Journal: bioRxiv

Article Title: Viral proteins and virus-like particles of the LTR5_Hs endogenous retrovirus in human primordial germ cell-like cells

doi: 10.1101/2022.09.24.509338

Figure Lengend Snippet: (A) Western blotting detection of HML2 GAG protein in cell lysate and cell culture supernatant. ACTB, β-actin. (B) Proteomics detection of HERVK GAG proteins (HERVK_9 and HERVK_21), pluripotency marker (SOX2), and PGCLC markers (SOX15 and CD38). Bars indicate mean ± SD of triplicated measurements. (C) Locations of detected peptides in HML-2 GAG, ENV, and PRO proteins. (D, E) Transmission electron microscopy images of VLPs formed at the surface of LTC-hPGCLCs. Areas shown with dotted rectangles in the low power images are magnified in the high-power image below. Scale bars in the low and high-power images indicate 500 nm and 100 nm, respectively. (E) Immunogold staining using an anti-HERVK GAG protein demonstrates specific enrichment of the gold particles at the VLPs.

Article Snippet: For immunodetection of HERV-K VLPs, grids were stained with an anti-HERVK capsid mouse monoclonal antibody (AUSTRAL Biologicals, HERM-1831-5, dilution 1:30) followed by secondary staining with protein A conjugated with gold particles.

Techniques: Western Blot, Cell Culture, Marker, Transmission Assay, Electron Microscopy, Staining

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Coassembly of HERV-K Gag changes properties of HIV-1 particles. HeLa cells expressing HIV-1 molecular clone ( a ), HERV-K Gag-FLAG ( b ), or HIV-1 molecular clone and HERV-K Gag-FLAG ( c ) were analyzed by transmission electron microscopy at 16 h post-transfection. Bars in frame 1, 200 nm; bars in frame 2 and 3, 100 nm. d , e The sizes of VLPs or mature particles, which are released from transfected cells, were measured using images of transmission electron microscopy. Total numbers of VLPs examined are 232, 239, and 256 for cultures expressing HIV-1 alone, HERV-K Gag-FLAG alone, and HIV-1 and HERV-K Gag-FLAG, respectively

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Expressing, Transmission Assay, Electron Microscopy, Transfection

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Coassembly with HERV-K Gag changes the size of particles containing HIV-1 p24 Gag. HeLa cells were separately transfected or cotransfected with the HIV-1 molecular clone pNL4-3 and indicated plasmids. For analysis of particles containing single Gag ( gray square ), the supernatants from separately transfected HeLa cells were collected and pooled. For coassembled particles ( black square ), the supernatant of cotransfected HeLa cells was used. These viruses were fractionated in rate-zonal gradient analysis. The amounts of HIV-1 Gag were measured by ELISA ( a ), and the amounts of HERV-K Gag were measured by immunoblotting using anti-Flag antibody ( b ). Representative data from five independent experiments are shown

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Coexpression of Gag chimeras containing HERV-K CA reduces HIV-1 release efficiency. a pCRVI/HeHeM, HeMHe, MHeHe, MMHe, MHeM and HeMM encode chimeras between HERV-K Gag and MLV Gag. All chimeric Gag constructs are tagged with the Flag epitope. b Cell and viral lysates from cotransfected cells were subjected to SDS-PAGE and analyzed by immunoblotting with HIV-Ig or anti-Flag antibody. c HeLa cells were cotransfected with the HIV-1 molecular clone pNL4-3 and indicated plasmids. The amount of p24 in the virus pellet was measured by ELISA. An empty vector pCRVI was used as a control. pCRVI/HERV-K Gag-Flag and pCRVI/MLV Gag-Flag express Flag-tagged HERV-K Gag and MLV Gag, respectively. Data from three independent experiments are shown as means ± standard deviations

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Construct, FLAG-tag, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Coexpression of Gag chimeras containing HERV-K CA-NTD reduces HIV-1 release efficiency. a pCRVI/HeHeMHe-1, HeMHeHe-1, MHeMM-1, MMHeM-1, HeHeMHe-2, HeMHeHe-2, MHeMM-2 and MMHeM-2 encode CA-domain chimeras between HERV-K Gag and MLV Gag. HERV-K MHR and MLV MHR are shown in green and blue , respectively. This same color coding is used to indicate constructs containing the different MHRs in the subsequent panels in Figs. 4, , and . b , d Cell and viral lysates from cotransfected cells were subjected to SDS-PAGE and analyzed by immunoblotting with HIV-Ig or anti-Flag antibody. c , e HeLa cells were transfected with pNL4-3 and indicated plasmids. The amount of p24 was measured as described in Fig. c. P values, compared with HERV-K Gag, were determined using a Student’s t test. * P < 0.01; ** P < 0.001; *** P < 0.0001; n.s. not significant

