Fig 1.
Construction and identification of recombinant viruses.
(A) Schematic diagram of the construction of the infectious AnHV-1-△UL51 clone, (I) genomic structure of AnHV-1-CHv50, (II) first red recombination replacing the UL51 gene with the kanamycin gene, (III) product after the first red recombination, (IV) second red recombination where the I-Sec I restriction site is recognized by recombinase to remove the Kan fragment, and (IV) the genomic structure of AnHV-1-△UL51. (B) Rescue and purification of AnHV-1-△UL51. (C) PCR identification of the recombinant virus. (D) Western blot analysis of UL51 gene-deficient viral protein expression. (E) IFA identification of the recombinant virus, representative images from three independent replicate experiments, scale bar, 40 μm.
Fig 2.
Deletion of pUL51 reduces the incidence and mortality of viral infection.
(A) Experimental design for assessing the effects of UL51 deletion on AnHV-1 pathogenicity in vivo. Fourteen-day-old ducklings were intramuscularly inoculated with 10⁷ TCID₅₀ of AnHV-1-ΔUL51, AnHV-1-ΔUL51-Rev, or AnHV-1-CHv50. Ducklings injected with MEM served as mock-infected controls. Ducklings in Group 1 (n = 10 per group) were monitored for 10 days post-infection for changes in body temperature, body weight, and survival. Ducklings in Group 2 (n = 6 per group) were euthanized at 5 days post-infection for histopathological assessment of organ lesions and viral load quantification. The schematic images were hand-drawn by the authors and edited manually (licensed under CC BY 4.0). The nonoriginal icon within these figures was sourced from https://openclipart.org/detail/326143. (B–D) Body temperature, body weight, and survival rate of ducks infected with AnHV-1-ΔUL51 over a 10-day period. (E) Macroscopic pathological changes in organs from AnHV-1-ΔUL51-infected ducks at 5 days post-infection. These images were taken by the authors of this manuscript and edited manually (licensed under CC BY 4.0). (F) Histopathological analysis of various tissues collected from AnHV-1-ΔUL51-infected ducks on day 5 post infection (hematoxylin and eosin staining; original magnification, 100×). (G) Viral DNA copies in the tissues and organs of ducks infected with AnHV-1-ΔUL51 at 5 days post-infection. Data are expressed as mean ± SD from three independent biological replicates. Statistical analysis was performed using two-way ANOVA (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Fig 3.
Multistep growth kinetics of recombinant viruses and the role of pUL51 in the viral life cycle.
(A) Multistep growth curves of AnHV-1-ΔUL51. Duck embryo fibroblasts (DEFs) were infected with AnHV-1-ΔUL51, AnHV-1-ΔUL51-Rev, or AnHV-1-CHv50 at an MOI of 0.01. Total cells and supernatants were harvested at the indicated time points, and viral titers were determined. (B) Effect of pUL51 on viral adsorption. DEFs were infected with a labeled virus at an MOI of 1 for 2 hours 4°C, after which the number of viral DNA copies was quantified to evaluate the adsorption efficiency. (C) Effect of pUL51 on viral invasion. Following the adsorption period, the cells were incubated at 37°C for 2 hours. Samples were collected, and viral entry efficiency was assessed via quantitative PCR. (D) Effect of pUL51 on viral genomic DNA replication. (E) Effect of pUL51 on viral release. DEFs were infected with viruses. After 16 hours, the medium was replaced with fresh maintenance medium, and both the cells and the supernatants were harvested at 1, 2, 3, and 4 hours. The ratio of extracellular to intracellular viral titers was used to assess viral release efficiency. (F) Effect of pUL51 on cell-to-cell spread. Plaque morphology was visualized by crystal violet staining. The diameters of 30 representative plaques per group were measured using ImageJ software; the results are summarized in the right panel. Data are expressed as mean ± SD from three independent biological replicates. Statistical analysis was performed using one-way/two-way ANOVA (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Fig 4.
Identification of viral proteins that interact with pUL51.
