Journal: Advanced Science

Article Title: FAM134B Restricts African Swine Fever Virus Capsid Assembly via Reticulophagy and Its Antiviral Activity is Antagonized by the Viral Virulence‐Associated Factor pMGF300‐2R

doi: 10.1002/advs.77741

Figure Lengend Snippet: FAM134B targets the ASFV proteins p72 and pA137R for degradation via reticulophagy. (A–C) FAM134B interacts with p72, and pA137R. HEK293T cells were cotransfected with pFlag‐p72 (A) or pFlag‐A137R (B) together with pHA‐FAM134B. The cells were lysed and whole cell lysates (WCL) were immunoprecipitated with an anti‐HA monoclonal antibody (mAb) at 36 h post‐transfection (hpt). The immunoprecipitates were examined by immunoblotting (IB). WSL cells were infected with ASFV‐WT at a multiplicity of infection (MOI) of 1. At 24 h post‐infection (hpi), the cells were lysed for immunoprecipitation (IP) with anti‐FAM134B. Anti‐IgG antibodies were used as a negative control (C). (D–G) FAM134B degrades p72 and pA137R via the autophagy‐lysosomal pathway. WSL cells were transfected with pFlag‐FAM134B, followed by infection with ASFV‐WT at an MOI of 1 and treatment with Lac (20 µM), MG132 (20 µM), CQ (50 µM), or BafA1 (100 nM) for 8 h at 16 hpi. The cell lysates were analyzed by IB (D, E). The ATG5 ‐knockout ( ATG5‐ KO) or wild‐type (WT) HEK293T cells were transfected with pFlag‐FAM134B, followed by infection with ASFV‐P31 at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (F, G). (H–L) The degradation of p72 and pA137R mediated by FAM134B depends on the LIR motif of FAM134B. WSL cells were transfected with pHA‐FAM134B or pHA‐FAM134B‐ΔLIR, followed by infection with ASFV‐WT at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (H, I). Wild‐type HEK293T cells, along with FAM134B ‐KO cells reconstituted with wild‐type FAM134B (WT‐ FAM134B ‐R) or the LIR mutant (ΔLIR‐ FAM134B ‐R), were infected with ASFV‐P31 at an MOI of 1. At 24 hpi, the expression levels of p72, pA137R, and FAM134B were analyzed by IB (J, K). Additionally, viral titers were quantified (L). (M–P) Co‐localization of FAM134B with LC3B and ASFV p72 (M, N) or pA137R (O, P). HEK293T cells were cotransfected with the indicated plasmids and protein colocalization was analyzed by confocal microscopy. The data are presented as the mean ± SD from three independent experiments. Statistical significance was determined by one‐way ANOVA for the data in panels (E, G, I, K, and L), and by two‐tailed unpaired t ‐test for the data in panels (N) and (P) (ns, not significant; * p < 0.05; *** p < 0.001; **** p < 0.0001).

Article Snippet: Mouse anti‐His (AE003), rabbit anti‐ β ‐tubulin (A12289), mouse anti‐GST (AE001), anti‐Flag (AE005), anti‐HA (AE008), anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (AC033), and anti‐GFP (AE012) and rabbit anti‐HA (AE105) antibodies were purchased from ABclonal Biotechnology Co., Ltd. Rabbit anti‐RFP (BGT‐ANT‐45233) antibodies were purchased from Biogradetech.

