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B
, the NF-B inhibitor
1 BBSRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK
2 Veterinary Laboratories Agency, New Haw, Addlestone, Surrey KT15 3NB, UK
Correspondence
Julian Seago
julian.seago{at}bbsrc.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B
, the inhibitor of NF-B, a transcription factor involved in the control of apoptosis, the immune response and IFN production. The Npro–IB interaction was confirmed using yeast two-hybrid analysis and additional co-precipitation assays. It was also shown that Npro localizes to both the cytoplasmic and nuclear compartments in stably transfected cells and in CSFV-infected cells. Following stimulation by tumour necrosis factor alpha, PK-15 cell lines expressing Npro exhibited transient nuclear accumulation of pIB, but no effect of CSFV infection on IB localization or NF-B p65 activation was observed.
Present address: Department of Virology, Faculty of Medicine, Imperial College London, St Mary's Campus, Norfolk Place, London W2 1PG, UK. 
Present address: University of East Anglia, Norwich NR4 7TJ, UK.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B (NF-B) is a collective name for a family of dimeric transcription factors that are normally sequestered within the cytoplasm as a latent complex by a family of inhibitory proteins, IBs, one of which is IB (Karin & Ben-Neriah, 2000
). The NF-B/IB complex is activated by phosphorylation in response to a variety of stimuli, including viral and bacterial pathogens (Pahl, 1999; Silverman, 2001); in the case of IB, the IB kinase complex phosphorylates Ser32 and Ser36, targeting it for ubiquitination and subsequent proteasomal degradation. Once activated, NF-B translocates to the nucleus and stimulates the expression of proteins that participate in the host immune response, but also those involved in oncogenesis and the regulation of programmed cell death (apoptosis). NF-B activation induces rapid resynthesis of IB, which translocates to the nucleus, dissociates NF-B from DNA and subsequently transports NF-B back into the cytoplasm (Arenzana-Seisdedos et al., 1997; Rodriguez et al., 1999; Sachdev et al., 1998; Turpin et al., 1999).
Due to its role as a central mediator of the immune response, the NF-B signalling pathway is a prime target for viruses that need to manipulate or evade the host's innate response in order to infect cells and replicate (Hiscott et al., 2001, 2006; Santoro et al., 2003).
Classical swine fever virus (CSFV) is a member of the genus Pestivirus in the family Flaviviridae and is the causative agent of classical swine fever, the clinical symptoms of which vary from mild to severe depending on the strain causing infection (Summerfield et al., 1998, 2000, 2001; van Oirschot, 1988). Pestiviruses such as CSFV and bovine viral diarrhea virus (BVDV) are able to cross the placenta and can establish an infection in the developing fetus, resulting in the birth of persistently infected animals (Charleston et al., 2001). In addition, pestiviruses can actively block type I interferon (IFN) induction and double-stranded (ds)RNA-mediated apoptotic responses (Baigent et al., 2002, 2004; Bensaude et al., 2004; Charleston et al., 2001; Horscroft et al., 2005; Ruggli et al., 2003, 2005; Schweizer & Peterhans, 2001).
CSFV has a positive-stranded RNA genome of about 12.5 kb in length that encodes 12 known proteins (Thiel et al., 1996). The CSFV genome, like those of other members of the genus Pestivirus, encodes an N-terminal cysteine-like autoprotease termed Npro that cleaves itself from the core protein and hence from the polyprotein chain. Npro has recently been shown to inhibit the production of type I IFNs by targeting IFN regulatory factor 3 (IRF-3) for proteasomal degradation (Bauhofer et al., 2007; Chen et al., 2007; Hilton et al., 2006; Seago et al., 2007).
To help elucidate new functions performed by the Npro product of CSFV, we carried out a yeast two-hybrid screen of a human library and found that (i) Npro physically interacts with IB, the inhibitor of NF-B, (ii) Npro is localized to both the cytoplasmic and nuclear compartments during CSFV infection and (iii) IB transiently accumulates in the nucleus of PK-15 cells constitutively expressing Npro after tumour necrosis factor alpha (TNF-) treatment.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Ser)–core mutant was created using a QuikChange mutagenesis kit (Stratagene). GST fusion constructs used for cell transfections were generated from existing DNA templates by PCR and ligation into pcDNA3.1/V5-His-Topo. The histidine fusion proteins His–pIB, His–hIB C terminus, His–
C249 pIB and His–253 pIB were created by PCR and ligation into pET28a (Novagene). The bait construct CSFV Npro–pGBKT7 was generated by PCR and ligation into pGBKT7 (carrying Gal4-BD; BD Biosciences). Prey constructs containing different regions of human IB (hIB) (clones F1–F7) were created by PCR and ligation into pGADT7 (BD Biosciences).
