Related Experiment Video
Updated: Apr 24, 2026
Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors
Published on: September 13, 2018
A CRISPR-Cas9 screen identifies LAPTM4A (lysosomal protein transmembrane 4 alpha) as a key host barrier against PRRSV
Zhan He1, Min Liu1, Nianqi Zhang1
1Guangdong Laboratory for Lingnan Modern Agriculture, State Key Laboratory of Animal Disease Control and Prevention, Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong, PR China.
Abstract:
Porcine reproductive and respiratory syndrome virus (PRRSV) manipulates host intracellular processes, particularly macroautophagy/autophagy and lysosomal function, to facilitate its replication and spread. However, the precise host factors and molecular mechanisms by which PRRSV remodels the autophagy-lysosome axis remain poorly defined. Here, we performed a CRISPR-Cas9 knockout screen targeting 1,332 genes involved in protein degradation, metabolism, and vesicular trafficking, and identified LAPTM4A (lysosomal protein transmembrane 4 alpha) as a critical antiviral factor involved in the lysosomal pathway. A yeast two-hybrid screen identified LAPTM4A as an interactor of PRRSV GP5 (glycoprotein 5). Mechanistically, GP5 recruits the E3 ubiquitin ligase NEDD4 and the autophagy receptor SQSTM1/p62 to promote K63-linked polyubiquitination of LAPTM4A, leading to its autophagic degradation. This selective degradation activates the AMPK-ULK1-MAP1LC3/LC3 signaling cascade, initiating autophagy while facilitating MTOR-lysosome colocalization, thereby suppressing TFEB nuclear translocation and transcription of lysosome-related genes. The resulting incomplete autophagic flux enhances viral replication. Additionally, in terms of host defense, LAPTM4A maintains lysosomal homeostasis by restraining excessive autophagy through AMPK-ULK1-LC3 signaling and promoting TFEB-dependent lysosomal gene expression by impairing the binding of RPTOR/raptor to MTOR, thus providing broad antiviral protection against multiple RNA viruses. Collectively, our findings identify LAPTM4A as a central regulator of lysosome-autophagy homeostasis and reveal a viral strategy that dismantles this defense axis to facilitate infection.Abbreviations: ATG5: autophagy related 5; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; Baf A1: bafilomycin A1; CHX: cycloheximide; Co-IP: co-immunoprecipitation; DMVT library: protein degradation, metabolism, and vesicular trafficking library; LAPTM4A: lysosomal protein transmembrane 4 alpha; MAGeCK: model-based analysis of genome-wide CRISPR-Cas9 knockout; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NC: negative control; PAMs: porcine alveolar macrophages; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; PRRSV: porcine reproductive and respiratory syndrome virus; qRT-PCR: quantitative real-time PCR; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TCID50: 50% tissue culture infective dose; TFEB: transcription factor EB; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.
Insights
Porcine reproductive and respiratory syndrome virus (PRRSV) degrades the LAPTM4A protein, a key regulator of autophagy and lysosomal function. This viral strategy disrupts host defense, promoting PRRSV replication and spread.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Porcine reproductive and respiratory syndrome virus (PRRSV) hijacks host cellular processes, including autophagy and lysosomal pathways, for replication.
- The specific host factors and mechanisms PRRSV employs to manipulate the autophagy-lysosome axis are not fully understood.
Purpose of the Study:
- To identify host factors involved in PRRSV's manipulation of the autophagy-lysosome axis.
- To elucidate the molecular mechanisms by which PRRSV disrupts host antiviral defense pathways.
Main Methods:
- Conducted a CRISPR-Cas9 knockout screen of 1,332 genes related to protein degradation, metabolism, and vesicular trafficking.
- Utilized yeast two-hybrid screening to identify interactions between viral and host proteins.
- Investigated the role of LAPTM4A in autophagy, lysosomal function, and antiviral defense.
Main Results:
- Identified Lysosomal Protein Transmembrane 4 Alpha (LAPTM4A) as a crucial antiviral factor in the lysosomal pathway.
- Demonstrated that PRRSV GP5 protein recruits NEDD4 and SQSTM1/p62 to induce K63-linked polyubiquitination and autophagic degradation of LAPTM4A.
- Showed that LAPTM4A degradation activates AMPK-ULK1-LC3 signaling, promotes MTOR-lysosome colocalization, suppresses TFEB activity, and enhances viral replication.
- Revealed that LAPTM4A maintains lysosomal homeostasis and provides broad antiviral protection by restraining excessive autophagy and promoting TFEB-mediated lysosomal gene expression.
Conclusions:
- LAPTM4A is a critical regulator of lysosome-autophagy homeostasis and acts as an antiviral factor.
- PRRSV employs a strategy to degrade LAPTM4A, thereby dismantling host antiviral defenses to facilitate infection.
- Understanding this mechanism offers potential targets for therapeutic interventions against PRRSV and other RNA viruses.
Related Concept Videos
01:23The Antiviral System of Bacteria and Archaea: CRISPR
01:28CRISPR/Cas9 Genome Editing
01:59CRISPR
02:53CRISPR and crRNAs
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...