1 Department of Veterinary Pathobiology, Life Sciences Center 471G, University of Missouri, Columbia, MO 65211-7310, USA
2 Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA
3 Novozymes, 1AS.31, Novo Alle, DK-2880 Bagsværd, Denmark
Correspondence
Christian L. Lorson
lorsonc{at}missouri.edu
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ABSTRACT |
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These authors contributed equally to this work.
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MAIN TEXT |
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The eukaryotic nucleus is highly organized into numerous distinct, yet highly dynamic, subnuclear structures (Lamond & Carmo-Fonseca, 1993; Lamond & Sleeman, 2003
; Lamond & Spector, 2003
; Matera, 2003
; Platani & Lamond, 2004
). A novel subnuclear structure has been identified following infection by the two highly related parvoviruses MVM and H-1 (Bashir et al., 2000
; Cziepluch et al., 2000
). These structures, termed autonomous parvovirus-associated replication (APAR) bodies, were identified at early time points (up to 15 h) post-infection and were found to be distinct from several of the classically described nuclear bodies, including Cajal bodies, PML bodies (PODs) and interchromatin granules (ICGs) (Bashir et al., 2001
; Cziepluch et al., 2000
). At later time points during infection, however, a dramatic nuclear reorganization occurs, in which formerly distinct nuclear structures merge into massive structures that contain NS1 (Young et al., 2002a
, b
). These structures are probably active sites of virus production (Young et al., 2002a
), but their role during the infection process is not yet known. In addition, the survival motor neuron (Smn) gene product is present in these large structures (hence the term Smn-associated APAR bodies or SAABs). The Smn protein is involved in a variety of cellular activities, including small nuclear ribonucleoprotein biogenesis and functioning as an RNA chaperone (Gubitz et al., 2004
; Paushkin et al., 2002
). Smn is the first cellular factor identified that interacts with NS1 and NS2 in vitro and in vivo during infection (Young et al., 2002a
, b
).
We recently reported a direct interaction between Smn and NS2, and our initial observations indicated that the two proteins colocalized within SAABs during MVM infection (Cotmore & Tattersall, 1986, 1987
; Young et al., 2002a
, b
). To characterize the nature of the NS2-positive subnuclear structures formed during infection more extensively, we performed confocal immunofluorescence assays (Young et al., 2002a
, b
) on highly synchronized A92L cells infected with wild-type MVM (Schoborg & Pintel, 1991
; Young et al., 2002a
). NS2 localization was monitored at 0, 10, 15, 20 and 30 h post-release and cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to identify nuclei. For all immunofluorescence experiments, parallel reactions were performed with primary antibody or secondary antibody alone to confirm the specificity of the staining (data not shown). At early time points, NS2 was predominantly cytoplasmic, although small nuclear foci were also detected, consistent with the induction of APAR bodies following wild-type MVM infection at early time points (Fig. 1a
). At later time points, although still significantly cytoplasmic, NS2 became enriched in the nucleus into subnuclear structures that increased in number and size as the infection proceeded (Fig. 1a
). The appearance and temporal formation of the NS2-positive nuclear bodies was consistent with our previous analysis of NS1 and SAABs (Young et al., 2002a
, b
).
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Only a limited number of cellular proteins have been shown to interact with the non-structural proteins of MVM. To determine the cellular distribution of a previously described NS2-interacting protein, Crm1 (Bodendorf et al., 1999; Brockhaus et al., 1996
), during wild-type MVM infection, similar confocal analyses were performed on highly synchronized MVM infected A92L cells. Typically, Crm1 is enriched at the nuclear periphery, consistent with its role as a nuclear/cytoplasmic transport factor (Fornerod et al., 1997
). In highly synchronized A92L cells at 0 h post-release, Crm1 was enriched at the nuclear periphery (Fig. 1d
), similar to previous descriptions (Brunet et al., 2002
; Fornerod et al., 1997
). However, in contrast to non-synchronized human cells (Brunet et al., 2002
; Fornerod et al., 1997
), the localization of Crm1 in highly synchronized A92L cells also appears to be localized diffusely throughout the cytoplasm (Fig. 1d
). NS2 and Crm1 remained localized diffusely in the cytoplasm up to 15 h post-release and, at this point, the accumulation of Crm1 at the nuclear periphery was reduced compared to the 0 h time point. However, at time points beyond 15 h post-release, Crm1 became enriched in SAABs (Fig. 1d
). At these later time points, Crm1 colocalized with the majority of NS2 within SAABs, demonstrating that these NS2-interacting proteins are also SAAB components. 14-3-3, an additional NS2-interacting factor, was examined similarly and was shown to redistribute to SAABs after 15 h time points (data not shown). Additional experiments were performed with anti-NS1 and anti-actin antibodies (Fig. 1e
). As expected, actin exhibited a typical cytoskeletal-staining pattern and did not localize within the NS1-staining SAABs at 30 h post-infection, demonstrating that specific cellular components are present in SAABs (Fig. 1e
).
