Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Schematic overview of structure-based engineering and functional validation of PFV Env mutants for enhanced heparan sulfate binding. (A) Structure-based strategy to identify key residues in the prototype foamy virus (PFV) Env protein responsible for heparan sulfate (HS) binding. The workflow begins with PFV Env amino acid sequence-based structure prediction, followed by docking simulations with HS to identify the interacting residues. Key amino acids predicted to mediate HS binding were selected and substituted to generate Env point mutants, which were subsequently cloned into expression vectors for functional testing. (B) Experimental workflow for PFV vector production and transduction assays. Transfer plasmid encoding EGFP reporter and Gag/Pol, together with the helper plasmid expressing either wild type or mutant Env were co-transfected into HEK293FT cells. The resulting viral particles were harvested and used to transduce the HT1080 cells. After 3 days of incubation, transduction efficiency and Env functionality were assessed using fluorescence microscopy, Western blotting, and flow cytometry.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Biomarker Discovery, Binding Assay, Virus, Sequencing, Clone Assay, Expressing, Plasmid Preparation, Transduction, Mutagenesis, Transfection, Incubation, Fluorescence, Microscopy, Western Blot, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Functional characterization of PFV Env upper domain point mutants affecting HS binding and viral entry efficiency. (A) The structure shown represents the receptor-binding domain (RBD) of the PFV Env surface subunit (SU), modeled using AlphaFold2. The molecular docking pose of heparan sulfate (HS) is displayed on the positively charged pocket of the RBD upper domain (UD), with key interacting residues (R298, R440, and E446) highlighted. The binding free energy which calculated from Autodock-vina, −7.0 kcal/mol is labeled. The molecular docking simulation revealed three key residues, Arg(R)298, Arg(R)440, and Glu(E)446, located in UD. These residues form hydrogen bonding with negatively charged sulfate group of HS and are highlighted in red and cyan for negatively charged and positively charged residues, respectively. Based on this structural analysis, R298A, R440A, and E446A substitutions were designed to disrupt the HS binding. The distance between key residues and HS are labeled with black arrow. (B) Western blot analysis was performed to confirm the protein expression of wild type and Env variants (R298A, R440A, and E446A) in HEK293FT cells. The cells were co-transfected with the PFV transfer vector (v3) and Env plasmids at a 30:1 ratio. All three mutants showed Env protein levels comparable to those of the wild type, indicating that the point mutations did not affect protein expression or stability. β -actin was used as a loading control. Mock, HEK293FT cell line served as a negative control; PC, HEK293FT cells transfected pCMV-Env were used as a positive control. (C) To assess the effect of each Env variant on viral infectivity, supernatants from transfected HEK293FT cells were used to transduce HT1080 cells. Transduction efficiency was monitored by EGFP expression using fluorescence microscopy after 3 days. Phase-contrast images confirmed the equivalent cell density across conditions. Scale bars = 100 μm. (D,E) Flow cytometry was used to quantitatively measure the proportion of EGFP-positive HT1080 cells transduced with each viral construct. Representative histograms and bar graph quantifications ( N ≥ 3) are shown in (D) and (E) , respectively. The average number from at least three independent experiments is shown at the top of each bar. Error bars represent the standard deviation of biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Labeling, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Negative Control, Positive Control, Variant Assay, Infection, Transduction, Fluorescence, Microscopy, Flow Cytometry, Construct, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Structure-guided refinement of PFV Env mutants to dissect the HS-binding determinants in the upper domain. (A) Two-dimensional interaction diagram showing the predicted hydrophobic and electrostatic interactions between selected residues and HS sulfate groups. (B) Schematic representation of the domain organization of PFV Env (1–988 aa), highlighting the receptor-binding domain (RBD; aa 217–570), including upper domains (UD; aa 244-312, 375-494) based on structural predictions. (C) Western blot validation of Env protein expression in HEK293FT cells transfected with the PFV transfer vector and one of the six-point mutant Env constructs. The co-transfection ratio (30:1) was consistent with that of previous experiments. β -actin served as a loading control. Mock, HEK293FT cell line served as a negative control. (D) Fluorescence microscopy showing the differential infectivity of PFV vectors with each Env variant in HT1080 cells, as measured by EGFP expression. Phase-contrast images confirmed similar cell confluency across all conditions. Scale bars = 100 μm. (E,F) Flow cytometry was used to quantitatively measure the proportion of EGFP-positive HT1080 cells transduced with each viral construct. Representative histograms and bar graph quantifications ( N ≥ 3) are shown in (E,F) , respectively. The average number from at least three independent experiments is shown at the top of each bar. Data are presented as mean ± SEM of biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Binding Assay, Western Blot, Biomarker Discovery, Expressing, Transfection, Plasmid Preparation, Mutagenesis, Construct, Cotransfection, Control, Negative Control, Fluorescence, Microscopy, Infection, Variant Assay, Flow Cytometry, Transduction

