Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins

Yu Kawasaki1,2, Shogo Matsumoto1 and Toshihiro Nagamine1

1 RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
2 Graduate School of Science and Engineering, Saitama University, Shimo-Okubo, Saitama City, Saitama 338-8570, Japan

Correspondence
Toshihiro Nagamine
tnaga{at}postman.riken.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
IE1, a principal transcriptional activator of the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), is an essential factor for viral DNA replication. During viral infection, IE1 accumulates in discrete subnuclear structures where viral DNA replication occurs. To analyse the dynamic properties of IE1, we monitored green fluorescent protein-tagged IE1 (IE1–GFP) in BmNPV-infected B. mori cells by live-cell microscopy. Time-lapse imaging showed that IE1-associated structures gradually expanded and occasionally fused with one another, while photobleaching experiments revealed that IE1–GFP was relatively immobile inside the IE1-associated structures. To investigate the spatial relationships between IE1 and viral structural proteins in infected cells, three GFP-tagged viral components were expressed together with DsRed-tagged IE1. Two structural proteins that constitute the occlusion-derived virus (ODV), P91–GFP and GFP–ODV-E25, localized to the periphery of the IE1-associated structures. While local accumulations of these proteins were often in contact with the IE1-associated structures, they did not extend beyond the boundaries of the structures. In contrast, the major capsid protein VP39–GFP predominantly accumulated within the IE1-associated structures. These data indicated, in conjunction with the finding of a high DNA content in the structures, that IE1 localizes to the virogenic stroma and therefore support the prediction previously proposed that the virogenic stroma is a site for viral DNA replication as well as for the assembly of nucleocapsids.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The expression product of the baculovirus immediate-early gene 1 (ie1), IE1, is a multifunctional protein that plays critical roles in baculovirus replication (reviewed by Friesen, 1997). IE1 was initially characterized using plasmid transfection assays as a transcriptional activator for baculovirus early gene expression (Guarino & Summers, 1986, 1987; Kovacs et al., 1991; Guarino & Smith, 1992; Nissen & Friesen, 1989; Pullen & Friesen, 1995; Blissard & Rohrmann, 1991; Theilmann & Stewart, 1991; Lu & Carstens, 1993; Ribeiro et al., 1994). In similar assays, IE1 has also been shown to be essential for the replication of plasmids carrying putative origins of baculovirus DNA replication (Kool et al., 1994; Ahrens & Rohrmann, 1995; Lu & Miller, 1995).

In Bombyx mori (BmN) cells, DNA replication of the baculovirus B. mori nucleopolyhedrovirus (BmNPV) occurs at discrete regions within the nucleus where IE1 and two DNA-binding proteins (DBP and LEF3) co-localize throughout the duration of DNA replication [<8 to approx. 24 h post-infection (p.i.); Okano et al., 1999]. Among these three proteins, IE1 initially localizes at small foci within the nucleus and, subsequently, the other two proteins associate with these foci prior to the onset of DNA replication to assemble viral DNA replication factories (Okano et al., 1999). During viral DNA replication within these IE1-associated structures, the structures gradually enlarge and ultimately occupy a large proportion of the infected-cell nuclei at the end of the replication period (24–36 h p.i.). Hence, temporal changes of IE1 localization seem to be correlated with viral DNA synthesis (Okano et al., 1999). Consequently, extensive studies on IE1 localization may serve to facilitate our understanding of the multifunctional roles of IE1 in virus replication. Previous experiments examining IE1 localization were performed using fixed cell samples; however, live-cell imaging has become a powerful tool for measuring intracellular dynamics and therefore would greatly facilitate analysis of IE1 and its dynamic properties. In living cells, time-lapse studies can track the movement of larger cellular structures, whereas fluorescence recovery after photobleaching (FRAP) is ideal for measuring the intracellular mobility of fluorescently tagged proteins (Lippincott-Schwartz & Patterson, 2003).

