Inhibition of USP14 influences alphaherpesvirus proliferation by degrading viral VP16 protein via ER stress-triggered selective autophagy

Abstract

Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical roles in immunity and other cellular functions. To investigate the role of USPs in alphaherpesvirus replication, we assessed 13 USP inhibitors for PRV replication. Our data showed that all the tested compounds inhibited PRV replication, with the USP14 inhibitor b-AP15 exhibiting the most dramatic effect. Ablation of USP14 also influenced PRV replication, whereas replenishment of USP14 in USP14 null cells restored viral replication. Although inhibition of USP14 induced the K63-linked ubiquitination of PRV VP16 protein, its degradation was not dependent on the proteasome. USP14 directly bound to ubiquitin chains on VP16 through its UBL domain during the early stage of viral infection. Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy. Ectopic expression of non-ubiquitinated VP16 fully rescued PRV replication. Challenge of mice with b-AP15 activated ER stress and autophagy and inhibited PRV infection in vivo. Our results suggested that USP14 was a potential therapeutic target to treat alphaherpesvirus-induced infectious diseases.Abbreviations ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; CCK-8: cell counting kit-8; Co-IP: co-immunoprecipitation; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR associated system 9; DDIT3/CHOP: DNA-damage inducible transcript 3; DNAJB9/ERdj4: DnaJ heat shock protein family (Hsp40) member B9; DUBs: deubiquitinases; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EP0: ubiquitin E3 ligase ICP0; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; FOXO1: forkhead box O1; FRET: Förster resonance energy transfer; HSPA5/BiP: heat shock protein 5; HSV: herpes simplex virus; IE180: transcriptional regulator ICP4; MAP1LC3/LC3: microtube-associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; PRV: pseudorabies virus; PRV gB: PRV glycoprotein B; PRV gE: PRV glycoprotein E; qRT-PCR: quantitative real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID₅₀: tissue culture infective dose; UB: ubiquitin; UBA: ubiquitin-associated domain; UBL: ubiquitin-like domain; UL9: DNA replication origin-binding helicase; UPR: unfolded protein response; USPs: ubiquitin-specific proteases; VHS: virion host shutoff; VP16: viral protein 16; XBP1: X-box binding protein 1; XBP1s: small XBP1; XBP1(t): XBP1-total.

Keywords: Alphaherpesvirus; ER stress; PRV VP16; USP14; selective autophagy.

Figure 1.

B-AP15 inhibits PRV infection in vitro. (A) PK-15 and 3D4/21 cells were treated with b-AP15 (0–1 μM) for 24–48 h. Cell viability was assessed using the CCK-8 cell counting assay. (B) PK-15 and 3D4/21 cells were infected with PRV-GFP (MOI = 0.01) and simultaneously treated with b-AP15 (0–1 μM) for 36 h. The fluorescence of GFP was detected by fluorescent microscopy. Scale bar: 200 μm. (C and D) Quantification of the percentage of GFP-positive cells from B by flow cytometry. (E) PK-15 and 3D4/21 cells were infected with PRV-QXX (MOI = 0.1) and simultaneously treated with b-AP15 (0–1 μM) for 24 h. PRV gB was assessed by immunoblot analysis. (F and G) PK-15 (F) and 3D4/21 (G) cells were infected with PRV-QXX (MOI = 0.1) and simultaneously treated with b-AP15 (0–1 μM) for 24 h. Viral titers were assessed by a TCID₅₀ assay. (H and I) PK-15 (H) and 3D4/21 (I) cells were infected with PRV-QXX (MOI = 0.01) and simultaneously treated with DMSO or b-AP15 (1 μM) for 0–48 h. One-step growth curves of PRV-QXX were assessed using a TCID₅₀ assay of viral titers. hpi, hour post infection. (J) Determination of IC₅₀ value of b-AP15 from G. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test.

Figure 2.

