IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites

WonKyung Kang1, Noriko Imai1, Yu Kawasaki1,2, Toshihiro Nagamine1 and Shogo Matsumoto1

1 Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan
2 Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan

Correspondence
WonKyung Kang
wkkang{at}riken.jp


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1–110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1–78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The transcription and replication of eukaryotic DNA viruses occur in specific regions of the nucleus (Maul, 1998). Recent evidence suggests that DNA replication of baculoviruses that are pathogenic for insects also occurs at specific regions in the nucleus. By visualizing the localization of replication factors such as IE1, LEF3 and DBP, Okano et al. (1999) demonstrated that DNA replication of Bombyx mori nucleopolyhedrovirus (BmNPV) occurs at discrete sites in the nucleus of BmN cells. They showed that IE1 initially forms foci in the nucleus; DBP and LEF3 then associate with these IE1 foci, which gradually enlarge as the infection proceeds (Okano et al., 1999). They also suggested that foci occupied by replication factors such as IE1 are the DNA replication sites, since the distribution of these proteins overlapped well with that of sites labelled with bromodeoxyuridine incorporation. In addition to an examination of fixed cells, IE1 localization was also investigated in living cells using green fluorescent protein tagged IE1 (IE1-GFP). Kawasaki et al. (2004) recently reported that IE1-GFP exhibits the same localization pattern as that of authentic IE1 in living cells, suggesting that focal formation of IE1 is absolute. However, in these experiments IE1-GFP focus formation followed infection by BmNPV. In contrast, transfection of DNA fragments containing the ie1 gene resulted in a diffused IE1 localization throughout the nucleoplasm rather than specific focal formation (Nagamine et al., 2005). Consequently, Nagamine et al. (2005) demonstrated, using an IE1 mutant protein defective in hr binding, that IE1 requires the hr elements of the viral genome for the focal distribution, and that direct binding of IE1 to hr is essential.

The orf8 gene of BmNPV is one of a set of genes unique to group I NPVs. The Autographa californica NPV (AcNPV) homologue, referred to as da26 or bv/odv-e26, has been well characterized (Beniya et al., 1998; O'Reilly et al., 1990). The da26 gene has been shown to stimulate late gene expression in cooperation with da41 in AcNPV (Guarino & Summers, 1988). O'Reilly et al. (1990) further showed that da26 is expressed as an early gene. In addition, Beniya et al. (1998) reported that da26 encodes an envelope protein common to budded virus and occlusion-derived virus. Although BmNPV ORF8 shares high identity to DA26 of AcNPV (96 % identical at the amino acid level), the function of BmNPV ORF8 appears quite different from its AcNPV counterpart. Imai et al. (2004) have recently demonstrated that the ORF8 protein is not associated with the BmNPV virion and that instead ORF8 colocalizes with IE1 to specific nuclear sites during infection. This suggests that ORF8 is one of the viral components that occupy nuclear-specific regions for viral DNA replication. To further examine this, we assessed the localization of GFP-fused ORF8 derivatives upon transient expression. In this study, we show that IE1 and hr elements facilitate the localization of ORF8 to specific nuclear sites where viral DNA replication occurs.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus, cell line, transfection and infection.
The BmN-4 (BmN) cell line was maintained in TC-100 with 10 % fetal bovine serum as described by Maeda (1989). The BmNPV T3 isolate (Maeda et al., 1985) was propagated on BmN cells as described by Maeda (1995). For confocal analysis, BmN cells (5x105) were seeded on 27 mm glass-bottomed dishes (Matsunami). Plasmid DNA (1 µg), described below, pSK-ie1 (Kang et al., 1999), and/or pBS-hr3 (Nagamine et al., 2005) were transfected into BmN cells using Lipofectin, according to the manufacturer's instructions (Gibco-BRL). At 24 h post-transfection, the cells were subjected to confocal analysis, infection (m.o.i. of 10), or Western blotting analysis.