It is unclear which aspect of the viral infection process induces parvovirus-associated nuclear-body formation (e.g. APAR bodies and SAABs). Whilst NS1 is responsible for the majority of MVM-induced cytotoxicity (Anouja et al., 1997; Corbau et al., 2000
), ectopic expression of NS1 and/or NS2 (data not shown) is not sufficient to induce SAAB formation. The following experiments were designed to determine whether NS2 was necessary for SAAB formation in the context of a viral infection. A92L cells were infected at relatively high m.o.i. by the mutant MVM NS2 : 1989; a virus that does not express NS2 due to a splice-site mutation that prevents the production of the NS2-encoding R2 mRNA (Naeger et al., 1990
). At 15 h post-release, little change is observed in the typical subcellular distributions of a number of SAAB factors, including Smn (Fig. 2a
), Sm core proteins (Fig. 2b
), p80 coilin, SR proteins and PML (data not shown). At this time, APAR bodies are detectable, as evidenced by the majority of NS1-positive nuclear foci clearly being separate from the Smn-positive nuclear gems (Fig. 2a
). However, at 30 h post-release, these formerly distinct nuclear components were beginning to merge, consistent with the formation of SAABs (Fig. 2a, b
). The number and size of these structures, however, were decreased compared to a wild-type MVM infection at the same time point post-release. SAAB formation has clearly been initiated at 30 h post-release, albeit at a slower rate. In parallel experiments, similar infections were allowed to proceed to 42 h post-release and, at this time point, SAAB formation and the nuclear organization of infected cells closely resembled those of a wild-type MVM infection at 2530 h post-release (data not shown). Taken together, these results demonstrate that NS2 is not essential for SAAB formation. MVM mutant viruses encoding subtle NS2 mutants that specifically disrupt the interactions with the cellular factors Crm1and 14-3-3 did not alter the nature or kinetics of SAAB formation (data not shown). These results are consistent with the notion that, whilst NS2 is required for efficient replication, it is not essential for SAAB formation.
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SAAB formation does not seem to be a general cellular response to viral infection, as other viral infections do not induce SAAB formation. Nuclear reorganization, however, is a well-characterized hallmark of a number of pathogens, such as adenovirus and herpesvirus. Whilst adenovirus infection results in the formation of virus replication centres, the molecular constituents are distinct from SAABs (data not shown).
This work also reports an analysis of NS2 subcellular distribution during a highly synchronized MVM infection. Our results are consistent with the previously described ability of NS2 to shuttle between the cytoplasm and the nucleus (Bodendorf et al., 1999; Cotmore & Tattersall, 1990
). We find that, early in infection, NS2 is predominantly cytoplasmic and becomes more nuclear at later time points in infection. However, it is unclear whether hyper- or hypophosphorylated NS2 accumulates in SAABs. NS2 redistribution could result from altered NS2 phosphorylation levels or from a global dysregulation of nuclear integrity.
It has become increasingly clear that subnuclear compartments are not merely storage sites, but serve critical cellular functions throughout the cell cycle (Matera & Frey, 1998; Matera, 1999
; Ogg & Lamond, 2002
). Whether these MVM-specific subnuclear structures represent a viral-induced reorganization that benefits the viral life cycle or whether SAABs represent a cellular defence mechanism in response to a viral infection is a critical question that is currently being investigated. Whilst the majority of the cytotoxic effects of an MVM infection have been attributed to NS1, it is interesting that ectopically expressed NS1 (and/or NS2) cannot induce SAAB formation (Young et al., 2002a
; data not shown), suggesting that these structures are not formed in response to these apoptotic or cytotoxic stimuli. Determining the nature of APAR bodies and SAABs and the roles that they play during the parvovirus life cycle will provide insight into the viral life cycle, as well as the interface between host and pathogen.
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ACKNOWLEDGEMENTS |
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Received 30 August 2004;
accepted 20 December 2004.
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