Knockout of USP14 inhibits PRV infection. (A) USP14 in sgControl and sgUSP14 PK-15 cells was assessed by immunoblot analysis. (B) sgControl and sgUSP14 PK-15 cells were cultured for 0–72 h. Cell proliferation was assessed using the CCK-8 cell counting assay. (C) sgControl and sgUSP14 PK-15 cells were infected with PRV-GFP (MOI = 0.01) for 36 h. The fluorescence of GFP was detected by fluorescent microscopy. Scale bar: 400 μm. (D) Quantification of the percentage of GFP-positive cells from C by flow cytometry. (E) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Viral titers were assessed by the TCID₅₀ assay. (F) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.01) for 0–48 h. One-step growth curves of PRV-QXX were assessed using a TCID₅₀ assay of viral titers. hpi, hour post infection. (G) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG (0–1.5 μg) as indicated for 24 h, and then infected with PRV-QXX (MOI = 0.1) for 24 h. PRV gB, USP14-FLAG and USP14 were assessed by immunoblot analysis. (H) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG (0–1.5 μg) as indicated for 24 h, and then infected with PRV-GFP (MOI = 0.01) for 36 h. The fluorescence of GFP was detected by fluorescent microscopy. Scale bar: 400 μm. (I) Quantification of the percentage of GFP-positive cells from H by flow cytometry. (J) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG (0–1.5 μg) as indicated for 24 h, and then infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Viral titers were assessed by the TCID₅₀ assay. (K) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG (1.5 μg) and USP14^S432A-FLAG (1.5 μg) as indicated for 24 h, and then infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Viral titers were assessed by the TCID₅₀ assay. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.

Figure 3.

B-AP15 influences PRV replication. (A) PK-15 cells were incubated with PRV-QXX (MOI = 0.1 and 1) combined with b-AP15 (0–1 μM) for 1 h at 4°C. After washing with cold PBS 3 times, cells were cultured in DMEM with 2% FBS for 24 h at 37°C. An attachment assay was assessed using the TCID₅₀ assay of viral titers. (B) PK-15 cells were incubated with PRV-QXX (MOI = 0.1 and 1) for 1 h at 4°C and then in DMEM with 10% FBS containing b-AP15 (0–1 μM) at 37°C. After 1 h to allow viral entry, cells were cultured in DMEM with 2% FBS for 24 h at 37°C. An entry assay was assessed using a TCID₅₀ of viral titers. (C) PK-15 cells were incubated with PRV-QXX (MOI = 0.1) combined with b-AP15 (1 μM) for 1 h at 4°C. After washing with cold PBS 3 times, cells were cultured in DMEM with 2% FBS combined with b-AP15 (1 μM) for 0–24 h at 37°C. PRV genome copy numbers were assessed by qRT-PCR analysis. (D) PK-15 cells were incubated with PRV-QXX (MOI = 0.1 and 1) at 4°C for 1 h and then in DMEM with 10% FBS at 37°C for 12 h. Cells were then cultured in DMEM with 2% FBS containing b-AP15 (1 μM) for 4–12 h at 37°C. A replication assay was assessed using the TCID₅₀ assay of viral titers. (E–G) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and simultaneously treated with DMSO or b-AP15 (1 μM) for 0–24 h. The mRNA levels of PRV IE180 (E), EP0 (F) and UL9 (G) were assessed by qRT-PCR analysis. hpi, hour post infection. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test.

Figure 4.