Plasmid constructions.
For the yeast two-hybrid system, a DNA fragment containing the complete open reading frame (ORF) of the orf8 gene was amplified by PCR using BmNPV genomic DNA and specific primers, POrf8-sal and POrf8-spe (Table 1), and then inserted into pDBLeu (Gibco-BRL) after digestion with SalI and SpeI (creating plasmid pDB-orf8). The deletion mutants were also generated by PCR amplification, using pDB-orf8 as the template. For the C-terminal deletions, either POrf8-dC1 or POrf8-dC2 (Table 1) was used with PDB-1 (Gibco-BRL), the amplified fragment was digested with Sal I and inserted into the SalI and StuI restriction sites of pDBLeu. For the N-terminal deletions, either POrf8-dN1 or POrf8-dN2 was used with PDB-2 (Gibco-BRL). The amplified fragment was inserted into pDBLeu after digestion with Sal I and NotI. The nucleotide sequences of all the constructs were confirmed using an ABI sequencer.


View this table:
[in this window]
[in a new window]
 
Table 1. Oligonucleotides used for PCR

 
To express fusion proteins with GFP, the plasmids p(hsp-EGFP)-C and p(hsp-EGFP)-N (Imai et al., 2005) were utilized. DNA fragments were amplified by PCR using BmNPV genomic DNA and the primers described in Table 1. For the full-length orf8 gene, PSt-fw and PEnd-rv were used. For the C-terminal deletions, either PN-rv1 or PN-rv2 was used with PSt-fw, while either PC-fw2 or PC-fw1 was used with PEnd-rv for the N-terminal deletions. The resulting PCR products were then digested with BamHI and SalI, and inserted into p(hsp-EGFP)-C and p(hsp-EGFP)-N. No differences were found between the C-terminal and N-terminal constructions regarding expression pattern, protein levels or localization (data not shown). The constructs used in this study were fused to the C-terminus of GFP. To transiently express ORF8, phsp-ORF8 was constructed by inserting the BamHI–SalI fragment of plasmid pGFP-ORF8, containing the intact orf8 gene, into the BamHI and SalI sites of phspMC (Imai et al., 2005).

To obtain a mutant IE1 protein that is deficient in hr binding, site-specific substitutions were introduced into the IE1-encoding DNA by overlapping PCR (Higuchi, 1990) using pSK-ie1, and the corresponding mutagenic primers shown in Table 1 (PIE1-m1 and PIE1-m2) and flanking pBluescript SK+ specific primers (T3 and T7). The resulting PCR products were digested with XbaI and KpnI, and inserted into the XbaI and KpnI sites of pBluescript SK+.

Yeast two-hybrid screening.
Yeast two-hybrid screening was performed using the ProQuest two-hybrid system (Gibco-BRL) as described previously (Kang et al., 2003). Primary transformants were selected for growth on histidine-dropout plates containing 25 mM 3-aminotriazole. His+ colonies were subsequently analysed for {beta}-galactosidase activity by filter-lift experiments. Plasmids from positive clones were rescued by transformation of Escherichia coli with total yeast DNA. Each plasmid DNA was amplified in E. coli and used for further analyses, including nucleotide sequencing and retransformation with pDB-orf8 into yeast to confirm reporter expression.

Confocal microscopy and Western blotting.
Confocal microscopy was performed as described by Okano et al. (1999), Kawasaki et al. (2004) and Nagamine et al. (2005). Confocal images were obtained with a Leica TCS NT using a 488 nm laser line for GFP. SDS-PAGE was performed in 10 % polyacrylamide gels as described by Laemmli (1970) with Precision Plus protein standards (Bio-Rad) as size markers. The Western blotting protocol, immunohistochemical experiment and anti-ORF8 polyclonal antibodies were described previously (Imai et al., 2004). Antibodies against GFP were purchased from Molecular Probes.


   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cytoplasmic localization of transiently expressed GFP-ORF8
Since the ORF8 protein of BmNPV has been shown to localize specifically in the nucleus during infection, we examined whether ORF8 is able to form foci when transiently expressed. To do this, we constructed a reporter plasmid in which the intact ORF for the orf8 gene was fused to the 3' end of the GFP-encoding gene and expressed from the Drosophila hsp70 promoter (pGFP-ORF8). Confocal microscopy showed that GFP-ORF8 fluorescence was distributed mainly in the cytoplasm and did not exhibit nuclear localization (Fig. 1a, c). This suggests that ORF8 requires other viral factor(s) for nuclear foci formation. To confirm this, pGFP-ORF8 transfected BmN cells were subsequently infected with BmNPV and the localization of GFP-ORF8 was then observed. As shown in Fig. 1 (b, d), subsequent infection with BmNPV resulted in nuclear localization of GFP-ORF8 as well as the formation of GFP-ORF8 foci. This pattern was reminiscent of the ORF8 localization observed in cells during the early stage of infection, supporting the theory that ORF8 does indeed require other viral factor(s) for focus formation.