(A) Schematic of the immunoprecipitation‒mass spectrometry (IP‒MS) workflow used to identify AnHV-1 pUL51-interacting proteins. Cells were transfected with a plasmid encoding pUL51-Flag. At 24 h posttransfection, the cells were infected with AnHV-1 at an MOI of 1 for another 24 h. The cell lysates were then subjected to immunoprecipitation using anti-Flag or control mouse IgG antibodies, followed by silver staining and Western blot analysis. (B) Silver staining and Western blot identification of pUL51-Flag IP-enriched samples. (C) Mass spectrometry results for pUL51-Flag. The raw mass spectrometry data results can be found at https://doi.org/10.6084/m9.figshare.31229161. (D) Co-IP experiments detecting interactions between pUL51 and pUL30, pUL38, pUL27, and pUL10 in DEFs. DEFs were cotransfected with 2 μg of pUL51-encoding plasmid and 2 μg of either pUL30, pUL38, pUL27, or pUL10-encoding plasmids. Samples were collected 36 h posttransfection for Co-IP analysis. (E) Coimmunoprecipitation experiments were performed to detect interactions between pUL51 and pUL30, pUL38, pUL27, and pUL10 during AnHV-1 infection. DEFs were infected with AnHV-1 at an MOI of 1. Cells were collected 36 hours post-infection and subjected to immunoprecipitation using a pUL51-specific rabbit polyclonal antibody and rabbit IgG as a control. Western blot analysis was performed using rabbit polyclonal antibodies specific to pUL51, pUL10, pUL27, and pUL30. DEFs were used as a control. (F) The colocalization of pUL51 with pUL30, pUL38, pUL27, and pUL10 in DEFs was detected by IFA. 0.5 μg of the pUL51-encoding plasmid was cotransfected with 0.5 μg each of pUL30, pUL38, pUL27, and pUL10 into DEFs. Samples were collected 24 h posttransfection for IFA analysis, representative images from three independent replicate experiments. Scale bar: 20 μm.
Fig 5.
pUL51 increases the expression of pUL10 and colocalizes with it in the Golgi apparatus.
(A) Transient expression of pUL51 and pUL27. DEFs were transfected with 1 μg of either empty pCAGGS vector or pCAGGS-UL51–3 × Flag, together with 1 μg of pCAGGS-UL27–3 × HA plasmid. Cells were harvested at 36 h posttransfection for Western blot analysis. (B) Transient expression of pUL51 and pUL10. DEFs were transfected with 1 μg of empty pCAGGS or pCAGGS-UL51–3 × Flag, along with 1 μg of pCAGGS-UL10–3 × HA plasmid. Lysates were collected at 36 h posttransfection for Western blot analysis. (C) Western blot analysis of pUL10 expression in cells infected with the UL51-deficient virus. DEFs were infected with AnHV-1-ΔUL51 or AnHV-1-CHv50 at an MOI of 0.1 and harvested at 18, 36, and 48 h post infection for Western blot. (D) DEF cells were cotransfected with the following plasmid combinations: (i) 0.5 μg of empty pCAGGS vector plus 0.5 μg of pCAGGS-UL27–3 × HA or (ii) 0.5 μg of pCAGGS-UL51–3 × Flag plus 0.5 μg of pCAGGS-UL27–3 × HA. Twenty-four hours post-transfection, the cells were treated with cycloheximide (CHX) and harvested at 0, 3, and 6 hours after CHX addition for Western blot analysis. (E) DEF cells were cotransfected with the following plasmid combinations: (i) 0.5 μg of empty pCAGGS vector plus 0.5 μg of pCAGGS-UL10–3 × HA or (ii) 0.5 μg of pCAGGS-UL51–3 × Flag plus 0.5 μg of pCAGGS-UL10–3 × HA. Twenty-four hours post-transfection, the cells were treated with cycloheximide (CHX) and harvested at 0, 3, 6, and 9 hours after CHX addition for Western blot analysis. (F) Colocalization of pUL51 and pUL10 with the Golgi marker GM130 in transfected DEFs. Cells were transfected with plasmids encoding UL51–3 × Flag and UL10–3 × HA. At 24 h posttransfection, immunofluorescence staining was performed using antibodies against GM130. Nuclei were stained with DAPI, representative images from three independent replicate experiments. Scale bar: 20 μm. Scale bar: 20 μm. (G) Colocalization of pUL51 and pUL10 with GM130 during AnHV-1 infection. DEFs were infected with AnHV-1-UL10HA. The proteins were labeled with anti-pUL51 (rabbit polyclonal) and anti-HA (mouse monoclonal) antibodies. The Golgi apparatus was stained with anti-GM130. The nuclei were counterstained with DAPI, representative images from three independent replicate experiments. Scale bar: 20 μm.