Inhibition of USP14 induces PRV VP16 ubiquitination and degradation that is not dependent on the proteasome. (A) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (0–1 μM) for 24 h. PRV VP16 was assessed by immunoblot analysis. (B) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (0–1 μM) for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (C) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.1) for 24 h. PRV VP16 was assessed by immunoblot analysis. (D) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding FLAG-VP16 for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (E) PK-15 cells were transfected with plasmids encoding FLAG-VP16, HA-UB, HA-UB^K48 and HA-UB^K63, and treated with b-AP15 (1 μM) as indicated for 24 h. Ubiquitination of FLAG-VP16 was assessed by the ubiquitination assay. (F) sgControl and sgUSP14 PK-15 cells were transfected with plasmids encoding FLAG-VP16, HA-UB, HA-UB^K48 and HA-UB^K63 as indicated for 24 h. Ubiquitination of FLAG-VP16 was assessed by the ubiquitination assay. (G) PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16^K168R, FLAG-VP16^K305R, and HA-UB and treated with b-AP15 (1 μM) as indicated for 24 h. Ubiquitination of FLAG-VP16 variants was assessed by the ubiquitination assay. (H) sgControl and sgUSP14 PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16^K168R and FLAG-VP16^K305R as indicated for 24 h. Ubiquitination of FLAG-VP16 variants was assessed by the ubiquitination assay. (I) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG and USP14-FLAG^S432A as indicated for 24 h, and then infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Ubiquitination of VP16 was assessed by the ubiquitination assay. (J) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM), MG132 (10 μM) and 3-MA (10 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (K) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (1 μM), MG132 (10 μM) and 3-MA (10 μM) as indicated for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (L) PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16^K168R and treated with b-AP15 (1 μM) for 24 h. FLAG-VP16 and FLAG-VP16^K168R were assessed by immunoblot analysis. (M) PK-15 cells were transfected with plasmids encoding FLAG-VP16 or FLAG-VP16^K168R for 24 h. Then, cells were infected with PRV-QXX (MOI = 0.1 and 1) and treated with b-AP15 (1 μM) as indicated for 24 h. Viral titers were assessed by the TCID₅₀ assay. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.

Figure 5.

USP14 interacts with PRV VP16 at the early stage of viral infection. (A) PRV-QXX (MOI = 10) was incubated with PK-15 cells at 4°C for 1 h. After three times washing with ice-cold PBS, the cells were incubated at 37°C for 0–20 min. Colocalization of USP14 and VP16 was assessed by the immunofluorescence analysis. Scale bar: 10 μm. (B) Cells were treated as in A. The interaction of USP14 with VP16 was assessed by co-IP assay. (C) Cells were treated as in A. PRV VP16 was assessed by immunoblot analysis. (D) Ubiquitination of VP16 in rPRV ΔUL48 rescued by VP16 and VP16^K168R was assessed by the ubiquitination assay. (E) rPRV (WT VP16, MOI = 10) and rPRV (VP16^K168R, MOI = 10) were incubated with PK-15 cells at 4°C for 1 h. After three times washing with ice-cold PBS, the cells were incubated at 37°C for 10 min. The colocalization of USP14 and VP16 variants was assessed by the immunofluorescence analysis. Scale bar: 10 μm. (F) De-ubiquitination of FLAG-VP16 by GST-USP14 was assessed by the in vitro de-ubiquitination assay. (G) The interactions of non-ubiquitinated and ubiquitinated FLAG-VP16 with GST-USP14 and GST-USP14ΔUBL were assessed by an in vitro affinity-isolation assay. (H) The interactions of non-ubiquitinated and ubiquitinated FLAG-VP16, FLAG-VP16^K168R, and FLAG-VP16^K305R with GST-USP14 were assessed by an in vitro affinity-isolation assay.

Figure 6.