View larger version (132K):
[in this window]
[in a new window]
 
Fig. 1. Distribution of GFP-ORF8. BmN cells were transfected with a plasmid expressing GFP-ORF8 (a, c) and subsequently infected with BmNPV (b, d). (a, b) GFP-ORF8 fluorescence images. (c, d) Differential interface contrast images of the same fields as shown in (a, b). Bar, 10 µm. p.i., post-infection.

 
Identification of BmNPV IE1 as an interacting protein
In order to identify viral factor(s) required for the nuclear localization of ORF8, the yeast two-hybrid system was used. The complete orf8 gene was fused to the DNA encoding the GAL4 DNA-binding domain in pDBLeu (pDB-orf8). We first confirmed that the GAL4–ORF8 fusion protein was not sufficient to activate expression of reporter genes by itself (data not shown). Using pDB-orf8 as bait, we screened a cDNA library constructed using mRNA purified from BmNPV-infected cells at 2 h post-infection. This time point was chosen because expression of orf8 begins in the early stage of infection. Yeast colonies that were first selected on medium lacking leucine, tryptophan and histidine were subsequently assayed for {beta}-galactosidase activity. Plasmids were then isolated from these positive clones and rescued by electroporation into an E. coli strain. These positive plasmids were then characterized by nucleotide sequencing and a database search for homologous sequences. The nucleotide sequences of two independent plasmids were homologous to ie1, suggesting an interaction between ORF8 and IE1. Since both plasmids included the full-length cDNA of ie1, only one of them was used for further examination.

In order to confirm the interaction between ORF8 and IE1, and to define the region of ORF8 that is responsible for the interaction with IE1, we constructed four deletion mutants containing aa 1–110, 1–78, 110–230 or 73–230 (referred to as pDB-orf8dC1, pDB-orf8dC2, pDB-orf8dN1 and pDB-orf8dN2, respectively). A coiled-coil domain is predicted to be present between residues 78–110 in ORF8; therefore, deletion mutants were designed for the purpose of either including or excluding this domain. The mutant constructs, pDB-orf8dC1 and pDB-orf8dN2, included the coiled-coil domain, while the other two constructs did not. Each of these constructs or pDB-orf8 was co-transformed with one of the positive plasmids encoding IE1 into the yeast and assayed for reporter expression. Reporter expression was reproduced using intact ORF8 and IE1, confirming the interaction between ORF8 and IE1 (Fig. 2). In addition, the C-terminal deletion mutant, ORF8dC1, containing residues 1–110, likewise activated reporter expression while the other deletions resulted in a loss of interaction with IE1 (Fig. 2). This suggests that the N-terminal region containing the coiled-coil domain is responsible for the interaction with IE1.



View larger version (8K):
[in this window]
[in a new window]
 
Fig. 2. Analysis of the interaction between ORF8 and IE1 using the yeast two-hybrid system. On the left are diagrams showing the structure of ORF8 and the portion of it encoded by each plasmid. The open box indicates ORF8, while the predicted coiled-coil domain (residues 78–110) within ORF8 is shown by a closed box. The thick black lines indicate the regions contained in the deletion mutants. On the right, the results of the {beta}-galactosidase assay are shown. + or – indicates positive or negative for interaction with IE1, respectively.

 
Construction of ORF8 deletion mutants fused with GFP
To examine whether IE1 plays a role in the nuclear localization of ORF8, we prepared ORF8 deletion mutant proteins fused to the C-terminus of GFP. The region included in each mutant was exactly the same as for the constructs used in the yeast two-hybrid analysis. To confirm that these constructs expressed properly, BmN cells transfected with one of the plasmids were harvested at 24 h post-transfection and subjected to Western blot analysis using antiserum against ORF8 or GFP. The anti-ORF8 antibody recognized all five ORF8 derivatives with immunoreactive bands at 55, 35, 40, 40 and 45 kDa, which likely correspond to GFP fused with intact ORF8, ORF8dC2, ORF8dC1, ORF8dN1 and ORF8dN2, respectively (Fig. 3). In addition, these immunoreactive bands were also recognized by an anti-GFP antibody (Fig. 3). These results confirmed that the ORF8 derivatives were adequately fused with GFP.