Yeast two-hybrid screen.
The Npro–pGBKT7 bait construct was used to screen 1x106 transformants from a human liver cDNA library (BD Biosciences) using a Matchmaker Yeast Two-hybrid System 3 kit (BD Biosciences) according to the manufacturer's protocol. Positive colonies were identified by growth on medium containing -X-Gal and without adenine, tryptophan, leucine or histidine. Plasmids from positive clones were rescued, sequenced and identified using web-based NCBI BLAST programs.
Generation of recombinant fusion proteins.
Recombinant proteins were expressed in Escherichia coli strain BL21(DE3)pLysS or BL21-AI. Mid-exponential-phase cells were harvested and lysed by sonication in lysis buffer [PBS containing 1 % Triton X-100, 1 mM PMSF and specific protease inhibitors (1 µg ml–1 each of leupeptin, pepstatin, chymostatin and antipain)]. GST fusion proteins were purified using glutathione–Sepharose beads (Amersham Biosciences) and His-tagged proteins were purified using a His-bead slurry (Sigma).
Cell culture and viruses.
All cells were maintained at 37 °C in 5 % CO2. The porcine kidney cell line PK-15 was maintained in minimum essential medium alpha (Gibco-BRL), 10 % fetal bovine serum (shown to be BVDV-free and anti-BVDV antibody-free) and penicillin/streptomycin. The Max kidney cell line was from an inbred NIH minipig major histocompatibility complex d/d haplotype and was grown in Iscove's modified Dulbecco's medium, 10 % fetal bovine serum (BVDV-free and anti-BVDV antibody-free) and penicillin/streptomycin. The virulent isolate of CSFV Brescia strain was used for all infections. Virus was isolated by freeze–thaw lysis, titrated by immunostaining with anti-E2 antibody WH303 (Bensaude et al., 2004) and used in experiments at an m.o.i. of 2 TCID50 per cell. Infected cells were not immunostained for each experiment, but individual CSFV virus stocks were routinely tested using WH303 to verify their infectivity.
Transfection and vaccinia virus infection.
Lipofectamine (Invitrogen) was used for all transfections. A vaccinia virus constitutively expressing the T7 RNA polymerase (MVA-T7) was used to enhance the expression of transfected constructs. When superinfection was performed, Max cells were first infected with CSFV for 24 h, then infected with MVA-T7 and finally transfected.
GST co-precipitation binding assays.
At 30 h after transfection with expression constructs, cells were lysed in PD buffer [50 mM Tris/HCl (pH 7.6), 1 % Triton X-100, 200 mM NaCl, 0.5 mM DTT, 1 mM PMSF and 1 µg specific protease inhibitors ml–1] with sonication. Clarified lysates were incubated with 30 µl 50 % glutathione–Sepharose bead slurry overnight at 4 °C. Protein–bead complexes were washed seven times with PD buffer and then reducing buffer [100 mM Tris/HCl (pH 6.8), 4 % SDS, 0.2 % bromophenol blue, 20 % glycerol, 200 mM β-mercaptoethanol] was added. In vitro co-precipitation assays were performed by incubating purified recombinant proteins in 1 ml PD buffer for 1 h at 4 °C, followed by the addition 30 µl 50 % glutathione–Sepharose bead slurry and incubation for a further 1 h at 4 °C. Protein–bead complexes were then washed as above.
Western blotting.
Proteins were separated by SDS-PAGE (10–15 % acrylamide) and transferred to nitrocellulose membranes (Hybond-C Extra; Amersham Biosciences). Membranes were blocked with 5 % (w/v) dried skimmed milk in PBS containing 0.5 % Tween 20. To produce primary antibodies, anti-Npro rabbit sera were generated by inoculating rabbits with the peptide KTNKQKPMGVEEPVYDATGKPLFGDPS corresponding to N-terminal aa 11–37 (DS14 serum). Mouse anti-
-tubulin (Sigma), mouse anti--tubulin (Santa Cruz Biotechnology), mouse anti-His (Amersham Biosciences), goat anti-GST (Amersham Biosciences), mouse or rabbit anti-IB (Santa Cruz Biotechnology), goat anti-p65 (Santa Cruz Biotechnology) and goat anti-p50 (Santa Cruz Biotechnology) antibodies were all used as indicated. Bound primary antibodies were detected by horseradish peroxidase-conjugated anti-mouse (Bio-Rad), anti-rabbit (Bio-Rad) or anti-goat (Promega) antibodies.
NF-B p65 activity assay.
