Molecular characterization of the duck enteritis virus US10 protein

Abstract

Background: There is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization.

Methods: Duck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance.

Results: The recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a γ2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein.

Conclusions: The DEV US10 gene is a γ2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.

Keywords: Duck enteritis virus; Kinetic class; Subcellular localization; True late gene; US10; Virion protein; γ2 Gene.

Fig. 1

Expression, identification, and purification of the recombinant US10 protein. a Expression of the recombinant protein. Total protein stain. Lane M: markers; lanes 1–3: the whole bacterial lysate, supernatant, and inclusion bodies of pET32a(+)/US10; lanes 4 and 5: the induced and uninduced pET32a(+). b Identification of the recombinant protein by western blotting with anti-DEV serum; lane M: markers; lane 1: pET32a(+); lanes 2–4: the whole bacterial lysate, supernatant, and inclusion bodies of pET32a(+)/US10. c Purification of the recombinant US10 protein. Detection with anti-DEV serum. Lane M: markers; lane 1: purified recombinant pET32a(+)/US10 protein

Fig. 2

Preparation and verification of the polyclonal antibody raised against DEV US10. a Agar diffusion reaction test. Middle well: purified recombinant pET32a(+)/US10 protein. b Cross-reactivity test; lane M: markers; lane 1: the cell lysates of pET32a(+); lane 2: the cell lysates of pET32a(+)/US10. c The DEV US10 was recognized by purified polyclonal antibody. Lane 1: mock-treated DEFs; lane 2: DEV-infected DEFs

Fig. 3

Expression of US10 protein and β-actin in DEV-infected cells. Proteins isolated from mock or DEV-infected cells at different times were subjected to western blot analysis with US10 or β-actin antiserum

Fig. 4

The melting curves and standard curves of DEV US10 gene and β-actin by qRT-PCR. a: The melting curve of the DEV US10 gene displayed a single peak at 84.0 °C, while β-actin displayed a single peak at 89.0 °C. b: The standard curves of DEV US10 and β-actin genes were calculated by iCycler IQ 5 software. Each dot represents the result of triplicate amplification of each dilution. The correlation coefficient and the slope of the regression curve were calculated and indicated. The standard curve equation of the DEV US10 gene is Y = −3.320X − 1.340, while that of the β-actin gene is Y = −3.241X − 1.801

Fig. 5

Transcriptional analysis of the DEV US10 gene. Total RNA was isolated from the DEV-infected DEF cells at each post-infection time point and converted to cDNA. Samples of cDNA were amplified using qPCR and SYBR green detection. Data are presented as the fold change in the expression of the DEV US10 gene. The transcriptional expression of the DEV US10 gene was normalized to that of a reference gene (β-actin)

Fig. 6

Pharmacological inhibition test showed that US10 is a true late gene. Lane M: markers; lanes 1, 4, and 7: DEV-infected cells, without any drugs; lanes 2, 5, and 8: DEV-infected cells treated with 100 μg/mL cyclohexamide (protein synthesis inhibitor); lanes 3, 6, and 9: DEV-infected cells treated with 300 μg/mL ganciclovir (DNA polymerase synthesis inhibitor). DEV UL55 and β-actin genes were used as a γ2 gene and a housekeeping gene, respectively

Fig. 7

Subcellular localization of DEV US10 (400×). DEF cells were infected with DEV for 12, 24, 36, and 48 h. The cells were fixed, permeabilized, and stained with anti-US10 serum and FITC-conjugated goat anti-rabbit antibody, followed by DAPI. Panels a, d, g, j: US10 protein expressed in DEV-infected DEF cells. Panels b, e, h, k: nucleus of DEV-infected DEF cells. Panels c, f, i, l: US10 protein is localized in the perinuclear region

Fig. 8

Mock-infected cells, blank control, preimmune serum control, and spontaneous fluorescence control subjected to immunofluorescence analysis (400×). Panels a, d, g, j: US10 protein was not detected in aboved control cells. Panels b, e, h, k: nucleus of aboved control cells. Panels c, f, i, l: There is no green fluorescence produced by false positives

Fig. 9

Western blot analysis of purified DEV virions. Virions purified from DEF cells were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against the US10 protein and a control glycoprotein C envelope protein (gC). Total mock-infected or infected cell lysates were also included as an antibody control