Characterization of subcellular localization of duck enteritis virus UL51 protein

The DEV UL51 gene (GenBank No. DQ072725), with a size of 759 bp, encoded a 252 amino acid protein, was identified in our laboratory [6]. Based on predicted amine acid sequence of DEV pUL51, various bioinformatics-aided tools: TargetP 1.1, SignalP 3.0 and TMHMM 2.0 server (from the search engine http://​www.​cbs.​dtu.​dk/​services/​) [16], PredictNLS server (from the search engine http://​www.​rostlab.​org/​services/​predictNLS/​) [17], CSS-Palm 2.0 online server (from the search engine http://​csspalm.​biocuckoo.​org/​online.​php) [18], and Golgi predictor (from the search engine http://​ccb.​imb.​uq.​edu.​au/​golgi/​golgi_​predictor.​shtml) [19], were used to analyze the possible localization of the pUL51.

DEV CHv strain is a high-virulence field strain isolated from china, obtained from Key Laboratory of Animal Disease and Human Health of Sichuan Province [20, 21]. Duck embryo fibroblasts (DEF) were cultured in MEM medium (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) (Gibco-BRL) at 37°C. For virus infection, MEM medium supplemented with 2–3% FBS was used.

A rabbit polyclonal UL51 antiserum (obtained from our Laboratory), raised against a recombinant 6-His-UL51 fusion protein expressed in E. coli, was purified using caprylic acid and ammonium sulfate precipitation and High-Q anion-exchange chromatography [22, 23]. The purified UL51 antiserum was subsequently used as primary antibody. Besides, a pre-immune rabbit serum was also obtained from our Laboratory and purified as described above. The purified pre-immune serum was used as a negative control.

DEF grown in the 6-well plates, were either mock-infected or infected with DEV CHv strain at a multiplicity of 5 PFU per cell, and harvested at 24 h p.i. Cells were lysed in SDS sample buffer, electrophoretically separated on SDS-polyacrylamide gels (SDS-PAGE) and electrically transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Japan). A nonspecific protein binding was blocked by treating membranes at 4°C overnight with TBST (25 mmol Tris-HCl, 150 mmol NaCl, pH 7.4, and 0.05% Tween-20) containing 5% bovine serum albumin (BSA). Then, the membranes were incubated at 37°C for 1 h with a 1:1000 dilution of the purified UL51 antiserum or pre-immune serum in TBST containing 0.1% BSA. After washing 3 times with TBST, the membranes were incubated at 37°C for 1 h with a 1:10000 dilution of goat anti-rabbit peroxidase-labeled second antibody (Sino-American Biotechnology Co., Shanghai, China). Washed 3 times with TBST again, the membranes were subsequently treated with an enhanced chemiluminescence (ECL) western blotting detection system (Amersham) and exposed to Hyperfilm-ECL (Amersham).

DEF, grown on coverslips in the 6-well plates, were either mock-infected or infected as described above. At different times (3, 6, 9, 12, 24, 36, 48 and 60 h p.i.), the cells were fixed with 4% paraformaldehyde for 20 min at 4°C and permeabilized with 0.1% Triton X-100 for 20 min at room temperature. The cells were then washed once with PBS and blocked for 1 h in PBS containing 10% BSA at 37°C. They were then incubated with a 1:100 dilution of the purified UL51 antiserum or pre-immune serum at 4°C overnight, washed 3 times for 10 min in PBS, and then treated with FITC conjugated goat anti-rabbit IgG (Sino-American Biotechnology Co.) for 45 minutes at 37°C. As described by Miller [24], the cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) counter-staining (5 μg/ml, Beyotime Institute of Biotechnology, Shanghai, China). Fluorescent images were examined under the Bio-Rad MRC 1024 imaging system.

DEF were grown in the 6-well plates and were either mock-infected or infected as described above. At different times (3, 6, 9, 12, 24, 36, 48 and 60 h p.i.), the cells were fixed with modified PLP fixative (10 mM NaIO4, 75 mM lysine, 37.5 mM phosphate buffer (PB) (pH 7.4), 4% paraformaldehyde, 0.1% glutaraldehyde) for 4 h and then washed with 0.1 M PB [25]. The cells were then harvested from the 6-well plates by scraping, resuspended in PB, and pelleted by low-speed centrifugation. The cell pellet was washed with PB, dehydrated through a graded series of ethanol, and embedded in LR White resin (London Resin Company) according to the manufacturer's instructions. Ultrathin sections were collected onto Formvar-coated nickel grids. The sections were incubated with 20% normal goat serum in PBS containing 1% BSA for 1 h at room temperature, washed five times in PBS, and incubated with the purified UL51 antiserum or pre-immune serum adequately diluted in PBS-1% BSA for 2 h. After five washes in PBS, the sections were incubated for 1 h with the secondary antibody goat anti-rabbit immunoglobulin conjugated with 10-nm-diameter gold particles (British BioCell International) and then washed five times in PBS and twice in double-distilled water. The sections were double stained with 4% uranyl acetate for 30 min followed by Reynold's lead citrate solution for 5 min [11]. Carbon-coated sections were examined with a Hitachi H-600 transmission electron microscope at 75 kV.