Fig 6.
The C9 residue of pUL51 is critical for its interaction with pUL10.
(A) Schematic representation of pUL51 mutant plasmids. (B) Interaction between the N-terminal fragment of pUL51 (amino acids 1–161) and pUL10. DEFs were transfected with 2 μg of pCAGGS-UL511–161-3 × Flag and 2 μg of pCAGGS-UL10-3 × HA. Coimmunoprecipitation (Co-IP) was performed at 36 h post transfection. (C) Interaction between the C-terminal fragment of pUL51 (amino acids 162–252) and pUL10. DEFs were transfected with 2 μg of pCAGGS-UL51162–252-3 × Flag and 2 μg of pCAGGS-UL10-3 × HA. Cells were harvested at 36 h posttransfection for Co-IP analysis. (D) Interaction between truncated pUL51 and pUL10. DEFs were transfected with 2 μg of a plasmid encoding a truncated pUL51 fragment and 2 μg of pCAGGS-UL10-3 × HA. Samples were collected at 36 h posttransfection for Co-IP. (E) Sequence analysis of the pUL51 C9 site. Site-directed mutagenesis was performed to substitute cysteine with alanine at position 9 (highlighted in red). Multiple sequence alignment of the N-terminal 20 amino acids of pUL51 from eight herpesviruses: HSV-1 (GenBank: NC_001806.2), VZV (GenBank: NC_001348.1), PRV (GenBank: MZ219273.1), BoHV-1 (GenBank: MH791338.1), AnHV-1 (GenBank: JQ647509.1), HCMV (GenBank: AH013698.2), EBV (GenBank: NC_009334.1), and KSHV (GenBank: NC_009333.1). Alignments were performed using MEGA 7.0. (F) Interaction between pUL51/C9A and pUL10. DEFs were transfected with 2 μg of plasmid encoding either wild-type pUL51 or pUL51/C9A, together with 2 μg of pCAGGS-UL10-3 × HA. Co-IP analysis was conducted 36 h posttransfection. (G) Interaction between pUL51/C9A and pUL27. DEFs were transfected with 2 μg of plasmid encoding either wild-type pUL51 or pUL51/C9A, together with 2 μg of pCAGGS-UL27-3 × HA. Co-IP analysis was conducted 36 h posttransfection. (H) Interaction between pUL51/C9A and pUL38. DEFs were transfected with 2 μg of plasmid encoding either wild-type pUL51 or pUL51/C9A, together with 2 μg of pCAGGS-UL38-3 × HA. Co-IP analysis was conducted 36 h posttransfection. (I) Interaction between pUL51/C9A and pUL30. DEFs were transfected with 2 μg of plasmid encoding either wild-type pUL51 or pUL51/C9A, together with 2 μg of pCAGGS-UL30-3 × HA. Co-IP analysis was conducted 36 h posttransfection. (J) Effect of pUL51/C9A on pUL10 stability. DEF cells were transfected with 0.5 μg of plasmid encoding wild-type pUL51 or pUL51/C9A, and 0.5 μg of pCAGGS-UL10-3 × HA plasmid was concurrently transfected. Twenty-four hours post-transfection, the cells were treated with cycloheximide (CHX) and harvested at 0, 3, and 6 hours after CHX addition for Western blot analysis. (K) DEFs were transfected with plasmids expressing pUL51 or pUL51/C9A, along with 0.5 μg of pCAGGS-UL10-3 × HA plasmid. Twenty-four hours post-transfection, the cells were fixed and analyzed by immunofluorescence to assess protein colocalization. The nuclei were counterstained with DAPI, representative images from three independent replicate experiments. Scale bar: 20 μm.
Fig 7.
C9 is crucial for the palmitoylation and Golgi localization of pUL51.
