Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles

doi: 10.20517/evcna.2025.106

Figure Lengend Snippet: Kinetics of extracellular release of Nef from h-microglia infected with VSV-G pseudotyped HIV-1. Immunoblot analysis of cell lysates and extracellular particles (EVs + VIR) from h-microglia cultures infected with VSV-G pseudotyped NL4-3 and collected at the indicated time points. Extracellular particles were enriched from culture media by ultracentrifugation through a sucrose cushion. Antibodies directed against viral proteins (gp120, Nef, p24), typical EV proteins (Alix, Flotillin), marker of efficient gradient separation (AChE) and impurity markers (Calnexin, Albumin and Cytochrome c) were used. CL: Cell lysate; EV: extracellular vesicles; VIR: virus; AChE: acetylcholinesterase; Nef: negative factor; h-microglia: human microglia; VSV-G: vesicular stomatitis virus glycoprotein; HIV-1: human immunodeficiency virus type 1; NL4-3: HIV-1 strain NL4-3; gp120: glycoprotein 120; p24: HIV-1 core protein p24; Alix: ALG-2-interacting protein X; Flotillin: flotillin protein; Calnexin: calnexin protein; Albumin: serum albumin; Cytochrome c: cytochrome c protein.

Article Snippet: For production of pseudotyped HIV-1 viral stocks, HEK293T cells were transfected with plasmids encoding the HIV-1 isolate YU-2 (ARP-1350), or molecular clones NL4-3 (ARP-114) and Nef-deleted NL4-3 (NL4-3 Δnef, ARP-12755) (all obtained from the BEI Resources Repository, USA), together with a plasmid encoding vesicular stomatitis virus envelope protein (VSV-G; pCMV-VSV-G; Addgene, USA).

Techniques: Infection, Western Blot, Marker, Virus

Journal: Extracellular Vesicles and Circulating Nucleic Acids

Article Title: HIV protein Nef expression in human microglia drives the release of distinct Nef-containing extracellular vesicles

doi: 10.20517/evcna.2025.106

Figure Lengend Snippet: HIV-1-infected h-microglia release Nef primarily via extracellular vesicles. (A) EVs and virions pelleted from 5-day-old h-microglia cultures infected with VSV-G pseudotyped YU-2, NL4-3, NL4-3Δnef, or left uninfected (-) were further separated by 6%-18% iodixanol density gradient ultracentrifugation, and eleven fractions were collected. To support efficient separation of EVs and virions on the gradient; (B) AChE activity (y-axis) of fractions (x-axis) was quantified by colorimetric assay and (C) p24 concentration (y-axis) in fractions (x-axis) was quantified by HIV-1 p24 ELISA assay; (D) Immunoblot analysis of precipitated fractions using antibodies against AChE, Nef, p24, and Flotillin. EV: Extracellular vesicles; AChE: acetylcholinesterase; Nef: negative factor; Flotillin: flotillin protein; HIV-1: human immunodeficiency virus type 1; h-microglia: human microglia; VSV-G: vesicular stomatitis virus glycoprotein; YU-2: HIV-1 strain YU-2; NL4-3: HIV-1 strain NL4-3; NL4-3Δnef: HIV-1 NL4-3 strain with Nef gene deletion; p24: HIV-1 core protein p24; ELISA: enzyme-linked immunosorbent assay.

Article Snippet: For production of pseudotyped HIV-1 viral stocks, HEK293T cells were transfected with plasmids encoding the HIV-1 isolate YU-2 (ARP-1350), or molecular clones NL4-3 (ARP-114) and Nef-deleted NL4-3 (NL4-3 Δnef, ARP-12755) (all obtained from the BEI Resources Repository, USA), together with a plasmid encoding vesicular stomatitis virus envelope protein (VSV-G; pCMV-VSV-G; Addgene, USA).