Observations of baculovirus-infected cells by electron microscopy have shown that progeny nucleocapsids assemble in a virus-induced subnuclear structure, called the virogenic stroma (reviewed by Williams & Faulkner, 1997). This stroma consists of two distinct regions: one is a fibrillar electron-dense area, while the other is an electron-lucent intrastromal space in which progeny nucleocapsids are formed (Summers, 1971; Harrap, 1972; Young et al., 1993). A major capsid protein of 39 kDa, VP39 (Pearson et al., 1988; Blissard et al., 1989; Thiem & Miller, 1989), has been shown to accumulate in this intrastromal space (Braunagel et al., 1996a). In contrast to the intrastromal space, the electron-dense region is considered to be DNA rich and can be intensively stained with DNA-specific fluorescent dyes (Williams & Faulkner, 1997; Rosas-Acosta et al., 2001). In addition to its role in the maturation of nucleocapsids, the virogenic stroma is assumed to be the most probable site of viral DNA replication because of its high DNA content (Williams & Faulkner, 1997). However, to date, no study has established a direct relationship between DNA replication sites and the virogenic stroma.

Following maturation within the virogenic stroma, the nucleocapsids leave the stroma, with some of them acquiring their envelopes within the peristromal regions of the nucleus (Kawamoto et al., 1977a, b; Fraser, 1986). This type of virion, called an occlusion-derived virus (ODV), is eventually encapsulated in an occlusion body within the nucleus after intranuclear envelopment (reviewed by Funk et al., 1997). Baculoviruses can also produce another type of virion, termed a budded virus (BV). Nucleocapsids destined to become BVs are transported from the nucleus to the plasma membrane and obtain BV envelopes by budding through the plasma membrane instead of the intranuclear envelopment (reviewed by Funk et al., 1997). In studies examining the intracellular localization of viral proteins by fluorescent microscopy, a number of ODV-associated proteins (e.g. ODV-E25, ODV-E66, P91 and P74) were shown to localize to the periphery of the nucleus (Russell & Rohrmann, 1997; Braunagel et al., 1999; Rosas-Acosta et al., 2001; Slack et al., 2001). More detailed observations with immunoelectron microscopy, however, have revealed that these proteins are mainly localized to the peristromal region in association with ODVs and intranuclear microvesicles (Russell & Rohrmann, 1993, 1997; Braunagel et al., 1996a, b; Hong et al., 1997). The intranuclear microvesicles are the proposed source of ODV envelopes (Hong et al., 1997). Thus, the peripheral regions of the nucleus in which ODV-associated proteins often accumulate are considered to be the peristromal regions for intranuclear envelopment and are assumed to be a subnuclear compartment distinct from the virogenic stroma (Hong et al., 1997).

In this report, we have described the use of green fluorescent protein-tagged IE1 (IE1–GFP) to demonstrate the dynamic behaviour of IE1 in BmN cells infected with BmNPV. We have also shown, using other GFP-tagged proteins, P91–GFP, GFP–ODV-E25 and VP39–GFP with DsRed-tagged IE1 (IE1–DsRed), that IE1 localized to subnuclear structures that were separate from the ODV-associated proteins, P91 and ODV-E25, but were associated with the major capsid protein, VP39. These subnuclear structures were also shown to contain large amounts of DNA. These data revealed the specific localization of IE1 in the virogenic stroma and this conclusion was consistent with a previous theory, which proposed that the virogenic stroma is a site for viral DNA replication, as well as for the assembly of nucleocapsids (Williams & Faulkner, 1997).


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
BmN cells were maintained in TC100 medium (Funakoshi Co.) supplemented with 10 % FBS (Okano et al., 1999). BmNPV wild-type isolate T3 (Maeda & Majima, 1990) was propagated in BmN cells.

