minute virus of mice ns1 interacts with the smn protein, and they

JOURNAL OF VIROLOGY, Apr. 2002, p. 3892–3904 Vol. 76, No. 80022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.8.3892–3904.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Minute Virus of Mice NS1 Interacts with the SMN Protein, and TheyColocalize in Novel Nuclear Bodies Induced by Parvovirus Infection

Philip J. Young,1 Klaus T. Jensen,2 Lisa R. Burger,2 David J. Pintel,2 and Christian L. Lorson1*Department of Biology, Arizona State University, Tempe, Arizona 85287,1 and Department of Molecular Microbiology

and Immunology, School of Medicine, University of Missouri, Columbia, Missouri 652122

Received 24 October 2001/Accepted 10 January 2002

The human survival motor neuron (SMN) gene is the spinal muscular atrophy-determining gene, and aknockout of the murine Smn gene results in preembryonic lethality. Here we show that SMN can directlyinteract in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minutevirus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically,SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression,SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails tocolocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclearalterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product(PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR)bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termedSMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These resultshighlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formedduring infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient toinduce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viralinfection process.

Minute virus of mice (MVM) is an autonomous parvovirusconsisting of a single-stranded genome approximately 5 kb inlength (12, 13). The genome contains two overlapping tran-scription units controlled by two viral promoters: P4 and P38(2, 16, 32, 33). Expression of the viral nonstructural proteins,NS1 and NS2, is driven by the P4 promoter, while P38-derivedtranscripts encode the viral structural proteins, VP1 and VP2(2, 16, 32, 33). NS1 performs critical functions in viral geneexpression and genome replication. NS1 is also required forMVM-associated cytotoxicity, although the mechanism(s) isunclear (3). Upon infection, parvoviruses cannot induce cellsinto S phase, so the virus must rely on actively dividing cells tonaturally progress through the cell cycle; when the cells enterS phase, a productive, lytic infection can progress (12, 13).Cells infected by autonomous parvoviruses are prevented fromfurther progress in the cell cycle (39–42) yet remain viable andsustain virus replication for many hours before they die (49).

Recently, a novel subnuclear compartment was identifiedfollowing infection by either of two highly related parvoviruses,MVM and H-1 (5, 14). This structure, termed autonomousparvovirus-associated replication (APAR) bodies, was identi-fied at 15 h postinfection and was found to be distinct frommost of the classically described nuclear bodies such as Cajalbodies, promyelocytic leukemia gene product (PML) onco-genic domains (PODs), and speckles (14). APAR bodies wereshown to be active sites of viral replication and to containcellular factors such as cyclin A, DNA polymerases � and �,

proliferating cell nuclear antigen (PCNA), and replication pro-tein A (4).

The eukaryotic nucleus is highly organized and containsnumerous subnuclear compartments (34). Although nuclearbodies have been identified and classified based on their con-stituents, mounting evidence suggests that nuclear bodies andtheir contents are highly dynamic and can fluctuate in responseto a variety of internal and external stimuli (28, 35, 46, 47). Thefunctions of the various nuclear structures have been partiallyascribed to their molecular constituents. For example, Cajalbodies are enriched in mature snRNPs and contain factorssuch as the survival motor neuron gene product (SMN), whichis involved in snRNP biogenesis (9, 22, 29). PODs are distinctnuclear structures that are linked to transcription regulationand apoptosis and contain the PML protein, retinoblastomaprotein Rb, Sp100, and PIC1/SUMO-1 (7, 45). Speckles orinterchromatin granules are highly enriched in splicing factors,although it is unclear if these structures are storage sites forsplicing factors or whether they actively participate in mRNAmaturation (34, 36).

Now we show that MVM NS1 specifically interacts with acomponent of Cajal bodies, the SMN protein, and that SMNand NS1 colocalize in Cajal bodies following transient expres-sion of NS1. As suggested by earlier results, at early time pointsfollowing MVM infection of synchronized cells, NS1 and SMNfail to colocalize. However, at later time points (20 to 30 hpostinfection) NS1 and SMN colocalize in large nuclear bodiesthat are the active sites of virus replication and viral capsidassembly. Unlike transfection of NS1 alone, at these later timepoints, MVM infection induces a massive nuclear reorganiza-tion in which constituents from Cajal bodies, PODs, interchro-matin granules, and APAR bodies accumulate in multiple

* Corresponding author. Mailing address: Department of Biology,Arizona State University, Tempe, AZ 85287. Phone: (480) 965-5444.Fax: (480) 965-2519. E-mail: [email protected].