View larger version (59K):
[in this window]
[in a new window]
 
Fig. 3. Western blotting analyses of GFP-fused ORF8 derivatives. Extracts of BmN cells transfected with plasmids containing GFP-fused ORF8 (lane 1), ORF8dC2 (lane 2), ORF8dC1 (lane 3), ORF8dN1 (lane 4) or ORF8dN2 (lane 5) were subjected to Western blotting analysis using either anti-ORF8 or anti-GFP antibodies. Plasmid p(hsp-EGFP)-C, expressing GFP only, was transfected and used as a control (lane 6). Closed circles indicate the immunoreactive bands. Size markers are indicated on the left.

 
IE1 and hr3 facilitate the nuclear localization of ORF8
We assessed the localization of the transiently expressed GFP-fused ORF8 derivatives with and without IE1. We first confirmed that the presence of IE1 did not alter the localization of GFP itself in transfected cells (Fig. 4a, panels i and ii). When a GFP-orf8 construct containing the intact orf8 gene was transiently expressed in BmN cells, GFP-ORF8 fluorescence was distributed throughout the cytoplasm and did not show nuclear localization (Fig. 4b, panel iv). This was also shown in Fig. 1(a). The presence of IE1, however, changed the distribution of GFP-ORF8 from the cytoplasm to the nucleus (Fig. 4b, panel v). GFP-ORF8 showed granular localization in the nucleus but significant focus formation was not observed. This suggests that IE1 is the factor mediating the nuclear localization of ORF8 and that focus formation of ORF8 requires some other factor(s).



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 4. Effect of IE1 and hr on the localization of ORF8. BmN cells were transfected with p(hsp-EGFP)-C (a), a plasmid encoding GFP-fused ORF8 (b) or phsp-ORF8 (c). Cotransfections with a plasmid expressing IE1, pSK-ie1 (panels ii, v, viii), or pSK-ie1 and pBS-hr3 (panels iii, vi, ix), or neither plasmid (panels i, iv, vii) were also performed. BmN cells transfected with phsp-ORF8 were subjected to immunohistochemistry at 24 h post-transfection using a polyclonal anti-ORF8 antiserum (c). The top panels are fluorescence images of GFP (i, ii, iv, v, vii, viii) or IE2 immunofluorescence images (iii, vi, ix) and the lower ones are differential interface contrast images of the same fields as shown in the top panels. Bar, 10 µm.

 
Recently, it has been reported that the hr region of the BmNPV genome is required for the focal distribution of IE1 (Nagamine et al., 2005). Therefore, we examined if GFP-ORF8 forms foci in the nucleus when transiently expressed in the presence of IE1 and hr. Prior to doing this, we confirmed that IE1 and hr3 did not alter the localization of GFP in transfected cells (Fig. 4a, panels i and iii). In the presence of IE1 and hr3, GFP-ORF8 showed focus formation (Fig. 4b, panel vi). To confirm this, ORF8 was transiently expressed by itself (Fig. 4c, panel vii) or in the presence of IE1 and hr3 and the localization was visualized using anti-ORF8 antibodies. This immunohistochemical experiment showed that ORF8 localized to the nucleus as well as the cytoplasm, but that the presence of IE1 resulted in nuclear accumulation of ORF8 (Fig. 4c, panel viii), and the combined presence of IE1 and hr3 showed focus formation (Fig. 4c, panel ix). Taken together, these data imply that IE1 facilitates nuclear localization of ORF8 and that the hr region of the viral genome is required for focus formation of ORF8.