Plasmid constructions.
To make a plasmid expressing GFP-tagged IE1 under the control of the ie1 promoter, its open reading frame (ORF) and 760 bp upstream region were inserted into a promoterless GFP vector, pEGFP-1 (Clontech). The termination codon of the ie1 coding sequence and the next 3 bp (TGAATT) in a plasmid containing the EcoRI-G fragment [map unit (m.u.) 90·5–96·4] of the BmNPV genome were replaced with the DNA sequence CTCGAG by means of site-directed mutagenesis for XhoI digestion. The mutagenized plasmid was digested with EcoRI and XhoI and the resulting fragment, which contained the ie1 ORF and its 760 bp upstream region, was inserted into the EcoRI–SalI sites of pEGFP-1. To align the two coding sequences of ie1 and GFP in the same reading frame, the recombinant plasmid was digested with BamHI, treated with mung bean nuclease and ligated intramolecularly. The resulting plasmid, pKm-IE1-GFP was partially sequenced to confirm that it was the desired construct. Plasmid pKm-IE1-DsRed was constructed by replacement of the KpnI–NotI fragment in pKm-IE1-GFP with the corresponding fragment of pDsRed (Clontech) and by alignment of the ie1 and DsRed coding sequences to the same reading frame. This was performed by AgeI digestion, mung bean nuclease treatment and intramolecular ligation. To construct plasmids expressing P91–GFP and P91–DsRed under the control of the p91 promoter (pPK-p91-GFP and pPK-p91-DsRed, respectively), the coding sequences of GFP and DsRed were inserted into the PstI-K fragment (m.u. 48·4–52·3) of the BmNPV genome that contains the p91 gene. The termination codon and the 3'-flanking region of the p91 coding sequence (TAAACATTTTATGT) in the PstI-K fragment were replaced with a DNA sequence containing XhoI and SpeI sites (CTCGAGTTACTAGT) by two cycles of site-directed mutagenesis. The resulting plasmid was digested with XhoI and SpeI and ligated with the XhoI–XbaI fragments of pEGFP-1 and pDsRed2-1 (Clontech) containing the coding sequences of GFP and DsRed, respectively. Plasmids expressing GFP–ODV-E25 and VP39–GFP under the control of their own promoter (pPES-oe25-GFP and pPES-vp39-GFP, respectively) were generated in a procedure similar to that for P91–GFP. The PstI–SphI subfragment (m.u. 52·3–57·0) in the PstI-E fragment of the BmNPV genome was mutagenized. For the production of pPES-oe25-GFP, the 12 bp 5'-flanking region (immediately in front of the initiation codon) of the ODV-E25 coding sequence (TTAAAACAAATC) was replaced with the NheI–XhoI-cutting sequence (GCTAGCCTCGAG). The NheI–SalI fragment of pEGFP-C1 (Clontech) containing the GFP ORF was subsequently inserted into the NheI–XhoI site. Plasmid pPES-vp39-GFP was constructed by replacing the termination codon of the vp39 coding sequence and the next 9 bp (TAAAAATGGAGT) with the XhoI–NheI-cutting sequence (CTCGAGGCTAGC) followed by insertion of the XhoI–XbaI fragment of pEGFP-1 containing the GFP ORF into the XhoI–NheI site.

Plasmid transfections and viral infections.
BmN cells (8x105) were seeded onto 27 mm glass-bottomed dishes (Matsunami) and left to stand for several hours to allow cell attachment. To introduce plasmids, the cells were transfected with 0·5 µg of each plasmid DNA sample using Lipofectin reagent (Invitrogen). The transfected cells were incubated at 28 °C for 24 h and directly analysed with a confocal microscope or infected with BmNPV at an m.o.i. of 10. In all experiments, time zero was defined as the time point at which fresh medium was added following the 1 h virus adsorption period. For DNA staining, BmN cells were plated onto 22x22 mm coverslips (no. 1; Matsunami) and allowed to attach over a period of several hours. Cells were transfected using Lipofectin reagent (Invitrogen) and subsequently infected with BmNPV. Infected cells were fixed in pre-chilled methanol at –20 °C for 6 min, incubated with propidium iodide (PI; 1 µg ml–1) and RNase A (100 µg ml–1) in PBS at 37 °C for 30 min and mounted with the Slow Fade light anti-fade kit (Molecular Probes).