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FIG. 1. SMN and NS1 interact directly both in vitro and in vivo. (A) Western blot analysis of recombinant polyhistidine (six-His-)-tagged humanSMN captured by GST-tagged NS1 immobilized on GST resin. The blot was developed with an anti-SMN monoclonal antibody. (B) Western blotanalysis of NS1 coimmunoprecipitated with endogenous SMN from transfected A92L cells. Anti-SMN coprecipitated endogenous SMN, andanti-NS1 rabbit polyclonal sera detected NS1 complexed with SMN. NS1-SMN binding was not detected when the immunoprecipitating antibody(�) was omitted or when cells were not transfected with NS1 (mock). mAb, monoclonal antibody. (C) (Top) BIA of NS1 coimmunoprecipitatedwith endogenous SMN from MVM-infected double-blocked A92L cells. Total protein extracts from A92L cells 30 h postinfection were used.Anti-SMN (MANSMA3) coprecipitated SMN, and anti-NS1 rabbit polyclonal sera detected NS1 complexed with SMN. Levels of the capturedanti-SMN antibody (A), SMN-NS1 complex (B), and anti-NS1 rabbit polyclonal antibody (C) are indicated. (Bottom) NS1 is not coprecipitatedwith SMN from double-blocked noninfected A92L cells. Total protein extracts from A92L cells 30 h after being released from the blocking processwere used. Anti-SMN (MANSMA3) precipitated SMN, and anti-NS1 rabbit polyclonal sera detected no NS1 complexed with SMN. Levels thecaptured anti-SMN antibody (A), SMN (B), and anti-NS1 rabbit polyclonal antibody (C) are indicated.

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large nuclear bodies, termed SMN-associated APAR bodies(SAABs). These results highlight the dynamic nature of nu-clear bodies and identify a novel structure generated in re-sponse to parvovirus infection. They also suggest that somefeature(s) of virus replication other than merely expression ofthe viral nonstructural proteins is required for the observednuclear reorganization following parvovirus infection.

MATERIALS AND METHODS

Virus propagation. Wild-type prototypic MVM (MVMp) was grown and ti-tered on NB324K cells and used to infect A92L and NB324K cells at a multiplicityof infection of 10. Cells were highly synchronized by growth in isoleucine-deficient media for 40 h, followed by incubation in the presence of 36 �Maphidicolin and MVM for 12 to 16 h. The cells were then washed in phosphate-buffered saline and released in complete Dulbecco’s modified Eagle’s medium.Viral replication is detectable 5 to 6 h postrelease (44). Neuraminidase at aconcentration of 0.05 U/ml was added 7 h after release to prevent reinfection. Atvarious time points cells were labeled for 20 min with bromodeoxyuridine(BrdU) at a concentration of 10 �M.

Recombinant proteins. NS1 cDNA was cloned into glutathione S-transferase(GST) expression vector pGEX3X (Pharmacia). Wild-type SMN cDNA wascloned into the pET32c vector (Novagen). Transformed bacteria were inducedwith 1 mM isopropyl-�-D-thiogalactopyranoside for 4 h at 37°C. Expressed re-combinant proteins were purified from inclusion bodies by sequential extractionwith increasing urea concentrations (2, 4, 6, and 8 M) in phosphate-bufferedsaline (PBS) as previously described (52).

Transient transfections. Approximately 105 A92L cells were transiently trans-fected with 2 �g of pCI:HA-NS1 by using calcium phosphate (32).

Cellular extracts. Total cellular extracts were derived from approximately 105

A92L cells infected with MVM at 0 and 30 h postrelease. Total extracts werenormalized, and equivalent levels were used for biomolecular interaction analysisexperiments.

BIA. The BIAcore biosensor detects changes in total mass at the surface of asensor chip by measuring variations of the critical angle needed to produce totalinternal refraction. The change in critical angle is proportional to the amount ofbound protein and is expressed as resonance units (RU). In Fig. 1C, the shift inRU is plotted against time and is displayed as a sensorgram. Injected samplescontain protein, urea, imidazole, and HEPES buffered saline (HBS) and there-fore are denser than HBS running buffer. This increase in medium density duringprotein injections produces the large vertical spikes seen on the sensorgram (the

FIG. 2. Colocalization of SMN, p80 coilin, SIP1, and NS1 in transfected A92L cells. SMN (primary antibody: MANSMA1; secondary antibody:TRITC [red]), SIP1 (primary antibody: MANSIP1A; secondary antibody: anti-mouse TRITC conjugate [red]), p80 coilin (primary antibody: 5P10monoclonal antibody; secondary: anti-mouse TRITC conjugate [red]), and NS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody:anti-rabbit FITC conjugate [green]) are indicated. Arrows, Cajal bodies containing NS1. Insets show nuclear regions of interest magnified by anadditional factor of 2. Bar � 30 �m.

