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. 2022 Jan 4:12:792361.
doi: 10.3389/fmicb.2021.792361. eCollection 2021.

UL11 Protein Is a Key Participant of the Duck Plague Virus in Its Life Cycle

Linjiang Yang  1   2   3 , Mingshu Wang  1   2   3 , Anchun Cheng  1   2   3 , Qiao Yang  1   2   3 , Ying Wu  1   2   3 , Juan Huang  1   2   3 , Bin Tian  1   3 , Renyong Jia  1   2   3 , Mafeng Liu  1   2   3 , Dekang Zhu  2   3 , Shun Chen  1   2   3 , Xinxin Zhao  1   2   3 , Shaqiu Zhang  1   2   3 , Xumin Ou  1   2   3 , Sai Mao  1   2   3 , Qun Gao  1   2   3 , Di Sun  1   2   3 , Yanlin Yu  1   2   3 , Ling Zhang  1   2   3
Affiliations

UL11 Protein Is a Key Participant of the Duck Plague Virus in Its Life Cycle

Linjiang Yang et al. Front Microbiol. .

Abstract

Tegument protein UL11 plays a critical role in the life cycle of herpesviruses. The UL11 protein of herpesviruses is important for viral particle entry, release, assembly, and secondary envelopment. Lipid raft is cholesterol-rich functional microdomains in cell membranes, which plays an important role in signal transduction and substance transport. Flotillin and prohibition, which are considered to be specific markers of lipid raft. However, little is known about the function of duck plague virus (DPV) UL11 in the life cycle of the viruses and the relationship between the lipid raft and UL11. In this study, an interference plasmid shRNA126 for UL11 was used. Results showed that UL11 is involved in the replication, cell to cell spread, viral particle assembly, and release processes. Furthermore, UL11 was verified that it could interact with the lipid raft through sucrose density gradient centrifugation and that function correlates with the second glycine of the UL11. When the lipid raft was depleted using the methyl-β-cyclodextrin, the release of the DPV was decreased. Moreover, UL11 can decrease several relative viral genes mRNA levels by qRT-PCR and Western blot test. Altogether, these results highlight an important role for UL11 protein in the viral replication cycle.

