Proviral changes in the autophagy status of infected cells differentially affect the outcome of secondary bacterial infection at the cellular level.

(A) A simplified representation of the ordered steps that constitute the autophagy flux (see main text) (AR: autophagy receptors). (B) Summary of available observations on the outcome of secondary bacterial infection of measles virus-infected epithelial cells (Salmonella Typhimurium or Shigella flexneri) or HIV-1-infected macrophages (Mycobacterium tuberculosis).</p

Syncytia formation induces autophagy in MeV-infected cells.

<p>(A) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz wild-type (Sch-MeV) or deficient for MeV-C expression (Sch-MeVΔC). 24 h post infection, the number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy in infected cells detected by staining for the viral nucleoprotein N (MeV-N). (B) GFP-LC3 HeLa cells were infected or not with Ed-MeV (MOI 3) or treated with rapamycin (Rapa) (125 nM), and treated or not with the FIP peptide (10 µg/mL). 24 h post infection, the number of GFP+ vesicles per cell was assessed by confocal microscopy. (C) HeLa cells were co-transfected with a vector encoding for the H protein of Ed-MeV and one encoding for the F protein. 24 h post transfection H/F-co-transfected HeLa cells (H⁺F⁺ HeLa) were co-cultured with GFP-LC3-HeLa cells to induce cell-cell fusion. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy after 18 h of co-culture. (D) GFP-LC3 HeLa cells and HeLa cells were treated with the indicated siRNA for 24 h. HeLa were then co-transfected with MeV-H/F as in (C) and co-cultured with siRNA treated GFP-LC3 HeLa cells. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy. (E) GFP-LC3 HeLa cells were treated with Polyethylene Glycol (PEG) and treated or not with 250 nM rapamycin (Rapa) for 2 h. The number of GFP+ vesicles within monocucleated or multinucleated cells was assessed by confocal microscopy 6 h after PEG treatment. For each experiment, representative profiles are shown and are accompanied by a graph representing the number of GFP+ vesicles per cell ( = GFP+ vesicles per one nucleus). For syncytia, the number of dots was normalized to the number of nuclei. Error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

The late wave of autophagy induced by attenuated MeV is productive.

<p>(A) HeLa cells were infected with Sch-MeV (MOI 1). At the indicated time points post infection, cellular p62 expression was determined by western blot. (B) HeLa cells were infected with Ed-MeV (MOI 1). At the indicated time points post infection, cellular p62 expression was determined by western blot. The graph represents the intensity of p62 over actin expression normalized to the control condition (non infected). Error bars, mean ± MD of two independent experiments. (C) HeLa cells were infected with Ed-MeV (MOI 3). 24 h p.i., cellular p62 expression was determined by western blot. Representative results are shown and are accompanied by a graph representing the intensity of p62 over actin expression normalized to the control condition (non infected). (D) mRFP-GFP-LC3 HeLa cells were infected with Ed-MeV (MOI 3) or treated with 250 nM rapamycin (Rapa) or 75 µm Chloroquine (CQ) during 2 h. 24 h p.i., the total number of autophagic vesicles (mRPP+) and the number of autophagosomes (mRFP+/GFP+) were assessed by confocal microscopy. The number of autolysosomes was determined by subtracting the number of autophagosomes (mRFP+/GFP+) from the total number of vesicles (mRPP+). Representative profiles are shown and are accompanied by a graph representing the number of autophagic vesicles (white), autophagosomes (yellow) and autolysosomes (red) per nuclear profile for each condition. (E–F) HeLa cells were treated with the indicated si-RNA for 48 h and infected with Ed-MeV (MOI 3 (E) or MOI 0.1 (F)). 24 h (E) or 48 h (F) post-infection, cells were lysed and anti-N and anti-P western blot were performed to reveal MeV-N and MeV-P, respectively. Representative results are shown and are accompanied by a graph representing the intensity of MeV-N or MeV-P expression over cellular actin normalized to the control condition. (C–F) : error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4⁺ T lymphocytes

HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes’ fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4+ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle. Abbreviations: BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4+ TL: CD4+ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.</p

The MeV-C protein is involved in MeV-induced autophagy.

