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

Sheng-Li Ming; Shuang Zhang; Qi Wang; Lei Zeng; Lu-Yu Zhou; Meng-Di Wang; Ying-Xian Ma; Li-Qiang Han; Kai Zhong; He-Shui Zhu; Yi-Lin Bai; Guo-Yu Yang; Jiang Wang; Bei-Bei Chu

doi:10.1080/15548627.2021.2002101

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

Autophagy. 2022 Aug;18(8):1801-1821. doi: 10.1080/15548627.2021.2002101. Epub 2021 Nov 25.

Authors

Sheng-Li Ming^{1

2

3}, Shuang Zhang^{1

2

3}, Qi Wang^{1

2

3}, Lei Zeng^{1

2

3}, Lu-Yu Zhou^{1

2

3}, Meng-Di Wang⁴, Ying-Xian Ma^{1

2

3}, Li-Qiang Han^{1

2

3}, Kai Zhong^{1

2

3}, He-Shui Zhu^{1

2

3}, Yi-Lin Bai⁵, Guo-Yu Yang^{1

2

3

6}, Jiang Wang^{1

2

3}, Bei-Bei Chu^{1

2

3

6}

Affiliations

¹ College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, People's Republic of China.
² Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs of the People's Republic of China, Zhengzhou, Henan Province, China.
³ Key Laboratory of Animal Growth and Development, Zhengzhou, Henan Province, People's Republic of China.
⁴ The Education Department of Henan Province Henan University of Animal Husbandry and Economy, Zhengzhou, Henan Province, People's Republic of China.
⁵ College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province, People's Republic of China.
⁶ International Joint Research Center of National Animal Immunology, Henan Agricultural University, Zhengzhou, Henan Province, People's Republic of China.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Alphaherpesvirinae* / pathogenicity
Alphaherpesvirinae* / physiology
Animals
Autophagy*
Cell Proliferation
Endoplasmic Reticulum Stress*
Herpes Simplex Virus Protein Vmw65* / metabolism
Macroautophagy
Mice
Sequestosome-1 Protein
Ubiquitin Thiolesterase* / metabolism

Substances

Herpes Simplex Virus Protein Vmw65
Sequestosome-1 Protein
Usp14 protein, mouse
Ubiquitin Thiolesterase

Grants and funding

This work was supported by grants from National Natural Science Foundation of China (32072858), the Natural Science Foundation of Henan (202300410213), and Outstanding Talents of Henan Agricultural University (30600773).