GENE-39676; No. of pages: 8; 4C: Gene xxx (2014) xxx–xxx

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Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus) Zhiming Xiang a,1, Shaoping Weng a, Hemei Qi a, Jianguo He a,b, Chuangfu Dong a,⁎ a b

State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China School of Marine Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, PR China

a r t i c l e

i n f o

Article history: Received 29 March 2014 Received in revised form 5 May 2014 Accepted 12 May 2014 Available online xxxx Keywords: Megalocytivirus Spotted knifejaw iridovirus DNA binding protein FstK-like protein (FLP) Transcription inhibitor

a b s t r a c t Prokaryotes contain many DNA binding proteins with large molecular weights and multiple domains. DNA binding proteins are involved in DNA replication, transcription, and other physiological processes. In this study, a DNA binding protein, containing an Ftsk-like protein (FLP) domain, was cloned and characterized from SKIV-ZJ07, a member of the RSIV-type megalocytivirus, using bioinformatics and molecular biology approaches. SKIV-FLP is 3762 base pairs long, encodes a viral protein of 1253 amino acid residuals, and contains an Ftsk (or EBV-NA3) and a Grx-2 domain. Virion localization indicated that SKIV-FLP is a major viral structural protein located below the major capsid protein. Laser confocal microscopy showed that SKIV-FLP is a cytoplasm-/nuclear-localized protein. However, the reconstruction experiments demonstrated that SKIV-FLP may contain three nuclear localization signals, each present in FLP-NT (1–380 aa), FtsK domain (380–880 aa), and Grx-2 domain (880–1253 aa). When SKIV-FLP was fused to the Gal-4 DNA-binding domain and co-transfected with L8G5-Luc, SKIV-FLP suppressed L8G5-Luc transcription. As a transcription inhibitor, SKIV-FLP also inhibited the transcription of NF-κB and IFN-γ (a type II IFN) promoter in HEK293T cells, suggesting that SKIV-FLP has a role in evading host immunity. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Iridoviruses are large double-stranded DNA viruses that infect poikilotherms, including fish, amphibians, reptiles, and insects (Jancovich et al., 2012; Williams et al., 2005). The viruses that infect vertebrates are collectively called piscine iridoviruses, which include lymphocytivirus, ranavirus, and megalocytivirus (Chinchar et al., 2005). In the past decade, megalocytivirus has become one of the most alarming disease causative agents in the bony fish aquaculture industry worldwide (Kurita and Nakajima, 2012; Marcos-López et al., 2011; Subramaniam et al., 2012; Waltzek et al., 2012). Megalocytivirus-associated disease outbreaks have resulted in significant economic losses in public aquaria, food fish, and ornamental fish industries, as well as wild fish stock endangerment (Chinchar et al., 2009; Kurita and Nakajima, 2012). In the past several years, great advancements have been achieved Abbreviations: aa, amino acid; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CPE, cytopathic effects; DBP, DNA binding protein; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; FLP, FtsK-like proteins; hpi, hour post infection; IFA, immunofluorescence assay; ISKNV, infectious spleen and kidney iridovirus; MCP, major capsid protein; MMP, myristylated membrane protein; NBT, nitroblue tetrazolium; RBIV, rock bream iridovirus; RSIV, red seabream iridovirus; SGIV, Singapore grouper iridovirus; TRBIV, Turbot reddish body iridovirus. ⁎ Corresponding author. E-mail address: [email protected] (C. Dong). 1 Present address: Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China.

