyme responsible for parvovirus DNA replica-tion (6). Here, we show that DNA polymerase d indeed accu-mulates in APAR bodies in MVM-infected NBE cells (Fig. 3ato c) and therefore at the sites of ongoing MVM DNA repli-cation. DNA polymerase d requires for processive DNA syn-thesis the presence of the cofactor PCNA, which is also in-volved in other processes, like DNA recombination and repair(15). PCNA is distributed throughout the nucleus except thenucleoli, but it specifically accumulates in the APAR bodies ofMVM-infected NBE cells (Fig. 3d to f). This finding corrobo-rates earlier in vitro data demonstrating that PCNA is indis-pensable for MVM DNA replication (1, 5).

The single-strand-DNA-binding protein replication proteinA (RPA) is required for MVM DNA replication in vitro (5)and cannot be replaced by other proteins with single-strand-DNA-binding activity (J. Christensen, personal communica-tion). Furthermore, a direct interaction between the 70-kDasubunit of RPA and NS1 was demonstrated in vitro (4). Inagreement with these in vitro findings we have observed thatRPA massively accumulates at the sites of ongoing parvovirusDNA replication in infected cells (Fig. 3g to i), which stronglysuggests its involvement in this process in vivo.

FIG. 1. MVM DNA replication colocalizes with NS1 in APAR bodies in the nuclei of infected A9 cells. Representative confocal optical sectionsthrough the nuclei of infected cells are shown. NS1 was localized with the SP8 polyclonal antiserum and a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (a). Replication was monitored by incorporation of BrdU and indirect immunofluorescence using a BrdU-specificantibody and a tetramethyl rhodamine isothiocyanate (TRITC)-conjugated secondary antibody (b). In a merged image, colocalized structures frompanels a and b appear yellow (c). By phase-contrast microscopy (Nomarski), the cells show no obvious sign of NS1-induced cytotoxicity at the timeof fixation (15 h postinfection) (d).

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Surprisingly, DNA polymerase a was also found at the sitesof MVM DNA replication (Fig. 3j to l). So far, there is noevidence for a contribution of DNA polymerase a to parvovi-rus DNA replication, since neutralizing antibodies against thisenzyme do not impair viral DNA replication in vitro (5, 19).Furthermore, the presence of preformed hairpin primersmakes a DNA polymerase a-dependent primase activity dis-pensable. However, it was shown that DNA polymerase a canbe isolated together with DNA polymerase d and the PCNAloading factor, replication factor C, from mammalian cells as astable complex that is replication competent when templateDNA, PCNA, and nucleotides are added (17). It could bedemonstrated that in this complex, DNA polymerase a is ac-

tive on single-stranded-DNA templates only when primer syn-thesis is a precondition for replication. On a primer-templatejunction, as present in parvovirus DNA, the DNA-binding af-finity of replication factor C is higher than that of DNA poly-merase a, thereby rendering the latter inactive (17, 27). SinceDNA polymerase a is required for chromosomal DNA repli-cation, it is tempting to speculate that MVM utilizes cellularreplication complexes that are at least partially preformed,exploiting only the DNA polymerase d activity and thus evad-ing the need for an energy-consuming dissociation of pre-formed cellular complexes. This would again highlight the op-portunistic and minimalistic strategies used by these viruses.

In summary, the data presented here show for the first time

FIG. 2. Cyclin A, but not cyclin E or cdk2, accumulates in APAR bodies. Representative confocal images of nuclei from double-labeledMVM-infected A9 (a to c) or NBE (d to i) cells are shown. NS1 was detected with FITC using the NS1-specific 3D9 antibody (a generous gift fromN. Salome and D. Pintel) (a, d, and g). Images of cyclin A (b), cyclin E (e), and cdk2 (h) were detected with TRITC in the same confocal planeas shown in the left column using the respective primary antisera for cyclin A (a generous gift from M. Pagano), cyclin E (Ab-1; Neomarkers,Fremont, Calif.), and cdk2 (Ab-3; Neomarkers). The superimposition of both channels from the left and middle columns reveals either thecolocalization (yellow signal [c]) or the lack of colocalization (distinct red and green signals [f and i]) of the respective factors in relationship toAPAR bodies.

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FIG. 3. Cellular replication factors DNA polymerases d and a, PCNA, and RPA accumulate in APAR bodies. Representative confocal imagesof nuclei from double-labeled MVM-infected NBE cells are shown. NS1 was detected with FITC using the SP8 antiserum (a, d, g, and j).Replication factors were detected with TRITC in the same confocal plane as shown in the left column, using the respective antibodies against DNApolymerase d (Transduction Laboratories) (b), PCNA (PC10; Upstate Biotechnology) (e), RPA (Ab-1; Oncogene) (h), and DNA polymerase a(SJK132-20 [26]) (k), respectively. A merged image of both channels from the left and center columns provides evidence for colocalization of thesefactors in APAR bodies (c, f, i, and l).

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the formation of APAR bodies in MVM-infected cells and theaccumulation of various cellular replication factors in thesestructures. Our data support the idea that the basic cellularreplication machinery is used by MVM for viral DNA replica-tion, including DNA polymerase d, PCNA, RPA, and cyclin A.DNA polymerase a, which is part of the cellular replicationmachinery, was unexpectedly found to accumulate in APARbodies but is most likely not active in parvovirus DNA repli-cation. For cyclin A, it will be of interest to identify the role thisfactor plays in parvovirus DNA replication, in addition to itsrole in the activation of the conversion reaction in vitro.

We acknowledge the generous gift of antibodies from D. Pintel andN. Salome (University of Missouri—Columbia) and antiserum fromM. Pagano (New York University, New York, N.Y.). The expert sup-port of H. Spring (DKFZ, Heidelberg, Germany) with acquisition ofdata by confocal microscopy is gratefully acknowledged.

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