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Functional screening of lower-domain Env variants. (A) Molecular docking simulation of the PFV Env RBD, highlighting candidate HS-binding residues in the lower domain (circled green). (B) Two-dimensional interaction diagram illustrating predicted hydrophobic and electrostatic interactions between selected lower domain residues and HS sulfate groups. (C) Schematic representation of PFV Env organization (1-988 aa), showing the localization of lower domain (LD) subregions (aa 217-243, 313-374, and 495-570), as determined by structural modeling. Guided by structure-based docking, seven candidate residues within the LD were selected for mutagenesis based on side-chain polarity and the potential for π-stacking interactions to enhance HS binding. The specific amino acid substitutions and their rationales are listed. (D) Western blot analysis of Env expression in HEK293FT cells co-transfected with vectors encoding each LD point mutant and the PFV packaging system. Mock, HEK293FT cell line served as a negative control. (E,F) EGFP fluorescence (E) and corresponding flow cytometry histograms (F) of HT1080 cells transduced with PFV particles carrying individual LD variants and representative flow cytometry histograms corresponding to these samples. (H,I) EGFP fluorescence imaging (H) and flow cytometry histograms (I) of HT1080 cells transduced with PFV particles containing double (LD var1,7; LD var5,6) or triple (LD var1,5,6; UD var5 and LD var5,6) variants. (G,J) Quantification ( N ≥ 3) of GFP-positive HT1080 cells by flow cytometry, summarizing means ± SEM from biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Mutagenesis, Western Blot, Expressing, Transfection, Negative Control, Fluorescence, Flow Cytometry, Transduction, Imaging

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Structure-based functional screening of chimeric PFV-SFV Env variants to identify critical residues in HS-mediated viral entry. (A) Structural alignment of the SFV RBD crystal structure (PDB: 8AEZ) with the PFV-SFV gorⅠⅠ chimera RBD predicted by Swiss-Model and AlphaFold2 (AF2). (B) Molecular docking simulation of the PFV-SFV chimera RBD, highlighting the potential HS-binding residues in the lower domain (circled in green). (C) Zoomed view of the interaction interfaces and key residues (upper panel) and two-dimensional interaction diagram (bottom panel) showing predicted hydrophobic and electrostatic interactions between selected residues and HS sulfate groups. (D) Schematic representation of the PFV Env domain organization, with the SFV gorⅠⅠ RBD (aa 208-557) replacing the PFV RBD (aa 217-570). Specific amino acid substitutions (var1-6) and their rationales are listed. (E) Western blot analysis confirmed the expression of wild type chimeric Env (PFV backbone with SFV RBD) and six chimeric Env point mutants (variants 1–6) in the HEK293FT cells. β-actin was used as a loading control. PFV wild type Env (PFV WT Env)-transfected HEK293FT cells were used as a positive control. Mock, HEK293FT cell line served as a negative control. (F) Infectivity of each construct was assessed by EGFP fluorescence in HT1080 cells transduced with the PFV vectors. (G) Flow cytometry analysis quantified the proportion of GFP-positive cells for PFV WT Env, chimeric WT Env, and chimeric Env.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Binding Assay, Western Blot, Expressing, Control, Transfection, Positive Control, Negative Control, Infection, Construct, Fluorescence, Transduction, Flow Cytometry