in understanding the epidemiology, pathology, and genomics of megalocytiviruses; however, knowledge of their viral protein profiles is still very limited (Shuang et al., 2013; Xu et al., 2010). Recently, structural proteins of two megalocytiviral strains have been determined using comprehensive proteomic approaches (Dong et al., 2011; Shuang et al., 2013). Both viral proteomic approaches have shown that the mature megalocytiviral virions contain 38 to 49 viral proteins in purified viral particles, which include approximately 20 highly abundant viral structural proteins. Among these viral proteins, a rock bream iridovirus ORF058L (RBIV-ORF058L) homologous protein has been identified as the largest abundant viral structural protein (Dong et al., 2011; Shuang et al., 2013). In RBIV, 118 potential non-overlapping open reading frames with coding capacities for polypeptides ranging from 50 to 1253 amino acids have been identified through computer-assisted analysis of its complete genomic DNA sequence; among which, RBIV-ORF058L encodes the largest viral protein, with 1253 aa length, which may act as a DNA-binding protein, according to a bioinformatics analysis (Do et al., 2004). The Blast program from the NCBI Conserved Domain Database indicates that RBIV-ORF058L contains an FtsK-like domain (380– 496AA) or an EBNA-3 domain (397–561AA) at a similar site. Furthermore, according to a domain forecast program (http://myhits.isb-sib. ch), RBIV-ORF058L also contains a Grx-2 domain (880–980AA). Given that RBIV-ORF058L and its homologs in other megalocytiviruses contain an FtsK-like domain, these viral proteins are designated as FtsK-like proteins (FLP) in this study.

http://dx.doi.org/10.1016/j.gene.2014.05.026 0378-1119/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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Z. Xiang et al. / Gene xxx (2014) xxx–xxx

Proteins with FtsK domains have multiple biological functions, such as the implementation of bacterial chromosome separation, cell division, viral packaging, and separation of particles (Capiaux et al., 2002; Goldberg et al., 2003; Wang et al., 2006). In phages and herpesviruses, Ftsk domain-containing proteins are evolutionarily related and belong to an FtsK-HerA superfamily of ATPases (Iyer et al., 2004; Przech et al., 2003). The advent of the direct testing of translocation models helps provide mechanistic insight into these systems and other systems of nucleic acid translocation (Maluf and Feiss, 2006). The EB-NA3 domain has a structure similar to that of EBNA-3 proteins in the Epstein–Barr virus. The protein families are nuclear antigens, including EBNA-3A, -3B, and -3C, and EBNA-3C, which have been reported to exhibit transcriptional regulation activity (Arad et al., 2011; Hickabottom et al., 2002). Studies have indicated that the proteins are essential for evading host immunity by sustaining proliferation and through the immunoblastic transformation of B-lymphocytes (Hickabottom et al., 2002). Glutaredoxin protein contains a Grx domain and functions as an electron carrier in the glutathione-dependent synthesis of deoxyribonucleotides through the enzyme ribonucleotide reductase. These proteins have been widely studied in prokaryotic and eukaryotic organisms (Guagliardi et al., 1995; McFarlan et al., 1992; Minakuchi et al., 1994; Morell et al., 1995). In the vaccinia virus, a glutaredoxin homolog participates in the form of a viral structure (Ahn and Moss, 1992). Although RBIV-ORF058L has been previously predicted to be a DNA-binding protein in megalocytiviral genome (Do et al., 2004), it is still poorly understood despite further studies on its protein property. In the current study, an RBIV-ORF058L homologous protein was cloned and characterized from a virulent megalocytiviral strain, SKIV-ZJ07, and was designed as SKIV-FLP. The virion and subcellular localizations of SKIV-FLP and its mutants were determined. The functions of SKIV-FLP were also primarily analyzed. This study provides information for further understanding viral protein characteristics and their functions in megalocytiviruses and other iridoviruses.