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initial vertical line indicates the start of an injection and the second vertical lineindicates the end of an injection). The amount of bound protein following aninjection is indicated by the increase in the horizontal baseline. A Cm5 sensorchip was covalently linked with rabbit anti-mouse immunoglobulin G by theamino-coupling protocol according to the manufacturer’s instructions (BIAcoreAB, Stevenage, Herts, United Kingdom). A BIAcore-X apparatus with an op-erating flow rate of 5 �l per min was used. All injected volumes were 10 �l.Concentrations of stock recombinant proteins for each reaction were determinedprior to injection. Proteins were diluted in HBS to 10 �g/ml unless otherwisestated. During biomolecular interaction analysis (BIA) experiments, nonspecificinteractions were ruled out by ensuring that neither SMN nor NS1 bound directlyto the chip in the absence of specific antibodies or protein binding partners.

Immunohistochemistry. Subconfluent A92L and NB324K cell lines werewashed three times with PBS, fixed for 1 min with 50% acetone–50% methanol,and then washed an additional three times with PBS. Cells were then analyzed bydouble-label immunofluorescence. Images were captured with tetramethyl rho-damine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) filter setsand a 100� oil immersion objective in conjunction with a Leica TCS-NT confocalmicroscope. All experiments were repeated in the absence of the primary anti-bodies to eliminate the possibility of FITC-TRITC cross-reaction.

Immunofluorescence experiments were repeated with various primary anti-bodies to eliminate epitope masking and cross-reaction during time courses.Western blotting was performed on total protein extracts from all infection timepoints to ensure that each NS1 and SMN antibody used was specific to theprotein it was generated against. All other antibodies were obtained from com-mercial vendors and have been shown to have no nonspecific cross-reactivity.The antibodies used were as follows: anti-SMN antibodies MANSMA1 (exon 2specific), MANSMA4 (exon 4 specific), and MANSMA3 (exon 5 specific); anti-NS1 (monoclonal antibody 3D9); anti-MVM VP1 (peptide polyclonal antibody);anti-capsid/assembled (monoclonal antibody D4H) (30); anti-SIP1 antibody(MANSIP1A); anti-p80 coilin antibody (5P10); anti-PML antibody (PG-M3;Santa Cruz Biotechnology); anti-cyclin A antibody (C-19; Santa Cruz Biotech-

nology); anti-cyclin E (N-20; Santa Cruz Biotechnology), anti-BrdU (ChemiconInternational); anti-SR (1H4; Zymed Laboratories); NS1 (rabbit polyclonal an-tibody M50); and anti-Sm (Y12; Neomarkers).

In vitro binding assays. GST and His-binding resins (Novagen) were washedthree times in binding buffer (500 mM NaCl, 20 mM Tris-HCl [pH 7.8], 0.2%NP-40) and incubated with recombinant proteins diluted to 100 �g/ml in bindingbuffer for 1 h at 4°C. The resins were washed three times with binding buffer andincubated with the respective binding proteins diluted to 100 �g/ml in bindingbuffer for 1 h at 4°C. Resins were washed three times with binding buffer, andbound fractions were resolved by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis.

Protein concentrations were confirmed by Coomassie blue staining

RESULTS

Identification of NS1 as an SMN-interacting protein. TheNS1 nonstructural protein of MVM and the bovine papilloma-virus transactivator E2 protein interact with similar subsets ofcellular transcription factors to activate viral gene expression,including TATA binding protein TBP and Sp1 (8, 32, 43, 48).Recently, E2 has been shown to interact with SMN, a proteinthat has reported functions in transcriptional activation andRNA-processing pathways (29, 48). Based on the previouslyobserved similarities between NS1 and E2 the following exper-iments were devised to determine whether NS1 and SMNassociate. Initially, a bacterially expressed and purified GST-NS1 fusion protein and polyhistidine (six-His-)-tagged SMNwere incubated together to determine whether an NS1-SMN

FIG. 3. SMN, p80 coilin, and NS1 colocalize in SAABs in MVM-infected A92L cells. SMN (primary antibody: anti-SMN monoclonal antibodyMANSMA1; secondary antibody: anti-mouse TRITC conjugate [red]), p80 coilin (primary antibody: 5P10 monoclonal antibody; secondaryantibody: anti-mouse TRITC conjugate [red]), and NS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody: anti-rabbit FITCconjugate [green]) are indicated. Synchronized infections were obtained by performing an isoleucine-aphidicolin double block on A92L cells priorto infection. Immunofluorescence experiments were performed on infected cells 15, 20, 25, and 30 h after entry into S phase. Insets show nuclearregions of interest magnified by an additional factor of 2. Bar � 30 �m.