Wells of a six-well plate were seeded with 0.5x106 cells. Where indicated, cells were infected with CSFV Brescia strain and stimulated with human recombinant TNF- (25 ng ml–1; Invitrogen) at 48 h post-infection (p.i.). Nuclear extracts were prepared and analysed for NF-B p65-binding activity using an ELISA-based TransAm kit (Active Motif) according to the manufacturer's protocol.
Immunohistochemistry and fluorescence microscopy.
Cells were fixed with 4 % paraformaldehyde for 15 min, permeabilized with 0.1 % Triton X-100 and blocked with 0.5 % BSA in PBS or 30 % normal goat serum. An anti-CSFV E2 monoclonal antibody (WH303) and anti-Npro rabbit serum (DS14) were used to stain CSFV-infected cells. Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies were used and nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI; Sigma).
Subcellular extracts.
CSFV-infected or uninfected PK-15 cells were harvested and resuspended in buffer A [10 mM HEPES/KOH (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF, 1 µg specific protease inhibitors ml–1, 0.1 % NP-40) for 10 min on ice. Lysates were centrifuged at 12 000 g for 1 min and the supernatants kept as the cytoplasmic extracts. The pellet was resuspended in buffer B [20 mM HEPES/KOH (pH 7.9), 25 % glycerol, 0.2 mM EDTA, 0.42 M NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM PMSF and 1 µg specific protease inhibitors ml–1) for 1 h on ice and then centrifuged at 12 000 g for 10 min. Supernatants were kept as the nuclear extracts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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B as an Npro-interacting proteinB, the inhibitor of NF-B. Alignment using CLUSTAL_W of the hIB open reading frame with that of the pig homologue (pIB) revealed an identity of 94 % [GenBank accession nos BC002601
[GenBank]
(human) and Z21968
[GenBank]
(pig)]. To confirm the specificity of the interaction between Npro and the C-terminal portion of hIB, yeast cells were co-transformed with a bait construct encoding Npro and either the isolated hIB C-terminal prey construct or control plasmids. Positive clones were identified by blue growth on selective minimal media containing the substrate -X-Gal (Fig. 1a).
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B, or hIB clones containing longer N-termini, were able to interact with Npro. Fig. 1(b)
shows that the clone with the longest N terminus able to interact with Npro in the yeast two-hybrid assay encoded aa 121–317 of hIB and that full-length hIkB was unable to interact with CSFV Npro.
CSFV Npro shares considerable homology with Npro of the related pestivirus BVDV. In fact, yeast two-hybrid analysis confirmed that a bait construct encoding BVDV Npro also interacted with the C-terminal portion of hIB isolated in the above screen (S. Goodbourn & L. Hilton, personal communication).
Npro binds to pIB in vitro in co-transfected cells and in CSFV-infected cells
To confirm our initial observations and to investigate whether Npro could bind full-length IB in a different assay, we expressed His-tagged hIB and pIB constructs and GST and GST–Npro constructs in bacteria and purified the respective proteins (Fig. 2b). To verify the auto-cleavage integrity, a recombinant GST–Npro protein was purified that had initially been expressed as a GST–Npro–core precursor (Fig. 2a). GST and the GST-tagged Npro proteins were then coupled to glutathione–Sepharose beads and used to investigate capture of the IB proteins.
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shows, initial assays revealed that GST–Npro was able to bind to the C-terminal portion of hIB that was identified in the yeast screen. In addition, GST–Npro was able to co-precipitate pIB (Fig. 2d, lanes 1–3).
The X protein product of hepatitis B virus has also been shown to bind IB, as well as IB with a specific C-terminal deletion (C253), but was unable to bind IB with a marginally shorter C-terminal deletion (C249) (Weil et al., 1999). In order to determine whether Npro had a similar requirement for its interaction with pIB, purified recombinant pIB C-terminally truncated proteins (C249 and C253) were used to perform further co-precipitation assays. Fig. 2(d) (lanes 4–9) shows that neither pIB deletion protein was able to co-precipitate with GST–Npro, suggesting that the C-terminal aa 249–317 of pIB are important for the observed interaction.
To determine whether Npro could interact with pIB inside cells in the presence of other proteins, Max cells were infected with a vaccinia virus strain encoding the T7 RNA polymerase (MVA-T7) and then co-transfected with pIB and GST–Npro constructs containing both the cytomegalovirus and T7 promoters. Fig. 3(a) (bottom panel) shows that, in comparison with the GST control, GST–Npro was able to co-precipitate the hIB C terminus (aa 213–317), pIB and V5–His-tagged pIB proteins, thus confirming the in vitro binding assays. To validate the interaction further, reciprocal co-precipitation assays were performed in which GST–pIB was able to sequester Npro from the myriad of cellular proteins (Fig. 3b, bottom).