Shuang et al., 2013). Rabbit anti-ISKNV-rMCP and -rMMP sera were prepared and kept in our laboratory (Dong et al., 2011). 2.2. Cloning and phylogenetic analysis of SKIV-FLP Total genomic DNA from SKIV-ZJ07 infected MFF-1 cells was prepared as template for RBIV-ORF058L homolog amplification using a primer set of FLP-NP-F and FLP-NP-R (Table 1). The PCR products were cloned into pGEM-T easy vector (Promega, USA) for sequencing using an ABI 3770 sequencer (Applied Biosystems, USA). Comparison and phylogenetic analysis of FLP were performed using the MegAlign program of the DNASTAR software. The Clustal method was used to correct the distances for multiple substitutions at a single site. Some megalocytiviruses whose whole genomes are known were selected for phylogenetic analysis. 2.3. Expression and antibody preparation of SKIV-FtsK (380–880 aa) fragment According to the obtained full length of SKIV-FLP (accession number: HM246185), a primer set (Ftsk-L & pET-FLP-R, Table 1) was designed to clone and express SKIV-FtsK (380–880 aa) in the pET32a expression vector, as previously described (Dong et al., 2011). The recombinant His-FtsK was subsequently purified through affinity chromatography on Ni-NTA Superflow resin (Qiagen, Germany), according to the instructions of the manufacturer. Protein concentration was determined using the Bradford assay; the purified proteins were stored at −80 °C until use. For the antibody preparation, 0.5 mg of purified recombinant His-SKIV-FtsK was emulsified with an equal volume of Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant (FIA) for the three following immunizations. A rabbit received the three subcutaneous immunizations at 2-week intervals. Blood samples were obtained on the tenth day after the last immunization for sera collection. 2.4. Purification and fractionation of SKIV-ZJ07

2. Materials and method 2.1. Cell lines, virus and antibodies Mandarin fish fry (MFF-1) cells were developed and characterized in our laboratory, and were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS, Gibco) at 25 °C (Dong et al., 2008). HeLa cells and HEK293T cells were cultured in DMEM with 10% FBS in an incubator at 37 °C, 95% humidity, and 5% CO2. Megalocytiviral strain SKIV-ZJ07 was isolated from diseased cagecultured spotted knifejaw (Oplegnathus punctatus) using MFF-1 cells, and then characterized and kept in our laboratory (Dong et al., 2010;

SKIV-ZJ07 was purified from infected-MFF-1 cells through gradient sucrose density centrifugation, as described previously (Shuang et al., 2013). The purified virus was resolved in TMN buffer [150 mM NaCl, 2 mM MgCl2, and 20 mM Tris–HCL (pH 7.5)], and then stored at −80 °C until use. The fractionation of the purified virions was prepared according to a previous description on the treatment of ISKNV (Dong et al., 2011). In brief, 100 μl of 2% Triton X-100 was added to an equal volume of purified SKIV-ZJ07 suspension, and was mixed thoroughly. After incubation for 3 min at room temperature, the mixture was centrifuged at 20,000 ×g for 30 min at 4 °C. The supernatant was removed and kept until use. The pellet was resolved in 200 μl of TMN buffer.

Table 1 Primer sets used in this work. Primer

Orientation

Nucleotide sequences

Targeta

FLP-NP-F FLP-NP-R FLP-QT-F FLP-QT-R Actin-QT-F Actin-QT-R MCP-QT-F MCP-QT-R FLP-NT-L pET-FLP-R Grx-R Ftsk-L FLP-NT-R Ftsk-R

Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Antisense Sense Antisense Antisense

5′-ATGGCAGAAGGTGGAATGAAGCCT-3′ 5′-CTACGACTCTTCGGCTGAGCTTTTAG-3′ 5′-AGCCAGGGACATACGACCACA-3′ 5′-CTCTGCCGCATAATCCTTCACA-3′ 5′-GTACGTCGCCCTGGACTTCG-3′ 5′-CTGTTGTAGGTGGTCTCGTGGATT-3′ 5′-GGTATCACCAACGGTCAGACTATGC-3′ 5′-GCTGGGTGCTCTGGCTGATGA-3′ 5′-AAAGAATTCATGTTACGGGGCCTGC-3′ 5′-TTTCTCGAGTAGGTCCCGCTTGTTG-3′ 5′-AAAGTCGACATGGTATTATCAAACCCATT-3′ 5′-AATGAATTCATGTGCAAGCGTCTGCT-3′ 5′-TTTGTCGACAGGTCCCGCTTGTT-3′ 5′-AAAGTCGACATGGTATTATCAAACCCATT-3′