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complex formed in vitro. GST-NS1-bound fractions were sep-arated with glutathione-linked agarose beads. Specific NS1-SMN in vitro binding was observed by Western analysis with ananti-SMN monoclonal antibody (Fig. 1A). SMN did not non-specifically interact with the agarose beads alone (Fig. 1A) orwith certain NS1-mutant GST fusions (P. J. Young, K. T.

Jensen, D. J. Pintel, and C. L. Lorson, unpublished data).Coimmunoprecipitations were then performed with extractsfrom murine A92L cells to determine whether the NS1-SMNcomplex formed in vivo. A92L cells were transfected with hem-agglutinin (HA)-tagged NS1, and coimmunoprecipitation re-actions were performed with a monoclonal antibody specific

FIG. 4. SAAB formation is neither a consequence of the blocking process nor restricted to murine cell lines. (A) Double-label experiments wereperformed on infected and uninfected blocked A92L cells at 30 h postrelease. SMN (primary antibody: anti-SMN monoclonal antibodyMANSMA1; secondary antibody: anti-mouse TRITC conjugate) and NS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody:anti-rabbit FITC conjugate) are indicated. Arrows indicate SMN-positive nuclear bodies. Insets show nuclear regions of interest magnified by anadditional factor of 2. Bar � 30 �m. (B) SMN (primary antibody: anti-SMN monoclonal antibody MANSMA1; secondary antibody: TRITC) andNS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody: FITC) are indicated. Experiments were performed on NB324K cells 0 and30 h postinfection. Insets show nuclear regions of interest magnified by an additional factor of 2. Bar � 30 �m.



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for the peptide encoded by SMN exon 5. Endogenous SMNcoprecipitated the HA-tagged NS1 protein (Fig. 1B). NS1 wasnot detected when cells were not transfected or when theanti-SMN monoclonal antibody was omitted from the immu-noprecipitation reactions (Fig. 1B).

BIA was performed to confirm and further characterize theNS1-SMN interaction in vivo. To determine whether SMN andNS1 form a complex during MVM infection, highly synchro-nized A92L cells (23) were harvested at 30 h postrelease. BIAwas used to immunoprecipitate endogenous SMN with an anti-SMN monoclonal antibody. At 30 h postinfection, NS1 copre-cipitated with endogenous SMN (Fig. 1C, top). NS1 is notcoprecipitated with SMN from double-blocked noninfectedA92L cells (Fig. 1C, bottom; compare the horizontal plateausfor peak C in the top and bottom sensorgrams). A thoroughanalysis of the SMN-NS1 interaction is the subject of a manu-script in preparation (P. J. Young, K. T. Jensen, D. J. Pintel,and C. L. Lorson, unpublished data). Taken together theseresults demonstrate that NS1 and SMN interact in vivo and invitro and form a complex during viral infection.

NS1 localizes in SAABs in the later stages of MVM infec-tion. Nuclear SMN accumulates within Cajal bodies and thenucleolus (51, 52). At early time points (pre-15 h) after infec-tion, however, NS1 has been reported to localize in APARbodies, nuclear structures distinct from Cajal bodies. SinceNS1 and SMN interact avidly in vitro and in vivo during infec-tion, we performed the following series of indirect double-labelimmunofluorescence experiments to address this apparent dis-crepancy. First, HA epitope-tagged NS1 was transiently trans-fected into A92L cells. Cajal bodies were identified using anti-bodies specific for SMN, SIP1, and the Cajal body markerprotein, p80 coilin. By 30 h after transient NS1 transfection,NS1 colocalized with all three proteins within Cajal bodies(Fig. 2). As expected, Cajal bodies were positively stained forSMN and SIP1 in mock-transfected cells.

To determine whether this localization pattern was a conse-quence of NS1 transfection or whether similar distributionswould be observed following viral infection, double-label im-munofluorescence was performed on a series of highly syn-chronized A92L cells infected with MVM. Cells were harvested

FIG. 5. PML colocalizes with NS1 in SAABs at 20 h postinfection but not in A92L cells transiently expressing HA-tagged NS1. Double-labelexperiments were performed on A92L cells transfected with HA-tagged NS1 (TF) and blocked A92L cells 15, 20, and 30 h after infection with MVM(IF). PML (primary antibody: anti-PML monoclonal antibody; secondary antibody: anti-mouse TRITC conjugate) and NS1 (primary antibody:rabbit anti-NS1 polyclonal; secondary antibody: anti-rabbit FITC conjugate) are indicated. Insets show nuclear regions of interest magnified by anadditional factor of 2. Bar � 30 �m.



