Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: (a) Representative western immunoblot analysis and quantification of HERV-K envelope (HERV-K env) relative to histone H3 (H3) in PAH vs. Con neutrophils (n=6 Con and n=10 PAH). (b) Immunofluorescence microscopy visualization of HERV-K env protein (green) and nuclei by DAPI staining (blue) in Con and PAH neutrophils. Antibodies used for staining are described in the “Methods”. A range of z-stack images was collected for image analysis. Mean fluorescence intensity (MFI) and cell area were quantified via image j from 3-5 randomly selected visual fields (n=3). Scale bar = 20µm (c-f) HL-60 cells were cultured in RPMI complete media and transfected using HL-60 Cell Avalanche transfection reagent per manufacturer’s protocol. (c) RT-qPCR analysis of HERV-K envelope, RIG-I , and PKR mRNA in HL-60 cells overexpressing HERV-K env (n=3). (d) Representative western immunoblot and quantification of NE relative to H3 in HL-60 cells overexpressing HERV-K env (n=3). (e) Western immunoblot analysis and quantification of HERV-K dUTPase (dUTPase) in plasma collected from Con or PAH patients. Plasma was depleted of albumin, measured by BCA, and run on a gradient gel (n=17). ( f ) Representative western immunoblot analysis and quantification of VCL relative to H3 in dHL-60 cells, treated with 10 µg/mL HERV-K dUTPase or Veh (HERV-K dUTPase elution buffer) for 24 h (n=4). Bars represent mean ±SEM. *p<0.05 and ***p<0.001 by by unpaired Student t-test.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence, Cell Culture, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: Exosomes were isolated from plasma of 17 healthy donor controls and 17 PAH patients, using the ExoTIC device described under ‘Methods’. CD66b positive neutrophil exosomes were pulled down using anti-CD66b beads, from pooled exosomes of Con and PAH pooled plasma. (a) Size distribution of the pooled exosomes, determined using Nanosight. (b) Representative transmission electron microscopy (TEM) images of CD66b positive neutrophil exosomes derived from pooled plasma of PAH vs. Con patients. Scale bar=100 nm (c) Western immunoblot analysis and quantification of NE and HERV-K envelope from pooled PAH vs. Con neutrophil exosomes, relative to the exosome marker CD9. H3 from PAH neutrophil total lysate was used as a negative control. (d) NE activity in PAH vs. Con exosomes after 120 min incubation. NE was assessed by the production of BODIPY FL labeled fluorescent elastin fragments from self-quenching BODIPY FL-conjugated bovine neck ligament elastin. Bars represent mean ± SEM n=3 technical replicates of the pooled exosomes. *p<0.05, **p<0.01 by unpaired Student t-test.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Marker, Negative Control, Activity Assay, Incubation, Labeling

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: The secretion of HERV-K dUTPase from monocytes results in the upregulation of vinculin thereby increasing neutrophil adhesion and reducing migration. An increase in HERV-K envelope (Env) in the neutrophil is likely via double strated (ds) RNA required for the interferon response and heightened neutrophil elastase. The increase in neutrophil elastase promotes neutrophil extracellular traps NETs. Elastase released from granules associates with HERV-K env in exosomes causing pathologic features of pulmonary arterial hypertension.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Migration

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: The kinetics of GALV replication in MDTF cells expressing HA epitope tagged PiT1 or PiT2K522E. MDTFPiT1-HA (upper three lines) or MDTFPiT2K522E-HA (lower three lines) cells were exposed to 1ml (o), 0.2ml (□) or 0.05ml (▵;) of GALV-GFP viral supernatant in the presence of polybrene. Cells were harvested for flow cytometry analysis at day 1, 2, 4, 7, 9, 11, 14 and 30. The percentage of GFP positive cells is displayed at each time point, day postinfection (dpi). The experiments were repeated three times, and values are presented as average standard deviation.

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Expressing, Flow Cytometry, Standard Deviation

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: The binding affinity of GALV RBD with MDTF cells expressing HA tagged PiT1, PiT2K522E or PiT2. GALV RBD tagged with both V5 and His was purified using high-performance nickel-NTA (Ni-NTA) agarose, serially diluted four times and applied to a binding assay for MDTFPiT1-HA ( A ) or MDTFPiT2K522E-HA and MDTFPiT2-HA cells ( B ). The kinetics of binding of GALV RBD was compared. The mean fluorescence intensity (MFI) for GALV RBD binding is indicated on the y -axis and GALVRBD concentration (mg) on the x -axis. Three independent experiments were performed and the mean ± standard deviation was presented in the curve.