Confocal microscopy and FRAP.
Confocal images were obtained with a Leica TCS NT using a 488 nm laser line for GFP and a 568 nm laser line for DsRed and PI. For double-labelling experiments, only one laser line was used for any single-channel recording to avoid cross talk between channels. To obtain merged figures, single-channel images were digitally superimposed with Photoshop 6.0 software. In FRAP experiments, Leica software (TCS NT version 1.6) was used to bleach a spot with a 100 ms pulse at high laser intensity and to acquire time-lapse images of the bleached cells. Quantification of fluorescence intensity was also done using Leica software.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Viral infection induces temporal changes in IE1–GFP localization, which resembles the behaviour of untagged IE1
To investigate the intracellular dynamics of IE1 in living cells, we constructed a reporter plasmid under the control of the authentic ie1 promoter expressing GFP fused to the C terminus of IE1. Observations of BmN cells transfected with this plasmid by confocal microscopy revealed that fluorescence signals from IE1–GFP were predominantly distributed within the nucleus and in particular within the nucleoplasm (Fig. 1a–c). While most cells exhibited nuclear fluorescence exclusively, faint cytoplasmic fluorescence could be detected simultaneously with strong nuclear fluorescence in some cells (data not shown). The IE1–GFP distribution (i.e. nucleoplasmic localization) was similar to that of an untagged IE1 produced by transfection with a plasmid carrying the entire ie1 gene (Prikhod'ko & Miller, 1999). This result demonstrated that IE1–GFP exhibited the same nuclear translocation ability as that of untagged IE1.



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Fig. 1. Dynamic localization patterns of IE1–GFP. BmN cells were transfected with a plasmid expressing IE1–GFP (a–c) and subsequently infected with BmNPV (d–o). Transfected and infected cells were directly analysed by confocal microscopy at 24 h post-transfection and at the indicated time points p.i., respectively. For each time point, IE1–GFP fluorescence images (a, d, g, j and m), differential interface contrast images (b, e, h, k and n) and merged images (c, f, i, l and o) are shown.

 
During the viral life cycle, the distribution of IE1 changes temporally depending on the infection process (Okano et al., 1999). To examine the localization of IE1–GFP in the context of viral infection, BmN cells expressing IE1–GFP were infected with BmNPV. As shown in Fig. 1(d–f), in addition to the overall nuclear fluorescence, small foci of IE1–GFP fluorescence could be detected in the nucleus of the infected cells at 2 h p.i. At 4 h p.i., IE1–GFP foci could be observed more clearly than those at 2 h p.i. (Fig. 1, compare g with d) and resembled IE1 foci stained with anti-IE1 antibody in the nuclei of BmNPV-infected cells (Okano et al., 1999). At 8 h p.i., when viral DNA synthesis begins and the foci of untagged IE1 undergo expansion (Okano et al., 1999), the IE1–GFP foci likewise began to expand (Fig. 1j–l), and at 24 h p.i., as with untagged IE1, they occupied a large proportion of the infected-cell nuclei (Fig. 1m–o). These results indicated that IE1–GFP exhibited the same dynamic localization as untagged IE1 and that it could be used as a reporter to trace IE1 localization in insect cells.

Time-lapse imaging of IE1–GFP during viral infection demonstrates the dynamics of IE1-associated structures
To follow the temporal changes of IE1 localization in a single cell, we performed time-lapse recording of IE1–GFP-expressing cells using a confocal microscope. At 4–5 h p.i., small IE1 foci were clearly observed in most of the BmNPV-infected BmN cells (see Fig. 1). As seen in Fig. 2, time-lapse monitoring of these cells revealed a progressive expansion of IE1-associated structures. The onset time of this expansion varied considerably (i.e. some started at 5–6 h p.i., while others started at 10–11 h p.i.) in cells that were infected for 1 h at an m.o.i. of 10. Our employment of an asynchronous culture for this experiment may have been the cause of the observed variation (Braunagel et al., 1998; Ikeda & Kobayashi, 1999). Typically, a stationary (pre-expansion) phase of the bright IE1-associated structures required 2–3 h, after which the IE1-associated structures began to expand. In cells possessing multiple IE1-associated structures, we observed a tendency among the structures to undergo simultaneous expansion, which often resulted in their fusing with one another. This highly dynamic stage of expansion and fusion usually persisted for 3–4 h and the subsequent enlargement of the IE1-associated structures was thereafter maintained, although the structure appeared to shrink after polyhedron formation (data not shown).