Techniques: Infection, Activity Assay, Colorimetric Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Virus

Journal: Advanced Science

Article Title: A Heparan Sulfate Mimetic RAFT Copolymer Inhibits SARS‐CoV‐2 Infection and Ameliorates Viral‐Induced Inflammation

doi: 10.1002/advs.202411737

Figure Lengend Snippet: HMSA‐06 attenuates the inflammatory reaction induced by SARS‐CoV‐2 infection. A) The macrophage‐like THP‐1 cells were induced by LPS+ATP to activate the inflammation reaction in the presence or absence of various concentrations of HMSA‐06‐20 or HMSA‐06‐05. The expression of the mature caspase‐1 and IL‐1β in the supernatant as well as the pro‐caspase‐1 and pro IL‐1β in the cell pellets were assessed by western blot. The representative western blots of two independent experiments were shown. B) Macrophage‐like THP‐1 cells were induced by LPS+ATP in the absence or presence of HMSA‐06‐20. The ASC puncta were observed under the microscope and the representative pictures were shown. HMSA‐06 significantly decreased the number of ASC puncta. Scale bar, 30 µm. C) The macrophage‐like THP‐1 cells were treated with LPS+ATP to activate the inflammation reaction with or without adding HMSA‐06‐20 (2 µ m ) or heparin (100 µg mL −1 ). The expression of pro‐ or mature caspase‐1 and IL‐1β in cell pellets or supernatants were assessed by western blot. D) Macrophage‐like THP‐1‐ACE2 cells were infected by the SARS‐CoV‐2 prototype (MOI = 0.2) in the absence or presence of HMSA‐06‐20 (1 or 2 µ m ) with or without prime by LPS. The western blots show the expression of the pro or mature caspase‐1 and IL‐1β in cell pellets or supernatant as well as the expression of the SARS‐CoV‐2 S protein. The representative western blots of two independent experiments were shown. E) The representative images illustrate the ASC puncta in the SARS‐CoV‐2 infected macrophage‐like THP‐1‐ACE2 cells with or without adding the HMSA‐06‐20. Scale bar, 30 µm. F) Quantification of the cells with ASC puncta. The Mean ± SD of the percentage of the cells with ASC puncta from 30 images randomly taken from each condition is shown ( n = 30). Statistical analysis was performed using Student's t‐test. **** p ≤ 0.0001. G) The representative images show the NLRP3 puncta in the SARS‐2 (SARS‐CoV‐2) infected macrophage‐like THP‐1‐ACE2 cells in the presence or absence of HMSA‐06‐20 (2 µ m ) or MCC950 (10 µ m ). Scale bar, 30 µm. H) The Mean ± SD of the percentage of the cells with NLRP3 puncta from 30 images randomly taken from each condition in (E) is shown ( n = 30). Statistical analysis was performed using Student's t‐test. **** p ≤ 0.0001. ns, not significant. I) The macrophage‐like THP‐1‐ACE2 cells were infected using a high titer of SARS‐CoV‐2 (MOI = 5). The expression of active caspase‐1 and mature IL‐1β in the supernatant and the expression of SARS‐CoV‐2 S protein in cell pellets in the presence or absence of HMSA‐06‐20 (2 µ m ) were examined by western blot.

Article Snippet: Goat anti‐Mouse IgG (H+L) Cross‐Adsorbed Secondary Antibody, Alexa Fluor 488 (Invitrogen, A‐11001); Adenosine 5′‐triphosphate disodium salt hydrate (Sigma, A2383); Lipopolysaccharide (LPS) Solution (Invitrogen, 00‐4976‐93); CQ (InvivoGen, 50‐63‐5); heparin (Hepalink, Inj010702, Shenzhen); StrataClean Resin (Agilent,400714‐61); MCC950 (MedChemExpress, HY‐12815). pMRX‐IP‐GFP‐LC3‐RFP‐LC3ΔG was a gift from Noboru Mizushima (Addgene plasmid # 84 572; http://n2t.net/addgene:84572; RRID:Addgene_84572). pSELECT‐GFP‐hLC3 was purchased from InvivoGen (France, psetz‐gfplc3).

Techniques: Infection, Expressing, Western Blot, Microscopy