To further investigate this, we observed whether the presence of IE1 and hr3 changed the localization of transiently expressed GFP-fused ORF8 mutant derivatives. When expressed alone the mutant constructs had the following localization patterns. GFP-ORF8dC1 (containing residues 1–110) and GFP-ORF8dN2 (containing residues 73–230) were distributed throughout the cell (Fig. 5a, d, panels i and x). GFP-ORF8dC2 (containing residues 1–78) was distributed throughout the cytoplasm (Fig. 5b, panel iv), while GFP-ORF8dN1 (containing residues 110–230) localized in the nucleus (Fig. 5c, panel vii). In the presence of IE1, GFP-ORF8dC1 accumulated within the nucleus (Fig. 5a, panel ii), while the presence of IE1 had no effect on the localization pattern of the other mutants (Fig. 5b, c, d, panels v, viii and xi). In addition, GFP-ORF8dC1 formed foci in the presence of IE1 and hr3 (Fig. 5a, panel iii). The combined presence of IE1 and hr3 had no effect on the localization of the other mutants (Fig. 5b,c,d, panels vi, ix and xii). Using the yeast two-hybrid system, the region included in ORF8dC1 was shown to be essential for interaction with IE1. Thus, we conclude that an interaction with IE1 is important for the nuclear localization of ORF8.



View larger version (78K):
[in this window]
[in a new window]
 
Fig. 5. Localization of GFP-fused ORF8 derivatives in the presence of IE1 and hr3. BmN cells were transfected with one of the plasmids containing GFP-fused ORF8dC1 (a), ORF8dC2 (b), ORF8dN1 (c) and ORF8dN2 (d). Cotransfections with either a plasmid expressing IE1, pSK-ie1 (panels ii, v, viii and xi), or pSK-ie1 and pBS-hr3 (panels iii, vi, ix and xii), or neither plasmid (i, iv, vii and x) were also performed. The top panels (i–xii) are fluorescence images of GFP-fused proteins, and the lower ones (i'–xii') are differential interface contrast images of the same fields. Bar, 10 µm.

 
Direct binding of IE1 to hr mediates the localization of ORF8 to specific regions in the nucleus
Nagamine et al. (2005) previously demonstrated using a mutant IE1 deficient in hr-binding activity that direct binding of IE1 to the hr element is essential for the focal localization of IE1. In this mutant, two basic residues (K162/K163) in the hr-binding domain were replaced with noncharged residues (G162/S163). Our results also demonstrated that the hr region is involved in the focus formation of ORF8. Therefore, we examined whether foci were observed when a mutant ie1 lacking hr-binding ability was cotransfected with GFP-orf8 and hr3 instead of wild-type (wt) ie1. As shown in Fig. 6(a), the presence of IE1 (both wt and mutant) appeared to mediate the nuclear localization of GFP-ORF8 (Fig. 6a, panels i and iii). However, focus formation of GFP-ORF8 was observed only in the presence of wt IE1 and hr3 (Fig. 6a, panel ii). Mutant IE1 failed to form foci despite the presence of hr3 (Fig. 6a, panel iv). To understand the population of foci in transfected cells, we counted the number of foci (Fig. 6b). GFP-ORF8 failed to form foci in approximately 90 % of the cells transfected with wt ie1 alone while more than one focus per nucleus was observed in approximately 50 % of the cells transfected with wt ie1 and hr3. In addition, focus formation of GFP-ORF8 was not observed in approximately 80 % of the cells transfected with mutant ie1, and the presence of hr3 had no effect on this. These data suggest that the direct binding of IE1 to hr is important for the focus formation of ORF8.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 6. Effect of mutant IE1 on the distribution of GFP-ORF8. (a) BmN cells were transfected with a plasmid containing GFP-fused ORF8. A plasmid containing wt or mutant (mt) IE1 was used for cotransfection in the absence (panels i and iii, respectively) or presence of hr3 (panels ii and iv, respectively). +hr3 or –hr3 indicates the presence or absence of hr3, respectively. (b) Population of transfected cells containing GFP-ORF8 foci. The transfected cells in (a) were used for counting the number of foci per nucleus. Black bars, wt IE1+hr3; grey bars, wt IE1–hr3; white bars, mt IE1+hr3; hatched bars, mt IE1–hr3.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we demonstrated that BmNPV ORF8 requires viral factors for focus formation in the nucleus. We also demonstrated that a direct interaction between ORF8 and IE1 is essential for mediating the nuclear localization of ORF8. In addition, we showed that IE1 and hr3 facilitate the localization of ORF8 to specific nuclear sites.

