Identification of Aquatic Birnavirus VP3 Death Domain and Its Dynamic Interaction Profiles in Early and Middle Replication Stages in Fish Cells

Infectious pancreatic necrosis virus (IPNV) is a fish pathogen and the prototype of the Birnaviridae virus family (Dobos et al., 1979). IPNV causes infectious pancreatic necrosis, an acute and serious disease in juvenile salmonid fish worldwide (Hill and Way, 1995) Birnaviruses possess a bi-segmented, double-stranded RNA genome contained within a medium-sized, unenveloped, icosahedral capsid. The protein products of four unrelated major genes undergo various post-translational cleavage processes to generate three to five different structural proteins (Dobos, 1995). The largest of these proteins (VP1; 90–110 kDa) is encoded by the smaller segment B RNA (Wu et al., 1998). The larger genome segment A contains a large open reading frame (ORF; which encodes VP3 [32-kDa; a minor capsid protein] [Wu et al., 1998], VP4 [28 kDa], and VP2 [the major capsid protein; 46 kDa] [Wu et al., 1998]) and a small ORF, which encodes VP5 (17 kDa; a non-structural protein with antiapoptotic activity) (Hong et al., 2002a; Hong and Wu, 2002c).


Introduction
Infectious pancreatic necrosis virus (IPNV) is a fish pathogen and the prototype of the Birnaviridae virus family (Dobos et al., 1979).IPNV causes infectious pancreatic necrosis, an acute and serious disease in juvenile salmonid fish worldwide (Hill and Way, 1995) Birnaviruses possess a bi-segmented, double-stranded RNA genome contained within a medium-sized, unenveloped, icosahedral capsid.The protein products of four unrelated major genes undergo various post-translational cleavage processes to generate three to five different structural proteins (Dobos, 1995).The largest of these proteins (VP1; 90-110 kDa) is encoded by the smaller segment B RNA (Wu et al., 1998).The larger genome segment A contains a large open reading frame (ORF; which encodes VP3 [32-kDa; a minor capsid protein] [Wu et al., 1998], VP4 [28 kDa], and VP2 [the major capsid protein; 46 kDa] [ Wu et al., 1998]) and a small ORF, which encodes VP5 (17 kDa; a non-structural protein with antiapoptotic activity) (Hong et al., 2002a;Hong and Wu, 2002c).
Previously, it was found that IPNV-induced apoptosis (Hong et al., 1998;Hong, et al., 1999a;Hong et al., 1999b;Hong and Wu, 2002) may be mediated through activation of caspase-8 and -3 (Hong et al., 2005) and requires new protein synthesis (Hong et al., 1999).This pathway may be triggered through NF-κB transcription factor transactivation of downstream effector genes such as Bad (Hong et al., 2008).Recently, it was found that IPNV-induced loss of ΔΨm can be blocked by the adenine nucleotide translocase (ANT) inhibitor bongkrekic acid (BKA) (Chen et al., 2009), and that IPNV-induced expression of annexin 1 can have an anti-death function (Hwang et al., 2007).IPNV was also found to induce apoptotic cell death and necrotic cell death in the same cells through TNF up-regulation (Wang et al., 2011).
The structures associated with IPNV replication and genome and particle assembly in infected cells have only started to be elucidated.Recently, viral particles of different sizes Viral Genomes -Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions 262 were identified during the IPNV infective cycle (Villanueva et al., 2004).Immediately after synthesis, non-infectious, immature particles 66 nm in diameter appeared.These provirion particles were detected simultaneously with viral double-stranded (ds) RNA in infected cells, suggesting that viral assembly occurs as soon as dsRNA replication has begun.Subsequent maturation into smaller virions (60 nm in diameter) was found to proceed through proteolytic cleavage of the viral precursors within the capsid.An early study of IPNV also suggested an association of VP3 with viral RNA.VP3-containing ribonucleoprotein core structures were identified by electron microscopy studies, and an association of the basic C-terminal end of the protein with the viral genome was proposed (Hudson et al., 1986).Furthermore, Pedersen found that VP3 can interact with itself, coprecipitate dsRNA, and interact with VP1 (Pedersen et al., 2007).The dynamics of VP3 interaction with other proteins or RNA are still unknown.
In the present study, we located the death domain of VP3 protein and examined its interactions with other viral molecules culminating in the formation of the VP3-VP1-RNA-VP2 complex during viral replication.