<p>(A–B) GFP-LC3-HeLa cells were infected or not with Ed-MeV (MOI 3) and treated or not with 0.5 µg/ml cycloheximide (CHX) during 1.5 h (A) or 24 h (B), in the presence of 125 nM rapamycin (Rapa) when indicated. (C) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz (Sch-MeV) at an MOI 1. Autophagy was monitored by the numeration of GFP+ autophagosomes at the indicated period of time post infection. (D) GFP-LC3-HeLa cells were infected with the attenuated strain of MeV Schwarz wild-type (Sch-MeV) or deficient for MeV-C expression (Sch-MeVΔC). 24 h post infection, the total number of GFP+ vesicles per cell was assessed by confocal microscopy in infected cells detected by staining for the viral nucleoprotein N (MeV-N). For each experiment, graph represents the number of GFP+ vesicles per cell ( = GFP+ vesicles per one nucleus). For syncytia, the number of dots was normalized to the number of nuclei. Error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

Ed-MeV exploits autophagy to replicate.

<p>(A) HeLa cells were treated with the indicated si-RNA for 48 h and infected with Ed-MeV (MOI 3). One, two or three days post infection infectious viral particles were titrated by plaque assay. (B–D) HeLa cells were treated or not with 125 nM rapamycin (Rapa) or 50 µM chloroquine (CQ) and infected with Ed-MeV (MOI 1 (B) or MOI 0.1 (C–D)). Three days post infection infectious viral particles were titrated by plaque assay (B) or 48 h post-infection, cells were lysed and MeV-N (C), MeV-P (D) expression were detected by western blot. (C–D) Representative results are shown and are accompanied by a graph representing the intensity of MeV-N (C) or MeV-P (D) expression over actin, normalized to the non-infected condition. For each experiment, error bars, mean ± SD of three independent experiments. Student's t test; ***p<0.005; **p<0.01; *p<0.05; #p>0.05.</p

Impaired reprogramming of the autophagy flux in maturing dendritic cells from crohn disease patients with core autophagy gene-related polymorphisms

Crohn disease (CD) is an inflammatory bowel disease whose pathogenesis involves inappropriate immune responses toward gut microbiota on genetically predisposed backgrounds. Notably, CD is associated with single-nucleotide polymorphisms affecting several genes involved in macroautophagy/autophagy, the catabolic process that ensures the degradation and recycling of cytosolic components and microorganisms. In a clinical translation perspective, monitoring the autophagic activity of CD patients will require some knowledge on the intrinsic functional status of autophagy. Here, we focused on monocyte-derived dendritic cells (DCs) to characterize the intrinsic quantitative features of the autophagy flux. Starting with DCs from healthy donors, we documented a reprogramming of the steady state flux during the transition from the immature to mature status: both the autophagosome pool size and the flux were diminished at the mature stage while the autophagosome turnover remained stable. At the cohort level, DCs from CD patients were comparable to control in term of autophagy flux reprogramming capacity. However, the homozygous presence of ATG16L1 rs2241880 A>G (T300A) and ULK1 rs12303764 (G/T) polymorphisms abolished the capacity of CD patient DCs to reprogram their autophagy flux during maturation. This effect was not seen in the case of CD patients heterozygous for these polymorphisms, revealing a gene dose dependency effect. In contrast, the NOD2 rs2066844 c.2104C>T (R702W) polymorphism did not alter the flux reprogramming capacity of DCs. The data, opening new clinical translation perspectives, indicate that polymorphisms affecting autophagy-related genes can differentially influence the capacity of DCs to reprogram their steady state autophagy flux when exposed to proinflammatory challenges. Abbreviation: BAFA1: bafilomycin A1, CD: Crohn disease; DC: dendritic cells; HD: healthy donor; iDCs: immature DCs; IL: interleukin; J: autophagosome flux; LPS: lipopolysaccharide; MHC: major histocompatibility complex; nA: autophagosome pool size; SNPs: single-nucleotide polymorphisms; PCA: principal component analysis; TLR: toll like receptor; τ: transition time; TNF: tumor necrosis factor.</p