Journal: Journal of Virology

Article Title: Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication

doi: 10.1128/jvi.01842-23

Figure Lengend Snippet: PRRSV Nsp2 induces GA fragmentation by degrading GRASP65 dependent on its cysteine protease activity. ( A ) HEK-293T cells were transfected with the plasmids encoding GRASP65-myc and Nsp2-HA or HA-tagged empty vector. The inhibitors 3-MA and MG132 or DMSO were added 12 h before the collection of the samples. GRASP65 protein abundance was detected by IB. ( B ) HeLa cells were transfected with the plasmid encoding Nsp2-HA for 6 h. The inhibitors 3-MA, MG132, or DMSO were added, and the samples were collected after 24 h. GM130 and Nsp2-HA were visualized with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The morphology of GA was assessed by confocal microscopy (scale bars = 10 µm). ( C ) The schematic diagram of Nsp2-mutant-HA. ( D ) HEK-293T cells were co-transfected with the plasmids encoding GRASP65-myc and Nsp2-HA or Nsp2-mutant-HA. At 24 h post-transfection, the cell lysates were collected for analysis of the GRASP65 protein level with IB. ( E ) HeLa cells were transfected with the plasmid encoding Nsp2-HA or Nsp2-mutant-HA for 24 h. GM130, Nsp2-HA, and Nsp2-mutant-HA were visualized with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. The morphology of GA was assessed by confocal microscopy (scale bars = 10 µm). White arrows indicated GA morphology.

Article Snippet: For the transfection, the plasmids were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. No. 2066194) or TransIntro PL Transfection Reagent (TransGen Biotech, Cat. No. FT301-01) according to the manufacturer’s instructions.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Quantitative Proteomics, Staining, Confocal Microscopy, Mutagenesis

Journal: Journal of Virology

Article Title: Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication

doi: 10.1128/jvi.01842-23

Figure Lengend Snippet: PRRSV Nsp2-induced GA fragmentation disassociates RAB2 from GM130 to enhance its interaction with ULK1. ( A and B ) HEK-293T or HeLa cells were transfected with the plasmids encoding RAB2-myc/GFP and Nsp2-HA or HA-tagged empty vector for 36 h. ( A ) Co-IP was performed with anti-myc magnetic beads, and IB was conducted with the specific antibodies. ( B ) The fluorescent signals were observed with confocal microscopy. The co-localization was assessed by determination of the Pearson’s correlation coefficient (scale bars = 10 µm). ( C ) MARC-145 cells were transfected with the plasmid encoding RAB2-GFP. At 24 h post-transfection, the MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected, and the fluorescent signals were observed with confocal microscopy. The co-localization was assessed by determination of the Pearson’s correlation coefficient (scale bars = 10 µm). ( D ) HEK-293T cells were co-transfected with the plasmids encoding RAB2-myc, ULK1-Flag, and Nsp2-HA or HA-tagged empty vector for 36 h. Co-IP was performed with anti-myc magnetic beads, and IB was conducted with the specific antibodies. ( E ) HeLa cells were co-transfected with the plasmids encoding RAB2-GFP, ULK1-Flag, and Nsp2-HA or HA-tagged empty vector for 36 h. The fluorescent signals were observed with confocal microscopy. The co-localization was assessed by determination of the Pearson’s correlation coefficient (scale bars = 10 µm). ( F ) HEK-293T cells were transfected with the plasmid encoding Nsp2-HA or HA-tagged empty vector for 36 h, followed by IP with anti-HA magnetic beads and IB analysis with the specific antibodies. ( G ) MARC-145 cells were infected with PRRSV at 0.1 MOI or mock infected for 24 h. The cell lysates were immunoprecipitated by the anti-ULK1 antibody pre-incubated with protein A/G magnetic beads. IB was conducted with the specific antibodies.

Article Snippet: For the transfection, the plasmids were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. No. 2066194) or TransIntro PL Transfection Reagent (TransGen Biotech, Cat. No. FT301-01) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Magnetic Beads, Confocal Microscopy, Infection, Immunoprecipitation, Incubation

Journal: Journal of Virology

Article Title: Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication

doi: 10.1128/jvi.01842-23

Figure Lengend Snippet: PRRSV Nsp2 induces GA fragmentation to promote autophagy by activating the RAB2-ULK1 pathway. ( A ) HEK-293T cells were transfected with the plasmid encoding Nsp2-HA or HA-tagged empty vector for 36 h. The cell lysates were collected for IB. ( B ) HEK-293T cells were co-transfected with the plasmids encoding Nsp2-HA and GRASP65-myc or myc-tagged empty vector. At 36 h post-transfection, the cell lysates were collected for IB. ( C and D ) HEK-293T or MARC-145 cells were transfected with siRAB2 or siNC. At 24 h post-transfection, the HEK-293T cells were transfected with the plasmid encoding Nsp2-HA or HA-tagged empty vector, and the MARC-145 cells were infected with PRRSV at an MOI of 1. The cell lysates were collected for IB. Data represent means ± SEM from three independent experiments. Statistical analysis was carried out using Student’s t test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: For the transfection, the plasmids were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. No. 2066194) or TransIntro PL Transfection Reagent (TransGen Biotech, Cat. No. FT301-01) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Infection

Journal: Journal of Virology

Article Title: Porcine reproductive and respiratory syndrome virus triggers Golgi apparatus fragmentation-mediated autophagy to facilitate viral self-replication

doi: 10.1128/jvi.01842-23

Figure Lengend Snippet: PRRSV triggers GA fragmentation-mediated autophagy to facilitate viral self-replication. ( A through C ) MARC-145 cells were transfected with the plasmid encoding GRASP65-myc or myc-tagged empty vector. At 24 h post-transfection, the MARC-145 cells were infected with PRRSV at an MOI of 5 for 10 h. ( A ) The fluorescent signal of PRRSV dsRNA abundance was detected using IFA with a mouse anti-dsRNA mAb. Nuclei were stained with DAPI. Scale bars = 50 µm. ( B ) PRRSV dsRNA abundance was detected using FCM with the mouse anti-dsRNA mAb. ( C ) PRRSV RNA abundance was detected using RT-qPCR. ( D ) MARC-145 cells were transfected with the plasmid encoding GRASP65-myc or myc-tagged empty vector for 24 h, and PRRSV infectivity was detected using IFA. IFA images were taken by confocal microscopy, and the numbers of PRRSV N protein-expressed and DAPI-stained cells were counted using ImageJ software. The infection rate was expressed as PRRSV N protein-expressed cells/DAPI-stained cells (scale bars = 50 µm). ( E and F ) MARC-145 cells were transfected with the plasmid encoding GRASP65-myc or myc-tagged empty vector. At 24 h post-transfection, the MARC-145 cells were infected with PRRSV at an MOI of 1 or mock infected for indicated time periods (12, 24, 36 h, or 48 h). ( E ) PRRSV N protein abundance was detected using IB. ( F ) PRRSV titers were measured by assessing TCID 50 . Data represent means ± SEM from three independent experiments. Statistical analysis was carried out using Student’s t test. *** P < 0.001 and **** P < 0.0001.

Article Snippet: For the transfection, the plasmids were transfected with Lipofectamine 2000 (Thermo Fisher Scientific, Cat. No. 2066194) or TransIntro PL Transfection Reagent (TransGen Biotech, Cat. No. FT301-01) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Infection, Staining, Quantitative RT-PCR, Confocal Microscopy, Software, Quantitative Proteomics