Whole length of SKIV-FLP

a

Q-PCR for SKIV-FLP Q-PCR for actin Q-PCR for SKIV-MCP F: pEGFP-N1, pCMV-Flag, pCMV-BD (1-1253 aa) and pEGFP-N1 (1-380 aa). R: pET32a (380-880 aa) R: pEGFP-N1, pCMV-Flag, pCMV-BD (1-1253 aa) and pEGFP-N1 (880-1253 aa). F: pEGFP-N1 (380-880 aa) and pET32a (380-880 aa). R: pEGFP-N1 (1-380 aa) R: pEGFP-N1 (380-880 aa)

F: forward primer; R: reverse primer.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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The two-fold diluted SKIV-ZJ07 suspension and Triton-X-100treated SKIV supernatant and pellets were subjected to Western blotting analysis, as described previously (Dong et al., 2011). Rabbit antiSKIV-His-FstK (380–880), -ISKNV-rMCP, and -ISKNV-rMMP sera were used as primary antibodies. AP-conjugated goat anti-rabbit antibody was used as the secondary antibody. The blots were visualized using fresh NBT/BCIP solution.

on circular glass coverslips in a 24-well plate and infected with SKIVZJ07 using an MOI of 1.0. After the entire CPE appearance, the infected cells were fixed with pre-cooled methanol (−20 °C) for 15 min at RT, followed by washing twice with sterile PBST. Rabbit anti-SKIV-HisFstK (380–880), -ISKNV-rMCP, and -ISKNV-rMMP sera were used as the primary antibodies and Alexa-fluor488-conjugated goat antirabbit antibody was used as the secondary antibody. The coverslips were observed under an LECA fluorescence microscope or LECA LSM 410 laser scanning confocal microscope.

2.6. Immunofluorescence assay (IFA)

2.7. Temporal pattern analysis through quantitative real-time PCR

IFA was performed on SKIV-infected MFF-1 cells, as previously described (Dong et al., 2008). In brief, confluent MFF-1 cells were grown

For temporal pattern analysis, MFF-1 cells were treated with SKIVZJ07 at an MOI of 1.0. The cells were collected at 0, 2, 4, 8, 12, 24, 36,

2.5. Western blotting analysis

Fig. 1. Multiple sequence alignment and phylogenetic analysis of SKIV-FLP-FtsK domain with those in other megalocytiviral strains. (A), multiple sequence alignment of SKIV-FLP-FtsK domain with those in four genome-sequenced megalocytiviral isolates, based on partial amino acid sequences of FLP. (B), phylogenetic relationship of SKIV-FLP-FtsK with its homologous proteins from rock bream iridovirus [AAT71873.1], orange-spotted grouper iridovirus [AAX82371], infectious spleen and kidney necrosis virus [NP_612284], and turbot reddish body iridovirus [ADE34402.1], based on neighbor-joining analyses of the partial amino acid sequences of FLP.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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48, 72, and 96 h post infection (hpi) for RNA extraction, according to the protocols of the manufacturer (Promega, USA). Gene-specific primer sets of FLP-QT-F and FLP-QT-R, MCP-QT-F and MCP-QT-R, and actinQT-F and actin-QT-R (Table 1) were designed to amplify FLP, MCP, and actin genes, respectively. Each gene was assayed in triplicate at each sample time point. Three-step quantitative real-time PCR was performed on an ABI PRISM 7900 Sequence detection system using SYBR Premix Ex-Taq™ (Takara, Soochow in China), according to the instructions of the manufacturer. Reactions were performed in a 20 μl reaction system (10 μl of 2× SYBR Green PCR Mast Mix, 1 μl of 10 μM primers, 8 μl of nuclease-free water, and 1 μl of cDNA). The cycling parameters were 95 °C for 5 min, followed by 40 cycles of 95 °C for 5 s, 60 °C for 10 s, and 72 °C for 15 s. The threshold cycles and relative fold inductions were calculated using the ABI PRISIM 7900HD SDS software.