Techniques: Immunoprecipitation, Transfection, Western Blot, Infection, Negative Control, Knock-Out, Expressing, Mutagenesis, Confocal Microscopy, Two Tailed Test

Journal: PLOS Pathogens

Article Title: Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway

doi: 10.1371/journal.ppat.1014569

Figure Lengend Snippet: (A) Schematic diagram of SADS-CoV E protein truncation mutants. The E protein consists of an N-terminal domain (residues 1–10), a transmembrane domain (residues 10–33), and a C-terminal domain (residues 33–75). Truncation mutants (mt1–mt7) were constructed by sequentially deleting residues from the C-terminus. (B) Co-IP analysis of the interaction between Rab10 and E protein mutants. HeLa cells were co-transfected with Myc-Rab10 and HA-tagged E-WT or mutants (mt1–mt7). Cell lysates were immunoprecipitated with anti-HA beads and analyzed by western blot with anti-Myc and anti-HA antibodies. GAPDH served as the loading control for input samples. The IP Myc/HA ratios, normalized to E-WT, are indicated below the blots. (C–D) Confocal microscopy analysis of the co-localization between Myc-Rab10 (green) and HA-tagged E protein mutants (red) in HeLa WT cells. Nuclei were stained with DAPI (blue). Scale bar, 5 μm. (E–F) Statistical quantification of co-localization using Mander's and Pearson's correlation coefficients. Panels (E) and (F) show the co-localization analysis between SADS-CoV E (WT, mt6, or mt7). (G) Measurement of Rab10 concentration in the cell culture supernatant. HeLa cells were transfected with HA-tagged E protein mutants, and the concentration of secreted Rab10 was quantified by ELISA. (H) Representative confocal images showing the co-localization of E-WT-HA or Emt7-HA (red) with the ERGIC marker LMAN1 (green) in HeLa WT cells. Nuclei were stained with DAPI (blue). Scale bar, 5 μm. (I–J) Statistical quantification of co-localization using Mander's and Pearson's correlation coefficients. Panels (I) and (J) show the co-localization analysis between E-HA (WT or mt7) and LMAN1. Data are presented as means ± SD from three independent experiments. E–F, G, one-way ANOVA with Dunnett's test. I–J, unpaired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-Rab10 (Abcam, ab237703, IFA: 1:100, WB: 1:1000); Alexa Fluor 488 Anti-Rab10 antibody MJF-R23 (Abcam, ab302654, IFA: 1:50); rabbit anti-SADS-CoV N; mouse anti-SADS-CoV N; mouse anti-SADS-CoV S were prepared and maintained in our laboratory; mouse anti-SARS-CoV-2 N (ABclonal, A20142, WB: 1:1000); rabbit anti-TGN46 (ABclonal, A19618, IFA: 1:100, WB: 1:1000); rabbit anti-LMAN1 (ABclonal, A4941, IFA: 1:100, WB: 1:1000); rabbit anti-LAMP1 (ABclonal, A22194, IFA: 1:100, WB: 1:1000); rabbit anti-Alix (ABclonal, A25326 , WB: 1:1000); rabbit anti-CD9 (ABclonal, A1703, WB: 1:1000); rabbit anti-Caveolin-1 (ABclonal, A22417, WB: 1:1000); rabbit anti-Calnexin (Abcam, ab22595, WB: 1:1000); mouse anti-dsRNA (Scicons, #10010500, IFA: 1:100)rabbit anti-GAPDH (Proteintech, #81640–5-AP, WB: 1:10000); rabbit anti-Flag (Proteintech, #80801–2-RR, WB: 1:5000); mouse anti-Flag (Proteintech, #66008–4-Ig, IFA: 1:200, WB: 1:5000); rabbit anti-Myc (Proteintech, #10828–1-AP, WB: 1:4000); mouse anti-HA (ABclonal, AE008, IFA: 1:100, WB: 1:1000).

