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State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of Chinese Academy of Sciences, Wuhan 430072, PR China
Correspondence
Qi-Ya Zhang
zhangqy{at}ihb.ac.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the Rana grylio virus 53R sequence determined in this work is EU358954.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Previous studies have revealed that RGV is a member of the family Iridoviridae and is closely related to frog virus 3 (FV3), the type species of the genus Ranavirus (Zhang et al., 1999, 2001, 2006). Recently, cellular changes and several viral proteins have also been investigated during RGV infection and replication (Huang et al., 2006, 2007; Sun et al., 2006; Zhao et al., 2007).
Iridoviruses are large, icosahedral, enveloped DNA viruses that contain circularly permutated and terminally redundant double-stranded DNA genomes (Fauquet et al., 2005; Williams et al., 2005). To date, 12 iridovirus genomes have been sequenced completely (Eaton et al., 2007), but the key genes and their functions remain to be elucidated, especially the viral envelope protein genes. Viral envelope proteins are particularly important because of their vital roles in virus assembly and infection (Chazal & Gerlier, 2003). Recently, two proteins were characterized as membrane-tropic structural proteins, but these are conserved only among members of the genus Megalocytivirus in the family Iridoviridae (Ao & Chen, 2006; Xu et al., 2008). To understand molecular mechanisms for iridovirus assembly and infection, we focused our attention on some of the transmembrane (TM) proteins that are shared by all sequenced iridoviruses. For this purpose, a TM protein gene corresponding to open reading frame (ORF) 53R of FV3 (Tan et al., 2004) was found to be conserved among all sequenced iridoviruses. In this study, we cloned and characterized the TM protein gene from RGV and revealed its possible functional role in virus assembly.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2006).
Gene cloning and protein sequence analysis.
Using the sequences of the conserved 53R genes of the FV3 and tiger frog virus genomes (He et al., 2002; Tan et al., 2004), a pair of primers (5'-TCCACATAAAATCTACTCCGAT-3' and 5'-GAAAGGAATGCAAGTCCTATC-3') located in the 5'- and 3'-flanking regions of the 53R ORF was designed and used to amplify RGV 53R from the genomic DNA. The fragment was cloned and sequenced, and the sequence data were compiled and analysed using DNASTAR software. The non-redundant protein sequence database of the National Center for Biotechnology Information (National Institutes of Health, MD, USA) was searched using BLASTP and iterative searches were performed using PSI-BLAST (Altschul et al., 1997). Multiple sequence alignments were constructed using CLUSTAL_X v1.83 and edited using GeneDoc.
Prokaryotic expression, protein purification and antibody preparation.
Because there are two hydrophobic domains in the middle region of RGV 53R, a fragment encoding the C-terminal 279 aa unique to RGV 53R was amplified using primers 5'-GTAGGATCCGAGGGCTTCTCGTCCG-3' and 5'-GTCCTCGAGATCCTTTACCCCTGTGG-3' and ligated into the prokaryotic vector pET-32a (Novagen). The recombinant plasmid, named pET32a/53R, was transformed into Escherichia coli BL21(DE3) and the bacteria were induced for 4 h with 1 mM IPTG at 37 °C to express the fusion protein. The fusion protein was purified from inclusion bodies under denaturing conditions using a HisBind purification kit (Novagen), mixed with an equal volume of Freund's adjuvant (Sigma) and used to immunize mice by hypodermal injection once every 7 days. After the fifth immunization, anti-RGV 53R serum was collected and tested by Western blotting with lysates from virus-infected cells. Its specificity was validated by a pre-adsorption experiment. Detection of cellular 
-tubulin was used as an internal control.
RT-PCR and Western blot analysis.
Total RNA and protein were isolated from cells infected with RGV at an m.o.i. of 1 at various times post-infection (p.i.) (0, 4, 8, 12, 16, 24, 36 and 48 h) or mock infected, and were subjected to RT-PCR and Western blot analysis, respectively, as described previously (Zhao et al., 2007). For RT-PCR, the specific primers 5'-CATCAGAACGGGAGGACAGA-3' and 5'-CGCCGTGTCGTCCTTGTAG-3' were used to monitor the transcription of RGV 53R. For Western blot analysis, anti-RGV 53R serum was used as the primary antibody at a dilution of 1 : 500, followed by alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibody at a dilution of 1 : 1000 (Vector Laboratories) as the secondary antibody. Internal controls were carried out simultaneously by detecting -tubulin mRNA and protein, respectively.