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Structure-guided engineering of prototype foamy virus Env identifies key residues for heparan sulfate binding and enhances transduction efficiency

doi: 10.3389/fbioe.2026.1716928

Figure Lengend Snippet: Establishment and functional validation of Tet-On–inducible PFV Env-expressing HEK293T cell lines. (A) Workflow for generating stable cell lines. HEK203FT virus producer cells were co-transfected with three plasmids (pCMV-Gag/Pol, pMD-VSV-G, and pLVX-Transgene). Lentiviral particles carrying the transgene were harvested and used to transduce the HEK293T cells. Stable cell populations were established through puromycin selection. (B) Schematic representation of the Tet-On doxycycline (Dox)-inducible system used to drive PFV Env expression in HEK293T cells. The Env cassette was stably integrated into the lentiviral vectors. (C) Western blot analysis confirmed the establishment of HEK293T cells harboring Tet-Env cassettes. Cell lysates were analyzed by Western blotting using anti-Env and anti-β-actin antibodies. The letters above the lanes indicate Env type and Dox treatment. NC, HEK293FT cell line served as a negative control; PC, PFV wild type Env (PFV WT Env)-transfected HEK293FT cells were used as a positive control. (D) Functional validation using an infection assay. HT1080 cells were infected with viral particles produced from Tet-Env-293T clones following Dox induction. Successful production of infectious viruses was confirmed by GFP expression observed under fluorescence microscopy. (E) Flow cytometry analysis of HT1080 cells infected with supernatants from individual TetEnv-293T clones with or without Dox treatment. (F) Quantification ( N ≥ 3) of GFP-positive HT1080 cells by flow cytometry, summarizing means ± SEM from biological triplicates.

Article Snippet: The human fibrosarcoma cell line HT1080 was obtained from the Korean Cell Line Bank, and the human embryonic kidney cell line HEK293FT was obtained from Thermo Fisher Scientific (USA).

Techniques: Functional Assay, Biomarker Discovery, Expressing, Stable Transfection, Virus, Transfection, Transduction, Selection, Western Blot, Negative Control, Positive Control, Infection, Produced, Clone Assay, Fluorescence, Microscopy, Flow Cytometry

Journal: Neuro-Oncology Advances

Article Title: Effects of short- and long-term mutant IDH1 inhibition on radiosensitivity across genetically diverse patient-derived IDH1-mutant glioma cells

doi: 10.1093/noajnl/vdag057

Figure Lengend Snippet: Short- and long-term AGI-5198 treatment does not alter cell viability responses to irradiation in IDH1-mutant fibrosarcoma cells. (a) Intracellular 2-hydroxyglutarate (2-HG) levels measured after 5-day or 5-week AGI-5198 (AGI) treatment (5 μM), and after washout following 5-week treatment in IDH1-mutant HT1080 cells. (b) Workflow of the AGI-5198 treatment and radiation experiment (created with BioRender.com) (c) Clonogenic assay of HT1080 cells treated with DMSO or AGI-5198 (5 μM) for 5 days prior to irradiation (0, 1, or 2 Gy). (d) Cell viability at 72 h after irradiation (0-10 Gy) following 5-day or 5-week AGI-5198 treatment (5 μM). Data are shown as mean ± SEM; ns indicates not significant; * P < .05; ** P < .01; **** P < .0001.

Article Snippet: The human fibrosarcoma cell line HT1080 (IDH1R132C) was obtained from ATCC and authenticated by STR profiling to match the ATCC dataset.

Techniques: Irradiation, Mutagenesis, Clonogenic Assay