2.8. Plasmid construction Several recombinant plasmids were constructed for mammalian cell transfection and functional analysis. The primer sets are listed in Table 1. (i) Construction of pCMV-BD-FLP and pCMV-tag2B-FLP. The pCMV-BD contains the coding region of the GAL4 DNA-binding domain (DBD) and expresses a fusion protein of GAL4-DBD and FLP. The pCMVtag2B-FLP expresses a FLAG-targeted FLP protein. The recombinant plasmids were constructed using the FLP-NT-L and Grx-2-R primer set, and contain the coding region of the full length (1 aa to 1253 aa) of FLP. (ii) Construction of pEGFP-N1-targeted FLP and its mutants. The DNA fragment containing the coding region of the full length (1 aa to 1253 aa) was amplified using the FLP-NT-L & Grx-2-R primer set, FLPNT (1 aa to 380 aa) was amplified using the FLP-NT-L & FLP-NT-R primer set, FtsK (380 aa to 880 aa) was amplified using the Ftsk-L & Ftsk-R primer set, and Grx-2 (880 aa to 1253 aa) using the Grx-2-L & Grx-2-R primer set. These target DNA fragments were amplified from the SKIV-ZJ07-infected MFF-1 cell DNA template to generate fusion proteins of FLP and its three mutants with enhanced green fluorescent protein (EGFP).

3. Results and discussion 3.1. Cloning and sequence analysis of SKIV-FLP Based on the PCR amplification and sequence analysis, the full length of SKIV-FLP was cloned from SKIV-ZJ07-infected MFF-1 cells. The nucleotide sequence of SKIV-FLP was deposited in the GenBank database, with accession number of HM246185. The sequence consists of 3762 nucleotides and encodes a putative viral protein with 1253 amino acid residuals (protein ID: ADL09362), and the polypeptide sequence contains an FtsK (380 aa to 496 aa) or EBV-NA3 (397 aa to 561 aa) domain at the N-terminus, and a Grx-2 (880 aa to 988 aa) domain at the Cterminus (Supplemental Fig. 1). Multiple sequence alignment analysis shows that SKIV-FLP is highly identical to RBIV-ORF58L, but their homologs, such as ISKNV-ORF062L, RSBIV-ORF077R, and TRBIV-ORF057L (Fig. 1A), differ. In addition, an SKIV-FLP phylogenetic tree was constructed using the different members of the homologs sampled from the megalocytiviral strains (Fig. 1B). SKIV-FLP is located at the top of the branches, indicating that SKIV-FLP is a conserved protein. According to literature, all megalocytiviruses can be subdivided into three main genetic clades, which are represented by ISKNV, RSIV, and TRBIV (Kurita and Nakajima, 2012). In previous studies, SKIV-ZJ07 has been confirmed to be an RBIV-closed RSIV-type megalocytivirus, both in nucleotide and proteomic levels (Dong et al., 2011; Shuang et al., 2013). Thus, SKIV-FLP unsurprisingly has the closest genetic relationship to RBIV-058L.

3.2. Localization of SKIV-FLP in purified SKIV-ZJ07 virions Using rabbit anti-SKIV-rFtsK (380–880 aa), -ISKNV-rMCP, and ISKNVrMMP sera as primary antibodies, Western blotting shows that SKIV-FLP exists completely in the pellet fraction of the TX-100-treated SKIV virions, whereas MMP (ISKNV-ORF007 target protein), a known envelope protein in megalocytiviruses (Dong et al., 2011), exists mainly in the supernatant

2.9. Transient transfection for subcellular localization and dual luciferase assays Transient transfection was performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. For subcellular localization, HeLa cells were seeded on sterile microscope cover-glasses in a 6-well plate, and then transfected with 1 μg of the pEGFP-N1-FLP vector and its various truncation vectors. At 48 h after transfection, cells were washed with PBS buffer for 5 min, fixed with 4% paraformaldehyde for 10 min, and then stained for the nuclear fractions with 4′,6′-diamidino-2-phenylindole hydrochloride. The cells transfected with fluorescent vectors were directly observed under fluorescent microscopy or laser confocal imaging, as described previously (Xiang et al., 2010). For the dual luciferase assay, HEK293T cells were directly seeded in a 24-well plate before transfection. The pCMV-BD-FLP or pCMV-BD vector was transiently co-transfected into the HEK293T cells, along with the pL8G5-Luc reporter and pLexA-VP-16, using the Lipofectamine 2000 reagent, as described previously in the analyses on zebrafish IFN-γ promoter (NM_212864.1) and NF-κB luciferase reporter genes (Xiang et al., 2010). Cells were transfected in a serum-free culture medium (Gibco, USA). After 4 to 6 h of transfection, the medium was replaced with a complete medium with 10% FBS and antibiotics. The transfected HEK293T cells were lysed for the luciferase assay (Xiang et al., 2010). The luciferase activities of the total cell lysates were measured using the luciferase reporter assay system (Promega, USA). The Renilla luciferase activity was expressed as the fold stimulation relative to the transfected empty vector. Values were expressed as the mean relative stimulations for a representative experiment from the three separate experiments, with each experiment performed in duplicate.