Keywords: Duck plague virus; UL11; lipid raft; prohibition; replication.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
DPV growth curve analysis of interfering UL11 in DEF cells. (A) In the transfection condition, DEF cells were transfected UL11 with shRNA37, shRNA126, shRNA218, and shRNAneo, respectively. After 24 h, cells were collected to do Western blot analysis. (B) Quantification of the UL11 downregulation ratio. **P < 0.01; nsP > 0.05. (C) The effect of the shRNAneo on the replication of DPV. nsP > 0.05. (D) Growth curve of the UL11 downregulated mutant. The DPV infection at 0.01 MOI. ****P < 0.0001.
FIGURE 2
FIGURE 2
UL11 in the viral adsorption, invasion, replication, release processes, and cell to cell spread. (A) DEF cells were transfected with shRNA126 and shRNAneo, then inoculated with 1 MOI DPV to undergo the adsorption experiment. The cell samples were collected for virus copy number detection. nsP > 0.05. (B) The shRNA126 and shRNAneo plasmids were transfected into the DEFs, then inoculated with 1 MOI DPV to detect invasion experiment. After incubation for 3 h, the cell samples were collected for virus copy number detection. nsP > 0.05. (C) The shRNA interference plasmid was transfected into the DEFs, and then the cells were incubated with 1 MOI DPV to detect DPV replication. Cells were cultured at 37°C for 6, 7, 8, 9, and 10 h, the cells samples were collected for virus copy number detection. ***P < 0.001; ****P < 0.0001. (D) The shRNA interference plasmids were transfected into the DEFs, then the cells were incubated with 1 MOI DPV to detect DPV egress. The supernatant was collected and tested TCID50 in the culture for 15, 30, 45, and 60 min. nsP > 0.05; *P < 0.05; **P < 0.01. (E) Green fluorescent plaques produced by the shRNA126 and shRNAneo. Statistical analysis of randomly selected viral green fluorescent plaques at the right. Plates were scanned, and plaque diameters were measured in Image J. ****P < 0.0001. (F) Crystal violet assay to test the cell to cell spread. Representative plaques are shown. 25 plaques per sample were measured to quantify the results at the right. Plates were scanned, and plaque diameters were measured in Photoshop. ****P < 0.0001.
FIGURE 3
FIGURE 3
Electron microscopy analysis of DPV, shRNA126, and shRNAneo. (A) Electron micrographs of a DEF cell for the UL11 interfering group shRNA126. (B) Higher magnifications with close-ups of selected areas show incompletely enveloped particles for shRNA126. (C) Electron micrographs of a DEF cell for the shRNAneo group. (D) Higher magnifications with close-ups of selected areas show fully enveloped particles for shRNAneo. (E) Electron micrographs of a DEF cell for the DPV group. (F) Higher magnifications with close-ups of selected areas show fully enveloped particles for DPV. The orange triangle indicates the viral particles with complete envelopes, and the green triangle indicates the viral particles with membrane-attached.
FIGURE 4
FIGURE 4
Effect on the other viral genes when downregulated the UL11. (A) mRNA level of UL11, UL16, UL21, gE, ICP22, ICP27, ICP4, US3, and gB. Transfected the shRNA126 into the DEF cells and then infected the cells with 1MOI DPV. After 24 h, the DEF cells were harvested and did the qRT-PCR. nsP > 0.05; ***P < 0.001; ****P < 0.0001. (B) Protein expression levels of UL11, UL16, UL21, gE, ICP22, ICP27, US3, and gB when interfering with the UL11 gene. Transfected the shRNA126 into the DEF cells and then infected the cells with 1MOI DPV. After 24 h, the DEF cells were harvested and did the Western blot to undergo grayscale value. nsP > 0.05; *P < 0.05.
FIGURE 5
FIGURE 5
UL11 can interact with the lipid raft marker, flotillin-1, and prohibition. (A) DEF cells were transfected with EGFP-N1-UL11 for 24 h, then cells were fixed and did the Immunofluorescence analysis. The primary antibody is an anti-FLOT-1 monoclonal antibody, and the secondary antibody is Alexa Fluor 594 Goat anti-Rabbit IgG. The nuclei were stained with DAPI. (B) The co-immunoprecipitation between the UL11 and PHB. HEK 293T cells were transfected with UL11 and pCAGGS for 24 h. Then the samples were collected to do the co-immunoprecipitation. (C) As described in materials and methods, HEK 293T cells were transfected with UL11 and collected 48 h post-transfection. After removed of cell nuclei, cell lysates were subjected to sucrose-gradient centrifugation. Fractions were collected from the top of the tube. Proteins in each fraction were separated by SDS-PAGE and analyzed by Western blotting. (D) As described in Materials and methods, HEK 293T cells were transfected with UL11G2A and fractionated at 48 h post-transfection. After removed of cell nuclei, cell lysates were subjected to sucrose-gradient centrifugation. Fractions were collected from the top of the tube. Proteins in each fraction were separated by SDS-PAGE and analyzed by Western blotting.
FIGURE 6
FIGURE 6
Lipid rafts take part in the DPV cell to cell spread and release processes. (A) Cell viability of adding MβCD. 1, 3, 5, 7, and 10 mM of MβCD was set, then the cell activity was detected. Moreover, 3 replicates were averaged. **P < 0.01. (B) MβCD was then diluted to a final concentration of 7 mM, a concentration that does not affect cells, and 1MOI DPV viruses were allowed to infect DEF cells. After incubation for 18 h at 37°C, the cell growth medium was replaced by a cell maintaining medium. The supernatant and cytoplasm were collected as samples, respectively, and the supernatant was put into 37°C for further culture. The supernatant and cytoplasm were collected and tested TCID50 separately in the culture for 15 and 30 min. The results presented are representative of three independent experiments. nsP > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. (C) MβCD was then diluted to a final concentration of 7 mM and different 10 x dilution gradient DPV were allowed to infect DEF cells for 5–6 days. After fixing, cells were stained with crystal violet. ****P < 0.0001. (D) Plates were scanned, and plaque diameters were measured in Adobe Photoshop. Data show plaque sizes on 25 plaques. ****P < 0.0001.
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