Inhibition of USP14 induces autophagy. (A) PK-15 cells were treated with b-AP15 (0–1 μM) for 24 h. LC3-I, LC3-II, SQSTM1, ATG5, ATG12 and BECN1 were assessed by immunoblot analysis. (B) LC3-I, LC3-II, SQSTM1, ATG5, and BECN1 were assessed by immunoblot analysis in sgControl and sgUSP14 PK-15 cells. (C) PK-15 cells were transfected with plasmid encoding GFP-LC3 and treated with DMSO or b-AP15 (1 μM) for 24 h. The fluorescence of GFP-LC3 was detected by fluorescent microscopy. Scale bar: 10 μm. (D) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding GFP-LC3 for 24 h. The fluorescence of GFP-LC3 was detected by fluorescent microscopy. Scale bar: 10 μm. (E) Quantification of GFP-LC3 puncta per cell from C and D using ImageJ software. (F) PK-15 cells were treated with DMSO or b-AP15 (1 μM) for 24 h. The autophagosome-like vesicles were detected by transmission electron microscope. Scale bar: 500 nm. (G) Quantification of autophagosome-like vesicles from F using ImageJ software. (H) PK-15 cells were transfected with plasmid encoding GFP-RFP-LC3 and treated with DMSO or b-AP15 (1 μM) for 24 h. The fluorescence of GFP and RFP was detected by fluorescent microscopy. Scale bar: 10 μm. (I) Quantification of autophagosomes and autolysosomes from H using ImageJ software. (J) sgControl and sgUSP14 PK-15 cells were treated with bafilomycin A₁ (10 μM) for 24 h. LC3-I, LC3-II and SQSTM1 were assessed by immunoblot analysis. (K) ATG5 in sgControl and sgATG5 PK-15 cells was assessed by immunoblot analysis. (L) BECN1 in sgControl and sgBECN1 PK-15 cells was assessed by immunoblot analysis. (M and N) sgControl, sgATG5 (M) and sgBECN1 (N) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (O) sgControl, sgATG5 and sgBECN1 PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with DMSO or b-AP15 (1 μM) as indicated for 24 h. Viral titers were assessed by the TCID₅₀ assay. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.

Figure 7.

Inhibition of USP14 induces ER stress. (A) PK-15 cells were treated with b-AP15 (0–1 μM) for 24 h. HSPA5, ATF6, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, ATF4, XBP1 and FOXO1 levels were assessed by immunoblot analysis. (B) XBP1, FOXO1, p-EIF2AK3, EIF2AK3, p-EIF2A and EIF2A levels were assessed by immunoblot analysis in sgControl and sgUSP14 PK-15 cells. (C) PK-15 cells were treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. HSPA5, ATF6, p-EIF2AK, EIF2AK3, p-EIF2A, EIF2A, XBP1, LC3-I, LC3-II, SQSTM1 and ATG5 were assessed by immunoblot analysis. (D) sgControl and sgUSP14 PK-15 cells were treated with GSK2606414 (10 μM) as indicated for 24 h. HSPA5, ATF6, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, XBP1, LC3-I, LC3-II, SQSTM1 and ATG5 and USP14 were assessed by immunoblot analysis. (E) sgControl and sgUSP14 PK-15 cells were mock infected or infected with PRV-QXX (MOI = 0.1) for 24 h. USP14, VP16, XBP1, FOXO1, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, SQSTM1, LC3-I and LC3-II were assessed by immunoblot analysis. (F) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (G) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (1 μM) and GSK2606414 (10 μM) as indicated for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (H) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with GSK2606414 (10 μM) for 24 h. PRV VP16 was assessed by immunoblot analysis. (I) PK-15 cells were infected with PRV-QXX (MOI = 0.1 and 1) and treated with DMSO, b-AP15 (1 μM), GSK2606414 (10 μM) and b-AP15 (1 μM) + GSK2606414 (10 μM) for 24 h. Viral titers were assessed by the TCID₅₀ assay. (J) PK-15 cells were transfected with siControl, siEIF2A-1, siEIF2A-2 and siEIF2A-3 for 48 h. EIF2A was assessed by immunoblot analysis. (K) PK-15 cells were transfected with siControl or siEIF2A-1 and treated with b-AP15 (1 μM) as indicated for 48 h. p-EIF2A, EIF2A, LC3-I, LC3-II, SQSTM1 and ATG5 were assessed by immunoblot analysis. (L) PK-15 cells were transfected with siControl or siEIF2A-1 for 24 h. Then, cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM) as indicated for 24 h. p-EIF2A, EIF2A, VP16, LC3-I, LC3-II, SQSTM1, and ATG5 were assessed by immunoblot analysis. Data were shown as mean ± SD based on three independent experiments. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.

Figure 8.

