Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Experimental workflow. HIV-1 NL4.3 virions were cultured in suspension (S) or embedded in collagen matrices (DC: Dense collagen; LC: Loose collagen). TZM-bl cells were infected and the relative infectivity determined. b Normalized infectivity of HIV-1 NL4.3 particles. Virions were cultured in S, DC or LC for 16 h prior to relative infectivity determination. Data is normalized to suspension condition (gray dotted line). c Kinetics of ERVI. Culture supernatants were harvested at 0, 4, 8 or 16 h post seeding/embedding, the relative infectivity of virions was determined as in b . d Normalized infectivity of differentially cultured HIV-1 NL4.3 virions. PA: polymerizing agent. e . One representative Western Blot analysis showing the gp120/p24 ratio from HIV-1 NL4.3 virions cultured in S, DC or LC. The gp120/p24 ratios are indicated below the blots. f Quantification of western blot analyses from three independent experiments as in e . g Saturation of ERVI. Increasing amounts of virions were cultured in S, DC or LC prior to infectivity determination. h Normalized infectivity of HIV-1 NL4.3 particles after seeding or embedding in different matrices for 16 h. Data are normalized to suspension condition (gray dotted line). i Conservation of sensitivity to ERVI across lentiviruses. The normalized infectivity of a panel of lentiviruses was assessed after ERVI as in a . Data are normalized to respective suspension condition (gray dotted line). Results represent the mean ± SD of three independent experiments. Symbols indicate data from individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA test; Dunnett’s post-test ( b , d , h , i ), or by matched two-way ANOVA test; Tukey’s post-test ( g ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Cell Culture, Suspension, Infection, Western Blot

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Normalized infectivity of HIV-1 NL4.3 virus containing Vpr.Int.GFP fusion proteins after exposure to LC. b Experimental workflow to assess collagen deposition at the surface of virions. HIV-1 NL4.3 Vpr.Int.GFP virions were collected from S, LC or LCAF647 cultures and analyzed by microscopy. c Representative confocal micrographs of HIV1 NL4.3 Vpr.Int.GFP virions within AlexaFluor 647 stained LC matrices. Yellow arrows depict fluorescent virions. Scale bar: 20 µm. d Representative TIRF-micrographs of HIV-1 NL4.3 Vpr.Int.GFP virions were harvested from the supernatants of fluorescently labeled collagen matrices as in ( c ). Scale bar: 5 µm. e Averaged computational slices of a tomogram showing HIV-1 NL4.3 virions after 16 h of culture in S (left panel), DC (middle panel), or LC (right panel). Scale bar = 50 nm. f Quantification of the diameter of virus particles treated as indicated in d . Violin plot shows individual data points with corresponding median, 25% and 75% quartiles. g Averaged computational slices of a tomogram showing disrupted dense (left panel) or loose (right panel) collagen fibers. Scale bar = 100 nm. h Experimental workflow. HIV-1 NL4.3 virions retrieved from suspension or collagen matrices after 16 h of culture were harvested, and equivalent amounts of RT units were incubated with RM-8 or EF-C infectivity-enhancing peptide, nanofibrils (PNFs) or left untreated (UT) prior to infection of TZM-bl reporter cells. i Impact of infectivity enhancers on the infectivity of suspension or collagen primed virions. Results represent the mean ± SD of three independent experiments. Symbols indicate individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA, Dunnett’s post-test ( a ), unpaired t-tests ( d ), or by two-way ANOVA test with Geisser Greenhouse correction; Tukey’s post-test ( i ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Infection, Virus, Microscopy, Staining, Labeling, Suspension, Incubation

Journal: Nature Communications

Article Title: A tissue-intrinsic mechanism sensitizes HIV-1 particles for TLR-triggered innate immune responses

doi: 10.1038/s41467-026-72586-3

Figure Lengend Snippet: a Experimental workflow. HIV-1 NL4.3 Vpr.mRuby2 virions were cultured in S, DC or LC for 16 h. Equivalent amounts of RT units from culture supernatants or untreated virions were then incubated with TZM-bl cells for 2 h at 4 °C. Cell membranes were then stained with Concanavalin-A AF-488, prior to microscopy processing. b Normalized infectivity of HIV-1 NL4.3 Vpr.mRuby2 virions after 16 h of culture as in a . c Representative spinning disk micrographs of cells incubated with HIV-1 NL4.3 Vpr.mRuby2 virions. Yellow arrows indicate Vpr.mRuby2+ spots detected at the surface of the target cells. Scale bar: 15 µm. d Average binding frequency of HIV-1 NL4.3 Vpr.mRuby2 virions to target cells as shown in c . e Average volume of Vpr.mRuby2 spots detected as shown in c . f Experimental workflow. HIV1 NL4.3 Vpr.BlaM virions were as in a . Equivalent amounts of RT units from culture supernatants or untreated virions were then incubated with TZM-bl cells for 4 h at 37 °C, in the presence or absence of T20 fusion inhibitor. Cells were then loaded with the CCF2-AM and processed by flow cytometry. g Normalized infectivity of HIV-1 NL4.3 Vpr.BlaM virions after16h of culture as in a . h Representative flow cytometry dot plots of CCF2 loaded cells. Gates identify cleaved-CCF2+ cells. See Sup. Figure. for gating strategy. i Quantification of the percentage of CCF2-product positive cells measured by flow cytometry. j Correlation between Relative Infectivity and levels of CCF2-product positive cells after ERVI by linear regression. Results represent the mean ± SD of three independent experiments. Symbols indicate individual experiments. Significance is indicated by p -values, and was calculated by one-way ANOVA test; Dunnets’s post-test ( b , g ), or by one-way ANOVA test; Tukey’s post-test ( d , e , i ). Source data are provided as a file.

Article Snippet: 293T cells (ATCC, CRL-3216) and TZM-bl reporter cells (NIH AIDS Reagent Program, courtesy of Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc. (ARP-8129)) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS, Capricorn) and 1% penicillin/streptomycin (Gibco).

Techniques: Cell Culture, Incubation, Staining, Microscopy, Infection, Binding Assay, Flow Cytometry