Using a yeast two-hybrid screen, we were able to identify IE1 as an interacting partner of BmNPV ORF8. In addition, we demonstrated that the N-terminal region of ORF8 is required for this interaction. A coiled-coil domain in this region might play an important role for IE1 interaction since a C-terminal deletion mutant lacking this domain did not interact with IE1. However, one of the N-terminal deletion mutant (residues 73–230) proteins failed to interact with IE1 despite the presence of the coiled-coil domain, suggesting that other domain(s) in the N-terminal half of the protein are also required for the interaction with IE1.

The BmNPV ORF8 protein localizes specifically in the nucleus and colocalizes with IE1 to specific nuclear sites during infection (Imai et al., 2004). However, transient expression of GFP-ORF8 resulted in cytoplasmic localization of the protein, but following BmNPV infection the localization of GFP-ORF8 changed from the cytoplasm to the nucleus. Since IE1 was identified as an ORF8 interacting factor using a yeast two-hybrid system, we investigated whether IE1 was one of the factors involved in the nuclear localization of ORF8. As expected, GFP-ORF8 localized to the nucleus in the presence of IE1. Transient expression of ORF8 showed that ORF8 was distributed throughout the cell, suggesting that ORF8 may be small enough to freely pass the nuclear pores whereas GFP-ORF8 may not be, so it is excluded. The presence of IE1, however, changed the localization of ORF8, as well as GFP-ORF8 to principally the nucleus, suggesting that IE1 facilitates the nuclear localization of ORF8. Furthermore, the presence of IE1 changed the subcellular localization of a deletion mutant protein (accumulated in the nucleus) that interacted with IE1 in the yeast two-hybrid assay, but had no effect on deletion mutant proteins that did not interact with IE1 in the assay. These results suggest that a direct interaction between ORF8 and IE1 is essential for the nuclear localization of ORF8.

Although GFP-ORF8 localized in the nucleus in the presence of IE1, focus formation was not observed, suggesting that ORF8 focus formation requires some other factor(s). Since Nagamine et al. (2005) earlier reported that IE1 is incapable of forming nuclear foci alone and that hr elements are required for focus formation, we examined whether the hr region is likewise involved, and demonstrated that GFP-ORF8 indeed forms foci in the presence of IE1 and hr3. The BmNPV ORF8 protein is capable of binding to nucleic acids (Imai et al., 2004); therefore, ORF8 could bind hr directly. However, our results using the IE1 mutant demonstrated that the focus formation of ORF8 is mediated by the direct binding of IE1 to hr rather than by ORF8 to hr. The nuclear-specific regions occupied by replication factors such as IE1 are the sites for viral DNA replication (Okano et al., 1999). Our transient-expression experiments also confirmed that ORF8 colocalizes with IE1 at such sites. These observations strongly suggest a possibility that BmNPV ORF8 may be involved in viral DNA replication.

It was suggested that BmNPV ORF8 is essential for virus replication since a null mutant virus was not isolated (Imai et al., 2004). However, a truncated mutant virus (BmD8) that still expresses the N-terminal half of ORF8 was isolated. It was shown that viral DNA replication was not affected in BmD8, but less progeny virus were produced than in wt BmNPV (Imai et al., 2004). Consequently, it was suggested that it is possible that ORF8 plays a role in viral DNA replication because the N-terminus of ORF8 is sufficient for this function. In our present study, we showed that the N-terminal region of ORF8 is essential for IE1 interaction, which is necessary for the nuclear localization of ORF8. The truncated protein expressed by BmD8 contains this IE1 interacting region, suggesting that the interaction is essential and that viable virus cannot be obtained if the region is deleted. This is also supported by our finding that the truncated protein has exactly the same localization pattern as the wt ORF8 does, and likewise colocalizes with IE1 in nuclear foci in BmD8 infected cells (unpublished data). On the basis of these findings, we suggest that ORF8 plays an important role in virus replication by localizing at specific sites for viral DNA replication. Further analyses, including identification of ORF8 interacting proteins, should provide more information regarding ORF8 function during viral infection.


