Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) Cell extracts from WT, atg7 −/− , and atg13 −/− U2OS cells were analyzed by WB with anti ATG7, ATG13, LC3, and β-actin antibodies. ( B ) Cells from (A) were infected with IAV at an MOI of 0.1 for 0, 16 and 24 hours before WB examination with antibodies against NP and β-actin (top). NP levels were quantified relative to WT cells infected for 16 hours (bottom). ( C ) Cells from (A) were infected with IAV at an MOI of 0.1 before being IF with the anti-NP antibody at 8 or 12 hpi to determine the percentage of infected cells using a TissueFAXS microscope. ( D ) ATG7- or ATG13-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 16 hours and analyzed by WB with anti-NP, ATG7, ATG13, and β-actin antibodies (top). NP levels were quantified relative to siCtrl cells (bottom). ( E ) The atg7 −/− cells were transfected with siCtrl or a pool of siRNA targeting the three members of the LC3 subfamily (siLC3s) before infection with IAV at an MOI of 0.1. Cells extracts were collected at 0, 8, 16, and 24 hpi and examined by WB with anti-NP, LC3, and β-actin antibodies (top). NP levels were quantified relative to siCtrl cells infected for 8 hours (bottom). ( F ) LC3TKO cells were transfected with pcDNA3.1, pCDN3.1-LC3B, pCDN3.1-LC3B G120A , pCDN3.1-LC3B G120A* , or pCDN3.1-LC3B G120* for 48 hours and then infected with IAV at an MOI of 0.1 for 16 hours before WB with anti-NP, LC3, and β-actin antibodies. ( G ) NP level quantification in (F). Bars represent amounts relative to infected LC3TKO cells transfected with the pCDN3.1-LC3B plasmid. Error bars represent SDs [ n = 3 in (B), (D), (E), and (G); n = 2 in (C)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Microscopy, Transfection, Plasmid Preparation

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa cells treated as in in the presence or absence of Baf were processed for IF with anti-M1 and anti-LC3 antibodies. Insets highlight the colocalization between LC3 and M1, and white arrowheads point to colocalization (in untreated cells) and noncolocalization (in Baf-treated cells). Images were collected using a DeltaVision microscope. Scale bars, 5 μm. ( B ) Quantification of the LC3-positive M1 puncta in (A). Error bars represent SDs. ( C ) IAV cytoplasm entry in sHeLa cells was carried out as in , except that IAV was at an MOI of 30. Baf (200 nM) was used to block IAV fusion at LEs. Cell extracts were subjected to IP with LC3 antibodies before separating the coisolated proteins and WB analysis with anti-LC3, NP, and immunoglobulin G (ΙgG) (control) antibodies. ( D ) Quantification of the NP bound to LC3 in (C), expressed relative to infected cells not treated with Baf. ( E ) Cell extracts from sHeLa cells treated and processed as in (C). WB membranes were probed with anti-LC3, HDAC6, DYNC1I1, KIF5B, and ΙgG antibodies (control). ( F ) Quantification of the DYNC1I1 bound to LC3s in (E), expressed relative to mock-treated cells. Error bars represent SDs [ n = 3 in (D) and (F); n = 3 in (B), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Microscopy, Blocking Assay, Control, Infection

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf or 100 nM Ctn before WB with the indicated antibodies. ( B ) NP levels in (A) relative to siCtrl. ( C ) PCNT-depleted sHeLa cells transfected with the GFP-PCNTB or GFP-PCNTS plasmid were infected with IAV at an MOI of 0.1 for 8 hours and examined by WB with the indicated antibodies. NP levels are relative to siCtrl. The GFP antibody was used for GFP-PCNTS. The PCNT antibody only detects PCNTB. ( D ) Cells as in (C) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to infected siCtrl cells. ( E ) PCNT-depleted sHeLa cells were transfected with the GFP-PCNTS or GFP-PCNTS ΔPACT plasmid before IAV infection at an MOI of 0.1 for 8 hours and examined by WB analysis with the indicated antibodies. NP levels are relative to siCtrl. ( F ) Cells as in (E) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to siCtrl. ( G ) DYNC1I1-, PCNT-, or LC3/PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified by RT-PCR, normalized to GAPDH, and expressed relative to siCtrl. ( H ) Cells from (G) were infected with IAV for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. ( I ) DYNC1I1-, PCNT-, HDAC6-, or PCNT/HDAC6-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified as in (G). ( J ) Cells as in (I) were infected for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. Error bars represent the SDs [ n = 3 in (B) to (J)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) PCNT-depleted atg7 −/− cells were infected with IAV at MOI 30 for 3 hours, and cell extracts were subjected to IP with an anti-LC3 antibody before examining the input and the coisolated proteins by WB with anti-LC3, NP, PCNT, DYNC1I1, and ΙgG (control) antibodies. ( B ) DYNC1I1, NP, and PCNT bound to LC3s in (A) relative to the infected siCtrl cells. ( C ) sHeLa APEX2KI and LC3 APEX2KI cells were infected with IAV as in , but 500 μM biotin phenol (BP) and 1 mM H 2 O 2 were added 30 and 1 min, respectively, before isolating biotinylated proteins. sHeLa APEX2KI cells without BP incubation were used as a negative control. The input and the affinity-purified proteins were analyzed by WB with antibodies against biotin, NP, PCNT, DYNC1I1, or β-actin. ( D and E ) Biotinylated DYNC1I1 (D) and NP (E) in (C) relative to the noninfected sHeLa APEX2KI cells. ( F ) PCNT-depleted atg7 −/− cells were processed for IF as in with antibodies against M1 and LC3. Insets highlight colocalization between M1 and LC3. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( G ) Percentage of the LC3-positive M1 puncta in (F). ( H ) Model for IAV host cytoplasm entry. The lower pH of LEs triggers the fusion between endocytoses IAV VPs at LEs. Uncoating and cytoplasmic vRNP release is mediated by two dynein-dependent systems that take advantage of the pulling force of MT-based motors. vRNPs are linked to dynein motors via the LC3-PCNT adaptor complex or HDAC6, which binds ubiquitin. It is unknown which vRNP components interact with LC3s and HDAC6. Error bars represent SDs [ n = 3 in (B), (D), and (E); n = 5 in (C), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Control, Incubation, Negative Control, Affinity Purification, Microscopy, Ubiquitin Proteomics