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Construct, SDS Page, Western Blot, Transfection

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Chimeric Gag constructs containing a part of CA partially colocalize with HIV-1 Gag at the PM. HeLa cells coexpressing YFP-tagged HERV-K Gag, MLV Gag or chimeric Gag ( green ) and mRFP-tagged HIV-1 Gag ( red ) proteins were examined using fluorescence microscopy at 16 h after cotransfection ( a ). Images acquired at the mid-section of the cells. b The R strength of correlation between fluorescence intensities of pairs of indicated Gag-fluorescent protein chimeras was calculated for cells coexpressing these Gag proteins. P values were determined using a Student’s t test. * P < 0.01; *** P < 0.0001; n.s. not significant

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Construct, Fluorescence, Microscopy, Cotransfection

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: HERV-K CA-NTD and MHR are not required for reduction of HIV-1 infectivity. HeLa cells were cotransfected with pNL4-3 and indicated plasmids. a Virus in the supernatant from cotransfected HeLa cells were collected and fractionated in rate-zonal gradient analysis. The amount of HIV-1 Gag were measured by ELISA. b Virus stocks were prepared from each fraction after rate-zonal gradient analysis. The viruses were purified and normalized by p24 ELISA. TZM-bl cells, which harbor an HIV-1 LTR-driven luciferase-reporter gene, were infected with the purified viruses. At 2 days post-infection, luciferase activities were measured by luminometor. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P < 0.01; ** P < 0.001; *** P < 0.0001; n.s. not significant

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Purification, Luciferase

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: HERV-K Gag reduces release efficiency of HIV-1 at a stage prior to particle pinching-off. HeLa cells were cotransfected with HIV-1/YP(−) and indicated pCMV plasmids and pCRVI plasmids. The amount of p24 in the virus pellet was measured by ELISA

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: HERV-K Gag reduces release efficiency of HIV-1 through inhibition of an early stage. HIV-1 mutants are defective for the HIV-1 assembly at the early stage. HeLa cells were cotransfected with indicated HIV-1 mutants and indicated pCRVI plasmids at 10:1 ratio. Two days later, the amount of p24 released into the supernatants was measured by ELISA. Data from three independent experiments are shown as means ± standard deviations. P values were determined using a Student’s t test. * P < 0.01; ** P < 0.001; *** P < 0.0001; n.s. not significant

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Retrovirology

Article Title: Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

doi: 10.1186/s12977-017-0351-8

Figure Lengend Snippet: Most Gag chimeras containing HERV-K CA colocalize with HIV-1 Gag at the plasma membrane (PM). HeLa cells coexpressing YFP-tagged HERV-K Gag, MLV Gag or chimeric Gag ( green ) and mRFP-tagged HIV-1 Gag ( red ) proteins were examined using fluorescence microscopy at 16 h after cotransfection ( a ). Images acquired at the mid-section of the cells. b The R strength of correlation between fluorescence intensities of pairs of indicated Gag-fluorescent protein chimeras was calculated for cells coexpressing these Gag proteins. Data from 11 to 33 cells are shown as means ± standard error of the mean (SEM). P values were determined using a Student’s t test. * P < 0.01; *** P < 0.0001; n.s. not significant. c For the analysis of HIV-1 Gag localization patterns, we acquired images of 11–33 cells per condition at the middle and top focal plains. If Gag puncta distributes over the half of the circumference of a cell, the cell is classified as “PM (High signals)”. If Gag distributes less than the half of the cell circumference, the cell is classified as “PM (Low signals)”. If there is no Gag-puncta signal at the plasma membrane and in the cytosol in both top and middle focal plains of a cell, the cell is classified as “Cytosol”. If there is Gag puncta in the cytoplasm in the middle focal plain, it is classified as “Intra + PM”. Data from 11 to 33 cells are shown

Article Snippet: Gag proteins in the cell and virion lysates were detected by immunoblotting using HIV-Ig (NIH AIDS Research and Preference Reagent Program), mouse monoclonal anti-Flag antibody (Wako), mouse monoclonal anti-HERV-K Gag antibody (HERM-1831-5) (Austral Biologicals) as primary antibodies.

Techniques: Fluorescence, Microscopy, Cotransfection