Cell line and virus
Chinook salmon embryo cells (CHSE-214 obtained from the American Type Culture Collection, ATCC, Manassas, VA, USA) were grown at 18C in plastic tissue-culture flasks (Nalge Nunc International, Rochester, NY, USA) containing Eagle's minimum essential medium (MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and gentamycin (25 µg/ml).ZLE cells were grown at 28C in Leibovitz's L-15 medium (GibcoBRL, Grand Island, NY) supplemented with 5% v/v FBS and 25 μg/ml of gentamycin.An isolate of the Ab strain of IPNV, designated E1-S, was obtained from Japanese eels in Taiwan (Wu et al., 1987).The virus was propagated in CHSE-214 cell monolayers at a multiplicity of infection (MOI) of 0.01 per cell.Infected cultures were monitored as described previously (Dobos, 1977) and Tissue Culture Infectious Dose 50 (TCID 50 ) assay was performed on confluent monolayers (Nicholson and Dunn, 1974).

Western blot analysis
CHSE-214 cells (4.0 ml) were seeded at 10 5 cells per ml per 60 mm Petri dish for at least 20 h prior to cultivation.The resulting monolayers were rinsed twice with PBS.Cells were infected with virus at an MOI of 1.0 and were incubated for 0, 4, 6, 8, 12, or 24 h.Culture media were aspirated at the end of each time point.Cells were washed with PBS and lysed in 0.3 ml of lysis buffer (10 mM Tris base, 20% glycerol, 10 mM sodium dodecyl sulfate, and 2% -mercaptoethanol; pH = 6.8).ZLE cells (4.0 ml) were seeded at 1x10 5 cells per ml and cultivated as previously described for VP3 to allow mutant gene overexpression.The cells after 20 h were transfected with pEGFP, pVP3-EGFP, pEGFP-VP3: 1-158, pEGFP-VP3: 80-237, pEGFP-VP3: 80-158, pEGFP-VP3: 1-79, and pEGFP-VP3: 159-237 plasmids (2 g added to each dish) using Lipofectamine-Plus (Life Technologies).Transfection was allowed to proceed for 4 h.ZLE cells were incubated at 28C for 0, 24, and 48 h.Culture media were aspirated at the end of each incubation period.The cells were washed with PBS and finally lysed in 0.3 ml of lysis buffer.
Chemiluminescence detection was performed using the Western Exposure Chemiluminescence Kit (Amersham) according to the manufacturer's instructions.Chemiluminescence was visualized on Kodak XAR-5 film (Eastman Kodak, Rochester, NY, USA), and protein expression level amounts were quantified using a Personal Densitometer (Molecular Dynamic, Sunnyvale, CA).

Radioactive labeling of infected cells for determination of VP3 interactions
Radioactive labeling of infected cells: A confluent CHSE-214 cell monolayer in a plastic tissue culture plate (60 mm diameter, Nunc) was infected with IPN virus at 18°C at an MOI of 1.After a 1-h adsorption period, the medium was removed, the monolayer was rinsed three times with phosphate-buffered saline (PBS), and starved for 2 h at 18°C.Then, growth medium containing 50 μCi ml -1 of [ 35 S]methionine was added.At different times in the www.intechopen.comViral Genomes -Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions 264 labelling period (3-20 h) or continuously labelled for 20 h as a positive control, the cell layer was washed with PBS, and the monolayer was lysed in 1 ml of lysis buffer (0.5% [v/v] Nonidet P-40 in phosphate-buffered saline) (Wu et al., 1998).

