Porcine reproductive and respiratory syndrome virus degrades DDX10 via SQSTM1/p62-dependent selective autophagy to antagonize its antiviral activity

ABSTRACT

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus devastating the global swine industry. DEAD-box helicases (DDXs) are a family of ATP-dependent RNA helicases that are predominantly implicated in modulating cellular RNA metabolism. Meanwhile, a growing number of studies have suggested that some DDXs are associated with innate immunity and virus infection, so they are considered potential antiviral targets. Herein, we screened 40 DDXs and found that ectopic expression of DDX10 exhibited a significant anti-PRRSV effect, while DDX10 knockdown promoted PRRSV proliferation. Further analysis revealed that DDX10 positively regulates type I interferon production, which may contribute to its anti-PRRSV effect. Interestingly, PRRSV infection promoted DDX10 translocation from the nucleus to the cytoplasm for macroautophagic/autophagic degradation to block the antiviral effect of DDX10. By screening PRRSV-encoded proteins, we found that the viral envelope (E) protein interacted with DDX10. In line with the autophagic degradation of DDX10 during PRRSV infection, E protein could induce autophagy and reduce DDX10 expression in wild-type cells, but not in ATG5 or ATG7 knockout (KO) cells. When further screening the cargo receptors for autophagic degradation, we found that SQSTM1/p62 (sequestosome 1) interacted with both DDX10 and E protein, and E protein-mediated DDX10 degradation was almost entirely blocked in SQSTM1 KO cells, demonstrating that E protein degrades DDX10 by promoting SQSTM1-mediated selective autophagy. Our study reveals a novel mechanism by which PRRSV escapes host antiviral innate immunity through selective autophagy, providing a new target for developing anti-PRRSV drugs.Abbreviations: ACTB: actin beta; ATG: autophagy related; co-IP: co-immunoprecipitation; CQ: chloroquine; DDX10: DEAD-box helicase 10; E: envelope; EGFP: enhanced green fluorescent protein; hpi: hours post infection; hpt: hours post transfection; IFA: indirect immunofluorescence assay; IFN-I: type I IFN; IFNB/IFN-β: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; mAb: monoclonal antibody; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; OPTN: optineurin; ORF: open reading frame; PRRSV: porcine reproductive and respiratory syndrome virus; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID₅₀: 50% tissue culture infective dose; WT: wild type.

KEYWORDS:

• DDX10
• E protein
• innate immunity
• PRRSV
• selective autophagy
• SQSTM1/p62
• type I interferon

Acknowledgments

We would like to thank Dr. Jiyong Zhou (Zhejiang University) and Dr. Jun Cui (Sun Yat-sen University) for providing ATG7-KO-HEK-293T cells, ATG5-KO-HEK-293T cells, and SQSTM1-KO-HEK-293T cells. We also thank Dr. En-Min Zhou (Northwest A&F University) for providing rPRRSV-EGFP and Dr. Hongbo Zhou (Huazhong Agricultural University) for providing plasmid GFP-LC3.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data will be made available on request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2179844

Additional information

Funding

This work was supported by the National Key Research and Development Program of China (2022YFD1800305) and the National Natural Science Foundation of China (32002279).