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FIG. 6. Cyclin A and cyclin E colocalize in SAABs. Double-label experiments were performed on A92L cells transfected with HA-tagged NS1(TF) and blocked infected A92L cells 15 and 30 h after entry into S phase (IF). (Top) Cyclin A (CyA) colocalizes with NS1 in transfected andinfected cells. Cyclin A (primary antibody: goat anti-cyclin A polyclonal; secondary antibody: anti-goat FITC conjugate) and NS1 (primaryantibody: rabbit anti-NS1 polyclonal; secondary antibody: anti-rabbit TRITC conjugate) are indicated. (Bottom) Cyclin E (CyE) accumulates inSAABs at around 20 h postinfection. Cyclin E (primary antibody: goat anti-cyclin E polyclonal; secondary antibody: anti-goat FITC conjugate) andNS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody: anti-rabbit TRITC conjugate) are indicated. Insets show nuclear regionsof interest magnified by an additional factor of 2. Bar � 30 �m.



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at multiple time points after release into S phase (0, 15, 20, and30 h). Consistent with previous reports, at 15 h postrelease,NS1-positive APAR bodies and Cajal bodies were independentnuclear structures (Fig. 3). In contrast, at 20 h postrelease,SMN, p80 coilin (Fig. 3), SIP1 (data not shown), and NS1 (Fig.3) colocalized in abnormally large nuclear structures. At sub-sequent time points (25 and 30 h), both the size and thenumber of these large nuclear bodies increased dramatically.Prior to release, nuclear structures displayed a normal distri-bution pattern.

To confirm that the observed nuclear body formation wasnot a consequence of the synchronization procedure, immu-nofluorescence was performed in parallel with synchronizedbut uninfected A92L cells. In highly synchronized uninfectedcells, SMN distribution was unaffected at 0 and 30 h postre-lease, resulting in small, infrequent nuclear bodies, consistentwith normal Cajal body staining and in contrast to the numer-ous large structures formed in A92L cells at 20 to 30 h postre-lease (Fig. 4A). These large nuclear bodies were also observedat late time points following infection of unblocked A92L cells,confirming that the distribution pattern observed was not adirect consequence of the blocking process (data not shown).This novel localization pattern was not simply a consequenceof a virus-induced overexpression of the various Cajal bodycomponents; BIA of total protein extracts from MVM-infectedA92L cells 0 and 30 h after the cells entered S phase demon-strated that there was no significant increase in the levels ofthese proteins (data not shown).

To determine whether these large nuclear bodies were spe-cific to murine cells, an MVM-permissive human cell line(NB324K) was also examined following MVM infection. At 0 hpostinfection, discrete Cajal bodies were observed (Fig. 4B).At 30 h postinfection, multiple large nuclear bodies formed,demonstrating that these large structures were not cell typespecific but rather were a consequence of viral infection. Sim-ilar to murine cells, the uninfected NB324K cells did not ex-hibit these large nuclear structures (data not shown).

PODs are nuclear structures originally identified in oncovi-rus infection which are distinct from Cajal bodies and othernuclear bodies such as interchromatin granules and gems. Todetermine the relationship between PODs and NS1-SMN-pos-itive bodies double-label immunofluorescence experimentswere performed on both HA-NS1-transfected and highly syn-chronized, MVM-infected A92L cells. In transfected A92L cells(30 h posttransfection) and in MVM-infected cells 15 h afterrelease into S phase, PODs and NS1 bodies were identified ascompletely independent structures (Fig. 5). However, at 20 and30 h postrelease, PML was present in the enlarged NS1-con-taining nuclear bodies (Fig. 5). Noninfected synchronized cellsexhibited normal POD nuclear localization patterns (data notshown). To distinguish the previously described APAR bodiesfrom the larger, POD-NS1-SMN-inclusive structures foundlater in infection, the latter have been termed SAABs. As withSMN and p80 coilin, BIA was used to determine whether theappearance of SAABs was accompanied by an increase in PMLexpression levels. Total protein extracts from A92L cells at 0

FIG. 7. Cyclin A nuclear distribution is not altered by the blocking process. Double-label experiments were performed on noninfected A92Lcells 0 and 30 h after release from the blocking process. Cyclin A (CyA; primary antibody: goat anti-cyclin A polyclonal; secondary antibody:anti-goat FITC conjugate) and NS1 (primary antibody: rabbit anti-NS1 polyclonal; secondary antibody: anti-rabbit TRITC conjugate) areindicated. In comparison, double-label experiments performed on blocked A92L cells 30 h postinfection are shown. Insets show nuclear regions ofinterest magnified by an additional factor of 2. Bar � 30 �m.