VP3 localization
Green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an invaluable tool for monitoring gene expression and fusion protein localization in vivo or in situ, as well as for determination of cellular morphological changes (Chalfie et al., 1994;Hong et al., 1999b;Maniak et al., 1995;Oparka et al., 1997).A variant type of GFP (EGFP), a marker for visualization of apoptotic cell morphological changes (Hong et al., 1999b), was used in the present study to trace VP3 localization and monitor interaction with other proteins.
The tracking of VP3 with EGFP in ZLE cells has revealed their presence in plasma (Espinoza et al., 2000).However, VP3 was absent from endoplasmic reticulum (ER) when tracked with an ER marker (data not shown).
Notes: P1, designed as for forward primer and P2 as for reverse primer.
Table 1.The primers were used to VP3 and different mutant constructions of EGFP-VP3 fusion genes in plasmids.

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EGFP served as a tracer to allow direct visualization of apoptotic changes in ZLE cells overexpressing VP3 and VP3 peptides at 48 h p.t.The site of the death domain was found to be in a 78-aa region between aa 80 and aa 157 (Fig. 3A).Compared to EGFP-VP3 fusion proteins without this region, those containing this domain induced a higher rate of apoptosis (Fig. 3B

Kinetics of VP3 interaction during IPNV infection
Although VP3 participated in many viral processes (apoptosis induction, binding to viral RNA, binding to RdRp, and self interaction), it did not associate directly with capsid protein VP2.So, we were interested in the mechanism of interaction between VP3 protein and other proteins.The binding of native VP3 to two VP3 monoclonal antibodies was assessed at 0, 2.5, 4, 6, 8, and 24 h p.i. (Fig. 4).VP3 protein was found to bind to both anti-VP3 B9 (lanes, 15-20) and anti-VP3 E7 (lanes, 21-26) Mabs at 6, 8, and 24 h p.i. but not to the negative controls protein A (lanes, 3-8) and secondary anti-rabbit Ig (lanes, 9-14).The positive control ([ 35 S]methionine continuously labelled for 20 h) is shown in lane 2 and protein markers labelled with [ 35 S]methionine are shown in lane 1.
Furthermore, we found just only anti-VP3 E7 that at 6 h p.i., VP3 associated with VP1, p85 protein, and RNA to form a complex capable of binding VP2 protein during the period 8-24 h p.i (Fig. 5), indicating that viral particle assembly begins as early as 6 h p.i.

The role of VP3 protein in cell death
The Bcl-2 family of proteins, including both anti-and pro-apoptotic molecules, act at a critical, intracellular decision point along the common death pathway (Newton and Strasser, 1998).The ratio of antagonist (Bcl-2, Bcl-x L , Mcl-1, Bcl-W, and A1) to agonist (Bax, Bak, Bclx s , Bid, Bik, Bad, PUMA, and NOXA) molecules dictates whether a cell responds to a proximal apoptotic stimulus (Newton and Strasser, 1998;Galluzzi et al., 2008).Homologues of and A1) reside in mitochondria and stabilize the barrier function of mitochondrial membranes.In contrast, pro-apoptotic proteins can shuttle between non-mitochondrial locations (the cytosol for Bax, Bad, and Bid) and mitochondrial membranes where they can insert and permeabilize the mitochondrial membrane (Zamzami and Kroemer, 2001;Galluzzi et al., 2008).In our system, the Bcl-2 family member, zebrafish Bcl-xL, was found to play a role in blocking both apoptotic (Yang et al., 1995) and necrotic cell death.Recently, VP3 overexpression was found to up-regulate Bad protein (a proapoptotic Bcl-2 family member), but how VP3 overexpression was induced remained unknown.Moreover, the interactions of the death domain (80-158 aa) of VP3 protein with other molecules also remained unknown.From the recently published literature, we found that VP3 protein contains a potential phosphokinase C phosphorylation site in Domain 2 corresponding to residues 122-124 (Domain A2; TGR) (Chiu et al., 2010), which could have an important role in cell death induction.