Fig. 2. Virion localization of SKIV-FLP by Western-blotting analysis. Supernatant and pellet fractions of purified SKIV virions were isolated by treatment with 1% Triton X-100, and separated by 12% SDS-PAGE. The proteins were analyzed by WB using purified rabbit anti-sera against SKIV-FLP-FtsK, ISKNV-rMMP and ISKNV-rMCP, respectively. M, prestained broad range protein molecular mass marker. T, total protein of purified SKIV virions. S, supernatant fraction of detergent-treated SKIV virions. P, pellet fraction of detergent-treated SKIV virions.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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Fig. 3. Indirect immunofluorescence analysis of SKIV-ZJ07-infected MFF-1 cells using rabbit anti-SKIV-FLP (A), -ISKNV-rMMP (B) and -ISKNV-rMMP (C) sera, respectively.

fraction. By contrast, MCP was predominantly observed in the pellet fraction, with minor existence in the supernatant fraction (Fig. 2). A similar result was also observed in a study of these homologous viral proteins in ISKNV (Dong et al., 2011). Iridoviruses have four major structural components that include a central DNA–protein core, an internal lipid membrane, an icosahedral capsid, and an outmost membrane component, the viral envelope (Whitley et al., 2010). In our previous report, ISKNVMMP has been identified to be a major viral envelope protein in purified ISKNV and SKIV virions (Dong et al., 2011; Shuang et al., 2013). In the current study, both MCP and MMP were used as essential viral protein markers to assess the localization of SKIV-FLP in purified SKIV virions. As expected, SKIV-FLP was located below MCP, which strongly suggests that SKIV-FLP may be a major component protein located in the SKIV DNA–protein core. In a ranavirus, Singapore grouper iridovirus (SGIV), twelve viral proteins from the SGIV DNA–protein core were identified as DNA-binding proteins through multi-biotechnological approaches (Wang et al., 2013). However, no other viral proteins, excluding SKIVFLP and its homologs, were identified to be DNA-binding proteins in the viral DNA–protein core of megalocytiviruses. Future studies should be performed to explore more DNA-binding proteins in megalocytiviruses. IFA shows that SKIV-ZJ07-infected MFF-1 cells can be recognized well by anti-ISKNV-rMCP, -ISKNV-rMMP, and -SKIV-rFtsK antibodies (Fig. 3). Megalocytiviruses induce unique CPE in susceptible cells, characterized by increasingly round and enlarged cells (Dong et al., 2008, 2010; Imajoh et al., 2007). However, not all round cells are virus carriers. Almost all round cells can be stained well using the antibody against a nonstructural viral protein of ISKNV-VP23; however, only a number of

these cells can be stained using the antibody against the major capsid protein (MCP), the most popular structural viral protein in megalocytiviruses (Xu et al., 2010). In this study, as one of the most abundant viral structural proteins, SKIV-FLP shows similar expression pattern in the MFF-1 cells infected through the IFA approach, as well as those of ISKNV-MCP and -MMP. Furthermore, FLP, MCP, and MMP represent three different localization models, which are characterized as the DNA-core, major nucleocapsid, and outermost envelope, respectively. These