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B during infection, Max cells were infected at an m.o.i. of 2 for 24 h with CSFV, superinfected with MVA-T7 and then transfected with a GST–pIB plasmid. As Fig. 3(c) (top right) shows, GST–pIB, but not the corresponding GST control, did indeed co-precipitate Npro from CSFV-infected cells.
Npro does not bind to NF-B p50 or p65
The finding that Npro interacts with IB raised the possibility that Npro could also interact with the NF-B/IB complex. To address this, GST–Npro, NF-B p50 or p65, and pIB expression constructs were co-transfected into Max cells and co-precipitation assays were performed. In agreement with our previous assays, GST–Npro was able to interact with pIB; however, we were unable to identify any association between GST–Npro and the NF-B proteins p50 and p65, in either the presence or absence of pIB (Fig. 3d and e).
pIB is not degraded during CSFV infection
In order to activate NF-B, some viruses have been shown to initiate proteasomal degradation of IB (Diao et al., 2005; Su & Schneider, 1996). In contrast, vaccinia virus wild-type strains prevent IB degradation and thus inhibit NF-B activation (Shisler & Jin, 2004). To determine whether CSFV influences the expression level of pIB, whole-cell extracts from different periods of infection were prepared. Western blot analysis revealed no change in the levels of pIB during CSFV infection in comparison with non-infected cells (Fig. 4a). Furthermore, there was also no apparent change in the level of NF-B p65 (Fig. 4a). This confirmed that the proteolytic activity of Npro does not target pIB. To investigate whether CSFV inhibits pIB degradation, PK-15 cells that had been infected with CSFV for 48 h were treated with the NF-B activator TNF- and whole-cell extracts were analysed by Western blotting. As shown in Fig. 4(b), pIB was degraded in cell extracts prepared 15 min after treatment with TNF- from both the uninfected control cells and the CSFV-infected cells. By 1 h post-treatment, pIB in extracts from uninfected and infected cells had returned to levels comparable to those seen before TNF- treatment.
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B and NF-B/IB complexes results from an equilibrium between nuclear import, nuclear export and cytoplasmic retention. In order for Npro to interact directly with pIB, the two proteins need to share the same subcellular compartment. To characterize Npro localization in more detail, Max cells were transfected with a fusion construct encoding GFP–Npro. Although GFP–Npro was present within the cytoplasmic compartment, surprisingly its localization was predominantly nuclear (Fig. 5a, top panel). In comparison, the GFP control was expressed uniformly throughout both the cytoplasm and nucleus (Fig. 5a, bottom panel). We observed a similar localization pattern of Npro in CSFV-infected PK-15 cells using rabbit serum raised to the N-terminal part of Npro (Fig. 5b) and also in uninfected PK-15 cells transfected with constructs encoding V5- or HA-tagged Npro (data not shown). To provide further evidence that Npro was indeed localized to both subcellular compartments, nuclear and cytoplasmic extracts prepared from PK-15 cells that had been infected with CSFV for 48 h were analysed by Western blotting. As Fig. 5(c) (top panel) confirms, Npro was present within both the cytoplasmic and nuclear extracts.
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B activation occurs during CSFV infection of PK-15 cellsB p65 at 1 and 18 h after CSFV infection (Bensaude et al., 2004). To determine whether a similar activation profile occurs in CSFV-infected PK-15 cells, nuclear extracts were prepared at different times p.i. An ELISA-based assay that determines the level of DNA-bound immunoreactive NF-B p65 was then used to investigate p65 activation in each extract (Fig. 6a). In contrast to the double peak observed with CSFV-infected porcine aortic endothelial cells, only a single peak of p65 activation (at 1 h p.i.) was observed during CSFV infection of PK-15 cells and was most likely due to virus binding (Bensaude et al., 2004).
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-induced NF-B p65 activationB p65 in uninfected and CSFV-infected PK-15 cells in response to TNF-. As the data in Fig. 6(b) show, the amount of DNA-bound immunoreactive p65 was marginally but not significantly reduced in CSFV-infected cells, in comparison with non-infected cells. In the nuclear extracts of unstimulated cells, there were comparable levels of NF-B p65 activation.