Techniques: Construct, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot, Control, Confocal Microscopy, Staining, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Marker

Journal: PLOS Pathogens

Article Title: Rab10 coordinates SADS-CoV non-lytic egress through the ERGIC-TGN-lysosome trafficking pathway

doi: 10.1371/journal.ppat.1014569

Figure Lengend Snippet: (A) Alanine scanning mutagenesis of the C-terminal residues (64–75) of SADS-CoV E protein. HeLa cells were co-transfected with Myc-Rab10 and HA-tagged E-WT or alanine mutants. Cell lysates were immunoprecipitated with anti-HA beads and analyzed by western blot. (B) Sequence alignment of the C-terminal tails of E proteins from diverse coronaviruses, including alphacoronaviruses (SADS-CoV, TGEV, PEDV), deltacoronaviruses (PDCoV), and betacoronaviruses (SARS-CoV-2, MHV). (C) Co-IP analysis of Rab10 with E proteins from the indicated coronaviruses. HeLa cells were co-transfected with Myc-Rab10 and HA-tagged E from the indicated coronaviruses. Cell lysates were immunoprecipitated with anti-HA beads and analyzed by western blot. (D) Rescue of Rab10 interaction with MHV E protein. HeLa cells were co-transfected with Myc-Rab10 and HA-tagged MHV E-WT or the I83V mutant. (E) SADS-CoV VLPs secretion assay. HeLa cells were transfected with VLPs plasmids (N/M/E) containing E-WT, ΔE, E-mt7, or E-V75A. Cell lysates and concentrated medium samples were analyzed by western blot for SADS-CoV N. The ratio of N in medium versus lysate is shown below. (F) Schematic representation and rescue of recombinant icSADS-CoV-GFP E-WT and E-V75A. Fluorescence images show the successful rescue and propagation of viruses at Passage 2 in Vero E6 cells. Scale bar, 275 μm. (G) Comparison of viral protein expression and release. HeLa cells were infected with icSADS-CoV-GFP E-WT or E-V75A (MOI = 1) for 16 h. Cell lysates and concentrated culture supernatants (Medium) were analyzed by western blot for SADS-CoV S and N proteins. (H–K) Quantitative analysis of SADS-CoV egress for E-WT and E-V75A viruses. Metrics include total viral RNA (H), relative intracellular mRNA (I), released viral RNA in the supernatant (J), and released infectious viral titers measured by TCID 50 (K). Data points represent individual biological replicates (n = 3). Data are presented as means ± SD from three independent experiments. Statistical significance was analyzed by unpaired t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant.

Article Snippet: The following primary antibodies were used in this study: rabbit anti-Rab10 (Abcam, ab237703, IFA: 1:100, WB: 1:1000); Alexa Fluor 488 Anti-Rab10 antibody MJF-R23 (Abcam, ab302654, IFA: 1:50); rabbit anti-SADS-CoV N; mouse anti-SADS-CoV N; mouse anti-SADS-CoV S were prepared and maintained in our laboratory; mouse anti-SARS-CoV-2 N (ABclonal, A20142, WB: 1:1000); rabbit anti-TGN46 (ABclonal, A19618, IFA: 1:100, WB: 1:1000); rabbit anti-LMAN1 (ABclonal, A4941, IFA: 1:100, WB: 1:1000); rabbit anti-LAMP1 (ABclonal, A22194, IFA: 1:100, WB: 1:1000); rabbit anti-Alix (ABclonal, A25326 , WB: 1:1000); rabbit anti-CD9 (ABclonal, A1703, WB: 1:1000); rabbit anti-Caveolin-1 (ABclonal, A22417, WB: 1:1000); rabbit anti-Calnexin (Abcam, ab22595, WB: 1:1000); mouse anti-dsRNA (Scicons, #10010500, IFA: 1:100)rabbit anti-GAPDH (Proteintech, #81640–5-AP, WB: 1:10000); rabbit anti-Flag (Proteintech, #80801–2-RR, WB: 1:5000); mouse anti-Flag (Proteintech, #66008–4-Ig, IFA: 1:200, WB: 1:5000); rabbit anti-Myc (Proteintech, #10828–1-AP, WB: 1:4000); mouse anti-HA (ABclonal, AE008, IFA: 1:100, WB: 1:1000).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Sequencing, Co-Immunoprecipitation Assay, Recombinant, Fluorescence, Comparison, Expressing, Infection