Fig. 4. Temporal analysis of mRNA expression of SKIV-FLP and -MCP in SKIV infected MFF-1 cells by real-time quantitative PCR at 0, 2, 4, 6, 8, 12, 24, 48, 60, 72, and 96 hpi. Transcription values were normalized to mandarin fish β-actin.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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three viral proteins can be used as essential marker proteins for assisting the localization of other viral structural proteins in megalocytiviral virions. 3.3. Temporal pattern of SKIV-FLP in infected MFF-1 cells To determine the temporal pattern of SKIV-FLP in infected MFF-1 cells, SKIV-MCP (accession number: GQ202216) and mandarin fish β-actin (accession number: AY885683) genes were used as controls for realtime qPCR analysis. The result indicates that the mRNA expression of SKIV-FLP shows a similar temporal pattern to that of SKIV-MCP (Fig. 4). MCP is a structural protein in megalocytiviruses and is expressed in the late stage of virus infection. Thus, as a major structural viral protein in SKIV, SKIV-FLP may also be a late expression structural protein. 3.4. Subcellular localization of SKIV-FLIP and its mutants To examine the subcellular localization of SKIV-FLP and its mutants, pEGFP-N1-FLP (1 aa to 1253 aa), pEGFP-N1-NT (1 aa to 380 aa), pEGFPN1-Ftsk (380 aa to 880 aa), and pEGFP-N1-Grx (880 aa to 1253 aa), which contain the full length of FLP, FLP-N terminal, FtsK domain, and

Grx-2 domain, respectively, were constructed and transfected into HeLa cells. EGFP-FLP was expressed both in the cytoplasm and nucleus, and partially expressed a heterogeneous fluorescent dot, suggesting that SKIV-FLP may be a multimeric protein (Fig. 5). Interestingly, GFPFLP-NT, GFP-FLP-Ftsk, and GFP-FLP-Grx fusion proteins were also expressed, especially in the nucleus (Fig. 5), indicating that at least three nuclear localization signals exist in SKIV-FLP. However, different localization patterns were also observed among the FLP mutants. For instance, most of the EGFP-FLP-NTs were expressed in the nucleus; only a small amount was expressed in the whole cell. By contrast, both EGFPFLP-FtsK and EGFP-FLP-Grx were expressed exclusively in the nuclei. EGFP-FLP-FtsK was expressed as bright dot patterns in the nucleus (Fig. 5), and localization model is similar to that of DAXX and PMAL, located in the nuclear body and involved in chromatin remodeling (Ishov et al., 2004). The proteins with FtsK domains are responsible for chromatin remodeling in bacteria, but few are homologous in viruses. According to bioinformatics analyses, FLP is a nucleus location protein. The nucleus location signal (NLS) exists at the 533th aa to the 550th aa in SKIV-FLP. The EGFP-FLP-FtsK (380 aa to 880 aa) contains the predicted NLS, and experimentally determined to be located in the nucleus. No NLS was predicted to exist in the FLP-NT and Grx domains; however,

Fig. 5. Subcellular location of SKIV-FLP and its three mutants in HeLa cells. FLP was localized in the whole of the cell and its three mutations were localized in nuclei.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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Fig. 6. Transcriptional activity analysis of SKIV-FLP in HEK293T cells. The FLP inhibits the transcriptional activity of L8G5-luc to 2/3. Over-expression of the FLP inhibited NF-κB transcriptional activity to 1/18 (B) and the promoter of IFN-γ transcriptional activity to 1/7 (C).