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and 30 h postrelease were collected and assayed on a sensorchip containing a bound anti-PML monoclonal antibody.There was no difference in PML protein level between the twotime points (data not shown), suggesting that our observationswere due to relocalization rather than increased expression ofPML.

Cyclin A and cyclin E accumulate in SAABs 30 h postinfec-tion. APAR bodies contain cyclin A but not cyclin E (5). Theseproteins are involved in the G1/S phase transition of the cellcycle (27). A role for cyclins in the viral life cycle has beensuggested (4). Similar to previous observations (4), at 15 hpostinfection, we found that APAR bodies contained cyclin Abut not cyclin E (Fig. 6). Following NS1 transfection, NS1colocalized with cyclin A and cyclin E in small NS1-positivenuclear bodies. However, at 30 h after release of MVM infec-tion both cyclin A and E accumulated within SAABs (Fig. 6).

The presence of SMN and p80 coilin within these SAABs wasconfirmed in parallel experiments (data not shown). Nuclearredistribution of cyclin A (Fig. 7) and cyclin E (data not shown)was not observed in blocked, noninfected A92L cells at all timepoints tested.

SAABs are likely sites of virus replication. Double-labelimmunofluorescence experiments were performed on synchro-nized A92L MVM-infected cells to determine whether the cap-sid proteins accumulate within SAABs. The smaller APARbodies identified at 15 h postinfection and the larger SAABsidentified at 30 h postinfection contained VP1, VP1-containingintermediates, and fully assembled MVM capsids (Fig. 8). Thecapsids are likely due to de novo synthesis since capsids are notdetected at earlier time points (data not shown). However,there were differences in the overall cellular distribution ofassembled capsids compared to those of VP1 and VP1-con-

FIG. 8. Viral capsid components (VP1) and the assembled MVM capsid accumulate within SAABs. Double-label experiments were performedon blocked infected A9 cells 15 and 30 h postrelease. (A) NS1 and VP1 colocalize at the 15- and 30-h time points. NS1 (primary antibody: anti-NS1polyclonal; secondary antibody: anti-rabbit FITC conjugate) and VP1 (primary antibody: anti-VP1 polyclonal peptide antibody; secondaryantibody: anti-mouse TRITC conjugate) are indicated. (B) NS1 and the assembled MVM capsid colocalize at the 15- and 30-h time points. NS1(primary antibody: anti-NS1 polyclonal; secondary antibody: anti-rabbit FITC conjugate) and the capsid (primary antibody: anticapsid monoclonalantibody [30]; secondary antibody: anti-mouse TRITC conjugate) are indicated. Insets show nuclear regions of interest magnified by an additionalfactor of 2. Bar � 30 �m.



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taining capsid assembly intermediates at 15 and 30 h postre-lease. At 15 h postrelease, assembled capsids were diffuselydistributed throughout the nucleoplasm, as were the capsid-positive APAR bodies (Fig. 8B). In contrast, preassembledVP1 capsid protein intermediates were localized mainly withinthe cytoplasm at this time, with lower levels colocalized withinnuclear APAR bodies (Fig. 8A). Both assembled and nonas-sembled capsids were exclusively confined to the larger SAABsat 30 h, with reduced nucleoplasmic and cytoplasmic staining.

Interchromatin granules or speckled domains are distinctnuclear bodies that contain a variety of RNA splicing factors,including U snRNPs and SR proteins (36, 47). Antibodiesagainst the SR proteins and the Sm core proteins were used indouble-label experiments with MVM-infected synchronizedA92L cells to determine whether SAABs contain factors essen-tial for RNA splicing. As expected, in synchronized uninfectedA92L cells (data not shown) and at 0 h postrelease, the Sm andSR proteins localized within interchromatin granules (Fig. 9).However, following MVM infection, SR and Sm core proteinscolocalized with NS1 in the small nuclear bodies at 15 h post-release and in SAABs at 20 and 30 h postrelease (Fig. 9).