   ACKNOWLEDGEMENTS
 
We thank J. J. Hull for critical reading of the manuscript, M. Kurihara for providing BmN cells, and Y. Ichikawa and R. Nakazawa (Bioarchitect Research Group, RIKEN) for DNA sequencing. This research was supported by grants from Grant-in-Aid for Scientific Research (C) (15580044) (W. K.), Grant-in-Aid for Young Scientists (B) (16780041) (N. I.) from The Ministry of Education, Culture, Sports, Science and Technology (N. I.), and the Bioarchitect program of the Science and Technology Agency of Japan.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Beniya, H., Braunagel, S. C. & Summers, M. D. (1998). Autographa californica nuclear polyhedrosis virus: subcellular localization and protein trafficking of BV/ODV-E26 to intranuclear membranes and viral envelopes. Virology 240, 64–75.[CrossRef][Medline]

Guarino, L. A. & Summers, M. D. (1988). Functional mapping of Autographa californica nuclear polyhedrosis virus genes required for late gene expression. J Virol 62, 463–471.[Medline]

Higuchi, R. (1990). Recombinant PCR. In PCR Protocol, a Guide to Methods and Application, pp. 177–183. Edited by M. A. Gelfand, J. J. Sninsky & T. J. White. San Diego, CA: Academic Press.

Imai, N., Kurihara, M., Matsumoto, S. & Kang, W. (2004). Bombyx mori nucleopolyhedrovirus orf8 encodes a nucleic acid binding protein that colocalizes with IE1 during infection. Arch Virol 149, 1581–1594.[Medline]

Imai, N., Matsumoto, S. & Kang, W. (2005). The formation of BmNPV IE2 nuclear foci is regulated by the functional domains for oligomerization and ubiquitin ligase activity. J Gen Virol 86, 637–644.[Abstract/Free Full Text]

Kang, W., Suzuki, M., Zemskov, E., Okano, K. & Maeda, S. (1999). Characterization of baculovirus repeated open reading frames (bro) in Bombyx mori nucleopolyhedrovirus. J Virol 73, 10339–10345.[Abstract/Free Full Text]

Kang, W., Imai, N., Suzuki, M., Iwanaga, M., Matsumoto, S. & Zemskov, E. A. (2003). Interaction of Bombyx mori nucleopolyhedrovirus BRO-A and host cell protein LAMININ. Arch Virol 148, 99–113.[CrossRef][Medline]

Kawasaki, Y., Matsumoto, S. & Nagamine, T. (2004). Analysis of baculovirus IE1 in living cells: dynamics and spatial relationships to viral structural proteins. J Gen Virol 85, 3575–3583.[Abstract/Free Full Text]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Maeda, S. (1989). Gene transfer vectors of a baculovirus, Bombyx mori, and their use for expression of foreign genes in insect cells. In Invertebrate Cell System Applications, vol. I, pp. 167–181. Edited by J. Mitsuhashi. Boca Raton, FL: CRC Press.

Maeda, S. (1995). Further development of recombinant baculovirus insecticides. Curr Opin Biotechnol 6, 313–319.[CrossRef][Medline]

Maeda, S., Kawai, T., Obinata, M., Fujiwara, H., Horiuchi, T., Saeki, Y., Sato, Y. & Furusawa, M. (1985). Production of human {alpha}-interferon in silkworm using a baculovirus vector. Nature 315, 592–594.[CrossRef][Medline]

Maul, G. G. (1998). Nuclear domain 10, the site of DNA virus transcription and replication. Bioessays 20, 660–667.[CrossRef][Medline]

Nagamine, T., Kawasaki, Y., Iizuka, T. & Matsumoto, S. (2005). Focal distribution of baculovirus IE1 triggered by its binding to the hr DNA elements. J Virol 79, 39–46.[Abstract/Free Full Text]

Okano, K., Mikhailov, V. S. & Maeda, S. (1999). Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories. J Virol 73, 110–119.[Abstract/Free Full Text]

O'Reilly, D. R., Passarelli, A. L., Goldman, I. F. & Miller, L. K. (1990). Characterization of the DA26 gene in a hyper variable region of the Autographa californica nuclear polyhedrosis virus genome. J Gen Virol 71, 1029–1037.[Abstract]

Received 21 June 2005; accepted 22 July 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Kang, W.
Articles by Matsumoto, S.
PubMed
PubMed Citation
Articles by Kang, W.
Articles by Matsumoto, S.
Agricola
Articles by Kang, W.
Articles by Matsumoto, S.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS