Molecular Entomology Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan
Correspondence
WonKyung Kang
wkkang{at}riken.jp
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ABSTRACT |
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INTRODUCTION |
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Baculovirus ie2 encodes a protein that functions as a transregulator for transcription of viral genes (Carson et al., 1988, 1991
; Theilmann & Stewart, 1992
; Yoo & Guarino, 1994a
, b
) and is also involved in viral DNA replication (Gomi et al., 1997
; Kool et al., 1994
; Lu & Miller, 1995
; Prikhod'ko et al., 1999
). In spite of these important functions, it is not well understood how IE2 works. Previously, we showed using a yeast two-hybrid system and a cross-linking assay that BmNPV IE2 interacts with itself (Imai et al., 2000
). The 80 aa C-terminal coiled-coil region was identified as an interacting region. We have also demonstrated that BmNPV IE2 has a ubiquitin ligase E3 activity that is dependent on its RING finger domain (Imai et al., 2003
). Therefore, these data suggest that IE2 is capable of performing its functions by forming oligomers and ubiquitylating target proteins.
Analysis of BmNPV IE2 distribution in BmN cells showed that IE2 formed nuclear foci during infection. In this study, we showed that BmNPV IE2 localization in such characteristic domains did not require any other viral factors. Furthermore, we demonstrated that IE2 focus formation was regulated by two functional regions involved in oligomerization and ubiquitin ligase activity.
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METHODS |
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Construction of ie2 derivatives.
To express ie2 derivatives transiently in BmN cells, we produced an expression vector, phspMC, containing the Drosophila melanogaster heat-shock protein (hsp) 70 promoter (GenBank accession no. J01104). The hsp fragment of the LacZ gene cassette (Gomi et al., 1997) was inserted into the BglII and HindIII sites of pEGFP-1 (Clontech) and the EGFP region was then removed by digestion with AgeI and NotI and self-ligation. To generate phsp-IE2wt, the ie2-coding region was amplified by PCR using pPC-ie2 (Imai et al., 2000
) as a template, which encodes the full-length ie2 gene, and primers pPC-1 (5'-GAATAAGTGCGACATCATCATC-3') and pPC-2 (5'-GTAAATTTCTGGCAAGGTAGAC-3'). The amplified product was digested using SalI and inserted into the SalI and SmaI sites of phspMC. Plasmids phsp-IE2N1, phsp-IE2N3 and phsp-IE2C3 were likewise constructed by PCR using pPC-ie2N1, pPC-ie2N3 and pPC-ie2C3 as templates, respectively (Imai et al., 2000
). Two other plasmids containing predicted nuclear localization signal (NLS) regions (phsp-IE2NLS-C1 and phsp-IE2NLS-C2) were constructed by truncation of the ie2 internal region using PCR and the primers pNLS-C1f (5'-GAGCGGAGTCAGGAGGATTTGTTTGACTCG-3') and pNLS-C1r (complement of pNLS-C1f), and pNLS-C2f (5'-GAGCGGAGTCAGGAGGATTCCATCAAACAG-3') and pNLS-C2r (complement of pNLS-C2f). Plasmid phsp-IE2C243S was generated by site-specific mutagenesis PCR using the primer 5'-GACTTCGATTGATTCTAACCATGCTG-3' and its complement (Imai et al., 2003
).
Antibody production and immunodetection.
The BmNPV ie2 gene was amplified by PCR using an upstream primer (5'-GAGCTAGCATGAGTCGCCAAATCAA-3') incorporating an NheI site (underlined) and a downstream primer (5'-GACTCGAGTTAAGGTTTAGACATCTCA-3') with a XhoI site (underlined). The amplified fragment was digested with NheI and XhoI and fused in frame with a hexa-histidine tag (His-tag) in the pET-28c(+) vector (Novagen). After expression of His-tagged IE2 in Escherichia coli, recombinant IE2 protein was purified using His Bind Resin (Novagen) and used to raise polyclonal antibodies in rats. Infected cells were harvested at designated times and subjected to Western blot analysis and IHC. Subcellular fractionation and immunoblotting were performed as described previously (Jarvis et al., 1991; Zemskov et al., 2000
). IHC and confocal microscopy were performed as described by Kang et al. (1999)
and Okano et al. (1999)
. Each analysis was repeated independently two to five times. For IHC, anti-IE2 antiserum (1 : 200 dilution) and Cy-5-conjugated goat anti-rat IgG (1 : 200 dilution; Jackson Immuno Research) or FITC-conjugated goat anti-rat IgG (1 : 500 dilution; Cappel) were used. IE1 was detected by using anti-IE1 antiserum (1 : 200 dilution; a generous gift from L. Guarino, Texas A&M University) and Cy-5-conjugated goat anti-rabbit IgG (1 : 200 dilution; Amersham Life Sciences). To quantify total intensity of immunofluorescence in foci or the nucleus, the scanned confocal image was analysed using the TCS NT v. 1.6 (Leica).
Proteasome inhibition assay.
BmN cells infected with BmNPV at an m.o.i. of 10 were treated with MG132 (20 µM; Calbiochem) at 0 h post-infection (p.i.). Cells were incubated in the presence of MG132 until they were fixed or harvested at 4 h p.i. This assay was repeated three times.
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RESULTS |
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However, IE2NLS-C1 and IE2NLS-C2 exhibited very different localization characteristics. IE2NLS-C1 formed small foci and seemed to gather on the nuclear membrane, while IE2NLS-C2 showed enlarged foci [Fig. 4b(vi) and (vii), respectively]. This difference may be due to the region between aa 186 and 294 where another coiled-coil domain and a RING finger domain are contained (Fig. 4a
). IE2NLS-C1 contained both regions, while IE2NLS-C2 had neither, suggesting that this region is also involved in focus formation.
Formation of IE2 foci is also regulated by the ubiquitin ligase activity of IE2
We further investigated the possibility that the different localization characteristics of IE2NLS-C1 and IE2NLS-C2 were due to the region containing the coiled-coil domain and RING finger domain. Involvement of the RING finger domain was tested first, since we have previously shown that the activity of BmNPV IE2 as a ubiquitin ligase E3 is dependent on the RING finger domain (Imai et al., 2003). IE2C243S contained a single amino acid change (Cys-243
Ser), which resulted in disruption of the RING finger structure (Fig. 4a
). Transient expression of IE2C243S showed focus formation in the nucleus. However, the foci appeared larger and brighter than those of wt IE2 at 24 and 72 h p.t. (Fig. 5
a). This localization was very similar to that of IE2NLS-C2 [Fig. 4b
(vii)]. Since there was only a single amino acid change in IE2C243S, we concluded that the RING finger domain is required for focus formation.
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IE2 accumulates in foci by inhibiting the proteasome pathway
It has been reported that some ubiquitin ligase E3s are auto-ubiquitylated and then degraded by the proteasome as a means of controlling E3 levels (Fang et al., 2000; Honda & Yasuda, 1999
; Nuber et al., 1998
; Yang et al., 2000
). Therefore, we speculated that IE2 was also being auto-ubiquitylated and degraded during infection, and that the brightness and enlargement of foci shown by IE2C243S and IE2NLS-C2 were caused by the accumulation of IE2 in foci. To confirm this, we treated BmNPV-infected cells with the proteasome inhibitor MG132 and analysed IE2 localization. As expected, IE2 foci were enlarged and brighter in the presence of MG132 (Fig. 5b
). In addition, Western blot analysis of BmNPV-infected cells treated with MG132 showed IE2 accumulation with several slower-migrating bands (data not shown), supporting the suggestion that IE2 is actively degraded by proteasome. These results suggested that the protein level of IE2 is controlled by its own E3 activity.
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DISCUSSION |
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We showed that two regions of BmNPV IE2, the RING finger domain and the C-terminal coiled-coil region, were involved in IE2 focus formation. IE2 mutants lacking the C-terminal coiled-coil region did not form foci, although they localized to the nucleus. Due to a lack of NLSs, we were not able to determine whether foci were formed only by the C-terminal coiled-coil region. However, foci were observed when the region containing NLSs was included, suggesting that the C-terminal region is necessary. We have previously shown that IE2 oligomerizes through this C-terminal coiled-coil region (Imai et al., 2000). Therefore, we concluded that IE2 self-interaction is required for focus formation. Using Western blot analysis, Yoo & Guarino (1994b)
showed that full-length AcMNPV IE2 also localized to the nucleus. They also found that the transactivation activity of C-terminal deletion mutants was reduced or lost, and suggested that reduction of transactivation might be due to dispersion of IE2 to the cytosol or loss of its activity domain. Our results indicated that C-terminal deletion mutants of BmNPV IE2 are not able to localize to specific sites in the nucleus correctly. Therefore, this suggests that similar mislocalization of AcMNPV IE2 mutants results in the reduction of transactivation. However, we did not examine the transactivation activities of BmNPV IE2 or its derivatives.
Deletion of a central region containing another coiled-coil domain and the RING finger domain showed enlarged and brightened foci. Previously, we reported that BmNPV IE2 could function as a ubiquitin ligase E3 dependent on this RING finger domain (Imai et al., 2003). Therefore, we examined focus formation of an IE2 mutant containing a mutation of the RING finger domain and found that foci of this mutant were also enlarged and brightened. Quantification of immunofluorescence showed that foci of this mutant were 10 times brighter than those of wt IE2. In addition, treatment of infected cells with a proteasome inhibitor resulted in a similar increase in size and brightness of foci. These results indicated that loss of E3 ligase activity and/or inhibition of proteasomal degradation result in brighter and larger foci due to an accumulation of IE2. This proteasomal degradation by auto-ubiquitylation may explain how the IE2 expression level is regulated. Our Western blot analysis showed that BmNPV IE2 exists only during the early phase of infection, which is similar to the expression profile of AcMNPV IE2 (Krappa et al., 1995
). However, it has been reported that the BmNPV ie2 transcript is present throughout infection (272 h p.i.) (Katsuma et al., 2004
). This suggests that BmNPV IE2 expression is post-translationally regulated by proteasomal degradation.
The ICP0 of Herpes simplex virus type 1 (HSV-1) is also known to be a RING-type viral E3 (Boutell et al., 2002; Hagglund & Roizman, 2002
; Hagglund et al., 2002
; Van Sant et al., 2001
). In addition, it has been shown that ICP0 forms nuclear foci associated with PML bodies (Everett & Maul, 1994
; Maul & Everett, 1994
; Maul et al., 1993
). ICP0 targets and disrupts PML bodies by inducing the degradation of PML body components such as PML and Sp100 through the proteasome pathway (Boutell et al., 2003
; Chelbi-Alix & de The, 1999
; Everett et al., 1998
; Muller & Dejean, 1999
; Parkinson & Everett, 2000
). Moreover, the disruption of PML bodies by ICP0 is dependent on the RING finger domain (Everett & Maul, 1994
; Maul & Everett, 1994
; Maul et al., 1993
). Therefore, it is likely that PML bodies are regulated by the ubiquitylation ability of ICP0 during infection and that PML-related protein(s) might be ubiquitylated by ICP0. On the other hand, it has been shown that AcMNPV IE2 occasionally co-localizes with transiently expressed human PML (Mainz et al., 2002
). IE2 foci could be the PML body-like domains of insect cells, suggesting that BmNPV IE2 localizes to foci in order to prepare the sites for replication by targeting and ubiquitylating host protein(s), as is the case with HSV-1 ICP0. This may explain why IE2 forms foci earlier than IE1. Based on our data, we propose the following working model for IE2. Immediately after infection occurs, IE2 is transported to specific sites in the cell nucleus and forms foci there. During this time, IE2 oligomerization is required for anchoring of IE2 at these specific nuclear sites. At these foci, IE2 ubiquitylates its substrate(s) to support infection of BmNPV and then is gradually removed by auto-ubiquitylation and the proteasomal degradation system. Further analysis, including identification of the proteins interacting with IE2, will help to validate this model.
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ACKNOWLEDGEMENTS |
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Received 13 August 2004;
accepted 12 November 2004.