APAR bodies have been shown to be the sites of ongoingviral DNA replication (14). To determine whether SAABs arethe sites of viral replication during the later stages of viralinfection, similar experiments were performed on infected cellspreviously labeled with BrdU. In this procedure, the lack of a

denaturation step ensures that, while replicating viral single-stranded DNA is detected, cellular chromosomal DNA is not(50). BrdU was identified with an anti-BrdU monoclonal an-tibody. BrdU colocalized with NS1 in both the smaller nuclearbodies identified at 15 h postinfection (Fig. 10) and the largerSAABs identified at 20 and 30 h postinfection (Fig. 10).

Taken together, these results identify a novel subnuclearstructure that is induced upon viral infection. Furthermore,this nuclear reorganization sequesters the components of sev-eral previously distinct nuclear bodies, suggesting that SAABsare the active site of viral replication and capsid assembly.

DISCUSSION

The eukaryotic nucleus is a highly organized organelle thatis composed of a number of discrete nuclear bodies (34). In-terest in the various nuclear bodies has increased recently as ithas become clear that these structures are not simply storagesites but rather are highly dynamic structures that can changein size, number, and constituents in response to internal andexternal stimuli (14, 31, 34, 45). Here we identify the SMNprotein as an MVM NS1-interacting protein. This was initiallysurprising since previous reports suggested that NS1 accumu-lated in nuclear structures that were independent of SMN-positive nuclear bodies (14). Nuclear SMN is normally a com-ponent of Cajal bodies and typically colocalizes with the Cajal

FIG. 9. RNA splicing factors accumulate within SAABs. Double-label analysis was performed on blocked infected A92L cells 0, 15, 20, and 30 hafter entry into S phase. Sm proteins (primary antibody: Y12; secondary antibody: anti-mouse TRITC conjugate), SR proteins (primary antibody:anti-SR monoclonal antibody; secondary antibody: anti-mouse TRITC conjugate), and NS1 (primary antibody: rabbit anti-NS1 polyclonal;secondary antibody: anti-rabbit FITC conjugate) are indicated. Insets show nuclear regions of interest magnified by an additional factor of 2. Bar� 30 �m.



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body marker protein p80 coilin (9, 28, 51, 52). To address thisapparent discrepancy, we investigated the distribution of NS1after transfection and following the progression of a highlysynchronized infection. Transiently expressed NS1 colocalizedwith SMN in Cajal bodies; however, nuclear bodies such asPODs and speckles remained separate. Consistent with theprevious reports of APAR bodies, at time points before 15 hpostinfection APAR bodies were SMN-independent structuresand were separate from PODs. However, this was in starkcontrast to the later time points, at which MVM infectioninduced dramatic nuclear changes, resulting in the accumula-tion of Cajal body, POD, speckle, and APAR componentswithin large SAABs. Similar structures were also seen at latetime points in infected, unsynchronized cells (data not shown).This dramatic reorganization of the nuclear bodies is likely aspecific response to viral infection since the transient expres-sion of NS1 alone (Fig. 2) or NS2 alone or NS1 and NS2combined (data not shown) did not result in SAAB formation.Additionally, in NS1-transfected cells infected with wild-typeMVM, transiently expressed NS1 and virus-derived NS1 arepresent in SAABs later than 15 h after release h after release(data not shown). Although the detailed kinetics of viral rep-lication is incomplete, significant amounts of DNA replication,capsid production, and encapsidation appear to occur at theselate times in infection when the cells have become arrested(49).

APAR bodies are previously described nuclear bodies thatappear immediately upon infection and that do not spatiallycolocalize with other known nuclear bodies such as Cajal bod-

ies, PODs, and speckles (14). Localization in APAR bodies islikely the initial step in an MVM infection. At early timepoints, a fraction of NS1 was found in APAR bodies but therewas also considerable diffuse cytoplasmic staining. As the in-fection progressed, however, essentially all detectable NS1 wasconcentrated within SAABs. Since NS1 is essential for severalphases of the viral life cycle, including replication and tran-scriptional regulation of the capsid promoter, these resultssuggest that SAABs may play a role in the viral life cycle. Thestatus of viral expression and replication past 20 h duringsynchronous infection, however, has not been well character-ized. Alternatively, this massive nuclear aggregation may rep-resent the beginnings of NS1-mediated cytotoxicity or a virus-induced cell death pathway.