Cytosine β-D-arabinofuranoside (AraC), a viral DNA replication inhibitor, was used to classify the transcription class of RGV 53R. Briefly, 100 µg AraC ml–1 was added to FHM cells for 1 h prior to virus infection and the pre-treated cells were then mock infected or infected with RGV at an m.o.i. of 1. Total protein was extracted at 24 and 48 h p.i. for Western blot analysis.
Immunofluorescence microscopy.
FHM cells, grown on coverslips in six-well plates, were mock infected or infected with RGV at an m.o.i. of 0.1 and then fixed in 70 % ethanol overnight at –20 °C at 24, 36 and 48 h p.i. After blocking in 10 % normal goat serum at room temperature for 1 h, the cells were incubated with anti-RGV 53R serum in 1 % normal goat serum for 2 h, rinsed three times for 10 min each with PBS containing 1 % normal goat serum and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibodies (Pierce). 4,6-Diamidino-2-phenylindole (DAPI; Sigma) staining was used to visualize DNA in nuclei and virus factories. All samples were examined under a Leica DM IRB fluorescence microscope.
Transient transfection and subcellular localization.
A recombinant eukaryotic vector, pEGFP-N3/53R, was constructed by cloning the entire 53R ORF into pEGFP-N3 (Clontech) using primers 5'-CTAAAGCTTTATGTAGGGAAAATGGGA-3' and 5'-ATGGATCCCTATCCATAACCCCTGT-3'. This vector expressed RGV 53R with a C-terminal fusion green fluorescent protein (GFP) tag. Plasmids pEGFP-N3/53R and pDsRed2-ER, an endoplasmic reticulum (ER)-specific marker (Clontech), were transiently co-transfected into FHM cells to evaluate the intracellular location of RGV 53R. The cells were fixed at 12 and 48 h post-transfection and examined by fluorescence microscopy. To track the fate of RGV 53R in transfected cells in more detail, cells were infected with RGV at the same time as transfection. After 48 h, the cells were fixed and stained with DAPI as described above and then examined by fluorescence microscopy.
Detergent extraction and phase separation of purified virions.
Membrane component was extracted from RGV virions with a non-ionic detergent as described previously (Ojeda et al., 2006b). In brief, purified virions were treated with 50 mM Tris/HCl (pH 7.4) containing 1 % NP-40 detergent in the presence or absence of 50 mM dithiothreitol (DTT). The mixture was incubated for 1 h at 37 °C, and insoluble and soluble materials were separated by centrifugation at 15 000 g for 1 h. Proteins from the pellet and supernatant were analysed by 12 % SDS-PAGE and transferred to PVDF membrane for Western blot analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, the C-terminal region was less well conserved than the N-terminal region and the C-terminal length was variable.
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). The fusion protein was purified and used to generate anti-RGV 53R polyclonal antibody in mice. To test the specificity of the antibody, lysates from RGV-infected and mock-infected cells were first subjected to Western blot analysis. As shown in Fig. 2(b), the anti-RGV 53R antibody specifically recognized a 54.7 kDa protein in RGV-infected cell lysate, which corresponded to the theoretical molecular mass of RGV 53R, whereas no protein band was detected in the mock-infected cell lysate. Moreover, when the same antiserum was pre-adsorbed with the purified fusion protein, the specific 54.7 kDa protein in the RGV-infected cell lysate could not be detected using the pre-adsorbed antiserum. As an internal control, the -tubulin content was shown to be consistent between the lysates from the RGV-infected and mock-infected cells (Fig. 2c). These data indicated that the antibody was specific to RGV 53R.
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). At the protein level, a 54.7 kDa protein band was detected by Western blotting from 16 h p.i. in RGV-infected cells and its level also increased up to 48 h p.i. (Fig. 3b). As an internal control, the mRNA and protein levels of -tubulin expression were consistent throughout the experiments. The 54.7 kDa protein band was not detected when viral DNA replication was inhibited by AraC (Fig. 3c), consistent with the temporal data, indicating that RGV 53R belongs to the late expression class of genes.