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Fig. 2. Time-lapse observation of a single cell expressing IE1–GFP. BmN cells were transfected with a plasmid expressing IE1–GFP and subsequently infected with BmNPV. At the indicated time points p.i. (hour : minute), one of the infected cells was analysed by confocal microscopy. Because maximum intensities of fluorescent signals were normalized among panels, direct comparisons of signal intensities among these panels could not be done.

 
Mobility of IE1–GFP is restrained within IE1-associated structures
We next examined whether the mobility of IE1–GFP varied depending on its location (i.e. whether it was in an IE1-associated structure or the nucleoplasm) using FRAP. After irreversible bleaching of a cluster of IE1–GFP molecules with a 100 ms pulse of the 488 nm laser line, we measured the recovery of fluorescence intensity in the bleached spot (i.e. influx of active IE1–GFP molecules from the outside of the bleached spot). IE1–GFP transiently expressed in uninfected cells could be detected throughout the nucleoplasm (Fig. 1a–c). Bleaching a spot of the nucleoplasm with the laser pulse resulted in a moderate decrease in fluorescence intensity, but did not drop it to background levels, even at the first scan (0·7 s) after the pulse (Fig. 3a and d). However, photobleaching following fixation with 4 % paraformaldehyde resulted in a clearly observable dark spot corresponding to the bleached area, which persisted for the duration of the experimental time period (Fig. 3b and d). Therefore, it was unlikely that the ambiguous spot in the non-fixed cell was due to insufficient power of the laser pulse. Rather, the fluorescence recovery seemed to be too quick to allow for detection of the early phase of the recovery kinetics using our laser microscope. In contrast, when spots in the IE1-associated structures that formed in BmNPV-infected cells at 8 and 24 h p.i. were bleached, their fluorescence recoveries were extremely slow (Fig. 3c and d), requiring approximately 30 min for full recovery (data not shown). This implied that IE1 molecules residing in IE1-associated structures are relatively immobile and are tethered within the structures via fairly stable associations.



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Fig. 3. FRAP on BmN cells expressing IE1–GFP. BmN cells were transfected with a plasmid expressing IE1–GFP (a) and then fixed with 4 % paraformaldehyde (b) or infected with BmNPV (c). Spots indicated by the arrowheads in pre-bleached cells (–5 s) were photobleached and the cells were imaged at the indicated time points. Time zero was defined as the time point when the first scan following the photobleach finished. (d) Quantitative FRAP analysis of IE1–GFP in uninfected cells ({blacktriangleup}), uninfected and fixed cells (x), infected cells at 8 h p.i. ({bullet}) and infected cells at 24 h p.i. ({blacksquare}). Relative fluorescence values represent the mean±SD of five independent photobleaching experiments.

 
Two ODV-associated proteins, P91–GFP and GFP–ODV-E25 become concentrated at the periphery of IE1-associated structures
In previous studies, a number of ODV-associated proteins, including P91 and ODV-E25, were shown to be localized around the periphery of the nucleus (Russell & Rohrmann, 1997; Braunagel et al., 1999; Rosas-Acosta et al., 2001; Slack et al., 2001). These distributions are in sharp contrast to the location of the IE1-associated structure that often occupies the central region of the nucleus at 24 h p.i. (Okano et al., 1999, and Fig. 1). To investigate the exact spatial relationship between the IE1-associated structure and the perinuclear region in which the ODV-associated proteins were localized, we constructed plasmid vectors to express either GFP-tagged P91 (P91–GFP) or GFP-tagged ODV-E25 (GFP–ODV-E25) under the control of their own promoters and compared their subcellular localization to that of DsRed-tagged IE1 (IE1–DsRed). Fluorescent signals from IE1–DsRed exhibited dynamics of IE1-associated structures similar to those of IE1–GFP, while IE1–DsRed foci were less distinct than IE1–GFP foci early in infection (data not shown). A tendency of DsRed to oligomerize may explain the indistinct appearance. Before making comparisons with the IE1-associated structure, we examined the subcellular localizations of P91–GFP and GFP–ODV-E25 in BmN cells transfected with a single vector expressing either P91–GFP or GFP–ODV-E25, instead of co-expressing with IE1–DsRed. When we observed the cells that were transfected with the P91–GFP construct and then infected with virus, P91–GFP was localized around the perimeter of the nucleus at 24 h p.i. (Fig. 4a–c) and subsequently polyhedra formed in the region accumulating P91–GFP (data not shown). This distribution of P91–GFP was similar to that of untagged P91 (Russell & Rohrmann, 1997), indicating that the GFP tag did not interfere in the intracellular localization of the P91 molecules and that P91–GFP could be employed as a marker for live-cell imaging. The distribution pattern of GFP–ODV-E25 was likewise similar to that of untagged ODV-E25 (Braunagel et al., 1999; Rosas-Acosta et al., 2001), i.e. GFP–ODV-E25 fluorescence concentrated at the periphery of the nucleus at 24 h p.i. (Fig. 4d–f). Since the two ODV-associated proteins showed similar distributions, we compared their perinuclear localizations directly using GFP–ODV-E25 and DsRed-tagged P91 (P91–DsRed). Despite a relatively weak signal, the P91–DsRed localization pattern was similar to that of P91–GFP (data not shown). As seen in Fig. 4(g–i), local accumulation of GFP–ODV-E25 and P91–DsRed exhibited some degree of overlap, suggesting that these two proteins were, as reported previously, co-ordinately assembled in the ODV envelopment sites to generate ODVs (Hong et al., 1997; Russell & Rohrmann, 1997).



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Fig. 4. Subcellular localization of P91–GFP and GFP–ODV-E25. BmN cells were transfected with plasmids expressing P91–GFP (a–c) or GFP–ODV-E25 (d–f), or were co-transfected with plasmids expressing GFP–ODV-E25 and P91–DsRed (g–i), P91–GFP and IE1–DsRed (j–l), or GFP–ODV-E25 and IE1–DsRed (m–o). Transfected cells were infected with BmNPV and analysed by confocal microscopy at 24 h p.i.

 
We then investigated the spatial arrangement of the IE1-associated structures in relation to the ODV envelopment sites by simultaneous observation of the two marker proteins, IE1–DsRed and either P91–GFP or GFP–ODV-E25. As seen in Fig. 4(j–o), P91–GFP and GFP–ODV-E25 concentrated in regions adjacent to the IE1-associated structures. While these regions were often in contact with the IE1-associated structures, they failed to extend beyond the boundaries of the IE1-associated structures. This observation suggested that ODV envelopment occurred outside the IE1-associated structures.

IE1-associated structures expand with progressive DNA concentration
As described above, the IE1-associated structures were surrounded by accumulations of the ODV-associated proteins, P91 and ODV-E25 (Fig. 4 j–o). Furthermore, previous studies have shown that the ODV-associated proteins accumulate at the periphery of the virogenic stroma (Russell & Rohrmann, 1993, 1997; Hong et al., 1997). Taken together, these two independent observations lead to speculation that IE1 associates with the virogenic stroma. To test this possibility, we stained IE1–GFP-expressing cells with the DNA-specific fluorescent dye, PI, which allows for visualization of the virogenic stroma given its high DNA concentration (Williams & Faulkner, 1997; Rosas-Acosta et al., 2001). In most of the uninfected interphase cells, both IE1–GFP and DNA were distributed throughout the nucleoplasm (Fig. 5a–c). A certain proportion of the uninfected cells were present in the mitotic phase, which could be distinguished by the alignment of condensed DNA at the metaphase plate (Fig. 5b, arrow). In these cells, IE1–GFP also concentrated at the same place (Fig. 5a and c, arrows), suggesting that IE1 could co-localize with cellular chromosomes independent of specific viral DNA sequences, such as hr (homologous region). When compared with the uninfected cells, the infected cells at 4 h p.i. showed no difference in the DNA staining pattern (Fig. 5e), whereas small IE1–GFP foci were visible (Fig. 5d). At 8 h p.i., DNA staining in the marginal regions of the nuclei was slightly emphasized (Fig. 5h). This pattern seemed to be consistent with previous observations by electron microscopy, which showed that the heterochromatin becomes progressively marginalized along the inner nuclear membrane (Williams & Faulkner, 1997). Although DNA replication in the IE1-associated structures occurs at this time, the increased concentration of DNA in the structures could not be detected by PI staining. At 24 h p.i., however, the increase in DNA within the structures was clearly evident (Fig. 5j–l). This result suggested that IE1 becomes associated with the virogenic stroma, i.e. as the DNA progressively accumulates, the IE1-associated structure develops into the virogenic stroma.



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Fig. 5. DNA accumulation in IE1-associated structures. BmN cells were transfected with a plasmid expressing IE1–GFP and then mock-infected or infected with BmNPV. Uninfected (a–c) and infected cells at 4 h (d–f), 8 h (g–i) and 24 h (j–l) p.i. were fixed and stained with the DNA-specific fluorescent dye, PI. For each time point, IE1–GFP fluorescence images (a, d, g and j), PI fluorescence images (b, e, h and k) and merged images (c, f, i and l) are shown. Arrows indicate cellular chromosomes on the metaphase plate.

 
The major capsid protein, VP39, accumulates predominantly within IE1-associated structures
To verify that IE1 was associated with the virogenic stroma, we investigated the intracellular localization of the major capsid protein fused to GFP, VP39–GFP, in comparison with IE1. In cells transfected with the VP39–GFP gene and subsequently infected with virus, the distribution of VP39–GFP at 24 h p.i. presented a fine lattice-like appearance within the subnuclear regions (Fig. 6a–c). This localization of VP39–GFP was consistent with a previous observation showing VP39 accumulation within the intrastromal space of the virogenic stroma (Braunagel et al., 1996a) and therefore indicated that VP39–GFP could be used as a marker for the virogenic stroma. We then performed co-expression experiments with VP39–GFP and IE1–DsRed in BmN cells. The result in Fig. 6(d–f) revealed that a large proportion of VP39–GFP co-localized with IE1–DsRed, suggesting that VP39 accumulated largely within the IE1-associated structures. Collectively, we concluded that IE1 localizes to the virogenic stroma.



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Fig. 6. Subcellular localization of VP39–GFP. BmN cells were transfected with a plasmid expressing VP39–GFP (a–c) or were co-transfected with plasmids expressing VP39–GFP and IE1–DsRed (d–f). Transfected cells were infected with BmNPV and analysed by confocal microscopy at 24 h p.i.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In the cycle of baculovirus infection, IE1 localizes to the viral DNA replication site, which expands concomitantly with DNA synthesis (Okano et al., 1999). To study the intracellular dynamics of IE1 in living cells, we constructed plasmid vectors expressing fluorescent protein-tagged IE1 molecules and employed these tagged proteins as markers of IE1 dynamics. In BmNPV-infected BmN cells, IE1–GFP and IE1–DsRed showed the same localization patterns as untagged IE1 protein. This result indicated that the fluorescent protein tags did not interfere with IE1 localization and that IE1–GFP and IE1–DsRed were viable markers for live-cell imaging. At present, however, details of their functionality are still unclear. Since IE1–GFP associates with cellular chromosomes at the metaphase plate during metaphase, it seems that IE1–GFP can bind DNA either directly or indirectly. Moreover, co-transfection with two vectors carrying genes for IE1–DsRed and GFP–ODV-E25 led to significant expression of GFP–ODV-E25, even in uninfected cells (data not shown), whereas no expression of GFP–ODV-E25 was observed in uninfected cells that had been transfected with a single vector carrying the GFP–ODV-E25 gene. This suggested that IE1–DsRed possessed an activator function at least for the promoter of the ODV-E25 gene. Furthermore, we have recently isolated a recombinant virus that carries the IE1–GFP gene instead of the endogenous, wild-type ie1 gene (unpublished result), implying that IE1–GFP is functionality equivalent to untagged IE1.