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Binding Assay, Expressing, Purification, Fluorescence, Concentration Assay, Standard Deviation

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: GALV is release efficiency from MDTF cells expressing PiT1 or PiT2K522E. MDTF cells expressing receptor PiT1 or PiT2K522E were incubated with 0.5ml GALV-GFP viral stock at 37°C for 1 hour, then the cells were extensively washed and maintained in culture. Media were collected at two time points, 36 and 48 hours after initial exposure to GALV. The titer of GALV-GFP viruses present in 36 hours or 48 hours samples, were assayed by exposing MDTF cells expressing PiT1 to supernatant and performing FACS analysis to determine the percentage of GALV-GFP infected cells at 30 hours after viral exposure. The experiments were repeated three times independently and averaged values are shown.

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Expressing, Incubation, Infection

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: MDTF cells expressing PiT2K522E cells fail to establish GALV superinfection resistance in. MDTF cells mock infected or infected with GALV-GFP for two weeks, were challenged with GALV enveloped retroviral vector expressing the fluorescent marker protein, cherry red (GALV-RFP). Two days later, cells were harvested for FACS analysis. MDTFPiT1 cells uninfected ( A ) and infected with GALV-GFP ( B ), MDTFPiT2K522E cells uninfected ( C ) or infected with GALV-GFP ( D ). The x- axis represents MFI of GFP expression and the y -axis represents MFI of RFP expression. Three experiments were performed and representative images were presented. Region statistics was also exhibited as R3, R4 and R5. R3 = GFP negative and RFP negative, R6 = GFP positive and RFP positive and R5 = GFP positive but RFP negative. The statistics are listed in the table below the figure. The percentage of cells that fail to establish superinfection resistance was calculated by the value in R6 divided by the total values in R6 and R5.

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Expressing, Infection, Retroviral, Plasmid Preparation, Marker

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: Evaluation of the efficiency of GALV cell-cell transmission in co-culture experiments with MDTFPiT1 cells productively infected with GALV expressing GFP (GALV-GFP). MDTFPiT1 cells were exposed to GALV-GFP and then maintained and cultured for one week before being analyzed by FACS for GFP expression ( A ). Next GALV-GFP producer cells were co-cultured with MDTF cells expressing an HA epitope tagged PiT1 and analyzed by FACS for GFP and HA expression ( B ) or MDTF cells expressing the HA epitope tagged mutant receptor PiT2K522E and analyzed by FACS for GFP and HA expression ( C ). Approximately 24 hours after co-culture, cells were harvested for FACS analysis. The MFI of HA expression is indicated on the y-axis and the MFI of GFP expression is on the x-axis. Three independent experiments were performed and representative images presented.

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Transmission Assay, Co-Culture Assay, Infection, Expressing, Cell Culture, Mutagenesis

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: Summary of coculture experiments

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Infection

Journal: Retrovirology

Article Title: A mutant retroviral receptor restricts virus superinfection interference and productive infection

doi: 10.1186/1742-4690-9-51

Figure Lengend Snippet: Syncytia formation observed in MDTFPiT2K522E cells infected with GALV-GFP. MDTFPiT2K522E ( A ) or MDTFPiT1 cells ( B ) were exposed to GALV-GFP viruses. Four days after viral exposure, Several GFP positive syncytia were observed in MDTFPiT2K522E cells and the number of syncytia gradually increased over time. The experiments were performed three times and the representative images from cells infected with GALV-GFP seven days after viral exposure are shown. Syncytia were defined as those cells containing three or more nuclei. The total number of nuclei in syncythia cells was then counted. Five random fields were counted from each well of triplicate samples, using a 10x objective .

Article Snippet: The GALV enveloped replication-competent retroviruses (GALV-GFP and GALV), GALV enveloped retroviral vectors as well as GALV RBD were produced by calcium phosphate transfection of 293 T cells (Promega Corporation) as previously described [ ].

Techniques: Infection