the experiments show that both FLP-NT (1 aa to 380 aa) and FLP-Grx (880 aa to 1253 aa) are also nuclear localization proteins, indicating that the FLP has new nucleus location signals or models. Unexpectedly, the full length of FLP is localized both in the cytoplasm and nucleus, forming some dots in the cytoplasm (Fig. 5), which may indicate that the protein is activated by certain signaling molecules or kinases and oligomerizes by self-interaction with the FtsK-domain. In addition, FLP possibly contains three NLSs, such that FLP can shuttle among the different subcellular organelles through a certain mechanism by regulating NLSs. The early chromosome replication of iridoviruses occurs in the nucleus, and the latter virus chromosome replication, that is packing, happens in the cytoplasm (Goorha and Dixit, 1984). This large multidomain protein may therefore function as a matrix or scaffold for the assembly of different complexes. Moreover, the structure and function of FLP are beneficial to virus replication and packaging at the different cell departments. 3.5. Functional analysis of SKIV-FLP through Luc assays A fusion protein of SKIV-FLP and the DNA-binding domain (DBD) of the yeast transcription factor GAL4 under a CMV promoter was constructed to examine the potential function of FLP in transcriptional regulation. The pCMV-BD-FLP and pL8G5-Luc were co-transfected into HEK293T cells. The result shows that FLP is a transcriptional inhibitor that represses the activity to 50% (Fig. 6A). SKIV-FLP has an Ftsk or an EBV-NA3 domain and a Grx-2 domain, which may participate in the duplication, nuclear-rebuilding, and transcription. As a major viral structural protein in SKIV, FLP is also involved in host immune response (Shuang et al., 2013). To detect the FLP protein function in cell signaling pathways, luciferase report assay was performed. The result shows that FLP represses the activation of NF-κB and INF-γ promoter to 1/18 and 1/7, respectively (Fig. 6B and C), suggesting that FLP may be involved in these two signal pathways. The proteins containing Ftsk, EBNA, or Grx-2 domains are involved in viral transcription, replication, and virion morphogenesis (Ambinder et al., 1991; Becnel and White, 2007; Hirota et al., 2000; Jiang et al., 2000). SKIV-FLP has been predicted to contain an Ftsk (or EBNA) and a Grx-2 domain, with chromatin remodeling functions. The basic chromatin remodeling complexes altered by specialized modifiers may be active at different promoters in different specialized cells, or in different parts of the cell cycle (Ishov et al., 2004). When the protein was transiently transfected with L8G5-Luc, the results indicate that SKIV-FLP acted as a transcription inhibitor, repressing the expression of L8G5-Luc.

Furthermore, the infection of cells with viruses always results in the overall inhibition of the transcription of some crucial host genes. Latent infection-associated genes always have important roles in evading host immunity by regulating the expression of NF-κB, INF-γ, or other immune-related genes (Doukas and Sarnow, 2011; Schweighardt et al., 2010). In this study, SKIV-FLP represses the expression of NF-κB and INF-γ (Fig. 6A–C), suggesting that SKIV-FLP may be involved in virus evasion from the host immune system. 4. Conclusion This current study was the first to characterize an FtsK-like protein from megalocytivirus and to demonstrate the potential of this protein to be a major component in the virus. In vitro experiments showed that the protein inhibited NF-κB and INF-γ transcriptional activities, indicating that the protein may be involved in immune evasion from the host. These studies provide a new insight for better understanding the structure and function of FtsK-like proteins in megalocytiviruses. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.026. Conflict of interests The authors declare that there is no conflict of interest. Acknowledgments This research was supported by the National Basic Research Program of China under grant no. 2012CB114406; the National Natural Science Foundation of China under grant no. 31202021 and the Technology Planning Project of Guangdong Province under grant number 2011A020102002. References Ahn, B.Y., Moss, B., 1992. Glutaredoxin homolog encoded by vaccinia virus is a virionassociated enzyme with thioltransferase and dehydroascorbate reductase activities. Proceedings of the National Academy of Sciences of the United States of America 89, 7060–7064. Ambinder, R.F., Mullen, M.A., Chang, Y.N., Hayward, G.S., Hayward, S.D., 1991. Functional domains of Epstein–Barr virus nuclear antigen EBNA-1. Journal of Virology 65, 1466–1478. Arad, G., Levy, R., Nasie, I., Hillman, D., Rotfogel, Z., Barash, U., Supper, E., Shpilka, T., Minis, A., Kaempfer, R., 2011. Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock. PLoS Biology 9, e1001149.

Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026

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Please cite this article as: Xiang, Z., et al., Identification and characterization of a novel FstK-like protein from spotted knifejaw iridovirus (genus Megalocytivirus), Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.026