The virogenic stroma is a virus-induced nuclear structure in which progeny nucleocapsids are assembled (Williams & Faulkner, 1997). In this study, we have provided a number of lines of evidence showing that IE1 localizes to the virogenic stroma (i.e. that IE1-associated structures are identical to the virogenic stroma): (i) ODV-associated proteins P91 and ODV-E25 accumulated at the periphery of IE1-associated structures; (ii) the IE1-associated structures could be intensively stained with a DNA-specific dye; and (iii) the major capsid protein, VP39, was almost exclusively distributed in the IE1-associated structures. While this is the first report describing the localization of IE1 to the virogenic stroma, it has been shown previously that IE1 localizes to the site of viral DNA replication (Okano et al., 1999). These two independent findings are consistent with the previously proposed idea that the virogenic stroma is the site of viral DNA replication (Williams & Faulkner, 1997). The virogenic stroma begins to form around the time when viral DNA replication is initiated (Williams & Faulkner, 1997) and the IE1-associated structure expands coincidently with DNA synthesis (Okano et al., 1999). These observations may also support the identity of the two subnuclear structures, the virogenic stroma and the IE1-associated structure. Hence, we concluded that the virogenic stroma provides the site not only for nucleocapsid assembly but also for viral DNA replication.

Formation of the virogenic stroma (IE1-associated structure) may serve as an intranuclear compartmentalization to promote baculovirus replication. We have recently found that IE1 focus formation is induced by the binding of IE1 to the hr sequences of viral genomes (Nagamine et al., 2004). If this is the case, then the IE1 foci containing parental genomes may provide initial scaffolds to facilitate assembly of the DNA replication complex or factory (Okano et al., 1999). With the onset of DNA synthesis, the IE1 foci begin to expand and develop into the virogenic stroma, which can hold the newly synthesized DNA within its own structure, thereby making it possible to accelerate the chain reaction of DNA replication. Furthermore, the concentration of DNA within the virogenic stroma may be advantageous to the assembly of nucleocapsids within the structure. After DNA synthesis, the nucleus is most likely divided into at least two subnuclear compartments, one being the virogenic stroma and the other the periphery of the virogenic stroma, namely the peristromal compartment (Williams & Faulkner, 1997). Formation of this compartment seems to be dependent on the virogenic stroma, since P91–GFP and GFP–ODV-E25 often accumulate at the edge of the IE1-associated structure (data not shown). At this late stage of the infection cycle, the virogenic stroma becomes the supplier of progeny nucleocapsids, while the peristromal compartment serves as the site of intranuclear envelopment of nucleocapsids and subsequently as the site of polyhedron formation. Hence, the partitioning of the nucleus into these compartments may be necessary for spatial segregation of the two steps of virion formation: nucleocapsid assembly and intranuclear envelopment. These two events may be unable to occur at the same place for as yet unknown reasons. The intranuclear membrane formation is a unique feature of baculoviruses and, therefore, nuclear partitioning may also be a unique requirement for baculovirus infection. The virogenic stroma may have evolved from this requirement but still maintained its roles for providing the site of DNA replication and nucleocapsid assembly.

IE1–GFP within the IE1-associated structure exhibited slow fluorescence recovery in FRAP experiments. This immobility of IE1–GFP may imply a structural role for IE1 in the virogenic stroma. While further experiments are still needed, our results may provide new insight into the structural aspect of the virogenic stroma and show the multifunctional nature of the nuclear structure that provides the site for DNA replication and nucleocapsid assembly.


   ACKNOWLEDGEMENTS
 
We thank Masaaki Kurihara for help with the culture of BmN cells and Jimmy Joe Hull for critical reading of the manuscript. This research was supported by the Bioarchitect Research Program and the Chemical Biology Research Program from RIKEN.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 5 July 2004; accepted 25 August 2004.