(A) Detection of palmitoylation modification of pUL51-Flag using the palmitoylation inhibitor 2-BP. (B) Measurement of pUL51 palmitoylation. DEFs were transfected with plasmids expressing pUL51-Flag or pUL51/C9A-Flag. At 36 h post transfection, the cell lysates were immunoprecipitated using anti-Flag magnetic beads and subjected to an ABE assay in the presence or absence of hydroxylamine (HAM) treatment, followed by Western blot analysis. (C) Subcellular localization of pUL51 and pUL51/C9A. DEFs were transfected with plasmids expressing pUL51-Flag or pUL51/C9A-Flag for 24 h and then treated with or without 30 μM 2-bromopalmitate (2-BP). Localization was assessed by immunofluorescence using an antibody against the Golgi marker GM130. The nuclei were counterstained with DAPI, representative images from three independent replicate experiments. Scale bar: 20 μm.
Fig 8.
Palmitoylation at the C9 site maintains pUL51 stability by affecting the ubiquitin–proteasome degradation pathway.
(A) DEFs were transfected with a pUL51-Flag plasmid for 24 h and then treated with increasing concentrations of 2-BP for 6 h. Protein expression levels were analyzed by Western blot. (B) DEFs transfected with pUL51-Flag for 12 h were treated with 30 μM 2-BP or DMSO for 12 h, followed by incubation with 100 mg/mL cycloheximide (CHX). Lysates were collected at the indicated time points and analyzed by Western blot. (C) DEFs expressing pUL51-Flag or pUL51/C9A-Flag were treated with CHX (100 mg/mL). Lysates were harvested at the indicated times and subjected to Western blot analysis. (D) DEFs expressing pUL51-Flag were treated for 6 h with 30 μM 2-BP alone or in combination with 50 μM MG132. DEFs expressing pUL51-Flag were treated for 6 h with 30 μM 2-BP alone or in combination with 50 μM chloroquine (CQ). Protein levels were assessed by Western blot. (E) DEFs were cotransfected with plasmids encoding pUL51-Flag or pUL51/C9A-Flag together with Ub-HA. After 36 h, the lysates were immunoprecipitated and analyzed by Western blot. (F) DEFs expressing pUL51/C9A-Flag were treated with or without 50 μM MG132 for 6 h, followed by CHX (100 mg/mL). Lysates were collected at the indicated times and analyzed by Western blot. The band intensity of pUL51-Flag was quantified using ImageJ software.
Fig 9.
The palmitoyltransferases DHHC9 and DHHC18 promote the palmitoylation of pUL51.
(A) DEFs were cotransfected with plasmids encoding pUL51-HA and Flag-tagged DHHC proteins for 36 hours. The cell lysates were subjected to immunoprecipitation followed by Western blot analysis. (B) DEFs were cotransfected with pUL51-HA and DHHC-Flag plasmids for 24 hours and then fixed and immunostained. The colocalization of the pUL51-HA and DHHC-Flag proteins was observed using confocal microscopy. Scale bar: 20 μm. (C–F) DEFs were transfected with different concentrations of plasmids encoding DHHC2, DHHC9, DHHC14, or DHHC18, along with 1 μg of the pCAGGS-UL51-3xHA plasmid. Samples were harvested 36 hours post-transfection for Western blot analysis of protein expression. (G–J) DEFs were cotransfected with plasmids encoding DHHC2, DHHC9, DHHC14, or DHHC18 together with 1 μg of the pCAGGS-UL51-3xHA plasmid for 24 hours. Cycloheximide (CHX, 100 mg/mL) was then added, and cell lysates were collected at 0, 2, 4, 6, and 8 hours post-treatment for Western blot analysis. The band intensity of pUL51-HA was quantified using ImageJ software. (K) Subcellular localization of DHHC9 and DHHC18. DEFs were transfected with plasmids expressing DHHC9-Flag and DHHC18-Flag for 24 h. Localization was assessed by immunofluorescence using an antibody against the Golgi marker GM130. The nuclei were counterstained with DAPI, representative images from three independent replicate experiments. Scale bar: 20 μm. (L) DEFs were cotransfected with plasmids encoding DHHC9 or DHHC18 along with the pUL51-HA expression plasmid for 36 hours. Cell lysates were immunoprecipitated using mouse anti-HA magnetic beads. The samples were analyzed by Western blot to detect palmitoylation with or without hydroxylamine (HAM) treatment. (M) DEFs were cotransfected for 36 hours with a pUL51-Flag plasmid along with shRNA plasmids (shRNA-NC, shRNA-DHHC9-687, or shRNA-DHHC18-1019). Cell lysates were immunoprecipitated using mouse anti-Flag magnetic beads. The samples were analyzed by Western blot to detect palmitoylation with or without hydroxylamine (HAM) treatment.
