Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δ nef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag -EGFP, EGFP -LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant siRNA (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δ nef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP -LC3B and HIV-1 NL4-3 Δ nef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation, Retroviral, Microscopy

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Residues 40–57 in the N-terminal domain of NL4-3 Nef are required to counteract autophagy initiation. A , D Schematic representation of the domains replaced for the generation HIV-1 NL4-3 and SIV mac 239 Nef chimeras. B , E HEK293T cells were co-transfected with EGFP -LC3B and the different nef constructs: SIV mac 239 nef , NL4-3 nef and the selected nef chimeras. 48 h post-transfection, cells were analyzed by flow cytometry for autophagosome-associated EGFP-LC3B. Data correspond to the mean and SEM of the percentage of EGFP + cells from three independent experiments. C , F HEK293T cells were transfected with NL4-3 nef , SIV mac 239 nef and the selected chimeras. 48 h later, cells were exposed for 4 h to increasing concentrations of rapamycin (0–4 μM). Next, cells were analyzed by western blot for the levels of GFP, LC3, and ACTB. Densitometric analyses were performed to determine the ratio of LC3-II over LC3-I relative to SIV mac 239 nef with no rapamycin treatment. All images are representative of three independent experiments. Significantly different values are indicated by asterisks * P ≤ 0.05; ** P ≤ 0.01

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Transfection, Construct, Flow Cytometry, Western Blot

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Nef uses residues 48–49 to prevent autophagosome biogenesis. A Alignment comprising residues 40–57 in NL4-3 Nef. Substitutions introduced in each mutant are indicated in red. B HEK293T cells were co-transfected with EGFP -LC3B and either an empty vector, NL4-3 nef or the selected nef mutants. 48 h post-transfection, cells were analyzed by flow cytometry for autophagosome-associated EGFP-LC3B. C HEK293T cells were transfected with NL4-3 nef or the selected mutants. 48 h later, cells were treated with rapamycin (4 μM) for 4 h and analyzed by western blot for HA, LC3, and ACTB. Densitometric analyses determine the ratio of LC3-II:I relative to NL4-3 nef are shown underneath the blots. D HEK293T cells were co-transfected with BECN1 and either an empty vector, NL4-3 nef or 48–49 NL4-3 nef . 48 h later, cells were harvested and BCL2 was immunoprecipitated. The pulldown fraction was examined for BCL2 and BECN1. Lysates were also analyzed by western blot for BECN1, HA, BCL2, LC3, and ACTB. Densitometric analyses indicate the relative BCL2-BECN1 interaction. E HEK293T cells stably expressing EGFP-LC3 were transfected with an empty vector, NL4-3 nef , a Nef mutant harboring alanine substitutions at the residues responsible for blocking autophagy maturation (36–39 Nef) or 48–49 NL4-3 nef . 48 h later, cells were treated with DMSO or rapamycin (4 μM) for 4 h prior to microscopy visualization of EGFP-LC3-coated autophagosomes. Graph: number of autophagosomes per cell from 15 randomly selected cells. Scale bar: 10 μm. Images are representative of three independent experiments. Significantly different values are indicated by asterisks * P ≤ 0.05; *** P ≤ 0.001; **** P ≤ 0.0001

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Flow Cytometry, Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Blocking Assay, Microscopy

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Nef uses residues 48–49 to prevent Gag redistribution to autophagosomes, restoring Gag and virion levels. A HEK293T cells stably expressing EGFP-LC3 were co-transfected with NL4-3 Δ nef and either HA-GST (irrelevant protein), NL4-3 nef , or 48–49 NL4-3 nef . 48 h later, cells were stimulated with rapamycin (4 μM) for 4 h and autophagosome formation was examined by fluorescence microscopy. B The Pearson’s correlation coefficient for the Gag-LC3 co-localization was analyzed from three biological replicates. Scale bar: 10 μm. Images are representative of three independent experiments. C HEK293T were co-transfected with NL4-3 Δ nef and either an empty vector, NL4-3 nef , or 48–49 NL4-3 nef . 24 h later, cells were treated with DMSO or rapamycin (4 μM) for 12 h. The percentage of maximal virus release was determined by p24 ELISA of the culture supernatants. D Lysates were also analyzed by western blot for Gag, HA and ACTB. Significantly different values are indicated by asterisks * P ≤ 0.05

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Virus, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: HIV-1 transmitted/founder (T/F) viruses from subtypes B and C conserve the ability to antagonize autophagy. A , B HEK293T cells were transfected with the proviral DNA of HIV-1 NL4-3, HIV-1 NL4-3 Δ nef or the selected T/F clones that belong to the pandemic HIV-1 subtype B ( A ) or subtype C ( B ). 24 h later, the cell medium was replaced and supplemented with rapamycin (6.5 μM) or DMSO. 18 h later, the percentage of maximal virus production was measured by the accumulation of HIV p24 in the culture supernatant relative to the DMSO treatment. Bottom blots: Cell lysates were also analyzed by western blot for p55 and ACTB. In each case, the percentage of maximal virus production is indicated as the mean and SEM from 3 independent biological replicates. C , D HEK293T cells were co-transfected with EGFP -LC3B and the proviral DNA of HIV-1 NL4-3, HIV-1 NL4-3Δ nef or the selected T/F clones that belong to the pandemic HIV-1 subtype B ( C ) and subtype C ( D ). 48 h post-transfection, cells were treated with DMSO or rapamycin and subsequently analyzed by flow cytometry for autophagosome-associated EGFP-LC3. Data correspond to the mean and SEM from three independent replicates. Significantly different values are indicated by asterisks * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Transfection, Clone Assay, Virus, Western Blot, Flow Cytometry

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Antibody sources and conditions

Article Snippet: The expression constructs pC3- EGFP - LC3B (#11546, Dr. Karla Kirkegaard’s lab) and pcDNA4-BECN1-Flag (#24388, Dr. Qing Zhong’s lab) were obtained through Addgene.

Techniques: Microscopy