Inhibition of USP14 induces the interaction of SQSTM1/p62 with ubiquitinated VP16. (A) PK-15 cells were co-transfected with plasmids encoding VP16-mCherry and SQSTM1-EGFP, or VP16^K168R-mCherry and SQSTM1-EGFP, and treated with DMSO or b-AP15 (1 μM) for 24 h. The colocalization of UB, SQSTM1 and VP16 variants was assessed by immunofluorescence of UB and fluorescent microscopy. Scale bar: 10 μm. (B) sgControl and sgUSP14 PK-15 cells were mock infected or infected with PRV-QXX (MOI = 0.1) for 24 h. The interaction of SQSTM1 with VP16 was assessed by co-IP assay. (C) PK-15 cells transfected with plasmids encoding SQSTM1-EGFP, FLAG-VP16 and FLAG-VP16^K168R were treated with b-AP15 (1 μM) and 3-MA (10 μM) as indicated for 24 h. The interaction of SQSTM1-EGFP with the FLAG-VP16 variants was assessed by co-IP assay. (D) PK-15 cells transfected with plasmids encoding FLAG-VP16, SQSTM1-EGFP and SQSTM1ΔUBA-EGFP were treated with b-AP15 (1 μM) and 3-MA (10 μM) as indicated for 24 h. The interaction of FLAG-VP16 with SQSTM1-EGFP variants was assessed by a co-IP assay. (E) The interactions of His₆-SQSTM1 with non-ubiquitinated and ubiquitinated FLAG-VP16, FLAG-VP16^K168R and FLAG-VP16^K305R were assessed by an in vitro affinity-isolation assay. (F) The interactions of non-ubiquitinated and ubiquitinated FLAG-VP16 with His₆-SQSTM1 and His₆-SQSTM1ΔUBA were assessed by an in vitro affinity-isolation assay. (G) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with DMSO or b-AP15 (1 μM) for 24 h. The cell lysates were subjected to iodixanol density gradient centrifugation. SQSTM1, LC3-II and PRV VP16 levels in each fraction were assessed by immunoblot analysis. (H) PK-15 cells were co-transfected with plasmids encoding VP16-mCherry and SQSTM1-EGFP and treated with DMSO or b-AP15 (1 μM) for 24 h. The interaction of SQSTM1 with VP16 was assessed by FRET analysis. Scale bar: 10 μm. (I) Quantification of FRET efficiency from H. Data were shown as mean ± SD based on three independent experiments. ** P < 0.01 determined by two-tailed Student’s t-test. ns, no significance.

Figure 9.

B-AP15 antagonizes PRV infection in vivo. Preventive strategy for PRV-QXX challenge and b-AP15 treatment in mice. (B) Mice were intraperitoneally injected with DMSO or b-AP15 (8 mg/kg) on day −4 and day −2. On day 0, mice were intraperitoneally injected with DMSO or b-AP15 (8 mg/kg) and intranasally infected with PRV-QXX (5 × 10³ TCID₅₀ per mouse). The survival rate was monitored daily for 10 days (n = 12 per group). (C) Mice were treated as in B. On day 3, VP16, FOXO1, XBP1, p-EIF2AK3, EIF2AK3, p-EIF2A, EIF2A, SQSTM1, LC3-I and LC3-II levels in the lung were assessed by immunoblot analysis (n = 3). (D and E) Mice were treated as in B. On day 3, PRV gE mRNA (D) and PRV genome copy numbers (E) in the lung were assessed by the qRT-PCR analysis (n = 4). (F) Sections of mouse lungs from D were stained by hematoxylin-eosin staining. Scale bar: 100 μm. (G) Quantification of alveolar numbers from F by ImageJ software. (H) Therapeutic strategy for PRV-QXX challenge and b-AP15 treatment in mice. (I) On day 0, mice were intranasally infected with PRV-QXX (5 × 10³ TCID₅₀ per mouse). Mice were intraperitoneally injected with DMSO or b-AP15 (8 mg/kg) on day 1 and day 3. The survival rate was monitored daily for 10 days. (n = 12 per group). (J) A schematic model showing inhibition of USP14 influenced alphaherpesvirus proliferation. Data were shown as mean ± SD based on three independent experiments. ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test.