Fig 10.
Effect of pUL51/C9A mutation on AnHV-1 replication.
(A) Multistep growth kinetics of AnHV-1-UL51C9A. DEFs were infected with AnHV-1-UL51C9A, AnHV-1-UL51C9A and AnHV-1-CHv50 at an MOI of 0.01. Total cell samples were collected at the indicated time points, and viral titers were determined. (B) Statistical analysis of viral titers at each time point. (C) Effect of the C9A mutation on viral lesion size. Viral spread was assessed by crystal violet staining. Thirty representative plaques were measured per group. Plaque diameters were quantified using ImageJ software. (D) DEFs were infected with AnHV-1-CHv50 or AnHV-1-UL51C9A at an MOI of 0.1 for 36 h. The cell lysates were immunoprecipitated using a rabbit anti-UL51 polyclonal antibody and analyzed by Western blot to detect pUL51 palmitoylation with or without hydroxylamine (HAM) treatment. (E) DEFs were infected with AnHV-1-CHv50, AnHV-1-UL51C9A, or AnHV-1-UL51C9A Rev at an MOI of 0.1 for 36 h and then fixed and immunostained with an antibody against GM130 (a Golgi marker). The nuclei were counterstained with DAPI. Scale bar: 20 μm. Data are expressed as mean ± SD from three independent biological replicates. Statistical analysis was performed using two-way ANOVA (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).
Table 1.
Effects of mutations in UL51 on the distribution of viral particles in AnHV-1-infected DEFs.
Fig 11.
Electron microscopy analysis of AnHV-1-CHv50-, AnHV-1-ΔUL51-, AnHV-1-UL51C9A- and AnHV-1-UL51C9A Rev-infected DEFs.
(A–D) Electron microscopy images of DEFs infected with AnHV-1-CHv50, AnHV-1-ΔUL51, AnHV-1-UL51C9A and AnHV-1-UL51C9A Rev are presented. (D–G) Close-up image of the enlarged area showing virions in the cytoplasm of AnHV-1-CHv50-, AnHV-1-ΔUL51-, AnHV-1-UL51C9A- and AnHV-1-UL51C9A Rev-infected cells. Red solid arrows indicate viral particles located within vesicles or those that are mature. Red hollow arrows point to viral particles with incomplete envelopes. The black hollow arrows highlight viral particles lacking a secondary envelope. Yellow triangles indicate MVB. The nucleus (N) and cytoplasm (C) are marked. Bars, 1 μm (A-D) and 500 nm (E-H). (I) A total of 200 to 300 viral particles were counted across five cells. The figure shows the percentage of viral particles at the morphogenesis stage of infection.
Fig 12.
Effect of the pUL51/C9A mutation on AnHV-1 virulence.
(A) Clinical signs in the heads of ducklings were observed following infection with different viruses. (B) Histopathological examination of tissues from 14-day-old ducks intramuscularly inoculated with 1 mL (10⁶ TCID₅₀) of AnHV-1-UL51C9A, AnHV-1-UL51C9A Rev, AnHV-1-CHv50, or MEM. Necropsy was performed on day 5 post infection to assess lesions in various tissues. These images were taken by the authors of this manuscript and edited manually (licensed under CC BY 4.0). (C) Replication kinetics of the pUL51/C9A mutant virus in ducks. 14-day-old ducks (n = 9) were intramuscularly inoculated with 1 mL (10⁶ TCID₅₀) of AnHV-1-UL51C9A, AnHV-1-UL51C9A Rev, or AnHV-1-CHv50. Heart, liver, spleen, gizzard, bursa of Fabricius, duodenum, cecum, and thymus tissues were collected on days 3, 5, and 10 post infection. Viral DNA copy numbers were quantified by quantitative PCR. Data are expressed as mean ± SD from three independent biological replicates. Statistical analysis was performed using two-way ANOVA (ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).