The DEV pUL51 contains no potential mitochondrial targeting peptide, signal peptides, transmembrane helices and nuclear localization signal (NLS). However, it possesses one potential palmitoylation site at the position 9 amine acid (cysteine) near the N-terminal of the pUL51 with a high score (4.217). Moreover, the pUL51 is predicted as a Golgi type II membrane protein (Golgi localised transmembrane protein) with index values (20.662) greater than the threshold (20.005).

The purified UL51 antiserum and pre-immune serum was examined by SDS-PAGE (Fig 1A, lanes 1 and 2). To examine the reactivity and specificity of the UL51 antiserum, SDS-PAGE and western blotting was performed (Fig 1B, lanes 1 to 6). The results of western blotting showed that the UL51 antiserum reacted strongly with an approximate 34 kDa protein in lysates of DEV-infected cells (Fig 1B, lane 5). This band was not detected in mock-infected cells (Fig 1B, lane 4), and the pre-immune serum did not recognize any proteins in lysates of DEV-infected cells (Fig 1B, lane 6). These results indicated that the UL51 antiserum specifically detected the primary translation product of the UL51 gene; therefore, we used this UL51 antiserum for further experiments to study the locations of the DEV pUL51.

A detailed analysis of the intracellular localization of DEV pUL51 was investigated using the purified UL51 antiserum or pre-immune serum by IIF staining of mock- and DEV-infected cells. As shown in Fig 2, a faint pUL51-specific fluorescence was first detected in the cytoplasm of DEV-infected cells at 9 h p.i. (Fig 2C), and then a strong fluorescence was observed mainly in the juxtanuclear region at 12 h p.i. (Fig 2D). After that, the pUL51-specific fluorescence in the juxtanuclear region was dense and localized on wide areas of the cytoplasm. At 36 h p.i. (Fig 2E), the pUL51-specific fluorescence was found widely distributed in the cytoplasm and especially was stronger in the juxtanuclear region; meanwhile, the nucleus of some DEV-infected cells also contained little fluorescence granular. Following by a series of morphological changes, the cytoplasm disintegration and nuclear fragmentation in DEV-infected cells, the intensity of the reaction increased at 48 and 60 h p.i. (Fig 2F), while the pUL51-specific fluorescence was mainly detected in the cytoplasm of infected cells and that one localized in the nuclear was faint. No pUL51-specific fluorescence could be detected in mock-infected cells reacted with the UL51 antiserum (Fig 2A) and in DEV-infected cells reacted with the pre-immune serum (Fig 2B).

To identify the precise localization of DEV pUL51 in DEV-infected cells, TIEM was carried out. Immunoelectron microscopy showed that at 6 h p.i., only a little pUL51-specific immuno-labeling was first observed in the cytoplasm of DEV-infected cells (data not shown). At 12 h p.i., intense pUL51-specific immuno-labeling was detected in the juxtanuclear region (Fig. 3B), probably associated with Golgi apparatus. After that, ultrastructural changes of DEV-infected cells were especially remarkable, an increasing number of virus particles were accumulated in the cytoplasm with expansion of endoplasmic reticulum and formation of specialized vesicles. Starting from 24 h p.i., some immuno-labeling was found being associated with cytoplasmic virions and also with some membranous structure that was observed near the pUL51-specific immuno-labeling cytoplasmic virions in the cytoplasmic vesicles (Fig. 3C), and thereafter increasingly until 48 p.i. (Fig. 3D). At later times (60 h p.i.), the positive labeling was mainly localized in the cytoplasm and especially was scattered near the plasma membrane of DEV-infected cell (data not shown). No pUL51-specific immuno-labeling was seen in the DEV-infected cells reacted with the pre-immune serum (Fig. 3A) and in the mock-infected cells reacted with the UL51 antiserum (data not shown).