The requirement for the infection process in the formationof SAABs suggests that nuclear reorganization occurs in re-sponse to a step within the viral life cycle rather than simply inresponse to the expression of an individual protein. Whetherthis is a consequence of an early stimulus, such as the enteringcapsid, viral single-stranded DNA, or the hairpin structures, orwhether the stimulus comes at some later time point is notknown. There are mutations within the MVM NS2 protein andcapsid genes that result in infections by viruses that are defi-cient in various stages of the viral life cycle, for example, theproduction of the monomer replicative form or progeny single-stranded template (10, 11, 38). Experiments to determinewhich of these steps is required for SAAB formation within theviral life cycle are ongoing.

PODs/PML bodies have been associated with transcription,

FIG. 10. SAABs are the sites of viral DNA replication. Double-label experiments were performed on BrdU-treated blocked infected A92L cells15, 20, and 30 h after entry into S phase. Cells were labeled for 20 min with BrdU at a concentration of 10 �M. BrdU (primary antibody: anti-BrdUmonoclonal antibody; secondary antibody: anti-mouse TRITC conjugate) and NS1 (primary antibody: rabbit anti-NS1 polyclonal; secondaryantibody: anti-rabbit FITC conjugate) are indicated. Insets show nuclear regions of interest magnified by an additional factor of 2. Bar � 30 �m.



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cell growth, and antiviral responses (17, 19, 26). DNA andRNA viruses also frequently target PODs, presumably to fa-cilitate the early stages of transcription and replication (1, 15,18, 20, 50). PODs can also be targeted for reorganizationfollowing viral infection (20). For example, adenovirus proteinE4-ORF3 localizes to PODs/PML bodies, thereby causing aphysical restructuring of the bodies from spherical to extendedfibril-like structures termed nuclear tracks (20). Additionally,many viral infections induce interferon expression, and theseinfections or treatment with exogenous interferon can increasethe size and number of PODs (21, 26). Although parvovirusinfection does not induce an interferon response, a dramaticrelocalization of PODs is seen late in MVM infection.

The functional significance of the interaction between SMNand NS1 is still unknown. However, as SMN has been impli-cated in RNA transcription and processing, cellular transpor-tation, and apoptosis (6, 9, 22, 25, 29), NS1 may sequesterSMN to facilitate viral RNA processing or to inhibit host cellapoptosis, thus enabling efficient viral turnover. Consistent withthe SMN-NS1 complex performing a role during the viral lifecycle is the recent finding that novel heterogeneous nuclear ribo-nucleoprotein-like protein NS1-associated protein 1 (NSAP1)interacts with SMN and NS1 (24, 37). The functional signifi-cance of these interactions has not been determined. SMN isnot required for the maintenance of nuclear structures such asCajal bodies (51, 52); therefore it is unlikely that SMN isrequired for SAAB formation. It is possible, however, thatSMN plays a role in targeting or retaining NS1 in SAABs.Alternatively, since SMN is involved in snRNP biogenesis andtranscriptional regulation, the NS1-SMN interaction may fa-cilitate an NS1 activity that is required at later time points ininfection.

We have identified SAABs, a novel nuclear structure re-quired for parvovirus infection, that contain factors typicallyassociated with other distinct nuclear bodies. The site of thisdramatic nuclear reorganization is also a site of viral replica-tion and contains the viral capsids, demonstrating that SAABsmay play a role in the viral life cycle. SAABs also contain cyclinA and cyclin E, suggesting that viral infection and viral cycleprogression may be dependent on the ability to control specificstages of the cell cycle. Although it has been suggested thatcyclin A is sequestered by APAR bodies early in viral infectionto arrest the cell in the G1 phase, thus enabling efficient ex-pression of NS1, the exact function of cyclin A and cyclin E inthe latter stages of infection within SAABs has yet to be de-termined. These results highlight the fluidity and dynamic na-ture of nuclear structures and their contents and help addressthe functional significance of nuclear compartmentalization. Inaddition, while this study has no direct link with spinal mus-cular atrophy, identifying the role SMN plays in facilitating thecourse of viral infection may shed light on the cellular functionof SMN.

ACKNOWLEDGMENTS

P.J. Young and K.T. Jensen contributed equally to this work.We thank Glenn Morris, Angus Lamond, and Colin Parish for an-

tibodies, Kori Wallace for technical assistance, and the W. M. KeckBioImaging Laboratory for confocal imaging.

P.J.Y. was supported by a fellowship from Families of SMA, andK.T.J. was supported by a postdoctoral fellowship from the Universityof Missouri Life Science Mission Enhancement Program. Funding for

these studies was provided by Families of SMA (C.L.L.), the MuscularDystrophy Association (C.L.L), and the National Institutes of Health(C.L.L., RO1 NS41584-01; D.J.P., RO1 AI21302 and RO1 AI46458).

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