Transient nuclear accumulation of pIB in PK-15 Npro cells following TNF- induction
In order to determine the role of Npro per se in modulating NF-B-dependent pathways, we used a luciferase reporter assay and potent NF-B activators, including lipopolysaccharide, dsRNA and TNF-, to determine NF-B activity in different stably transfected cell lines (293, 3T3 and PK-15) constitutively expressing Npro protein (data not shown). This biochemical approach did not reveal consistent data to suggest that NF-B p65 activity is modulated by Npro. To extend the analysis of TNF- stimulation, we then prepared nuclear and cytoplasmic extracts of control PK-15 cells and a PK-15 cell line expressing His–Npro at various time points after stimulation with TNF- and analysed them by Western blotting for the presence of pIB. Fig. 6(c) shows that, following TNF- treatment, pIB was degraded and resynthesized within the cytoplasmic extracts of both cell types. In contrast, at 1 h after treatment with TNF-, a much higher level of pIB was present in the nuclear extract of the His–Npro cell line in comparison with the control cells. This demonstration of nuclear pIB retention was reproducible and occurred in two independent cell lines constitutively expressing His–Npro. However, similar nuclear accumulation of pIB could not be demonstrated in cells infected with CSFV and treated with TNF- 48 h later.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The roles performed by the N-terminal protease Npro, namely the suppression of IFN production via the degradation of IRF-3 and suppression of dsRNA-induced apoptosis, have recently been described (Bauhofer et al., 2007; Chen et al., 2007; Gil et al., 2006; Hilton et al., 2006; Meyers et al., 2007; Ruggli et al., 2003, 2005; Schweizer & Peterhans, 2001; Seago et al., 2007). The CSFV genome encodes a limited number of viral proteins and it is reasonable to expect that Npro interacts and modulates the host-cell machinery at different levels to ensure virus survival.
Here, we have provided conclusive biochemical evidence that CSFV Npro interacts directly with IB, an inhibitor of the transcription factor NF-B, which is a critical regulator of the early pathogen response. Using co-transfected cells, we were able to reciprocally co-precipitate pIB with Npro; however, we were unable to co-precipitate endogenous pIB in similar assays. Likewise, the NF-B subunits p65 and p50 in mammalian cells did not co-precipitate with Npro, suggesting that Npro only binds pIB that is not in a complex with the NF-B subunits. Furthermore, our data showed that lower amounts of pIB were precipitated by Npro in the presence of NF-B, suggesting that Npro and NF-B may compete with each other for unbound pIB. Our results clearly showed that Npro interacts with aa 213–317 of the C terminus of pIB. Various residues within this C-terminal region of IB, encompassed between aa 214 and 280, physically contact NF-B (Huxford et al., 1998; Jacobs & Harrison, 1998). It is feasible to suggest that when the NF-B subunits p65 and p50 are bound to pIB they may mask the Npro-binding site on the C-terminal end of pIB.
We also showed for the first time that Npro is present in both the cytoplasm and the nucleus during CSFV infection and that it is feasible that Npro may exhibit a discrete interaction with pIB in either compartment.
In the present study, we observed no statistical difference in the capacity of p65 to bind specific promoter sequences in CSFV-infected PK-15 cells in comparison with mock-infected cells. However, CSFV predominantly targets monocytes and macrophages, in which the effect of the interaction may be more pronounced. In addition, NF-B-binding sites have been identified in the promoter regions of >150 cellular genes, the majority of which participate in the immune response (Pahl, 1999). It is feasible that Npro may modulate the expression of particular genes within this repertoire. NF-B p65 activity in response to TNF- was also investigated in PK-15 cell lines expressing Npro and clearly demonstrated a transient nuclear accumulation of pIB at 1 h post-treatment. This observation may result from Npro sequestering newly synthesized IB and requires further investigation.
Hilton et al. (2006) showed that Npro from BVDV strain Pe515 or strain CP7 was unable to block the activation of an NF-B-dependent reporter gene in response to dsRNA. We also observed no statistical difference in NF-B activity between parental PK-15 cells and a PK-15 Npro stable cell line in response to dsRNA treatment (data not shown).
The role of IB is still not completely understood and other functions have been reported, including interaction with p53, a tumour suppressor protein involved in cellular proliferation and apoptosis (Chang, 2002; Dreyfus et al., 2005) and participation in the transcriptional regulation of specific promoters through the recruitment of repression elements (Aguilera et al., 2004).
In this study, we have provided new evidence that Npro interacts with the regulatory host protein IB and results in its transient nuclear accumulation. Further studies are required to unravel the functional significance of the interaction between IB and Npro; in particular, comparing the effect of wild-type and Npro-deleted viruses in monocytes and macrophages may reveal the significance of the interaction. Furthermore, microarray analysis of NF-B-dependent gene expression may reveal specific effects of Npro on the transcriptional profile of the host cell in response to specific stimuli.
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ACKNOWLEDGEMENTS |
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Received 6 December 2007;
accepted 8 April 2008.
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