Novel role for VP3 in the replication cycle
Viruses infect specific target cells, replicate in them to produce large numbers of progeny virions, which then spread to other susceptible cells to initiate new rounds of infection.They also encode proteins that are highly efficient for the optimization of such replication.However, target organisms use both systemic and cell-based defence mechanisms to limit the extent of viral infection, including immune and inflammatory processes and the execution or suicide of infected cells (Benedict et al., 2002).When we investigated the involvement of viral protein (VP3) in host cell death later in the viral replication cycle (VP3, apoptosis), we found that VP3 interacts with VP1, p85, and RNA initially (Fig. 4, lane 24; Fig. 5), then the VP1-p85-RNA-VP3 complex recruits VP2 at 8 h (Fig. 4, lane 25; Fig. 5) and 24 h (Fig. 4, lane 26; Fig. 5), suggesting that VP3 protein performs many functions.For example, VP3 may stimulate VP1 expression and prime viral assembly.In the present study, VP2 interacted with VP3 protein and a new protein p85 (which may have a role in viral replication), but the role of p85 remains to be elucidated.

Conclusion
IPNV E1-S is a fish pathogen of the IPNV Ab strain.It induces apoptotic cell death in CHSE-214 cells (Hong et al., 1998;Hong et al., 1999a;Hong et al., 1999b) and zebrafish ZLE cells (Hong et al., 2005).In summary, we provided evidence that IPNV minor capsid proteins were specifically involved in the induction of necrotic cell death.The death domain of VP3 was found to be located within the stretch 80-158 aa, which contains a protein kinase C phosphorylation site.We also found that the VP3 protein can interact with VP1-p85-RNA complex early in the replicative cycle (at 6 h p.i.), and that VP3-VP1-p85-RNA binds VP2 at 8 h and 24 h p.i. thereby affecting either viral replication or viral assembly.Our study adds important new information concerning the IPNV VP3-host cell interaction and provides the basis for study of the viral pathogenesis of VP3-mediated necrotic cell death and viral assembly.

Summary
Aquatic birnavirus induces secondary necrotic cell death through the synthesis of new protein.Very recently we found that the viral genome-encoded minor capsid protein VP3 can induce cell death in fish and mouse cells.In the present study, we identified the death domain of VP3 and the role of VP3 in processes such as interaction with VP1 and viral assembly during late stages of replication.Aquatic birnavirus-encoded VP3 was mildly expressed in CHSE-214 cells at 4 h post-infection (p.i.), but its expression increased up to 3.5to 4-fold by 6 h p.i. Furthermore, using a deletion series, the VP3 death domain was localized to a 78-amino acid (aa) segment (80-158 aa), which was separated from the VP3 self-binding domain (1-101 aa) and VP1 binding domain (the so-called RNA-dependent RNA polymerase, RdRp binding domain; 171-236 aa).Using two anti-VP3 monoclonal antibodies, VP3 was also found to interact with VP1, VP2, viral RNA, and host protein-85.Our results suggested that aquatic birnavirus VP3 not only triggers Bad-mediated cell death, but also stabilizes viral RNA, and promotes viral particle assembly.Thus, VP3 may be a good target for antiviral drug-therapy.

ABFig. 3 .
Fig. 3. Identification of the death domain of VP3 in ZLE cells.Expression of EGFP, EGFP-VP3, and EGFP-VP3 deletion mutants was assessed in ZLE cells.The pEGFP, pEGFP-VP3, and different pEGFP-VP3 mutants were transfected into ZLE cells with Lipofectamine-Plus, and the cells were incubated for 48 h post-transfection (p.t.).(A) Phase-contrast and fluorescence micrographs of transfected and untransfected apoptotic ZLE cells at 48 h p.t. Rounded up cells and plasma membrane blebbing are indicated by arrows (Bar = 10 µm).(B) The percentage of apoptotic cells containing EGFP, EGFP-VP3, or the different EGFP-VP3 deletion mutant constructs at 48 h p.t.The number of apoptotic cells per 200 cells per sample was assessed.Each point represents the mean of three independent experiments and the vertical bars indicate ± the standard error of the mean (SEM).Data were analyzed using either paired or unpaired Student's t-test as appropriate.Statistical significance was defined at P < 0.05.
www.intechopen.comIdentification of Aquatic Birnavirus VP3 Death Domain and Its Dynamic Interaction Profiles in Early and Middle Replication Stages in Fish Cells 271