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, when FHM cells were infected with RGV for 24 h, RGV 53R initially appeared in the cytoplasm and aggregated mainly at the two poles of the cells. At later times p.i., the protein converged at the perinuclear region and co-localized with the virus factories for virus assembly. At 48 h p.i., the distribution of the protein was more compacted around the virus factories than at 36 h p.i.
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), indicating that RGV 53R was targeted to ER membranes in transfected cells. However, as the time post-transfection increased, the synthesized 53R–GFP became more compacted and accumulated into spots at 48 h post-transfection. No co-localization with the ER marker was observed at that time (Fig. 5a). Interestingly, GFP fluorescence and DAPI staining revealed that the accumulated spots of 53R–GFP fluorescence were the sites of virus factories for virus assembly (Fig. 5b). In addition, ER marker labelling and DAPI staining also showed that the virus factory sites excluded the tested ER marker components, but they were closely surrounded by the ER components (Fig. 5c).
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, when the purified RGV virions were extracted using buffer without 1 % NP-40 or 50 mM DTT, no RGV 53R was detected in the soluble components. However, when the purified RGV virions were extracted using buffer with 1 % NP-40, RGV 53R could be detected equally in the supernatant and in the pellet. Moreover, if the purified RGV virions were extracted using buffer with 1 % NP-40 and 50 mM DTT, the protein was dissolved more efficiently in the supernatant. These results indicated that RGV 53R is associated with the virion membrane and is a viral envelope protein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) were revealed in the RGV 53R protein, implying that RGV 53R may be involved in virus infection and assembly (Ravanello & Hruby, 1994; Wolffe et al., 1995; Martin et al., 1997; Ojeda et al., 2006a, b). Therefore, the RGV 53R gene was selected for further characterization to understand its possible functional role in virus infection and assembly.
Iridoviruses have been found to display a complex gene regulation strategy in which genes are expressed in three main temporal kinetic stages: immediate-early, early and late (Nalcacioglu et al., 2003, 2007; Lua et al., 2005; Chen et al., 2006). Late genes are expressed after the onset of viral DNA replication and their expression can be blocked by viral DNA replication inhibitors (Chambers et al., 1999; Ebrahimi et al., 2003). In this study, our data showed that the transcriptional and translational products of RGV 53R were not produced prior to 12 h p.i. or in the presence of AraC, indicating that this gene belongs to the late gene expression class.
Replication and assembly of iridoviruses often take place in specific intracellular compartments known as the ‘viromatrix’ or ‘virus factories’ where viral components concentrate, including structural proteins and genomic DNA, as well as different types of membranous structure (Novoa et al., 2005; Huang et al., 2006; Netherton et al., 2007). Significantly, the association of RGV 53R with virus factories was not only revealed by immunofluorescence localization in RGV-infected cells, but also confirmed by expressing the 53R–GFP fusion protein in pEGFP-N3/53R-transfected cells. Finally, the RGV 53R protein was confirmed as being associated with the virion membrane by detergent extraction and Western blot detection. Therefore, the current data suggest that RGV 53R is a viral envelope protein.
In addition, the intracellular distribution and dynamic changes of RGV 53R in pEGFP-N3/53R-transfected cells revealed an interesting phenomenon. RGV 53R initially co-localized with the ER components at an early stage post-transfection, but as the post-transfection time increased, the ER marker components were excluded from the virus factories. Why? A possible explanation is that RGV 53R may play a role in recruiting ER-derived membranes into virus factories as the precursors of the virus inner envelope. Moreover, the process of recruitment appeared to modify or damage the cellular ER components so that the ER components were excluded from the virus factories. African swine fever virus (ASFV), the only member of the family Asfarviridae, shares close similarities in morphology and morphogenesis with iridoviruses (Cobbold et al., 2001). Previously, it has been reported that the ASFV envelope protein p54 is critical for the recruitment and transformation of collapsed ER membranes as the precursors of the inner viral envelope (Rodríguez et al., 2004). Therefore, another key aspect of future work is to clarify the function of RGV 53R and its interaction with cellular ER proteins.
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ACKNOWLEDGEMENTS |
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Received 24 January 2008;
accepted 28 March 2008.
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